靶向VEGF-A基因的2-甲氧乙基修饰的反义寡聚核苷酸对卵巢癌细胞SKOV3的抑制效应的研究
|
摘要
卵巢癌是妇科最常见的恶性肿瘤之一,因其在女性群体中发病率高、局部侵袭和远处转移能力强,多年来一直是妇产科研究的热点问题。肿瘤血管形成是指新生血管由瘤外长入瘤内或在瘤内扩布的过程,是肿瘤生长侵袭所必不可少的条件,对于肿瘤的发生、发展和预后起着至关重要的作用。在参与这一过程的多种因子中,血管内皮生长因子被认为是其中最关键的一类。血管内皮生长因子(vascular endothelial growth factor, VEGF)亦称血管通透因子(vascular permeability factor, VPF),是一种特异地作用于血管内皮细胞的生长因子,广泛分布于人和动物体内的大脑、肾脏、肝脏、脾脏、胰腺和骨骼等器官和组织中,1989年由Ferrara等人从牛垂体滤泡星状细胞的体外培养液中首先纯化出来并命名。血管内皮生长因子(VEGF)是血小板来源生长因子家族中的一种多肽类细胞因子,是迄今发现的最强的促血管生长因子之一。其特异性作用于血管内皮细胞,通过促进内皮细胞增殖、增加血管通透性来诱导肿瘤血管生成,在肿瘤的发生及发展过程中发挥着不可替代的作用。如何通过抑制血管内皮生长因子(VEGF)来治疗恶性肿瘤已经成为肿瘤治疗研究领域的新方向。反义寡聚核苷酸(antisense oligonucleotide, ASODN)技术作为目前新兴且日益成熟的基因沉默技术,被认为是目前基因工程学领域非常有效的工具性研究技术。该技术可以特异、高效且稳定地沉默靶基因的表达,从而达到相对比较理想的沉默效果。目前,以ASODN为代表的小分子靶向干扰技术已被广泛地应用于基因功能研究、疾病病因与治疗研究等多种生命科学领域,并初见成效。本实验研究目的是：①检测VEGF在卵巢癌组织中的基因和蛋白表达,探讨VEGF与卵巢癌侵袭性的相关性以及它们之间的内在联系。②设计并构建能有效转导进入SKOV3卵巢癌细胞的针对VEGF-A基因的2-甲氧乙基修饰的ASODN;③研究该寡聚核苷酸对SKOV3卵巢癌细胞中VEGF-A基因的沉默效果,并进一步明确沉默前后SKOV3卵巢癌细胞的增殖及凋亡水平变化,评估2-甲氧乙基修饰的ASODN能否作为一种靶向VEGF-A基因的卵巢癌的基因治疗与研究的有效手段。④构建SKOV3卵巢癌细胞的裸鼠皮下种植模型,观察利用超声微泡技术介导2-甲氧乙基修饰的ASODN对裸鼠皮下卵巢癌的生长抑制效应,并对肿瘤抑制率进行测定。评估超声微泡技术介导2-甲氧乙基修饰的ASODN对卵巢癌的抑制作用。
第一部分VEGF-A在卵巢癌中的表达及与生物学行为的关系
目的
血管内皮生长因子(VEGF)是目前已知作用最强的一种促血管生长因子,其基因在肿瘤中的表达与肿瘤的侵袭性和降低的患者生存率关系密切。本研究目的是检测VEGF在卵巢癌中的基因和蛋白的表达,并探讨VEGF-A与卵巢癌恶性程度和侵袭性的相关性以及它们之间的内在联系。
方法
1.标本
收集2008年7月-2010年4月期间在山东省立医院妇科住院并行手术治疗的患者132例。①卵巢癌组68例,年龄29-77岁,平均年龄(55.3±6.3)岁；其中浆液性腺癌31例,子宫内膜样腺癌23例,粘液性腺癌14例；临床分期为Ⅰ、Ⅱ期为25例,Ⅲ、Ⅳ期为43例。所有患者均为第一次接受手术治疗,术前无化疗史,无其它肿瘤病史。②卵巢良性肿瘤患者组34例,年龄29-70岁,平均年龄(39.7±7.5)岁；其中浆液性囊腺瘤22例,粘液性囊腺瘤12例。③正常对照组患者30例,年龄42-70岁,平均年龄(54.1±7.1)岁；来自子宫肌瘤、功血、子宫脱垂等切除子宫同时行卵巢切除,病理证实卵巢无异常发现者。以上所有诊断均经术后病理证实。
2.免疫组化染色方法
采用VEGF-A鼠抗人单克隆抗体,采用免疫组化链霉菌抗生物素蛋白-过氧化物酶连接(S-P)法,按标准实验流程,检测VEGF-A基因在卵巢癌、卵巢良性肿瘤和正常卵巢组织中的蛋白表达。
3.结果判定标准
根据半定量免疫组织化学评价方法评价切片中VEGF-A反应阳性细胞显色强度。在高倍视野(×400)下,每张切片随机取5个视野进行观察,计算500个肿瘤细胞免疫反应阳性的细胞百分比。每张切片分别有五名实验者进行单独评价,对于有分歧的应进行重复评价,直至达到一致。
4. VEGF-A表达评估
强阳性(+++)：阳性细胞大于50%或显色深；中度阳性(++)：阳性细胞30%至50%之间或染色略深；弱阳性(+)：阳性细胞小于30%或显色浅；阴性(-)：无阳性细胞染色。
5. FQ-PCR检测VEGF-AmRNA的表达差异
取出150mg冰冻卵巢癌组织标本,按标准实验流程,抽提RNA并进行逆转录。利用FQ-PCR技术检测目的基因VEGF-A的mRNA表达情况。
6. Wertem-blot检测VEGF-A蛋白水平的表达差异
取200mg冰冻组织标本,按照实验过程,提取VEGF-A蛋白,利用Wertem-blot检测目的基因VEGF-A的蛋白水平的表达差异。
6.统计学处理
所有统计学分析均在SPSS13.0软件上进行。对免疫组化结果用卡方检验进行统计分析；对FQ-PCR半定量结果及Wertem-blot结果进行单因素方差分析,设定p<0.05有统计学意义。
结果
1.免疫组织化学结果
VEGF-A在卵巢癌中以细胞质着色为主,仅有散在的细胞核着色。VEGF-A在卵巢癌组织中的阳性(率)为58(85.3%)；在卵巢良性肿瘤中VEGF-A的阳性(率)为12(35.3%)；而在正常卵巢组织中VEGF-A的阳性(率)仅为4(13.3%)。卡方检验示卵巢癌与卵巢良性肿瘤之间的差异有统计学意义(χ2=22.310,P=0.0001<0.05)；卵巢良性肿瘤与正常卵巢组织之间的差异有统计学意义(χ2=5.074,P=0.024<0.05)；卵巢癌与正常卵巢组织之间的差异有统计学意义(χ2=46.382,P=0.00002<0.05)。临床分期为Ⅰ、Ⅱ期的卵巢癌组织中VEGF-A的阳性率为21(84.0%),Ⅲ、Ⅳ期的卵巢癌组织中VEGF的阳性率为37(86.0%)。VEGF在Ⅰ、Ⅱ期与Ⅲ、Ⅳ期的卵巢癌组织中的表达之间的差异没有统计学意义(χ2=0.053,P=0.818)。在免疫组化反应为阳性的卵巢癌组织中以强阳性和中度阳性为主(60.3%,35/58),而在卵巢良性肿瘤中以弱阳性为主(72.7%,8/11)。
2.FQ-PCR结果
经方差分析,卵巢癌、卵巢良性肿瘤及正常卵巢组织之间VEGF-A的mRNA表达差异明显：卵巢癌vs卵巢良性肿瘤(P<0.05),卵巢癌vs正常卵巢组织(P<0.05),卵巢良性肿瘤vs正常卵巢组织(P<0.05),其中VEGF-A在Ⅰ、Ⅱ期与Ⅲ、Ⅳ期的卵巢癌的之间的表达无明显差异(P=0.78)。
3. Wertern-blot
经方差分析,卵巢癌、卵巢良性肿瘤及正常卵巢组织之间VEGF-A的蛋白水平表达差异明显：卵巢癌vs卵巢良性肿瘤(P<0.05),卵巢癌vs正常卵巢组织(P<0.05),卵巢良性肿瘤vs正常卵巢组织(P<0.05)
结论
1.VEGF-A在正常卵巢组织、卵巢良性肿瘤和卵巢癌中均有表达。
2.VEGF-A在卵巢癌、卵巢良性肿瘤和正常卵巢组织中的表达有显著性差异,因此VEGF-A与卵巢癌的恶性程度密切相关。
3.VEGF-A可以作为判断卵巢癌侵袭性生长的诊断性指标和用做卵巢癌基因治疗研究的靶点。
第二部分设计靶向VEGF-A基因的2-甲氧乙基修饰的反义寡聚核苷酸并测定其转染效率
目的
设计针对VEGF-A基因的2-甲氧乙基修饰的ASODN,并验证其转染效率,为下一步的研究做准备。
方法
1.设计针对VEGF-A基因的2-甲氧乙基修饰的ASODN
本实验所用的A、B、C三种不同的靶向VEGF-A基因的ASODN (ASODN)均由美国Oligos Etc Inc(Oregon)设计并合成后免费馈赠。
2.2-甲氧乙基修饰的ASODN转染效率的测定
取培养中生长状态良好的SKOV3卵巢癌细胞,在转染前一天将细胞种在6-孔培养板进行培养,按实验设计的分组情况在转染当天加入含有针对VEGF-A基因的2-甲氧乙基修饰的不同ASODN进行细胞的转染实验。然后在转染48小时后在荧光显微镜下观察红色荧光蛋白RFP的表达情况,观测载体的转染效率。
结果
在3组针对VEGF-A基因的2-甲氧乙基修饰的ASODN中,1#、2#、3#转染效率分别86.5±4.5%、91.2±5.7%、92.1±2.8%
结论
1.本部分实验说明,针对VEGF-A的2-甲氧乙基修饰的ASODN可以有效的转染目的细胞.
2.通过该部分实验,为下一步用反义寡居核苷酸对卵巢癌细胞SKOV3进行实验提供了可靠的基础。
第三部分利用2-甲氧乙基修饰的反义寡聚核苷酸沉默VEGF-A基因并检测该抑制作用对卵巢癌细胞SKOV3增殖及凋亡水平的影响
目的
利用构建好的针对VEGF-A基因的2-甲氧乙基修饰的ASODN,研究不同RNA ASODN对卵巢癌SKOV3细胞VEGF-A基因的抑制情况以及对卵巢癌细胞增殖和凋亡的影响及其意义。
方法
1.实验分组
1)：空白对照组：SKOV3 cells (no ASODNs and no lipofectin),
2)：阴性对照组：SKOV3/Silence (-) cells(no ASODNs),
3)：实验组1#：SKOV3/Silencel (+) cells,
4)：实验组2#：SKOV3/Silence2 (+) cells,
5)：实验组3#：SKOV3/Silence3 (+) cells,
其中,实验组1#、2#、3#为分别转染1#、2#、3#ASODN的卵巢癌细胞组,阴性对照组不含ASODN,空白对照组不含ASODN及脂质体,其余处理同对照组。
2.按实验分组,分别用含有针对VEGF-A基因的新型ASODN1#、2#、3#转染卵巢癌细胞株SKOV3细胞。
首先培养生长状态良好的SKOV3细胞；在转染前一天将SKOV3细胞种入6-孔培养板进行培养,转染当天,按实验设计的组别分别将含有反义寡聚合甘酸1#、2#、3#转入卵巢癌细胞,阴性对照组不含ASODN,空白对照组不含ASODN及脂质体,其余处理同对照组。
3.测定VEGF基因的沉默效果
用FQ-PCR技术检测目的基因VEGF-A的mRNA表达情况,同时通过Western-blot方法检测VEGF-A蛋白表达,测定VEGF-A基因的沉默效果。
4.MTT检测细胞增殖水平
转染前一天,将生长状态良好的细胞种入96-孔培养板培养。第二天按实验分组分别进行转染,转染24小时后,进行MTT分析,检测不同ASODN对卵巢癌细胞株SKOV3细胞增殖水平的影响。
5流式细胞计数检测SKOV3细胞的凋亡情况
将生长状态良好稳定转染反义寡聚合甘酸的SKOV3细胞按实验设计的要求(空白对照组、实验组1#、实验组2#、实验组3#)进行流式细胞分析,检测经过实验处理的SKOV3细胞的细胞周期及凋亡情况。
结果
1.FQ-PCR结果显示,和对照组相比,反义寡居核苷酸1#、2#、3#对卵巢癌SKOV3细胞中VEGF-A基因的表达有显著沉默作用,它们的沉默效率分别为68.4%、69.4%、55.7%,Western-blot结果显示了类似的沉默效果。
2.MTT检测细胞增殖水平实验结果表明,实验组1#,2#,3#的增殖抑制率(PIR)分别为42.5%、38.7%和44.5%,与阴性对照组和空白对照组相比差异有显著性(P<0.05),这一结果提示在VEGF-A基因被沉默后,卵巢癌细胞的增殖能力明显减弱,肿瘤细胞的生长能力明显被抑制。
3.流式细胞计数检测SKOV3细胞的凋亡结果说明,通过流式细胞计数分析不同处理的细胞群体中各时期细胞的比例,结果发现三个转染反义寡聚合甘酸SKOV3细胞的实验组与对照组比较,其沉默VEGF-A后,SKOV3细胞群中凋亡细胞显著增加(P<0.05)。
结论
1.2-甲氧乙基修饰的ASODN对SKOV3卵巢癌细胞株的生长和增殖能力具有明显的抑制作用；利用针对VEGF-A基因的ASODN特异性沉默VEGF-A基因能引起卵巢癌细胞生长周期迟缓、诱发卵巢癌细胞凋亡,VEGF-A基因能够作为卵巢癌瘤基因治疗的有效靶点。
2.针对VEGF-A基因的ASODN能够特异性沉默VEGF-A基因,在卵巢癌基因靶向治疗方面具有非常重要的研究及应用价值。
第四部分’超声微泡介导的靶向VEGF-A基因的2-甲氧乙基修饰的反义寡聚核苷酸抑制裸鼠卵巢癌生长的实验研究
目的
在体内验证靶向VEGF-A基因的2-甲氧乙基修饰的反义寡聚核苷酸对卵巢癌生长的抑制作用,并探讨超声微泡介导基因转染作用的有效性。
方法
30只Balb/c裸鼠皮下接种人卵巢癌细胞SKOV3后,待所有皮下种植瘤瘤体体积均超过300mm3后,随机将30只成瘤裸鼠分为3组,每组10只。分别为
①超声+脂质体微泡+靶向VEGF的2-甲氧乙基修饰的反义寡聚核苷酸组,即UM+ASODN组。该组中10只裸鼠每只均给予400μL脂质体/optison(?)微泡混合液／3#寡聚核苷酸(含2.5ug反义寡聚核苷酸)瘤中心注射。
②超声+脂质体微泡组即UM组,该组中10只裸鼠每给予100μLD-Hanks液+300μLoptison(?)微泡皮下种植瘤瘤中心注射
③空白对照组即BC组,该组中10只裸鼠,每只裸鼠均给予400μL D-Hanks液瘤中心注射。
所有30只裸鼠接受各自相应处理后,立即在瘤体表面皮肤涂以超声耦合剂,并给予1 MHz超声辐射,强度为1.0W/cm2,每次30s,间隔30s,共2次60s照射。伺候每3天用游标卡尺测量皮下肿瘤长径(a)及短径(b)变化,肿瘤体积根据公式V=πab2/6计算。15天后采用颈椎脱位法处死小鼠,称取瘤重。
统计学分析采用统计学软件SPSS16.0英文版进行,方差齐时多组均数比较采用方差分析,两两比较采用LSD检验；方差不齐时多组均数比较采用方差分析,两两比较采用DUNNETT's T3检验。设定p<0.01具有极显著性差异,p<0.05具有显著性差异。
结果
1.肿瘤的体积、瘤重与抑瘤率
(1) UM+ASODN组、UM组与BC组观察至超声照射后15天,裸鼠皮下种植瘤体积分别为108.2±26.42mm3,297.8±44.22mm3,339.7±41.32mm3。各组种植瘤体积不同(F=104.67,p=0.00<0.01)。LSD法两两比较UM+ASODN组与其他两组均有极明显差异(p=0.00<0.01)。表明UM+ASODN组裸鼠皮下种植瘤体积较其他两组较小且差异极为明显。UM组与BC组相比有明显差异(p=0.021<0.05),表明与BC组相比,UM组裸鼠皮下种植瘤体积明显较小。
(2) UM+ASODN组、UM组与BC组观察至超声照射后15天,裸鼠皮下种植瘤体积分别为0.70±0.08g,1.45±0.14g,1.66±0.23g。各组瘤重不同(F=93,87,p=0.00<0.01). DUNNETT's T3法两两比较UM+ASODN组瘤重较其他两组有极明显差异(p=0.00<0.01), UM+ASODN组裸鼠皮下种植瘤瘤重较其他两组较小且差异极为明显;UM组与BC组差异不具有统计学意义(p=0.078)0.05)。
(3)超声辐射15天后处死裸鼠时,可见UM+ASODN组裸鼠皮下种植瘤肿瘤体积较小,颜色苍白,与周围组织边界较为清楚,局部存在类似包膜组织包裹；而UM组与BC组裸鼠皮下种植瘤瘤体较大,色红,血管组织丰富,呈浸润性生长,与周围组织边界不清。
结论
1.本实验成功构建了SK0V3人卵巢癌皮下种植的裸鼠模型。
2.实验中成功利用超声联合optison(?)造影剂微泡介导VEGF-A基因的2-甲氧乙基修饰的反义寡聚核苷酸转染处理了SKOV3人卵巢癌裸鼠皮下种植的裸鼠模型
3.实验结果验证了体内环境下超声联合微泡造影剂optison(?)介导靶向VEGF-A的的2-甲氧乙基修饰的反义寡聚核苷酸转染卵巢癌SKOV3细胞极大的抑制了肿瘤的生长
Ovarian cancers, one of the most common malignancies in gynecological cancers, have been the focus of gynecological research due to its high incidence rate, invasion and migration. Tumor angiogenesis is a process involving the growth of new blood vessels within or toward the tumor and is essential for tumor itself to grow and metastasize. Vascular endothelial growth factor (VEGF), also called vascular permeability factor (VPF), is a growth factor acting on the membrane of vascular endothelial cells. VEGF is widely expressed in human and animal tissues such as the brain, kidney, liver, spleen and bones. It was first purified from the bovine pituitary follicular stellate cells in vitro culture medium and was named by Ferrara and others in 1989. VEGF, a polypeptide cytokine of platelet-derived growth factor family, is one of the strongest vascular growth factor ever found. VEGF acts specifically on vascular endothelial cells and plays a key role in tumor genesis and growth by promoting endothelial cell proliferation and increasing the vascular permeability to induce tumor angiogenesis. Targeted tumor therapy by inhibiting VEGF expression has been widely investigated recently.
