内皮抑素联合异环磷酰胺对小鼠移植瘤抑制作用的实验研究
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摘要
第一部分背景
肿瘤是一种严重危害人体健康的疾病,目前临床上有手术治疗、化疗、放疗、生物治疗等多种治疗方法,但疗效并不理想,死亡率仍然较高。如何在21世纪彻底攻克这一疾病,寻找更为有效的治疗方法和新的无毒副作用的治疗药物,已成为广大医务科研人员研究的热点之一。许多研究表明肿瘤的形成、生长、浸润和转移的关键是肿瘤的血管化,肿瘤形成后即进入无血管的浸润前期,此时肿瘤的直径一般小于2-3mm,细胞数一般小于1×107,肿瘤细胞主要靠弥散供给营养,当肿瘤直径超过2-3mm时,肿瘤就需要新生的血管维持营养供给、排泄代谢产物和提供转移的通道,而肿瘤血管的形成是一个及其复杂过程,这一过程有许多种相关的血管细胞因子参与,共同影响新生血管的形成。因此,研究开发抑制肿瘤血管生成的抗肿瘤药物是抑制肿瘤生长、防止肿瘤转移的有效策略。自20世纪70年代Folkman提出肿瘤的生长依赖新血管生成,抗血管生成可治疗实体性肿瘤以来,许多临床前的实验研究都想将针对某一血管生成环节的抗血管生成药物应用于动物原位瘤模型或皮下移植肿瘤的治疗,为抗肿瘤血管生成的临床应用提供一定的理论和实验依据,而目前抗肿瘤血管生成的治疗方法主要有3大方向：一是中和或者抑制促进血管形成的因子分泌,例如血管内皮生长因子和纤维细胞生长因子等；二是给予抑制血管形成的因子,例如血管抑制索和内皮抑制素等；三是用对肿瘤血管系统有特异作用的有毒物质或者抗体使肿瘤梗死,而目前对抑制血管生成的相关因子的研究进展最快,多种抗血管生成药物都已进人临床试验阶段,已取得了令人鼓舞的结果,其中又以血管抑素和内皮抑素抗血管生成作用最引人瞩目。分子生物学研究表示：内皮抑素(Endostatin. ES)能通过诱导血管内皮细胞凋亡、阻断血管内皮细胞生长因子(Vascular endothelial cell growth factor. VEGF)和基质金属蛋白酶(Matrix metalloproteinase. MMP)的活性等多种途径抑制新生血管生长,从而抑制肿瘤的生长和转移,是目前公认的最好最强的血管生成抑制因子,具有很强烈的抗肿瘤血管生成活性,能够明显降低肿瘤组织内的微血管密度,增加肿瘤细胞凋亡。内皮抑素对肿瘤血管内皮细胞生长具有强烈的抑制作用,并且能够有效抑制60种以上的肿瘤,能够调节12%的人类基因组表达,进而下调病理性的血管发生,而且没有毒副作用,也无耐药性；多项研究也报道了ES在甲状腺癌、肝癌、乳腺癌、直肠癌、宫颈癌及肺腺癌等多种肿瘤中均显示了良好的抗肿瘤效果,并且能够阻止肿瘤转移的形成。目前内皮抑素正在Ⅲ期临床试验,多项实验都表明内皮抑素具有特异性抑制血管内皮细胞作用,对多种依赖血管生成的实体瘤都有较强的抑制作用,为肿瘤的临床治疗开辟了一条新路。重组人血管内皮抑素注射液是我国专家学者自主创新研发的新型人血管内皮抑素,能够特异性的强烈抑制血管内皮细胞的增殖和生长,是一种全新的抑制血管形成的抑制剂,与化疗药物联合有协同作用,随着分子生物学研究的深入,化疗联合分子靶向治疗已成为肿瘤治疗研究的热点。
第二部分重组人血管内皮抑素对肿瘤血管生成抑制作用的实验研究
目的
1.探讨内皮抑素(Endostatin. ES)对小鼠肉瘤皮下移植瘤血管生成及肿瘤生长的抑制作用。
2.探讨内皮抑素(Endostatin. ES)抑制肿瘤血管生成及肿瘤生长作用的量效关系。
材料和方法。
1.建立小鼠肉瘤模型
将S180肉瘤腹水种鼠传代,无菌条件下抽取传代至第4天的S180肉瘤小鼠腹水,1000r/min,离心5min,PBS稀释,反复三次,最后DMEM液稀释,制成肿瘤细胞悬液,细胞计数为5×107个/ml,将细胞悬液在小鼠右前肢腋下注射0.1ml,然后观察小鼠皮下移植瘤成瘤情况,S180细胞接种后5天,75只荷瘤小鼠全部成瘤,测量肿瘤最大径(a)和最小径(b),计算肿瘤体积：V=ab2/2,将肿瘤体积过大及过小者排除,肿瘤体积约90mm3的60只荷瘤小鼠进入实验。