As a new and fastly developing gene silencing technology, antisense oligonucleotides (ASODN) technology is regarded as a very effective tool in the field of genetic engineering. Antisense oligonucleotides technology can inhibit the expression of target genes in a specific, efficient and stable manner and can achieve relatively satisfactory gene silencing effect. Currently, ASODN has been widely and successfully applied to study the function of genes, the etiology and treatment of a variety of diseases.
The purpose of this study is to:①determine the gene and protein expression of VEGF in ovarian cancers, in order to clarify the correlation between VEGF and the migration of ovarian cancers;②design and synthesize stable and efficient 2' -Methoxyethyl modified antisense oligonucleotides which can be transferred into ovarian cancer cells against VEGF gene;③study the silencing effects of the antisense oligonucleotides against VEGF-A expression in SKOV3 ovarian cancer cells, and further clarify the change of proliferation and apoptosis levels of SKOV3 cells before and after transfection, in order to investigate whether VEGF-A can be used as a potentially effective therapeutic target in the ovarian cancer gene therapy.④construct a nude mice model bearing human ovarian cancer cell line SKOV3 and observe the inhibitory effect of ASODN transfected using ultrasound irradiation treatment to endermic tumors cells of transferred ovarian cancers ascites in mice and evaluate tumor suppressive rate to evaluate the suppressive effects of ultrasound irradiation treatment and ASODN against ovarian cancers.
Part I Expression of VEGF and its relationship with biological features in ovarian cancers Obejectve
Vascular endothelial growth factor (VEGF) is one of the strongest factors promoting the growth of blood vessels, its genetic expression is closely related to the increased invasion of tumor and decreased survival rates. The aim of this research is to determine the gene and protein expression of VEGF in ovarian cancers, clarifying the relationship between VEGF and the invasion and malignancy of ovarian cancers.
Methods
1 Samples preparation
All samples were obtained during 132 excision surgeries at Shandong Provicial Hospital affiliated to Shandong University during July 2008 and April 2010, including:①ovarian cancers (n=32), which include serous cystadenocarcinomas (n=31), endometrioid adenomas (n=23) and mucoid adenocarcinomams (n=14). The subjects' ages were ranged 29-77 years (mean age 55.3±6.3 years), they all received operations for the first time, no chemotherapy history, no tumor history, and all the diagnoses were confirmed by pathologicy.②benign ovarian tumors (n=34), including serous cystadenomas (n=22) and mucinous cystadenomas (n=12). The ages of the subjects were ranged 29-70 years old (mean age 39.7±7.5years).③normal control group (n=30), obtained from womb and ovary excison sugeries for hysteromyoma, dysfunctional uterine bleeding and hysteroptosis, and the ovary were confirmed as normal by histopathological studies. The ages of the subjects were ranged 42-70 years (mean age 54.1±7.1 years). All the diagnoses are confirmed by histopathological studies.
2 Histochemistry
The expression of VEGF protein in ovarian cancer, benign ovarian tumors and normal ovary tissues are determined by immunohistochemical staining with specific mouse anti-VEGF monoclonal antibody by streptavidin-peroxidase method (abbreviation:S-P).
3. Analysis of immunohistochemical results
VEGF immunoreactivity is evaluated semiquantitatively based on the intensity of staining.5 fields at a magnification of 400×at every slice are observed at random. The percent of positive staining cells in 200 tumor cells is calculated. The slides are analysed independently by four observers blinded for expression of VEGF.For the discrepancies a second evaluation course is running to reach agreement.
4. Evaluation of B\VEGF protein expression
The VEGF expression is scored as:strong (+++):if more than 50% of cells are positive or a strong diffuse reaction is seen; moderate (++):if less than 50% of cells are positive, or a moderate diffuse reaction is observed; slightly positive (+):if immunoreactions are found in less than 30% of tumour cells or the diffuse reaction is weak; and negative(-):if no VEGF is stained.
5. Fluorescent quantitive PCR (FQ-PCR)
Total RNA are extracted from frozen tissues obtained from ovary excision surgeries. cDNA is synthesized from total RNA by reverse transcription. mRNA expression of VEGF-A is determined by FQ-PCR.
6. Western-blot to examine the protein expression of VEGF-A
VEGF-A protein is extracted from 200mg of frozened ovarian cancer tissues according to standard experiment procedures, and then the difference of protein expression of VEGF-A is determined using western-blot.
7. Statistical treatment
All data are analyzed by statistical package for the social science 13 (SPSS 13.0). The data of histochemistry are treated by chi-square test, the semi-quantitive data of FQ-PCR and Wstern-blot were treated by one-way ANOVA, P<0.05 is considered significant value.
Results
1. Result of histochemistry
In the ovarian tissues, a cytoplasmic VEGF reaction predominates and only scattered cells exhibited nuclear reaction. VEGF is expressed in 58(85.3%) ovarian cancers, in 12(35.3%) benign ovarian neoplasms and 4(13.3%) normal ovarian tissues. The chi-square test demonstrates a statistically difference in VEGF expression between vicious ovarian tumors and benign ovarian neoplasms (x2=22.310, P=0.000<0.05), between benign ovarian neoplasms and normal ovarian tissues(x 2=5.074, P=0.024<0.05), between vicious ovarian tumors and normal ovarian tissues(x 2=46.382, P=0.000<0.05), respectively. Among vicious ovarian tumors whose clinical stage are stageⅢand stageⅣVEGF staining is positive 41 (95.3%), while 23 (92%) specimens are positive stageⅠand stageⅡvicious ovarian tumors. No statistical deifferece is found between stageⅠ&Ⅱand stageⅢ&Ⅳvicious ovarian tumors (x 2=0.32, P=0.571). Furthermore, the strong and moderate positives (60.3%, 35/58) are most common in vicious ovarian tumors, while in benign ovarian neoplasms is slight (72.7%,8/11).
2 FQ-PCR results
The onw-way analysis of variance(ANOVA) result showed the statistical difference of the expression of VEGF among ovarian cancers, benign ovarian neoplasms and normal ovarian tissues:ovarian cancers vs benign ovarian neoplasms(P<0.05), benign ovarian neoplasms vs normal ovarian tissue (P<0.05), ovarian cancers vs normal ovarian tissue (P<0.05). No statistical difference of expression of VEGF was found between stageⅠ&Ⅱand stageⅢ&Ⅳovarian cancers (P=0.78).
3.Western-blot analysis
The results of ANOVA showed that the differences of protein expression of VEGF-A among ovarian cancers, benign ovarian neoplasms and normal ovarian tissues were statistically significant:ovarian cancers vs benign ovarian neoplasms (P<0.05), benign ovarian neoplasms vs normal ovarian tissue (P<0.05), ovarian cancers vs normal ovarian tissue (P<0.05).
Conclusions
1. There is high expression of VEGF in ovarian cancers.
2. There is a statistically difference in VEGF expression between vicious and benign ovarian neoplasms. VEGF is highly associated with malignant pituitary adenomas.
3. VEGF can serve as a useful tool for confirmation of ovarian tumors and VEGF gene also may be an effective target for ovarian cancer gene therapy.
PartⅡDesign and synthesis of 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene and the assessment of the transfection efficiency
Objective
Design and synthesize 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene, and test the inhibitory efficiency of the antisense oligonucleotides.
Methods
1. Design and synthesize 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A
2'-Methoxyethyl modified antisense oligonucleotides were designed and synthesized by Oligos Etc Inc (Oregon),3 groups of ASODN targeting VEGF-A gene were synthesized and marked as 1#,2# and 3#, respectively.
2. Test the efficiency of antisence oligonucleotides transfection in SKOV3 ovarian cancer cells)
Firstly, SKOV3 ovarian cancer cells were cultured, and the cells in good growth condition were spread into a six-well cell culture plate one day before transfection. Secondly,2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A was delivered into cultured cells according to previous groups design. Then we measured RFP expression under fluorescence microscope 48 hours after transfection to observe the transfectory potency of the recombined vectors.
Results
In 3 test groups transfected with 2'-Methoxyethyl modified antisense oligonucleotides, the transfection potency values of 1#,2#,3# were 86.5±4.5%,91.2±5.7% and 92.1±2.8%, respectively.
Conclusions
In this part,3 groups of ASODN 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A were successfully designed and synthesized.
PartⅢThe effect of 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene on the proliferation and apoptosis level of SKOV3 ovarian cancer cells
Objective
Study the influence and significance of 2'-Methoxyethyl modified antisense oligonucleotides 1#,2# and 3#, targeting VEGF-A gene on the proliferation and apoptosis level of SKOV3 ovarian cancer cells.
Methods
1. Transfect SKOV3 cells with ASODN 1#,2# and 3#
Firstly, SKOV3 ovarian cancer cells were cultured in good growth condition, and then the cells were spread into the six-well culture plate one day before transfection. On the day of transfection, the experiment was performed with ASODN group 1#,2# and 3#, the negative control group and the blank control group according to the design of experiment.
2. Measurement of the silencing effect on VEGF-A gene expression
Real-time PCR was performed to measure the mRNA expression of VEGF-A and Western-blot was performed to measure the protein expression of VEGF-A, testing the silencing effect of VEGF-A gene.
3. Measurement of the proliferation level of cells with MTT
SKOV3 cells which were in good growth condition and were seeded in 96-well culture plate before. On the second day after transfection, MTT was performed using standard methods to measure the changes of proliferation level before and after transfection.
4. Investigation of pituitary tumor cell apoptosis level variation by PI dyeing FCM
Stable tansfecion SKOV3 are cultured in good condition. According to designed groups (ASODN 1#,2#,3# and controls), cells are collected. PI dyeing FCM method is employed to contrast the changes of cell apoptosis levels.
Results
1. Realtime-PCR results showed that, ASODN 1#,2#,3# had significant gene silencing effect against VEGF-A gene expression of SKOV3, and their silencing efficiencies were 68.4%、69.44%、55.7%, respectively. Western-blot showed a similar result.
2. MTT-detected cellular proliferation level test results showed that, the inhibition rate (IR) of experimental group 1#,2#,3# were 42.5%,38.7% and 44.5%, respectively, which had significant differences with the negative control group and the blank control group (P<0.05). The result indicated that when the VEGF-A gene were silenced, the proliferation of the ovarian cancer cells were significantly decreased, and the growth ability of the cancer cells were significantly inhibited.
3. Analyzing the proportion of all period cells in cell colony by FCM, we found that, after VEGF-A was silenced, the proportion of apoptosis cell increased in the three experimental groups, comparing with control group.
Conclusions
1.2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene can significantly silence VEGF-A mRNA and protein expression in SKOV3 ovarian cancer cells. Besides,2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene can significantly inhibit the the proliferation and growth potency in SKOV3 ovarian cancer cells. It may indicate that VEGF-A gene can act as an effective target in the ovarian cancer gene therapy.
2. The above results also indicate that 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A shows a promising potential in targeted gene therapy and research of ovarian cancers.
Part IV Exploiting Inhibitory Effects of the 2'-O-Methoxyethyl-Modified Antisense oligonucleotides targeting VEGF-A gene in Ovarian Cancer modle of nude mice by a novel combination:liposome microbubbles and site-specific ultrasound exposure
Objective
To validate the inhibitory effects of the 2'-O-Methoxyethyl-Modified Antisense oligonucleotides targeting VEGF-A gene in Ovarian Cancer in vivo and to exploit the effectiveness of the transfection mediated by liposome microbubbles and ultrasound exposure
Method
methods:30 Balb/c nude mice were subcutaneously inoculated human ovarian cancer cell line SKOV3. When the endermic tumors were all larger than 300mm3 in size,the mice were then randomly divided into 3 groups (10 mice each group):
①UM+ASODN (liposome microbubbles+ASODN+site-specific ultrasound exposure) group:400μL mixture of microbubble/ASODN/(with 2.5ug ASODN) /liposome was injected into the center of the tumor of every mice
②; UM (liposome microbubbles+site-specific ultrasound exposure, no ASODN) group:300μL of microbubbles and 100μL of D-Hanks were injected into the center of the tumor of every mice
③BC (blank control,no ASODN,no microbubbles,no ultrasound exposure) group: 400μL of D-Hanks was injected into the center of the endermic tumors.
After all these have been done,site-specific ultrasound irradiation was carried out to each mouse with the parameter settting on a frequency 1 MHz and the intensity 1.0w /cm2.The exposure was given 30 seconds twice with an 30 seconds interval.
The tumor volume was measured every 3 days from the irradiation on and the size of the endermic tumor was calculated according to the formula V=πab2/6 (a=length, b=width) 15 days after the irradiation, the nude mice were killed and the weight of the tumor was weighed.
All data are analyzed by statistical package for the social science 16 (SPSS 16.0). The data of the size and the volume of the endermic tumors are treated by Anova analysis,and the comparison of the size between every two groups were carried out by LSD test. While the comparison of the weight of the endermic tumors between groups were carried out by DUNNETT T3 test, p<0.01 is considered to be of extremely significant value and p<0.05 is considered to be of significant value.
Result
(1)The mean volume of endermic tumor of UM+ASODN group, UM group and BC group was 108.2±26.42mm3,297.8±44.22mm3,339.7±41.32mm3 respectively. The ANOVA analysis demonstrates an extremely significant differnec between groups (F=104.67,p=0.00<0.01). LSD test told us that the volume of the ASODN+UM group was obviously statistically smaller than UM group and BC group (p=0.00<0.01) and the volume was statistically smaller in UM group than BC group (p=0.021<0.05).
(2)the mean weight of nude mice tumor were 0.70±0.08g,1.45±0.14g,1.66±0.23g respectively. The weight of the ASODN+UM group was obviously statistically smaller than UM group and BC group (DUNETT T3 method,p=0.00<0.01).The analysis shows no significant difference between UM group and BC group(DUNETT T3 method,p=0.078>0.01).
(3)The autopsy of the nude mice demonstrated that something like membrane surrounded the pale, smaller-sized tumors of ASODN+UM group. The boundary between tumors and normal tissues was obvious;While in UM group arid BC group, the tumor was red,huger in size, abundant in blood supply and mingled with the normal tissue that cannot be easily dissected.
Conclusion
(1) We successfully set up the Balb/c nude mice model bearing human ovarian cancer cell line SKOV3.
(2) We successfully disposed of the nude mice model bearing human ovarian cancer cell line SKOV3 with Optison(?) ultrasound contrast agent microbubbles and ultrasound exposure.
(3) We found that the 2'-O-Methoxyethyl-Modified Antisense oligonucleotides targeting VEGF-A gene greatly decrease the growth of the tumor of the SKOV3 ovarian cancer cells in vivo with site-specific ultrasound exposure and Optison(?) contrast agent microbubbles.
第一部分VEGF-A在卵巢癌中的表达及与生物学行为的关系
目的
血管内皮生长因子(VEGF)是目前已知作用最强的一种促血管生长因子,其基因在肿瘤中的表达与肿瘤的侵袭性和降低的患者生存率关系密切。本研究目的是检测VEGF在卵巢癌中的基因和蛋白的表达,并探讨VEGF-A与卵巢癌恶性程度和侵袭性的相关性以及它们之间的内在联系。
方法
1.标本
收集2008年7月-2010年4月期间在山东省立医院妇科住院并行手术治疗的患者132例。①卵巢癌组68例,年龄29-77岁,平均年龄(55.3±6.3)岁；其中浆液性腺癌31例,子宫内膜样腺癌23例,粘液性腺癌14例；临床分期为Ⅰ、Ⅱ期为25例,Ⅲ、Ⅳ期为43例。所有患者均为第一次接受手术治疗,术前无化疗史,无其它肿瘤病史。②卵巢良性肿瘤患者组34例,年龄29-70岁,平均年龄(39.7±7.5)岁；其中浆液性囊腺瘤22例,粘液性囊腺瘤12例。③正常对照组患者30例,年龄42-70岁,平均年龄(54.1±7.1)岁；来自子宫肌瘤、功血、子宫脱垂等切除子宫同时行卵巢切除,病理证实卵巢无异常发现者。以上所有诊断均经术后病理证实。
2.免疫组化染色方法
采用VEGF-A鼠抗人单克隆抗体,采用免疫组化链霉菌抗生物素蛋白-过氧化物酶连接(S-P)法,按标准实验流程,检测VEGF-A基因在卵巢癌、卵巢良性肿瘤和正常卵巢组织中的蛋白表达。
3.结果判定标准
根据半定量免疫组织化学评价方法评价切片中VEGF-A反应阳性细胞显色强度。在高倍视野(×400)下,每张切片随机取5个视野进行观察,计算500个肿瘤细胞免疫反应阳性的细胞百分比。每张切片分别有五名实验者进行单独评价,对于有分歧的应进行重复评价,直至达到一致。
4. VEGF-A表达评估
强阳性(+++)：阳性细胞大于50%或显色深；中度阳性(++)：阳性细胞30%至50%之间或染色略深；弱阳性(+)：阳性细胞小于30%或显色浅；阴性(-)：无阳性细胞染色。
5. FQ-PCR检测VEGF-AmRNA的表达差异
取出150mg冰冻卵巢癌组织标本,按标准实验流程,抽提RNA并进行逆转录。利用FQ-PCR技术检测目的基因VEGF-A的mRNA表达情况。
6. Wertem-blot检测VEGF-A蛋白水平的表达差异
取200mg冰冻组织标本,按照实验过程,提取VEGF-A蛋白,利用Wertem-blot检测目的基因VEGF-A的蛋白水平的表达差异。
6.统计学处理
所有统计学分析均在SPSS13.0软件上进行。对免疫组化结果用卡方检验进行统计分析；对FQ-PCR半定量结果及Wertem-blot结果进行单因素方差分析,设定p<0.05有统计学意义。
结果
1.免疫组织化学结果
VEGF-A在卵巢癌中以细胞质着色为主,仅有散在的细胞核着色。VEGF-A在卵巢癌组织中的阳性(率)为58(85.3%)；在卵巢良性肿瘤中VEGF-A的阳性(率)为12(35.3%)；而在正常卵巢组织中VEGF-A的阳性(率)仅为4(13.3%)。卡方检验示卵巢癌与卵巢良性肿瘤之间的差异有统计学意义(χ2=22.310,P=0.0001<0.05)；卵巢良性肿瘤与正常卵巢组织之间的差异有统计学意义(χ2=5.074,P=0.024<0.05)；卵巢癌与正常卵巢组织之间的差异有统计学意义(χ2=46.382,P=0.00002<0.05)。临床分期为Ⅰ、Ⅱ期的卵巢癌组织中VEGF-A的阳性率为21(84.0%),Ⅲ、Ⅳ期的卵巢癌组织中VEGF的阳性率为37(86.0%)。VEGF在Ⅰ、Ⅱ期与Ⅲ、Ⅳ期的卵巢癌组织中的表达之间的差异没有统计学意义(χ2=0.053,P=0.818)。在免疫组化反应为阳性的卵巢癌组织中以强阳性和中度阳性为主(60.3%,35/58),而在卵巢良性肿瘤中以弱阳性为主(72.7%,8/11)。
2.FQ-PCR结果
经方差分析,卵巢癌、卵巢良性肿瘤及正常卵巢组织之间VEGF-A的mRNA表达差异明显：卵巢癌vs卵巢良性肿瘤(P<0.05),卵巢癌vs正常卵巢组织(P<0.05),卵巢良性肿瘤vs正常卵巢组织(P<0.05),其中VEGF-A在Ⅰ、Ⅱ期与Ⅲ、Ⅳ期的卵巢癌的之间的表达无明显差异(P=0.78)。
3. Wertern-blot
经方差分析,卵巢癌、卵巢良性肿瘤及正常卵巢组织之间VEGF-A的蛋白水平表达差异明显：卵巢癌vs卵巢良性肿瘤(P<0.05),卵巢癌vs正常卵巢组织(P<0.05),卵巢良性肿瘤vs正常卵巢组织(P<0.05)
结论
1.VEGF-A在正常卵巢组织、卵巢良性肿瘤和卵巢癌中均有表达。
2.VEGF-A在卵巢癌、卵巢良性肿瘤和正常卵巢组织中的表达有显著性差异,因此VEGF-A与卵巢癌的恶性程度密切相关。
3.VEGF-A可以作为判断卵巢癌侵袭性生长的诊断性指标和用做卵巢癌基因治疗研究的靶点。
第二部分设计靶向VEGF-A基因的2-甲氧乙基修饰的反义寡聚核苷酸并测定其转染效率
目的
设计针对VEGF-A基因的2-甲氧乙基修饰的ASODN,并验证其转染效率,为下一步的研究做准备。
方法
1.设计针对VEGF-A基因的2-甲氧乙基修饰的ASODN
本实验所用的A、B、C三种不同的靶向VEGF-A基因的ASODN (ASODN)均由美国Oligos Etc Inc(Oregon)设计并合成后免费馈赠。
2.2-甲氧乙基修饰的ASODN转染效率的测定
取培养中生长状态良好的SKOV3卵巢癌细胞,在转染前一天将细胞种在6-孔培养板进行培养,按实验设计的分组情况在转染当天加入含有针对VEGF-A基因的2-甲氧乙基修饰的不同ASODN进行细胞的转染实验。然后在转染48小时后在荧光显微镜下观察红色荧光蛋白RFP的表达情况,观测载体的转染效率。
结果
在3组针对VEGF-A基因的2-甲氧乙基修饰的ASODN中,1#、2#、3#转染效率分别86.5±4.5%、91.2±5.7%、92.1±2.8%
结论
1.本部分实验说明,针对VEGF-A的2-甲氧乙基修饰的ASODN可以有效的转染目的细胞.
2.通过该部分实验,为下一步用反义寡居核苷酸对卵巢癌细胞SKOV3进行实验提供了可靠的基础。
第三部分利用2-甲氧乙基修饰的反义寡聚核苷酸沉默VEGF-A基因并检测该抑制作用对卵巢癌细胞SKOV3增殖及凋亡水平的影响
目的
利用构建好的针对VEGF-A基因的2-甲氧乙基修饰的ASODN,研究不同RNA ASODN对卵巢癌SKOV3细胞VEGF-A基因的抑制情况以及对卵巢癌细胞增殖和凋亡的影响及其意义。
方法
1.实验分组
1)：空白对照组：SKOV3 cells (no ASODNs and no lipofectin),
2)：阴性对照组：SKOV3/Silence (-) cells(no ASODNs),
3)：实验组1#：SKOV3/Silencel (+) cells,
4)：实验组2#：SKOV3/Silence2 (+) cells,
5)：实验组3#：SKOV3/Silence3 (+) cells,
其中,实验组1#、2#、3#为分别转染1#、2#、3#ASODN的卵巢癌细胞组,阴性对照组不含ASODN,空白对照组不含ASODN及脂质体,其余处理同对照组。
2.按实验分组,分别用含有针对VEGF-A基因的新型ASODN1#、2#、3#转染卵巢癌细胞株SKOV3细胞。
首先培养生长状态良好的SKOV3细胞；在转染前一天将SKOV3细胞种入6-孔培养板进行培养,转染当天,按实验设计的组别分别将含有反义寡聚合甘酸1#、2#、3#转入卵巢癌细胞,阴性对照组不含ASODN,空白对照组不含ASODN及脂质体,其余处理同对照组。
3.测定VEGF基因的沉默效果
用FQ-PCR技术检测目的基因VEGF-A的mRNA表达情况,同时通过Western-blot方法检测VEGF-A蛋白表达,测定VEGF-A基因的沉默效果。
4.MTT检测细胞增殖水平
转染前一天,将生长状态良好的细胞种入96-孔培养板培养。第二天按实验分组分别进行转染,转染24小时后,进行MTT分析,检测不同ASODN对卵巢癌细胞株SKOV3细胞增殖水平的影响。
5流式细胞计数检测SKOV3细胞的凋亡情况
将生长状态良好稳定转染反义寡聚合甘酸的SKOV3细胞按实验设计的要求(空白对照组、实验组1#、实验组2#、实验组3#)进行流式细胞分析,检测经过实验处理的SKOV3细胞的细胞周期及凋亡情况。
结果
1.FQ-PCR结果显示,和对照组相比,反义寡居核苷酸1#、2#、3#对卵巢癌SKOV3细胞中VEGF-A基因的表达有显著沉默作用,它们的沉默效率分别为68.4%、69.4%、55.7%,Western-blot结果显示了类似的沉默效果。
2.MTT检测细胞增殖水平实验结果表明,实验组1#,2#,3#的增殖抑制率(PIR)分别为42.5%、38.7%和44.5%,与阴性对照组和空白对照组相比差异有显著性(P<0.05),这一结果提示在VEGF-A基因被沉默后,卵巢癌细胞的增殖能力明显减弱,肿瘤细胞的生长能力明显被抑制。
3.流式细胞计数检测SKOV3细胞的凋亡结果说明,通过流式细胞计数分析不同处理的细胞群体中各时期细胞的比例,结果发现三个转染反义寡聚合甘酸SKOV3细胞的实验组与对照组比较,其沉默VEGF-A后,SKOV3细胞群中凋亡细胞显著增加(P<0.05)。
结论
1.2-甲氧乙基修饰的ASODN对SKOV3卵巢癌细胞株的生长和增殖能力具有明显的抑制作用；利用针对VEGF-A基因的ASODN特异性沉默VEGF-A基因能引起卵巢癌细胞生长周期迟缓、诱发卵巢癌细胞凋亡,VEGF-A基因能够作为卵巢癌瘤基因治疗的有效靶点。
2.针对VEGF-A基因的ASODN能够特异性沉默VEGF-A基因,在卵巢癌基因靶向治疗方面具有非常重要的研究及应用价值。
第四部分’超声微泡介导的靶向VEGF-A基因的2-甲氧乙基修饰的反义寡聚核苷酸抑制裸鼠卵巢癌生长的实验研究
目的
在体内验证靶向VEGF-A基因的2-甲氧乙基修饰的反义寡聚核苷酸对卵巢癌生长的抑制作用,并探讨超声微泡介导基因转染作用的有效性。
方法
30只Balb/c裸鼠皮下接种人卵巢癌细胞SKOV3后,待所有皮下种植瘤瘤体体积均超过300mm3后,随机将30只成瘤裸鼠分为3组,每组10只。分别为
①超声+脂质体微泡+靶向VEGF的2-甲氧乙基修饰的反义寡聚核苷酸组,即UM+ASODN组。该组中10只裸鼠每只均给予400μL脂质体/optison(?)微泡混合液／3#寡聚核苷酸(含2.5ug反义寡聚核苷酸)瘤中心注射。
②超声+脂质体微泡组即UM组,该组中10只裸鼠每给予100μLD-Hanks液+300μLoptison(?)微泡皮下种植瘤瘤中心注射
③空白对照组即BC组,该组中10只裸鼠,每只裸鼠均给予400μL D-Hanks液瘤中心注射。
所有30只裸鼠接受各自相应处理后,立即在瘤体表面皮肤涂以超声耦合剂,并给予1 MHz超声辐射,强度为1.0W/cm2,每次30s,间隔30s,共2次60s照射。伺候每3天用游标卡尺测量皮下肿瘤长径(a)及短径(b)变化,肿瘤体积根据公式V=πab2/6计算。15天后采用颈椎脱位法处死小鼠,称取瘤重。
统计学分析采用统计学软件SPSS16.0英文版进行,方差齐时多组均数比较采用方差分析,两两比较采用LSD检验；方差不齐时多组均数比较采用方差分析,两两比较采用DUNNETT's T3检验。设定p<0.01具有极显著性差异,p<0.05具有显著性差异。
结果
1.肿瘤的体积、瘤重与抑瘤率
(1) UM+ASODN组、UM组与BC组观察至超声照射后15天,裸鼠皮下种植瘤体积分别为108.2±26.42mm3,297.8±44.22mm3,339.7±41.32mm3。各组种植瘤体积不同(F=104.67,p=0.00<0.01)。LSD法两两比较UM+ASODN组与其他两组均有极明显差异(p=0.00<0.01)。表明UM+ASODN组裸鼠皮下种植瘤体积较其他两组较小且差异极为明显。UM组与BC组相比有明显差异(p=0.021<0.05),表明与BC组相比,UM组裸鼠皮下种植瘤体积明显较小。
(2) UM+ASODN组、UM组与BC组观察至超声照射后15天,裸鼠皮下种植瘤体积分别为0.70±0.08g,1.45±0.14g,1.66±0.23g。各组瘤重不同(F=93,87,p=0.00<0.01). DUNNETT's T3法两两比较UM+ASODN组瘤重较其他两组有极明显差异(p=0.00<0.01), UM+ASODN组裸鼠皮下种植瘤瘤重较其他两组较小且差异极为明显;UM组与BC组差异不具有统计学意义(p=0.078)0.05)。
(3)超声辐射15天后处死裸鼠时,可见UM+ASODN组裸鼠皮下种植瘤肿瘤体积较小,颜色苍白,与周围组织边界较为清楚,局部存在类似包膜组织包裹；而UM组与BC组裸鼠皮下种植瘤瘤体较大,色红,血管组织丰富,呈浸润性生长,与周围组织边界不清。
结论
1.本实验成功构建了SK0V3人卵巢癌皮下种植的裸鼠模型。
2.实验中成功利用超声联合optison(?)造影剂微泡介导VEGF-A基因的2-甲氧乙基修饰的反义寡聚核苷酸转染处理了SKOV3人卵巢癌裸鼠皮下种植的裸鼠模型
3.实验结果验证了体内环境下超声联合微泡造影剂optison(?)介导靶向VEGF-A的的2-甲氧乙基修饰的反义寡聚核苷酸转染卵巢癌SKOV3细胞极大的抑制了肿瘤的生长
Ovarian cancers, one of the most common malignancies in gynecological cancers, have been the focus of gynecological research due to its high incidence rate, invasion and migration. Tumor angiogenesis is a process involving the growth of new blood vessels within or toward the tumor and is essential for tumor itself to grow and metastasize. Vascular endothelial growth factor (VEGF), also called vascular permeability factor (VPF), is a growth factor acting on the membrane of vascular endothelial cells. VEGF is widely expressed in human and animal tissues such as the brain, kidney, liver, spleen and bones. It was first purified from the bovine pituitary follicular stellate cells in vitro culture medium and was named by Ferrara and others in 1989. VEGF, a polypeptide cytokine of platelet-derived growth factor family, is one of the strongest vascular growth factor ever found. VEGF acts specifically on vascular endothelial cells and plays a key role in tumor genesis and growth by promoting endothelial cell proliferation and increasing the vascular permeability to induce tumor angiogenesis. Targeted tumor therapy by inhibiting VEGF expression has been widely investigated recently.
As a new and fastly developing gene silencing technology, antisense oligonucleotides (ASODN) technology is regarded as a very effective tool in the field of genetic engineering. Antisense oligonucleotides technology can inhibit the expression of target genes in a specific, efficient and stable manner and can achieve relatively satisfactory gene silencing effect. Currently, ASODN has been widely and successfully applied to study the function of genes, the etiology and treatment of a variety of diseases.
The purpose of this study is to:①determine the gene and protein expression of VEGF in ovarian cancers, in order to clarify the correlation between VEGF and the migration of ovarian cancers;②design and synthesize stable and efficient 2' -Methoxyethyl modified antisense oligonucleotides which can be transferred into ovarian cancer cells against VEGF gene;③study the silencing effects of the antisense oligonucleotides against VEGF-A expression in SKOV3 ovarian cancer cells, and further clarify the change of proliferation and apoptosis levels of SKOV3 cells before and after transfection, in order to investigate whether VEGF-A can be used as a potentially effective therapeutic target in the ovarian cancer gene therapy.④construct a nude mice model bearing human ovarian cancer cell line SKOV3 and observe the inhibitory effect of ASODN transfected using ultrasound irradiation treatment to endermic tumors cells of transferred ovarian cancers ascites in mice and evaluate tumor suppressive rate to evaluate the suppressive effects of ultrasound irradiation treatment and ASODN against ovarian cancers.
Part I Expression of VEGF and its relationship with biological features in ovarian cancers Obejectve
Vascular endothelial growth factor (VEGF) is one of the strongest factors promoting the growth of blood vessels, its genetic expression is closely related to the increased invasion of tumor and decreased survival rates. The aim of this research is to determine the gene and protein expression of VEGF in ovarian cancers, clarifying the relationship between VEGF and the invasion and malignancy of ovarian cancers.
Methods
1 Samples preparation
All samples were obtained during 132 excision surgeries at Shandong Provicial Hospital affiliated to Shandong University during July 2008 and April 2010, including:①ovarian cancers (n=32), which include serous cystadenocarcinomas (n=31), endometrioid adenomas (n=23) and mucoid adenocarcinomams (n=14). The subjects' ages were ranged 29-77 years (mean age 55.3±6.3 years), they all received operations for the first time, no chemotherapy history, no tumor history, and all the diagnoses were confirmed by pathologicy.②benign ovarian tumors (n=34), including serous cystadenomas (n=22) and mucinous cystadenomas (n=12). The ages of the subjects were ranged 29-70 years old (mean age 39.7±7.5years).③normal control group (n=30), obtained from womb and ovary excison sugeries for hysteromyoma, dysfunctional uterine bleeding and hysteroptosis, and the ovary were confirmed as normal by histopathological studies. The ages of the subjects were ranged 42-70 years (mean age 54.1±7.1 years). All the diagnoses are confirmed by histopathological studies.
2 Histochemistry
The expression of VEGF protein in ovarian cancer, benign ovarian tumors and normal ovary tissues are determined by immunohistochemical staining with specific mouse anti-VEGF monoclonal antibody by streptavidin-peroxidase method (abbreviation:S-P).
3. Analysis of immunohistochemical results
VEGF immunoreactivity is evaluated semiquantitatively based on the intensity of staining.5 fields at a magnification of 400×at every slice are observed at random. The percent of positive staining cells in 200 tumor cells is calculated. The slides are analysed independently by four observers blinded for expression of VEGF.For the discrepancies a second evaluation course is running to reach agreement.
4. Evaluation of B\VEGF protein expression
The VEGF expression is scored as:strong (+++):if more than 50% of cells are positive or a strong diffuse reaction is seen; moderate (++):if less than 50% of cells are positive, or a moderate diffuse reaction is observed; slightly positive (+):if immunoreactions are found in less than 30% of tumour cells or the diffuse reaction is weak; and negative(-):if no VEGF is stained.
5. Fluorescent quantitive PCR (FQ-PCR)
Total RNA are extracted from frozen tissues obtained from ovary excision surgeries. cDNA is synthesized from total RNA by reverse transcription. mRNA expression of VEGF-A is determined by FQ-PCR.
6. Western-blot to examine the protein expression of VEGF-A
VEGF-A protein is extracted from 200mg of frozened ovarian cancer tissues according to standard experiment procedures, and then the difference of protein expression of VEGF-A is determined using western-blot.
7. Statistical treatment
All data are analyzed by statistical package for the social science 13 (SPSS 13.0). The data of histochemistry are treated by chi-square test, the semi-quantitive data of FQ-PCR and Wstern-blot were treated by one-way ANOVA, P<0.05 is considered significant value.
Results
1. Result of histochemistry
In the ovarian tissues, a cytoplasmic VEGF reaction predominates and only scattered cells exhibited nuclear reaction. VEGF is expressed in 58(85.3%) ovarian cancers, in 12(35.3%) benign ovarian neoplasms and 4(13.3%) normal ovarian tissues. The chi-square test demonstrates a statistically difference in VEGF expression between vicious ovarian tumors and benign ovarian neoplasms (x2=22.310, P=0.000<0.05), between benign ovarian neoplasms and normal ovarian tissues(x 2=5.074, P=0.024<0.05), between vicious ovarian tumors and normal ovarian tissues(x 2=46.382, P=0.000<0.05), respectively. Among vicious ovarian tumors whose clinical stage are stageⅢand stageⅣVEGF staining is positive 41 (95.3%), while 23 (92%) specimens are positive stageⅠand stageⅡvicious ovarian tumors. No statistical deifferece is found between stageⅠ&Ⅱand stageⅢ&Ⅳvicious ovarian tumors (x 2=0.32, P=0.571). Furthermore, the strong and moderate positives (60.3%, 35/58) are most common in vicious ovarian tumors, while in benign ovarian neoplasms is slight (72.7%,8/11).
2 FQ-PCR results
The onw-way analysis of variance(ANOVA) result showed the statistical difference of the expression of VEGF among ovarian cancers, benign ovarian neoplasms and normal ovarian tissues:ovarian cancers vs benign ovarian neoplasms(P<0.05), benign ovarian neoplasms vs normal ovarian tissue (P<0.05), ovarian cancers vs normal ovarian tissue (P<0.05). No statistical difference of expression of VEGF was found between stageⅠ&Ⅱand stageⅢ&Ⅳovarian cancers (P=0.78).
3.Western-blot analysis
The results of ANOVA showed that the differences of protein expression of VEGF-A among ovarian cancers, benign ovarian neoplasms and normal ovarian tissues were statistically significant:ovarian cancers vs benign ovarian neoplasms (P<0.05), benign ovarian neoplasms vs normal ovarian tissue (P<0.05), ovarian cancers vs normal ovarian tissue (P<0.05).
Conclusions
1. There is high expression of VEGF in ovarian cancers.
2. There is a statistically difference in VEGF expression between vicious and benign ovarian neoplasms. VEGF is highly associated with malignant pituitary adenomas.
3. VEGF can serve as a useful tool for confirmation of ovarian tumors and VEGF gene also may be an effective target for ovarian cancer gene therapy.
PartⅡDesign and synthesis of 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene and the assessment of the transfection efficiency
Objective
Design and synthesize 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene, and test the inhibitory efficiency of the antisense oligonucleotides.
Methods
1. Design and synthesize 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A
2'-Methoxyethyl modified antisense oligonucleotides were designed and synthesized by Oligos Etc Inc (Oregon),3 groups of ASODN targeting VEGF-A gene were synthesized and marked as 1#,2# and 3#, respectively.
2. Test the efficiency of antisence oligonucleotides transfection in SKOV3 ovarian cancer cells)
Firstly, SKOV3 ovarian cancer cells were cultured, and the cells in good growth condition were spread into a six-well cell culture plate one day before transfection. Secondly,2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A was delivered into cultured cells according to previous groups design. Then we measured RFP expression under fluorescence microscope 48 hours after transfection to observe the transfectory potency of the recombined vectors.
Results
In 3 test groups transfected with 2'-Methoxyethyl modified antisense oligonucleotides, the transfection potency values of 1#,2#,3# were 86.5±4.5%,91.2±5.7% and 92.1±2.8%, respectively.
Conclusions
In this part,3 groups of ASODN 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A were successfully designed and synthesized.
PartⅢThe effect of 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene on the proliferation and apoptosis level of SKOV3 ovarian cancer cells
Objective
Study the influence and significance of 2'-Methoxyethyl modified antisense oligonucleotides 1#,2# and 3#, targeting VEGF-A gene on the proliferation and apoptosis level of SKOV3 ovarian cancer cells.
Methods
1. Transfect SKOV3 cells with ASODN 1#,2# and 3#
Firstly, SKOV3 ovarian cancer cells were cultured in good growth condition, and then the cells were spread into the six-well culture plate one day before transfection. On the day of transfection, the experiment was performed with ASODN group 1#,2# and 3#, the negative control group and the blank control group according to the design of experiment.
2. Measurement of the silencing effect on VEGF-A gene expression
Real-time PCR was performed to measure the mRNA expression of VEGF-A and Western-blot was performed to measure the protein expression of VEGF-A, testing the silencing effect of VEGF-A gene.
3. Measurement of the proliferation level of cells with MTT
SKOV3 cells which were in good growth condition and were seeded in 96-well culture plate before. On the second day after transfection, MTT was performed using standard methods to measure the changes of proliferation level before and after transfection.
4. Investigation of pituitary tumor cell apoptosis level variation by PI dyeing FCM
Stable tansfecion SKOV3 are cultured in good condition. According to designed groups (ASODN 1#,2#,3# and controls), cells are collected. PI dyeing FCM method is employed to contrast the changes of cell apoptosis levels.
Results
1. Realtime-PCR results showed that, ASODN 1#,2#,3# had significant gene silencing effect against VEGF-A gene expression of SKOV3, and their silencing efficiencies were 68.4%、69.44%、55.7%, respectively. Western-blot showed a similar result.
2. MTT-detected cellular proliferation level test results showed that, the inhibition rate (IR) of experimental group 1#,2#,3# were 42.5%,38.7% and 44.5%, respectively, which had significant differences with the negative control group and the blank control group (P<0.05). The result indicated that when the VEGF-A gene were silenced, the proliferation of the ovarian cancer cells were significantly decreased, and the growth ability of the cancer cells were significantly inhibited.
3. Analyzing the proportion of all period cells in cell colony by FCM, we found that, after VEGF-A was silenced, the proportion of apoptosis cell increased in the three experimental groups, comparing with control group.
Conclusions
1.2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene can significantly silence VEGF-A mRNA and protein expression in SKOV3 ovarian cancer cells. Besides,2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene can significantly inhibit the the proliferation and growth potency in SKOV3 ovarian cancer cells. It may indicate that VEGF-A gene can act as an effective target in the ovarian cancer gene therapy.
2. The above results also indicate that 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A shows a promising potential in targeted gene therapy and research of ovarian cancers.
Part IV Exploiting Inhibitory Effects of the 2'-O-Methoxyethyl-Modified Antisense oligonucleotides targeting VEGF-A gene in Ovarian Cancer modle of nude mice by a novel combination:liposome microbubbles and site-specific ultrasound exposure
Objective
To validate the inhibitory effects of the 2'-O-Methoxyethyl-Modified Antisense oligonucleotides targeting VEGF-A gene in Ovarian Cancer in vivo and to exploit the effectiveness of the transfection mediated by liposome microbubbles and ultrasound exposure
Method
methods:30 Balb/c nude mice were subcutaneously inoculated human ovarian cancer cell line SKOV3. When the endermic tumors were all larger than 300mm3 in size,the mice were then randomly divided into 3 groups (10 mice each group):
①UM+ASODN (liposome microbubbles+ASODN+site-specific ultrasound exposure) group:400μL mixture of microbubble/ASODN/(with 2.5ug ASODN) /liposome was injected into the center of the tumor of every mice
②; UM (liposome microbubbles+site-specific ultrasound exposure, no ASODN) group:300μL of microbubbles and 100μL of D-Hanks were injected into the center of the tumor of every mice
③BC (blank control,no ASODN,no microbubbles,no ultrasound exposure) group: 400μL of D-Hanks was injected into the center of the endermic tumors.
After all these have been done,site-specific ultrasound irradiation was carried out to each mouse with the parameter settting on a frequency 1 MHz and the intensity 1.0w /cm2.The exposure was given 30 seconds twice with an 30 seconds interval.
The tumor volume was measured every 3 days from the irradiation on and the size of the endermic tumor was calculated according to the formula V=πab2/6 (a=length, b=width) 15 days after the irradiation, the nude mice were killed and the weight of the tumor was weighed.
All data are analyzed by statistical package for the social science 16 (SPSS 16.0). The data of the size and the volume of the endermic tumors are treated by Anova analysis,and the comparison of the size between every two groups were carried out by LSD test. While the comparison of the weight of the endermic tumors between groups were carried out by DUNNETT T3 test, p<0.01 is considered to be of extremely significant value and p<0.05 is considered to be of significant value.
Result
(1)The mean volume of endermic tumor of UM+ASODN group, UM group and BC group was 108.2±26.42mm3,297.8±44.22mm3,339.7±41.32mm3 respectively. The ANOVA analysis demonstrates an extremely significant differnec between groups (F=104.67,p=0.00<0.01). LSD test told us that the volume of the ASODN+UM group was obviously statistically smaller than UM group and BC group (p=0.00<0.01) and the volume was statistically smaller in UM group than BC group (p=0.021<0.05).
(2)the mean weight of nude mice tumor were 0.70±0.08g,1.45±0.14g,1.66±0.23g respectively. The weight of the ASODN+UM group was obviously statistically smaller than UM group and BC group (DUNETT T3 method,p=0.00<0.01).The analysis shows no significant difference between UM group and BC group(DUNETT T3 method,p=0.078>0.01).
(3)The autopsy of the nude mice demonstrated that something like membrane surrounded the pale, smaller-sized tumors of ASODN+UM group. The boundary between tumors and normal tissues was obvious;While in UM group arid BC group, the tumor was red,huger in size, abundant in blood supply and mingled with the normal tissue that cannot be easily dissected.
Conclusion
(1) We successfully set up the Balb/c nude mice model bearing human ovarian cancer cell line SKOV3.
(2) We successfully disposed of the nude mice model bearing human ovarian cancer cell line SKOV3 with Optison(?) ultrasound contrast agent microbubbles and ultrasound exposure.
(3) We found that the 2'-O-Methoxyethyl-Modified Antisense oligonucleotides targeting VEGF-A gene greatly decrease the growth of the tumor of the SKOV3 ovarian cancer cells in vivo with site-specific ultrasound exposure and Optison(?) contrast agent microbubbles.
引文
[1]Jemal A, Siregel R, Ward E, et al. Cancer statistics [J]. CA Cancer J Clin, 2008,58:71-96.
[2]Yancopoulos G D, Davis S, Gale N W, et al. Vascular-specific growth factors and blood vesselformation[J]. Nature,407:242-248,2000.
[3]Carmeliet P, Jain R K. Angiogenesis in cancer and other diseases [J]. Nature,2000,407:249-257.
[4]Folkman J. Tumor angiogenesis:therapeutic implications [J]. N Engl J Med, 1971,285:1182-1186.
[5]Kerbel R S. Tumor angiogenesis:past, present, and the near future.Carcinogenesis (Lond.),2000,21:505-515.
[6]Neufeld G, Cohen T, Gengrinovitch S, et al. Vascularendothelial growth factor (VEGF) and its receptors. FASEB J,1999,13:9-22.
[7]Daniel T C, Deborah M H, Rick N, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest,1989, 84:1470.
[8]Vrbikova J, Cibula D, Dvorakova K,et al. Insulin sensitivity in women with polycystic ovary syndrome[J]. J Clin Endocrinol Metab,2004,89(60):2942.
[9]Czernobilsky B. Common epithelial tumors of the ovary. Blaustein's pathology of the female genital tract, R J Kurman, ed. New York: Springer-Verlag,1987.
[10]Kristensen G B, Trope C. Epithelian ovarian carcinoma [J]. Lancet,1997, 349:113-17.
[11]Land A. Ovulation, ovulation induction and ovarian carcinoma. Ballieres Clin Obstet Gynaecol,1993,7:455.
[12]Russell P, Bannatyne P. Surgical pathology of the ovaries, Appendix B. Edinburgh:Churchill Livingstone,1989.
[13]Serove S, Sculy R, Sobin L. The molecular pathogenesis of ovarian cancer. In:International histological classification of tumours. No.9: Histological typing of ovarian tumours. Geneva:World Health Organisation, 1973:46-50.
[14]Krigman H, Bentley H, Robboy SJ. Pathology of epithelian ovarian tumours. Clin Obstet Gynecol 1994;37:475-91.
[15]Serov S F, Scully R E, Sobin L H. Histological typing of ovarian tumours. In: International histological classification of tumours, No.9. Geneva:World Health Organisation:17-54.Cannistra S A. Cancer of the ovary. N Engl J Med 1993:329:1550-9.
[16]Fox H, Singh N. Pathology of epithelian ovarian cancer. In:Jacobs IJ, Shepherd J H, Oram D H, et al, eds. Ovarian cancer,2nd ed. Oxford University Press,2002:57-66.
[17]Kerbel R S. Tumor angiogenesis [J]. N Engl J Med,2008, 358(19):2039-2049.
[18]Vermeulen P B, van Golen K L, Dirix L Y. Angiogenesis, lymphangiogenesis, growth pattern, and tumor emboli in inflammatory breast cancer:a review of the current knowledge [J]. Cancer,2010,116(11 Suppl):2748-2754.
[19]Charlesworth P J, Harris A L. Mechanisms of disease:angiogenesis in urologic malignancies [J]. Nat Clin Pract Urol,2006,3(3):157-169.
[20]Lazar D, Raica M, Sporeal, et al. Tumor angiogenesis in gastric cancer [J]. Rom J Morphol Embryol,2006,47(1):5-13.
[21]Abounader R, Laterra J. Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis [J]. Neuro Oncol,2005,7(4):436-451.
[22]Bochner B H, Cote R J, Weidner N, et al. Angiogenesis in bladder cancer: relationship between microvessel density and tumor prognosis [J]. J Natl Cancer Inst,1995,87(21):1603-1612.
[23]Weidner N, Folkman J, Pozza F, et al. Tumor angiogenesis:a new significant and independent prognostic indicator in early-stage breast carcinoma [J]. J Natl Cancer Inst,1992,84(24):1875-1887.
[24]Connolly D T, Heuvelman D M, Nelson R, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest,1989,84(5):1470.
[25]Kato T, Kameoka S, Kimura T, et al. Prognostic significance of angiogenesis associated with long-term survival in 377 Japanese patients with breast cancer [J]. Breast Cancer Res Treat,2000,64:132.
[26]Gehani S A, Parbhoo S P, Hatter T, et al. Is angiogenesis a predictor ofthe development of bone metastasis? Results of immunohistochemical study using CD31 and factor VIII for angiogenesis assessment in primary breast cancer [J]. Breast Cancer Res Treat,2000,64:131.
[27]Cantarella G, Risuglia N, Delleva R, et al. Trial inhibits angiogenesis stimulated by VEGF expression in human glioblastoma cells [J]. Br Cancer, 2006,22,94:1428-1435.
[28]Abounader R, Laterra J. Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis [J]. Neuro-Oncology,2005,7(4):436-451.
[29]Ferrara N. Molecular and biological properties of vascular endothelial growth factor [J]. J Mol Med,1999,77(7):527-543
[30]Olofsson B, jeltsch M, Ericsson U, et al. Current biology of VEGF-B and VEGF-C. Curr Opin Biotechnol,1999; 10(6):528-535.
[31]Connolly D T, Heuvelman D M, Nelson R, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest,1989,84(5):1470.
[32]Harper S J, Bates D O. VEGF-A splicing:the key to anti-angiogenetic therapeutics [J]? Nat Rev Cancer,2008,8(11):880-887
[33]Shinkaruk S, Bayle M, Latin G, et al. Vascular endothelial cell growth factor (VEGF), an emerging target for cancer chemotherapy [J]. Curr Med Chem Anticancer Agents,2003,3(2):95-117.
[34]Yabushita H, Shimazu M, Noguchi M, et al. Vascular endothelial growth factor activating matrix metalloproteinase in ascetic fluid during peritoneal dissemination of ovariana cancer [J]. Oncol Rep,2003,10:89-95.
[35]Murohara T, Horowitz R, SilverM, et al. Vascular endothelial growth factor vascular permeability factor enhances vascular permeability via nitricoxide and prOstacychlin [J]. Circulation,1998,97:99-107.
[1]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998,391:806-811.
[2]Guo S, Kemphues K J. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed [J]. Cell,1995,81:611-620.
[3]Chal Fie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression [J]. Science,1994,263:8022805.
[4]Zamecnik P C, Stephenson M L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligonucleotide [J]. Proc Natl Acad Sci U S A,1978,75:280-284.
[5]Mette M F, Matzke A J, Matzke M A. Resistance of RNA-mediated TGS to HC-Pro, a viral suppressor of PTGS, suggests alternative pathways for dsRNA processing [J]. Curr Biol,2001,11:1119-1123.
[6]Bernstein E, Caudy A A, Hammond S M, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference [J]. Nature,2001, 409:363-366.
[7]Hannon G J. RNA interference [J].Nature,2002,418:244-251.
[8]Paterson B M, Roberts B E, Kuff E L. Structural gene identificationand mapping by DNA-mRNA hybrid-arrested cell-free translation [J]. Proc Natl Acad Sci USA,1977; 74:4370-4.
[9]Couzin J. Breakthrough of the year. Small RNAs make big splash [J]. Science,2002,298:2296-7.
[10]First-and second-generation antisense oligonucleotide inhibitors targeted against human c-raf kinase.
[11]Drug properties of second-generation antisense oligonucleotides:how do they measure up to their predecessors?
[12]Dermal delivery of topically applied oligonucleotides via follicular transport in mouse skin.
[13]Comparing in vitro and in vivo activity of 2'-O-[2-(methylamino)-2-oxoethyl]-and 2'-O-methoxyethyl-modified antisense oligonucleotides.
[14]Volm M, Koomagi R, Mattern J. PD-ECGF, bFGF, and VEGF expression in non-small cell lung carcinomas and their association with lymph node metastasis [J]. Anticancer Res,1999,19:651-655.
[15]Maehara Y, Kabashima A, Koga T, et al. Vascular invasion and potential for tumor angiogenesis and metastasis in gastric carcinoma [J]. Surgery,2000, 128:408-416.
[16]Fujisaki K, Mitsuyama K, Toyonaga A, et al. Circulating vascular endothelial growth factor in patients with colorectal cancer [J]. Am J Gastroenterol,1998,93:249-252.
[17]Ferrara N, Houck K, Jakeman L, et al. Molecular and biological properties of the vascular endothelial growth factor family of proteins [J]. Endocr Rev, 1992,13:18-32.
[1]Kurman R J, Visvanathan K, Roden R, et al. Early detection and treatment of ovarian cancer:shifting from early stage to minimal volume of disease based on a new model of carcinogenesis [J]. Am J Obstet Gynecol,2008,198: 351-356.
[2]Potgens A J, We stphal H R, de Waal RM, et al. The role of vascular permeability factor and basic fibroblast growth factor in tumor angiogenesis [J]. Biol Chem Hoppe Seyler,1995,376:57-70.
[3]Inoue M, Hager J H, Ferrara N, et al. VEGF-A has a critical, nonredundant role in angiogenic switching and pancreatic beta cell carcinogenesis [J]. Cancer Cell,2002,1:193-202.
[4]Kargiotis O, Rao J S, Kyritsis A P. Mechanisms of angiogenesis in gliomas [J]. J Neurooncol,2006,78:281-293.
[5]Zhang A, Meng L, Wang Q, et al. Enhanced in vitro invasiveness of ovarian cancer cells through up-regulation of VEGF and induction of MMP-2 [J]. Oncol Rep,2006,15:831-836.
[6]Brockmann M A, Ulbricht U, et al. Glioblastoma and cerebral microvascular endothelial cell migration in response to tumor-associated growth factors [J]. Neurosurgery,2003,52:1391-1399.
[7]O'Byrne K J, Koukourakis M I, Giatromanolaki A, et al. Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small-cell lung cancer [J]. Br J Cancer,2000,82:1427-1432.
[8]Lange T, Guttmann-Raviv N, Baruch L, et al. VEGF162, a new heparin-binding vascular endothelial growth factor splice form that is expressed in transformed human cells [J]. J Biol Chem,2003,278: 17164-17169.
[9]Robinson C J, Stringer S E. The splice variants of vascular endothelial growth factor (VEGF) and their receptors [J]. J Cell Sci,2001,114:853-865.
[10]Stimpfl M, Tong D, Fasching B, et al. Vascular endothelial growth factor splice variants and their prognostic value in breast and ovarian cancer [J]. Clin Cancer Res,2002,8:2253-2259.
[11]Alvarez A A, Krigman H R, Whitaker R S, et al. The prognostic significance of angiogenesis in epithelial ovarian carcinoma [J]. Clin Cancer Res 1999,5: 587-591.
[12]Gasparini G, Bonoldi E, Viale G, et al. Prognostic and predictive value of tumour angiogenesis in ovarian carcinomas [J]. Int J Cancer,1996,69: 205-211.
[13]Shen G H, Ghazizadeh M, Kawanami O, et al. Prognostic significance of vascular endothelial growth factor expression in human ovarian carcinoma [J]. Br J Cancer,2000,83:196-203.
[14]Sonmezer M, Gungor M, Ensari A, et al. Prognostic significance of tumor angiogenesis in epithelial ovarian cancer:in association with transforming growth factor beta and vascular endothelial growth factor [J]. Int J Gynecol Cancer,2004,14:82-88.
[15]Ptaszynska M M, Pendrak M L, Bandle R W, et al. Positive feedback between vascular endothelial growth factor-A and autotaxin in ovarian cancer cells [J]. Mol Cancer Res,2008,6:352-363.
[16]Hefler L A, Mustea A, Konsgen D, et al. Vascular endothelial growth factor gene polymorphisms are associated with prognosis in ovarian cancer [J]. Clin Cancer Res,2007,13:898-901
[17]Gadducci A, Ferdeghini M, Fanucchi A, et al. Serum preoperative vascular endothelial growth factor (VEGF) in epithelial ovarian cancer:relationship with prognostic variables and clinical outcome [J]. Anticancer Res,1999, 19:1401-1405.
[18]Obermair A, Hefler L, Nather A, et al. Correlation of the serum concentration of vascular endothelial growth factor (VEGF) and hemoglobin levels in patients with epithelial ovarian cancer [J]. Ann Oncol,1999,10:998.
[19 Tempfer C, Obermair A, Hefler L, et al. Vascular endothelial growth factor serum concentrations in ovarian cancer [J]. Obstet Gynecol,1998, 92:360-363.
[20]Jewell E L, Darcy K M, Hutson A, et al. Association between the N-terminally truncated (DeltaN) p63alpha (DeltaNp63alpha) isoform and debulking status, VEGF expression and progression-free survival in previously untreated, advanced stage epithelial ovarian cancer:A Gynecologic Oncology Group study [J]. Gynecol Oncol,2009,115:424-429.
[21]Wong C, Wellman T L, Lounsbury K M. VEGF and HIF-1 alpha expression are increased in advanced stages of epithelial ovarian cancer [J]. Gynecol Oncol,2003,91:513-517.
[22]Zhang A, Meng L, Wang Q, et al. Enhanced in vitro invasiveness of ovarian cancer cells through up-regulation of VEGF and induction of MMP-2 [J]. Oncol Rep,2006,15:831-836.
[23]Shin J E, Jung S A, Kim S E, et al. Expression of MMP-2, HIF-1 alpha and VEGF in colon adenoma and colon cancer [J]. Korean J Gastroenterol,2007, 50:9-18.
[24]Jobim F C, Schwartsmann G, Xavier N L, et al. Expression of MMP-9 and VEGF in breast cancer:correlation with other prognostic indicators [J]. Rev Bras Ginecol Obstet,2008,30:287-293.
[25]Calvo A, Catena R, Noble MS, et al. Identification of VEGF-regulated genes associated with increased lung metastatic potential:functional involvement of tenascin-C in tumor growth and lung metastasis [J]. Oncogene, 2008,27:5373-5384.
[26]Kim T J, Landen C N, Lin Y G, et al. Combined anti-angiogenic therapy against VEGF and integrin alphaVbeta3 in an orthotopic model of ovarian cancer [J]. Cancer Biol Ther,2009,8:2263-2272.
[27]Ghosh S, Maity P. VEGF antibody plus cisplatin reduces angiogenesis and tumor growth in a xenograft model of ovarian cancer [J]. J Environ Pathol Toxicol Oncol,2010,29:17-30.
[28]Wang B, Kaumaya P T, Cohn D E. Immunization with synthetic VEGF peptides in ovarian cancer [J].,2010, Epub ahead of print.
[29]Hannon G J, Rossi J J. Unlocking the potential of the human genome with RNA interference [J]. Nature,2004,431:371-378.
[30]Mello C C, Conte D, Jr.. Revealing the world of RNA interference [J]. Nature,2004,431:338-342.
[31]Scherr M, Battmer K, Winkler T, et al. Specific inhibition of bcr-abl gene expression by small interfering RNA[J]. Blood,2003,101:1566-1569.
[32]Carron C P, Meyer D M, Pegg J A, et al. A peptidomimetic antagonist of the integrin alpha(v)beta3 inhibits Leydig cell tumor growth and the development of hypercalcemia of malignancy [J]. Cancer Res,1998, 58:1930-1935.
[33]Yamamoto S, Konishi I, Mandai M, et al. Expression of vascular endothelial growth factor (VEGF) in epithelial ovarian neoplasms:correlation with clinicopathology and patient survival, and analysis of serum VEGF levels [J]. Br J Cancer,1997,76:1221-1227.
[1]Aoi A.Watanabe Y.Mori S,et al. Herpes simplex virus thymidine kinase-mediated suicide gene therapy using nano/microbubbles and ultrasound[J].Ultrasound Med Biol,2008,34(3):425-434.
[2]Fischer A, Hacein-Bey-Abina S, Cavassana-Calvo M. Gene therapy of metabolic disease [J]. J Inherit Metab Dis,2006,29:409-412.
[3]Qasim W, Gaspar H B, Thrasher A J. Update on clinical gene therapy in childhood [J]. Arch Dis Child,2007,92:1028-1031.
[4]Zaldumbide A, Hoeben R C. How not to be seen:immune-evasion strategies in gene therapy [J]. Gene Ther,2008,15:239-246.
[5]Heller L C, Heller R. In vivo electroporation for gene therapy [J]. Human GeneTher,2006,17:890-897.
[6]Liang H D.Lu Q L.Xue S A, et al. Optimisation of ultrasound-mediated gene transfer (sonoporation) in skeletal muscle cells [J]. Ultrasound Med Biol,2004, 30 (11):1523-9.
[7]ter Haar G. Therapeutic applications of ultrasound [J]. Prog Biophys Mol Biol,2007,93:111-129.
[8]Yamashita T, Sondo S, Suzuki R,et al. A novel bubble liposome and ultrasound-mediated gene transfer to ocular surface.RC-1 cells in vitro and conjuctiva in vivo.Exp [J]. Eye Res,2007,85:741-748.
[9]Rahim A A, Taylor S L.Bush N L, et al. Spatial and acoustic pressure dependence of microbubble mediated gene delivery targeted using focused ultrasound [J]. J Gene Med,2006,8:1347-1357.
[10]Sundaram J, Mellein BR, Mitragotri S. An experimental and theoretical analysis of ultrasound-induced permeabilization of cell membranes [J]. Biophys J,2003,84:3087-3101.
[11]Sun Y, Kruse D E, Dayton P A, et al. High-frequency dynamics of ultrasound contrast agent s [J]. IEEE Trans Ultrason Ferroelectr Freq Control, 2005,52(11):1981-1991.
[12]Didenko Y T, Susliek K S. The energy efficiency of formation of Photons, radicals and ions during single 2 bubble cavitation. Nature,2002,418 (7):394-397.
[13]Marmottant P, Hilgenfeldt S. Controlled vesicle deformation and lysis by single oscillating bubbles [J]. Nature,2003,423(6936):153-156.
[14]Skyba D M, Price R J, LinkaAZ, et al. Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue[J]. Circulation,1998,98(4):290-293.
[15]Price R J, Skyba D M, KAUL S, et al. Delivery of collodal particles and red blood cells to tissue through microvessel ruptures created by targeted microbubble destruction with ultrasound[J]. Circulmion,1998,98(13): 1264—1267.
[16]冉海涛,任红,王志刚等,超声波空化效应对体外培养细胞细胞膜作用的实验研究中华超声影像学杂志[J].2003,12(8)：499-501.
[17]Bekeredjian R, Kroll R D. Ultrasound targeted microbubble destruction increases capillary permeability in hepatomas [J].Ultrasound Med Biol, 2007,33 (10):1592-1598.
[18]Joseph S, Olbrich C, Kirsch J, et al. A real-time in vitro assay for studying functional characteristics of target-specific ultrasound contrast agents [J].Pharm Res.2004,21(6):920-926.
[19]Rychak J, Lindner JR, Ley K, et al. Deformable gas-filled microbubbles targeted to P-selectin[J]. J Control Release,2006,114, (3):288-99.
[20]Fischer D, Bieber T, Brusselbach S, et al.Cationized human serum albumin as a non-viral vector system for gene delivery? Characterization of complex formation with plasmid DNA and transfection efficiency [J]. Int J Pharm,2001,225(1-2):97-111.
[21]Morris M C, Chaloih L, Heitz F, et al.Translocating peptides and proteins and their use for gene delivery [J].2000,11(5):461-6.
[22]Greenleaf W J, Bolander M E, Sarkar G, et al. Artificial cavitation nuclei significantly enhance acoustically induced cell transfection [J]. Ultrasound Med Biol,1998,24(4):587-95.
[23]Wu W, Ning X B, Jiang Z, et al. Formation of micro-vessel embo-lization in rabbit liver induced by low power ultrasound irradiation combined with LEVOVIST agent [J]. Southeast Univ(Nat Sci),2003,33(3):300-302.
[24]Zhang H, Jiang Z, wu W, et al. An experiment study on formation of oncology-vessel thromembolia induced by ultrasound iraadition microbubble agent [J]. Southeast Univ(Med Sci Edi),2004,23(3):195-198.
[25]Worthington AE, Thompson J, Rauth AM, et al. Mechanism of ultrasound-enhanced porphyrin cytotoxicity. Part I:A search for free radical effects[J]. Ultrasound Med Biol,1997,23(7):1095-1105.
[26]Barteria M, Diociautib M, Palac A, et al. Low frequency ultrasound induces aggregation of porcine lumarase by free radicals production[J]. Biophysical Chemistry,2004,111(1):35-42.
[27]Fuclaelli A F, Sisk E C, Thomas R M. Induction of base damage in DNA solutions by ultrasonic cavitation [J]. Free Radical Biol Med,1995, 18(2):231-238.
[28]Feril L B Jr, Kondo T, Zhao Q L,et al. Enhancement of ultrasound-induced apoptosis and cell lysis by echo-contrast agents [J]. Ultrasound Med Biol,2003, 29(2):331-7.
[29]Crowder K C, Hughes M S, Marsh J N,et al. Sonic activation of molecularly-targeted nanoparticles accelerates transmembrane lipid delivery to cancer cells through contact-mediated mechanisms:implications for enhanced local drug delivery [J]. Ultrasound Med Biol,2005,31 (12):1693-1700.
[30]Haag P, Frauscher F, Gradl J, et al. Microbubble2enhanced ultrasoundto deliver an antisense oligodeoxynucleotide targeting the human andro2gen recep tor into p rostate tumours [J]. J Steroid Biochem Mol Biol,2006,102 (1-5):103-113.
[31]Hauff P, Seemann S, Reszka R, et al. Evaluation of gas-filled microparticles and sonoporation as gene delivery system:feasibility study in rodent tumor models. Radiology,2005,236 (2):572-578.
[32]Leong-Poi H, Kuliszewski M A, Lekas M, et al. Therapeutic arteriogenesis by ultrasound-mediated VEGF 165 plasmid gene delivery to chronically ischemic skeletal muscle. Circ Res,2007,101 (3):295-303.
[33]Meijering B D, Henning R H, Van Gilst W H, et al. Optimization of ultrasound and microbubbles targeted gene delivery to cultured primary endothelial cells [J]. J Drug Target.2007,15(10):664-71.
[34]Siu T, Rohling RN, Chiao M. Power density requirement of a 4MHz micro-ultrasonic transducer for sonodynamic therapy.Biomed Microdevices [J]. 2008,10(1):89-97.
[35]Chen Z, Liang K, Liu J, et al. Enhancement of survivin gene downregulation and cell apoptosis by a novel combination:liposome microbubbles and ultrasound exposure [J]. Med Oncol.2009,;26(4):491-500.
[1]Leung D W, Cachianes G, Kuang W, et a1. Vascular endothelial growth factor is a secreted angiogenic mitogen [J]. Science,1989, 246(4935):1306-1310.
[2]Neufeld G, Cohen T, Gengrinovitch S. et al. Vascular endothelial growth factor(VEGF)and its receptors. [J], FASEB,1999,13:9-22.
[3]Yla-herttuala S, Martin J F. Cardiovascular gene therapy [J]. Lancet,2000, 355:213-22.
[5]Houck K A, Ferrara N, Winer J. et al. The vascuar endothelial growth factor family:identification of a fourth molecular pecies and characterization of alternative splicing of RNA[J]. Molecular Endocrinology,1991,5:1806-14
[6]Townson S, Lageremn-J, Gr-immond S, et al. characterization of the murine VEGF-related factor gene[J]. Biochem Biophys Res Commun,1996, 220:922-8.
[7]01ofsson-B, Pajusola-k, von-Euler-G, et al. Genomic organization of the mouse and human genes for vascular endothelial growth factor B (VEGF-B) and characterization of a second splice isoorm. [J]. Biol—Chem.1996, 271:19310-7.
[8]Paavonen K, Horelli-Kuitunen N, Chilov D, et al. Novel human vascular endothelial growth factor genes VEGF-Band VEGF-C localize to chromosomes 1lq13 and 4q34, respectively[J]. Circulation,1996,93:1079-82.
[9]Joukov V, Pajusola K, Kaipainen A, et a1. A, et al. A novel vascular endothelial growth factor. VEGF-C, is a ligand for the Flt4(VEGFR-3)and KDR(VEGFR-2)receptor tyrosine kinases[J]. EMBO,1996,15:1751.
[10]Cao Y, Linden P, Farnebo J, et a1. Vascular endothelial growth factor C induces angiogenesis in vivo [J]. Proc Natl Acad Sci U S A,1998, 95:14389-14394.
[11]chen MG, Jeltsch M, Kukk E, et al. Vascular endothelial growth factor D(VEGF-D)is a ligand for the tyrosine kinases VEGF receptor2(Flk1)and VEGF receptor(Hlt4)[J]. Proc Natl Acad Sci USA,1998,95:548-553.
[12]Veikkola T, Alitalo K. VEGFs, receptors and angiogenesis[J]. Cancer Biology,1999,9:211-220.
[13]Iyer S, Leonidas DD, Swarninathan GJ, et al. The crystal structure of human placenta growth factor-1(PIGF-1), an angiogenic protein, At 2.OA resolution[J]. J Biol Chem,2001,276:12153-12161.
[14]Park JE, Chen HH, Winer J, et al. Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo. and high affinity binding to Fit-1 but not to Flk-1KDR[J]. J Biol Chem,1994, 269:25646-256454.
[15]Kodama J, Seki N, Tokumo K, et a1. Placenta growth factor is abundantly expressed in human cervical squamous cell carcinoma[J]. EurJ Gynaecol Oncol,1997,18:508-510.
[16]Sasaki R. Microvessel count and vascular endothelial growth factor in renal cell carcinoma[J]. Nippon Hittyokika Gakkai Zasski,1996, 87(8):1032-1040.
[17]Baker EA, Bergin FG, Leaper DJ. Plasminogen activator system vascular endothelial growth factor and colorectal cancer progression[J]. Mol Pathol, 2000,53(6):307-312.
[18]Harris SR, Schoeffuer DJ, Yohiji H, et al. Tumor growth enhancing efects of vascular endothelial growth factor associated with increased nitric oxide synthase activity and inhibition of apoptosis in human breast carcinoma xenografts[J]. Cancer Lett,2002,179(1):9-101.
[19]Beierle EA, Strande LF, Chen MK. VEGF upregulates BCL-2 expression and is associated with decreased apoptosis in neuroblastoma cells[J]. Pediatr Surg,2002,37(3):467-471.
[20]Liao F, Li Y, Conner W, et a1. Moneclonal antibody to vascular endothelial-caherin is a potent inhibitor of angiogenesis, tumor growth, and metastasis[J]. Cancer Res,2000,60(24):6805-6810.
[21]Simona P, Pascal B. Molecular characterization of lymphatic endothelial cells[J]. Cell Biology,2002,99(25):16069-16074.
[22]Von Marschall Z, Scholz A, Stacker SA, et a1. Vascular endothelialgrowth factor-D induces lymphangiogenesis an lymphatic metastasis in models of ductal pancreatic cancer[J].1nt J Oncol,2005,27(3):669-679.
[23]Veikkola, Jussila L, Makinen T, et al. Signaling via vascular endothelial growth factor receptor 3 is sufficient for lymphangiogenesis in transgenic mice[J]. J EMBO,2001,20(6):1223-1231.
[24]Jayson GC, Mulatero C, RansonM, et al. Phase I investigation of recombinant anti-human vascular endothelial growth factor antibody in patients with advanced cancer[J]. Eur J Cancer,2005,41 (4):555-63.
[25]Ruegg C, Hasmim M, Lejeune FJ, et al. Antiangiogenic peptides and proteins:from experimental tools to clinical drugs[J]. Biochim Biophys Acta, 2006,1765 (2):155-77.
[26]Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis [J]. Cancer Res,2004,64 (19):7099-109.
[27]Manley PW, Bold G, Bruggen J, et al. Advances in the structural biology, design and clinical development of VEGF-R kinase inhibitors for the treatment of angiogenesis[J]. Biochim Biophys Acta,2004,1697 (1-2):17-27.
[28]Sofer SZ, Kim E, Huaug J, et al. Resistance of a VEGF—producing tumor to anti-VEGF antibidy.unimpeded growth of human rhabdoby tumor xenografls [J]. J Pediatr Surg,2002,37:528-532.
[1]Jamal A, Tiwari RC, Murray T, et al. Cancer statistics [J]. CA Cancer J Clin,2004,54 (1):8-29.
[2]Partridge EE, Barnes MN. Epithelial ovarian cancer:prevention, diagnosis and treatment[J]. CA Cancer J Clin,1999,49(5):297.
[3]Risch HA, Jain M, Marrett LD, et a1. Dietary fat intake and risk of epithelial ovarian cancer[J]. J Natl Cancer Inst,1994,86(18):1409.
[4]Hynes N E, Stern D E. The biology of erbB-2/neu/HER-2 and its role in cancer [J]. Bioch Bioph Acta,2004,1198(2-3):165-184.
[5]Kleinberg L, Florenes V A, Silin I, et al. Nuclear expression ofVEGFg is associated with improved survival in metastatic ovarian carcinoma[J]. Cancer, 2007,109(2):228-238.
[6]Anderson MH, Pedersen L, Jurgen C, et a1. Identification of a cytotosis T lymphocyte response to the apoptosis inhibitor protein VEGF in cancer patients[J]. Cane Res,2000,61(12):869-872.
[7]sui L, Dong Y, Ohno M, et al. VEGF expression and its correlation with cell proliferation and prognosis in epithelial ovarian tumors[J]. Int J Oncol,2002, 21(3):315-320.
[8]Zeis M, Siegel S, Wagner A, Schmitz M, et al. Generation of cytotoxic responses in mice and human individuals against hematological malignancies hematological malignancies using VEGFg-RNA-transfected dendritic cells[J]. Immunol,2003,170(11):5391—5397.
[9]Casti C, Dalerba P, Rivohini L, et a1. The apotosis inhibitor protein VEGF induces tumors specific CD8+and CD4+T cells in colorectal patients[J]. Canc Res,2003,63(8):4507-4515.
[10]Yin JQ, Gao J, Shao R, et al. ASODN agents inhibit oncogene expression and attenuate human tumor cell growth[J]. J Exp Ther Oncol,2003,3(4): 194-204.
[11]Berchuck A, Kamel A, Whitaker R, et al. Overexpression of Her-2/neu is associated with poor survival in advanced epothelial ovarian cancer [J]. Cancer Res,1990,50:4087-4091.
[12]Verri E, Guglielmini P, Puntoni M, et al. HER2/neuin oncoprotein Over expression in epithelial ovarian cancel:Evaluation of its prevalence and prognostic significance[J]. Oncology,2005,68(2-3):154—161.
[13]Nakayama K, Kanzaki A, Terada K, et a1. Prognostic value of the Cutrans-porting ATPase in ovarian carcinoma patients receiving Cisplatin-based chemotherapy[J].clin Cancer Res,2004,108:2804-2811.
[14]Wrinht T G, Tsai J, Jia Z, et al Inhibition by copper (1l)binding of hepatocyte growth factor(HGF)interaction with its receptor Met and blockade of HGF/Met function[J]. Biol Chem,2004,279 (31):32499-32506.
[2]Yancopoulos G D, Davis S, Gale N W, et al. Vascular-specific growth factors and blood vesselformation[J]. Nature,407:242-248,2000.
[3]Carmeliet P, Jain R K. Angiogenesis in cancer and other diseases [J]. Nature,2000,407:249-257.
[4]Folkman J. Tumor angiogenesis:therapeutic implications [J]. N Engl J Med, 1971,285:1182-1186.
[5]Kerbel R S. Tumor angiogenesis:past, present, and the near future.Carcinogenesis (Lond.),2000,21:505-515.
[6]Neufeld G, Cohen T, Gengrinovitch S, et al. Vascularendothelial growth factor (VEGF) and its receptors. FASEB J,1999,13:9-22.
[7]Daniel T C, Deborah M H, Rick N, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest,1989, 84:1470.
[8]Vrbikova J, Cibula D, Dvorakova K,et al. Insulin sensitivity in women with polycystic ovary syndrome[J]. J Clin Endocrinol Metab,2004,89(60):2942.
[9]Czernobilsky B. Common epithelial tumors of the ovary. Blaustein's pathology of the female genital tract, R J Kurman, ed. New York: Springer-Verlag,1987.
[10]Kristensen G B, Trope C. Epithelian ovarian carcinoma [J]. Lancet,1997, 349:113-17.
[11]Land A. Ovulation, ovulation induction and ovarian carcinoma. Ballieres Clin Obstet Gynaecol,1993,7:455.
[12]Russell P, Bannatyne P. Surgical pathology of the ovaries, Appendix B. Edinburgh:Churchill Livingstone,1989.
[13]Serove S, Sculy R, Sobin L. The molecular pathogenesis of ovarian cancer. In:International histological classification of tumours. No.9: Histological typing of ovarian tumours. Geneva:World Health Organisation, 1973:46-50.
[14]Krigman H, Bentley H, Robboy SJ. Pathology of epithelian ovarian tumours. Clin Obstet Gynecol 1994;37:475-91.
[15]Serov S F, Scully R E, Sobin L H. Histological typing of ovarian tumours. In: International histological classification of tumours, No.9. Geneva:World Health Organisation:17-54.Cannistra S A. Cancer of the ovary. N Engl J Med 1993:329:1550-9.
[16]Fox H, Singh N. Pathology of epithelian ovarian cancer. In:Jacobs IJ, Shepherd J H, Oram D H, et al, eds. Ovarian cancer,2nd ed. Oxford University Press,2002:57-66.
[17]Kerbel R S. Tumor angiogenesis [J]. N Engl J Med,2008, 358(19):2039-2049.
[18]Vermeulen P B, van Golen K L, Dirix L Y. Angiogenesis, lymphangiogenesis, growth pattern, and tumor emboli in inflammatory breast cancer:a review of the current knowledge [J]. Cancer,2010,116(11 Suppl):2748-2754.
[19]Charlesworth P J, Harris A L. Mechanisms of disease:angiogenesis in urologic malignancies [J]. Nat Clin Pract Urol,2006,3(3):157-169.
[20]Lazar D, Raica M, Sporeal, et al. Tumor angiogenesis in gastric cancer [J]. Rom J Morphol Embryol,2006,47(1):5-13.
[21]Abounader R, Laterra J. Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis [J]. Neuro Oncol,2005,7(4):436-451.
[22]Bochner B H, Cote R J, Weidner N, et al. Angiogenesis in bladder cancer: relationship between microvessel density and tumor prognosis [J]. J Natl Cancer Inst,1995,87(21):1603-1612.
[23]Weidner N, Folkman J, Pozza F, et al. Tumor angiogenesis:a new significant and independent prognostic indicator in early-stage breast carcinoma [J]. J Natl Cancer Inst,1992,84(24):1875-1887.
[24]Connolly D T, Heuvelman D M, Nelson R, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest,1989,84(5):1470.
[25]Kato T, Kameoka S, Kimura T, et al. Prognostic significance of angiogenesis associated with long-term survival in 377 Japanese patients with breast cancer [J]. Breast Cancer Res Treat,2000,64:132.
[26]Gehani S A, Parbhoo S P, Hatter T, et al. Is angiogenesis a predictor ofthe development of bone metastasis? Results of immunohistochemical study using CD31 and factor VIII for angiogenesis assessment in primary breast cancer [J]. Breast Cancer Res Treat,2000,64:131.
[27]Cantarella G, Risuglia N, Delleva R, et al. Trial inhibits angiogenesis stimulated by VEGF expression in human glioblastoma cells [J]. Br Cancer, 2006,22,94:1428-1435.
[28]Abounader R, Laterra J. Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis [J]. Neuro-Oncology,2005,7(4):436-451.
[29]Ferrara N. Molecular and biological properties of vascular endothelial growth factor [J]. J Mol Med,1999,77(7):527-543
[30]Olofsson B, jeltsch M, Ericsson U, et al. Current biology of VEGF-B and VEGF-C. Curr Opin Biotechnol,1999; 10(6):528-535.
[31]Connolly D T, Heuvelman D M, Nelson R, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest,1989,84(5):1470.
[32]Harper S J, Bates D O. VEGF-A splicing:the key to anti-angiogenetic therapeutics [J]? Nat Rev Cancer,2008,8(11):880-887
[33]Shinkaruk S, Bayle M, Latin G, et al. Vascular endothelial cell growth factor (VEGF), an emerging target for cancer chemotherapy [J]. Curr Med Chem Anticancer Agents,2003,3(2):95-117.
[34]Yabushita H, Shimazu M, Noguchi M, et al. Vascular endothelial growth factor activating matrix metalloproteinase in ascetic fluid during peritoneal dissemination of ovariana cancer [J]. Oncol Rep,2003,10:89-95.
[35]Murohara T, Horowitz R, SilverM, et al. Vascular endothelial growth factor vascular permeability factor enhances vascular permeability via nitricoxide and prOstacychlin [J]. Circulation,1998,97:99-107.
[1]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998,391:806-811.
[2]Guo S, Kemphues K J. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed [J]. Cell,1995,81:611-620.
[3]Chal Fie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression [J]. Science,1994,263:8022805.
[4]Zamecnik P C, Stephenson M L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligonucleotide [J]. Proc Natl Acad Sci U S A,1978,75:280-284.
[5]Mette M F, Matzke A J, Matzke M A. Resistance of RNA-mediated TGS to HC-Pro, a viral suppressor of PTGS, suggests alternative pathways for dsRNA processing [J]. Curr Biol,2001,11:1119-1123.
[6]Bernstein E, Caudy A A, Hammond S M, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference [J]. Nature,2001, 409:363-366.
[7]Hannon G J. RNA interference [J].Nature,2002,418:244-251.
[8]Paterson B M, Roberts B E, Kuff E L. Structural gene identificationand mapping by DNA-mRNA hybrid-arrested cell-free translation [J]. Proc Natl Acad Sci USA,1977; 74:4370-4.
[9]Couzin J. Breakthrough of the year. Small RNAs make big splash [J]. Science,2002,298:2296-7.
[10]First-and second-generation antisense oligonucleotide inhibitors targeted against human c-raf kinase.
[11]Drug properties of second-generation antisense oligonucleotides:how do they measure up to their predecessors?
[12]Dermal delivery of topically applied oligonucleotides via follicular transport in mouse skin.
[13]Comparing in vitro and in vivo activity of 2'-O-[2-(methylamino)-2-oxoethyl]-and 2'-O-methoxyethyl-modified antisense oligonucleotides.
[14]Volm M, Koomagi R, Mattern J. PD-ECGF, bFGF, and VEGF expression in non-small cell lung carcinomas and their association with lymph node metastasis [J]. Anticancer Res,1999,19:651-655.
[15]Maehara Y, Kabashima A, Koga T, et al. Vascular invasion and potential for tumor angiogenesis and metastasis in gastric carcinoma [J]. Surgery,2000, 128:408-416.
[16]Fujisaki K, Mitsuyama K, Toyonaga A, et al. Circulating vascular endothelial growth factor in patients with colorectal cancer [J]. Am J Gastroenterol,1998,93:249-252.
[17]Ferrara N, Houck K, Jakeman L, et al. Molecular and biological properties of the vascular endothelial growth factor family of proteins [J]. Endocr Rev, 1992,13:18-32.
[1]Kurman R J, Visvanathan K, Roden R, et al. Early detection and treatment of ovarian cancer:shifting from early stage to minimal volume of disease based on a new model of carcinogenesis [J]. Am J Obstet Gynecol,2008,198: 351-356.
[2]Potgens A J, We stphal H R, de Waal RM, et al. The role of vascular permeability factor and basic fibroblast growth factor in tumor angiogenesis [J]. Biol Chem Hoppe Seyler,1995,376:57-70.
[3]Inoue M, Hager J H, Ferrara N, et al. VEGF-A has a critical, nonredundant role in angiogenic switching and pancreatic beta cell carcinogenesis [J]. Cancer Cell,2002,1:193-202.
[4]Kargiotis O, Rao J S, Kyritsis A P. Mechanisms of angiogenesis in gliomas [J]. J Neurooncol,2006,78:281-293.
[5]Zhang A, Meng L, Wang Q, et al. Enhanced in vitro invasiveness of ovarian cancer cells through up-regulation of VEGF and induction of MMP-2 [J]. Oncol Rep,2006,15:831-836.
[6]Brockmann M A, Ulbricht U, et al. Glioblastoma and cerebral microvascular endothelial cell migration in response to tumor-associated growth factors [J]. Neurosurgery,2003,52:1391-1399.
[7]O'Byrne K J, Koukourakis M I, Giatromanolaki A, et al. Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small-cell lung cancer [J]. Br J Cancer,2000,82:1427-1432.
[8]Lange T, Guttmann-Raviv N, Baruch L, et al. VEGF162, a new heparin-binding vascular endothelial growth factor splice form that is expressed in transformed human cells [J]. J Biol Chem,2003,278: 17164-17169.
[9]Robinson C J, Stringer S E. The splice variants of vascular endothelial growth factor (VEGF) and their receptors [J]. J Cell Sci,2001,114:853-865.
[10]Stimpfl M, Tong D, Fasching B, et al. Vascular endothelial growth factor splice variants and their prognostic value in breast and ovarian cancer [J]. Clin Cancer Res,2002,8:2253-2259.
[11]Alvarez A A, Krigman H R, Whitaker R S, et al. The prognostic significance of angiogenesis in epithelial ovarian carcinoma [J]. Clin Cancer Res 1999,5: 587-591.
[12]Gasparini G, Bonoldi E, Viale G, et al. Prognostic and predictive value of tumour angiogenesis in ovarian carcinomas [J]. Int J Cancer,1996,69: 205-211.
[13]Shen G H, Ghazizadeh M, Kawanami O, et al. Prognostic significance of vascular endothelial growth factor expression in human ovarian carcinoma [J]. Br J Cancer,2000,83:196-203.
[14]Sonmezer M, Gungor M, Ensari A, et al. Prognostic significance of tumor angiogenesis in epithelial ovarian cancer:in association with transforming growth factor beta and vascular endothelial growth factor [J]. Int J Gynecol Cancer,2004,14:82-88.
[15]Ptaszynska M M, Pendrak M L, Bandle R W, et al. Positive feedback between vascular endothelial growth factor-A and autotaxin in ovarian cancer cells [J]. Mol Cancer Res,2008,6:352-363.
[16]Hefler L A, Mustea A, Konsgen D, et al. Vascular endothelial growth factor gene polymorphisms are associated with prognosis in ovarian cancer [J]. Clin Cancer Res,2007,13:898-901
[17]Gadducci A, Ferdeghini M, Fanucchi A, et al. Serum preoperative vascular endothelial growth factor (VEGF) in epithelial ovarian cancer:relationship with prognostic variables and clinical outcome [J]. Anticancer Res,1999, 19:1401-1405.
[18]Obermair A, Hefler L, Nather A, et al. Correlation of the serum concentration of vascular endothelial growth factor (VEGF) and hemoglobin levels in patients with epithelial ovarian cancer [J]. Ann Oncol,1999,10:998.
[19 Tempfer C, Obermair A, Hefler L, et al. Vascular endothelial growth factor serum concentrations in ovarian cancer [J]. Obstet Gynecol,1998, 92:360-363.
[20]Jewell E L, Darcy K M, Hutson A, et al. Association between the N-terminally truncated (DeltaN) p63alpha (DeltaNp63alpha) isoform and debulking status, VEGF expression and progression-free survival in previously untreated, advanced stage epithelial ovarian cancer:A Gynecologic Oncology Group study [J]. Gynecol Oncol,2009,115:424-429.
[21]Wong C, Wellman T L, Lounsbury K M. VEGF and HIF-1 alpha expression are increased in advanced stages of epithelial ovarian cancer [J]. Gynecol Oncol,2003,91:513-517.
[22]Zhang A, Meng L, Wang Q, et al. Enhanced in vitro invasiveness of ovarian cancer cells through up-regulation of VEGF and induction of MMP-2 [J]. Oncol Rep,2006,15:831-836.
[23]Shin J E, Jung S A, Kim S E, et al. Expression of MMP-2, HIF-1 alpha and VEGF in colon adenoma and colon cancer [J]. Korean J Gastroenterol,2007, 50:9-18.
[24]Jobim F C, Schwartsmann G, Xavier N L, et al. Expression of MMP-9 and VEGF in breast cancer:correlation with other prognostic indicators [J]. Rev Bras Ginecol Obstet,2008,30:287-293.
[25]Calvo A, Catena R, Noble MS, et al. Identification of VEGF-regulated genes associated with increased lung metastatic potential:functional involvement of tenascin-C in tumor growth and lung metastasis [J]. Oncogene, 2008,27:5373-5384.
[26]Kim T J, Landen C N, Lin Y G, et al. Combined anti-angiogenic therapy against VEGF and integrin alphaVbeta3 in an orthotopic model of ovarian cancer [J]. Cancer Biol Ther,2009,8:2263-2272.
[27]Ghosh S, Maity P. VEGF antibody plus cisplatin reduces angiogenesis and tumor growth in a xenograft model of ovarian cancer [J]. J Environ Pathol Toxicol Oncol,2010,29:17-30.
[28]Wang B, Kaumaya P T, Cohn D E. Immunization with synthetic VEGF peptides in ovarian cancer [J].,2010, Epub ahead of print.
[29]Hannon G J, Rossi J J. Unlocking the potential of the human genome with RNA interference [J]. Nature,2004,431:371-378.
[30]Mello C C, Conte D, Jr.. Revealing the world of RNA interference [J]. Nature,2004,431:338-342.
[31]Scherr M, Battmer K, Winkler T, et al. Specific inhibition of bcr-abl gene expression by small interfering RNA[J]. Blood,2003,101:1566-1569.
[32]Carron C P, Meyer D M, Pegg J A, et al. A peptidomimetic antagonist of the integrin alpha(v)beta3 inhibits Leydig cell tumor growth and the development of hypercalcemia of malignancy [J]. Cancer Res,1998, 58:1930-1935.
[33]Yamamoto S, Konishi I, Mandai M, et al. Expression of vascular endothelial growth factor (VEGF) in epithelial ovarian neoplasms:correlation with clinicopathology and patient survival, and analysis of serum VEGF levels [J]. Br J Cancer,1997,76:1221-1227.
[1]Aoi A.Watanabe Y.Mori S,et al. Herpes simplex virus thymidine kinase-mediated suicide gene therapy using nano/microbubbles and ultrasound[J].Ultrasound Med Biol,2008,34(3):425-434.
[2]Fischer A, Hacein-Bey-Abina S, Cavassana-Calvo M. Gene therapy of metabolic disease [J]. J Inherit Metab Dis,2006,29:409-412.
[3]Qasim W, Gaspar H B, Thrasher A J. Update on clinical gene therapy in childhood [J]. Arch Dis Child,2007,92:1028-1031.
[4]Zaldumbide A, Hoeben R C. How not to be seen:immune-evasion strategies in gene therapy [J]. Gene Ther,2008,15:239-246.
[5]Heller L C, Heller R. In vivo electroporation for gene therapy [J]. Human GeneTher,2006,17:890-897.
[6]Liang H D.Lu Q L.Xue S A, et al. Optimisation of ultrasound-mediated gene transfer (sonoporation) in skeletal muscle cells [J]. Ultrasound Med Biol,2004, 30 (11):1523-9.
[7]ter Haar G. Therapeutic applications of ultrasound [J]. Prog Biophys Mol Biol,2007,93:111-129.
[8]Yamashita T, Sondo S, Suzuki R,et al. A novel bubble liposome and ultrasound-mediated gene transfer to ocular surface.RC-1 cells in vitro and conjuctiva in vivo.Exp [J]. Eye Res,2007,85:741-748.
[9]Rahim A A, Taylor S L.Bush N L, et al. Spatial and acoustic pressure dependence of microbubble mediated gene delivery targeted using focused ultrasound [J]. J Gene Med,2006,8:1347-1357.
[10]Sundaram J, Mellein BR, Mitragotri S. An experimental and theoretical analysis of ultrasound-induced permeabilization of cell membranes [J]. Biophys J,2003,84:3087-3101.
[11]Sun Y, Kruse D E, Dayton P A, et al. High-frequency dynamics of ultrasound contrast agent s [J]. IEEE Trans Ultrason Ferroelectr Freq Control, 2005,52(11):1981-1991.
[12]Didenko Y T, Susliek K S. The energy efficiency of formation of Photons, radicals and ions during single 2 bubble cavitation. Nature,2002,418 (7):394-397.
[13]Marmottant P, Hilgenfeldt S. Controlled vesicle deformation and lysis by single oscillating bubbles [J]. Nature,2003,423(6936):153-156.
[14]Skyba D M, Price R J, LinkaAZ, et al. Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue[J]. Circulation,1998,98(4):290-293.
[15]Price R J, Skyba D M, KAUL S, et al. Delivery of collodal particles and red blood cells to tissue through microvessel ruptures created by targeted microbubble destruction with ultrasound[J]. Circulmion,1998,98(13): 1264—1267.
[16]冉海涛,任红,王志刚等,超声波空化效应对体外培养细胞细胞膜作用的实验研究中华超声影像学杂志[J].2003,12(8)：499-501.
[17]Bekeredjian R, Kroll R D. Ultrasound targeted microbubble destruction increases capillary permeability in hepatomas [J].Ultrasound Med Biol, 2007,33 (10):1592-1598.
[18]Joseph S, Olbrich C, Kirsch J, et al. A real-time in vitro assay for studying functional characteristics of target-specific ultrasound contrast agents [J].Pharm Res.2004,21(6):920-926.
[19]Rychak J, Lindner JR, Ley K, et al. Deformable gas-filled microbubbles targeted to P-selectin[J]. J Control Release,2006,114, (3):288-99.
[20]Fischer D, Bieber T, Brusselbach S, et al.Cationized human serum albumin as a non-viral vector system for gene delivery? Characterization of complex formation with plasmid DNA and transfection efficiency [J]. Int J Pharm,2001,225(1-2):97-111.
[21]Morris M C, Chaloih L, Heitz F, et al.Translocating peptides and proteins and their use for gene delivery [J].2000,11(5):461-6.
[22]Greenleaf W J, Bolander M E, Sarkar G, et al. Artificial cavitation nuclei significantly enhance acoustically induced cell transfection [J]. Ultrasound Med Biol,1998,24(4):587-95.
[23]Wu W, Ning X B, Jiang Z, et al. Formation of micro-vessel embo-lization in rabbit liver induced by low power ultrasound irradiation combined with LEVOVIST agent [J]. Southeast Univ(Nat Sci),2003,33(3):300-302.
[24]Zhang H, Jiang Z, wu W, et al. An experiment study on formation of oncology-vessel thromembolia induced by ultrasound iraadition microbubble agent [J]. Southeast Univ(Med Sci Edi),2004,23(3):195-198.
[25]Worthington AE, Thompson J, Rauth AM, et al. Mechanism of ultrasound-enhanced porphyrin cytotoxicity. Part I:A search for free radical effects[J]. Ultrasound Med Biol,1997,23(7):1095-1105.
[26]Barteria M, Diociautib M, Palac A, et al. Low frequency ultrasound induces aggregation of porcine lumarase by free radicals production[J]. Biophysical Chemistry,2004,111(1):35-42.
[27]Fuclaelli A F, Sisk E C, Thomas R M. Induction of base damage in DNA solutions by ultrasonic cavitation [J]. Free Radical Biol Med,1995, 18(2):231-238.
[28]Feril L B Jr, Kondo T, Zhao Q L,et al. Enhancement of ultrasound-induced apoptosis and cell lysis by echo-contrast agents [J]. Ultrasound Med Biol,2003, 29(2):331-7.
[29]Crowder K C, Hughes M S, Marsh J N,et al. Sonic activation of molecularly-targeted nanoparticles accelerates transmembrane lipid delivery to cancer cells through contact-mediated mechanisms:implications for enhanced local drug delivery [J]. Ultrasound Med Biol,2005,31 (12):1693-1700.
[30]Haag P, Frauscher F, Gradl J, et al. Microbubble2enhanced ultrasoundto deliver an antisense oligodeoxynucleotide targeting the human andro2gen recep tor into p rostate tumours [J]. J Steroid Biochem Mol Biol,2006,102 (1-5):103-113.
[31]Hauff P, Seemann S, Reszka R, et al. Evaluation of gas-filled microparticles and sonoporation as gene delivery system:feasibility study in rodent tumor models. Radiology,2005,236 (2):572-578.
[32]Leong-Poi H, Kuliszewski M A, Lekas M, et al. Therapeutic arteriogenesis by ultrasound-mediated VEGF 165 plasmid gene delivery to chronically ischemic skeletal muscle. Circ Res,2007,101 (3):295-303.
[33]Meijering B D, Henning R H, Van Gilst W H, et al. Optimization of ultrasound and microbubbles targeted gene delivery to cultured primary endothelial cells [J]. J Drug Target.2007,15(10):664-71.
[34]Siu T, Rohling RN, Chiao M. Power density requirement of a 4MHz micro-ultrasonic transducer for sonodynamic therapy.Biomed Microdevices [J]. 2008,10(1):89-97.
[35]Chen Z, Liang K, Liu J, et al. Enhancement of survivin gene downregulation and cell apoptosis by a novel combination:liposome microbubbles and ultrasound exposure [J]. Med Oncol.2009,;26(4):491-500.
[1]Leung D W, Cachianes G, Kuang W, et a1. Vascular endothelial growth factor is a secreted angiogenic mitogen [J]. Science,1989, 246(4935):1306-1310.
[2]Neufeld G, Cohen T, Gengrinovitch S. et al. Vascular endothelial growth factor(VEGF)and its receptors. [J], FASEB,1999,13:9-22.
[3]Yla-herttuala S, Martin J F. Cardiovascular gene therapy [J]. Lancet,2000, 355:213-22.
[5]Houck K A, Ferrara N, Winer J. et al. The vascuar endothelial growth factor family:identification of a fourth molecular pecies and characterization of alternative splicing of RNA[J]. Molecular Endocrinology,1991,5:1806-14
[6]Townson S, Lageremn-J, Gr-immond S, et al. characterization of the murine VEGF-related factor gene[J]. Biochem Biophys Res Commun,1996, 220:922-8.
[7]01ofsson-B, Pajusola-k, von-Euler-G, et al. Genomic organization of the mouse and human genes for vascular endothelial growth factor B (VEGF-B) and characterization of a second splice isoorm. [J]. Biol—Chem.1996, 271:19310-7.
[8]Paavonen K, Horelli-Kuitunen N, Chilov D, et al. Novel human vascular endothelial growth factor genes VEGF-Band VEGF-C localize to chromosomes 1lq13 and 4q34, respectively[J]. Circulation,1996,93:1079-82.
[9]Joukov V, Pajusola K, Kaipainen A, et a1. A, et al. A novel vascular endothelial growth factor. VEGF-C, is a ligand for the Flt4(VEGFR-3)and KDR(VEGFR-2)receptor tyrosine kinases[J]. EMBO,1996,15:1751.
[10]Cao Y, Linden P, Farnebo J, et a1. Vascular endothelial growth factor C induces angiogenesis in vivo [J]. Proc Natl Acad Sci U S A,1998, 95:14389-14394.
[11]chen MG, Jeltsch M, Kukk E, et al. Vascular endothelial growth factor D(VEGF-D)is a ligand for the tyrosine kinases VEGF receptor2(Flk1)and VEGF receptor(Hlt4)[J]. Proc Natl Acad Sci USA,1998,95:548-553.
[12]Veikkola T, Alitalo K. VEGFs, receptors and angiogenesis[J]. Cancer Biology,1999,9:211-220.
[13]Iyer S, Leonidas DD, Swarninathan GJ, et al. The crystal structure of human placenta growth factor-1(PIGF-1), an angiogenic protein, At 2.OA resolution[J]. J Biol Chem,2001,276:12153-12161.
[14]Park JE, Chen HH, Winer J, et al. Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo. and high affinity binding to Fit-1 but not to Flk-1KDR[J]. J Biol Chem,1994, 269:25646-256454.
[15]Kodama J, Seki N, Tokumo K, et a1. Placenta growth factor is abundantly expressed in human cervical squamous cell carcinoma[J]. EurJ Gynaecol Oncol,1997,18:508-510.
[16]Sasaki R. Microvessel count and vascular endothelial growth factor in renal cell carcinoma[J]. Nippon Hittyokika Gakkai Zasski,1996, 87(8):1032-1040.
[17]Baker EA, Bergin FG, Leaper DJ. Plasminogen activator system vascular endothelial growth factor and colorectal cancer progression[J]. Mol Pathol, 2000,53(6):307-312.
[18]Harris SR, Schoeffuer DJ, Yohiji H, et al. Tumor growth enhancing efects of vascular endothelial growth factor associated with increased nitric oxide synthase activity and inhibition of apoptosis in human breast carcinoma xenografts[J]. Cancer Lett,2002,179(1):9-101.
[19]Beierle EA, Strande LF, Chen MK. VEGF upregulates BCL-2 expression and is associated with decreased apoptosis in neuroblastoma cells[J]. Pediatr Surg,2002,37(3):467-471.
[20]Liao F, Li Y, Conner W, et a1. Moneclonal antibody to vascular endothelial-caherin is a potent inhibitor of angiogenesis, tumor growth, and metastasis[J]. Cancer Res,2000,60(24):6805-6810.
[21]Simona P, Pascal B. Molecular characterization of lymphatic endothelial cells[J]. Cell Biology,2002,99(25):16069-16074.
[22]Von Marschall Z, Scholz A, Stacker SA, et a1. Vascular endothelialgrowth factor-D induces lymphangiogenesis an lymphatic metastasis in models of ductal pancreatic cancer[J].1nt J Oncol,2005,27(3):669-679.
[23]Veikkola, Jussila L, Makinen T, et al. Signaling via vascular endothelial growth factor receptor 3 is sufficient for lymphangiogenesis in transgenic mice[J]. J EMBO,2001,20(6):1223-1231.
[24]Jayson GC, Mulatero C, RansonM, et al. Phase I investigation of recombinant anti-human vascular endothelial growth factor antibody in patients with advanced cancer[J]. Eur J Cancer,2005,41 (4):555-63.
[25]Ruegg C, Hasmim M, Lejeune FJ, et al. Antiangiogenic peptides and proteins:from experimental tools to clinical drugs[J]. Biochim Biophys Acta, 2006,1765 (2):155-77.
[26]Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis [J]. Cancer Res,2004,64 (19):7099-109.
[27]Manley PW, Bold G, Bruggen J, et al. Advances in the structural biology, design and clinical development of VEGF-R kinase inhibitors for the treatment of angiogenesis[J]. Biochim Biophys Acta,2004,1697 (1-2):17-27.
[28]Sofer SZ, Kim E, Huaug J, et al. Resistance of a VEGF—producing tumor to anti-VEGF antibidy.unimpeded growth of human rhabdoby tumor xenografls [J]. J Pediatr Surg,2002,37:528-532.
[1]Jamal A, Tiwari RC, Murray T, et al. Cancer statistics [J]. CA Cancer J Clin,2004,54 (1):8-29.
[2]Partridge EE, Barnes MN. Epithelial ovarian cancer:prevention, diagnosis and treatment[J]. CA Cancer J Clin,1999,49(5):297.
[3]Risch HA, Jain M, Marrett LD, et a1. Dietary fat intake and risk of epithelial ovarian cancer[J]. J Natl Cancer Inst,1994,86(18):1409.
[4]Hynes N E, Stern D E. The biology of erbB-2/neu/HER-2 and its role in cancer [J]. Bioch Bioph Acta,2004,1198(2-3):165-184.
[5]Kleinberg L, Florenes V A, Silin I, et al. Nuclear expression ofVEGFg is associated with improved survival in metastatic ovarian carcinoma[J]. Cancer, 2007,109(2):228-238.
[6]Anderson MH, Pedersen L, Jurgen C, et a1. Identification of a cytotosis T lymphocyte response to the apoptosis inhibitor protein VEGF in cancer patients[J]. Cane Res,2000,61(12):869-872.
[7]sui L, Dong Y, Ohno M, et al. VEGF expression and its correlation with cell proliferation and prognosis in epithelial ovarian tumors[J]. Int J Oncol,2002, 21(3):315-320.
[8]Zeis M, Siegel S, Wagner A, Schmitz M, et al. Generation of cytotoxic responses in mice and human individuals against hematological malignancies hematological malignancies using VEGFg-RNA-transfected dendritic cells[J]. Immunol,2003,170(11):5391—5397.
[9]Casti C, Dalerba P, Rivohini L, et a1. The apotosis inhibitor protein VEGF induces tumors specific CD8+and CD4+T cells in colorectal patients[J]. Canc Res,2003,63(8):4507-4515.
[10]Yin JQ, Gao J, Shao R, et al. ASODN agents inhibit oncogene expression and attenuate human tumor cell growth[J]. J Exp Ther Oncol,2003,3(4): 194-204.
[11]Berchuck A, Kamel A, Whitaker R, et al. Overexpression of Her-2/neu is associated with poor survival in advanced epothelial ovarian cancer [J]. Cancer Res,1990,50:4087-4091.
[12]Verri E, Guglielmini P, Puntoni M, et al. HER2/neuin oncoprotein Over expression in epithelial ovarian cancel:Evaluation of its prevalence and prognostic significance[J]. Oncology,2005,68(2-3):154—161.
[13]Nakayama K, Kanzaki A, Terada K, et a1. Prognostic value of the Cutrans-porting ATPase in ovarian carcinoma patients receiving Cisplatin-based chemotherapy[J].clin Cancer Res,2004,108:2804-2811.
[14]Wrinht T G, Tsai J, Jia Z, et al Inhibition by copper (1l)binding of hepatocyte growth factor(HGF)interaction with its receptor Met and blockade of HGF/Met function[J]. Biol Chem,2004,279 (31):32499-32506.