2.实验分组
采用随机数字表法将60只荷瘤小鼠随机等分为四组：生理盐水组15只：给予等量生理盐水；ES1组15只：给予ES剂量5mg/kg；ES2组15只：给予ES剂量10mg/kg；ES3组15只：给予ES剂量15mg/kg。ES及生理盐水瘤周皮下注射,连续给药14天。
3.检测指标
观察肿瘤生长情况,从内皮抑素用药的第一天算起,每3天用游标卡尺测量肿瘤最大径(a)和最小径(b),直至药物干预结束后第二天,计算每次测量肿瘤的体积,取平均值,绘制肿瘤生长曲线。在药物干预结束后第二天处死小鼠,处死前1分钟尾静脉注射DiOC7(3)(1mg/kg),然后迅速将肿瘤组织完整剥离取出,称瘤重,测量肿瘤最大径(a)和最小径(b),计算肿瘤体积和抑瘤率：抑瘤率(%)=(对照组瘤体平均体积-实验组瘤体平均体积)/对照组瘤体平均体积x100%.将剥离的肿瘤组织一半制作冰冻切片：新鲜标本用OCT包埋后,4-5μm连续切片,荧光显微镜下观察组织的血管标记,摄取DiOC7(3)标记血管图片(绿色荧光),将肿瘤血管图片用于定量分析,经相关软件计算灌注血管平均距离；另一半肿瘤组织立即用10%福尔马林固定,石蜡包埋、5μm切片、免疫组化染色检测肿瘤微血管密度(microvessel density. MVD):切片观察先在低倍光镜下(40倍)确定肿瘤内新生血管最密集区作为“热点”,进行血管计数,然后在高倍镜视野(200倍)计数5个视野,每个视野的血管平均数值为该切片的MVD值。
4.统计学分析
所有数据采用SPSS13.0软件进行统计学分析。定量数据采用均数±标准差(x±s)表示。组间比较采用单因素方差分析,组间多重比较根据各组间方差齐性检验结果选择不同的方法,按0.05检验水准,若组间方差齐同,则采用LSD-t检验,若组间方差不齐,则采用Tamhane's T2检验。以P≤0.05认为差别有统计学意义。
结果
在整个实验过程中,所有荷瘤小鼠均生长良好,进食、饮水正常,未出现明显消瘦等表现。小鼠在植瘤5天后,接种部位可触及小结节,以后渐呈类圆形或椭圆形生长,至实验结束时(用药2周后)接种部可见明显膨隆肿瘤瘤结块。在整个实验过程中无小鼠死亡,小鼠于接种第6天开始给药并记录肿瘤大小,各组肿瘤生长曲线见对照组肿瘤生长曲线陡直,各给药组生长曲线均位于对照组曲线下方,相对对照组略微平缓,且ES中、高剂量组比ES低剂量组小鼠肿瘤生长曲线平缓。接种后第6天,开始给予内皮抑素药物治疗,药物干预结束后第二天处死小鼠称瘤重及计算肿瘤体积。各治疗组平均瘤重及体积与对照组比较差别有统计学意义(P<0.05),各治疗组平均瘤重及体积小于对照组(P<0.05)；各治疗组之间平均瘤重及体积比较差别也有统计学意义(P<0.05),ES高剂量治疗组平均瘤重及体积小于ES中、低治疗组(P<0.05)；ES中剂量治疗组平均瘤种及体积也小于ES低治疗组(P<0.05),ES低、中、高治疗组肿瘤生长抑制率分别为24.91%和45.83%、65.30%。对照组、ES低剂量组、中剂量组、高剂量组MVD及灌注血管平均距离分别为43.13±9.86、30.27±9.95、17.87±7.69、9.60±4.31和43.33±8.21、66.06±14.98、84.94±6.99、108.16±18.10,在荷瘤鼠各组中灌注血管(Di0C7(3))的距离比较中,各治疗组灌注血管距离与对照组相比差别有显著统计学(P<0.01),对照组灌注血管平均距离小于各治疗组(P<0.01)；各治疗组间灌注血管平均距离比较差别也有显著统计学意义(P<0.01),ES高剂量组灌注血管平均距离大于中、低剂量组；ES中剂量组灌注血管平均距离明大于低剂量组(P<0.01)。免疫组化检测微血管密度,经内皮抑素治疗的肿瘤血管生成减少,各治疗组微血管密度与对照组比较差别有显著统计学意义(P<0.01),ES高、中、低剂量组的微血管密度低于对照组(P<0.01)；各治疗组之间微血管密度比较差别也有显著统计学意义(P<0.01),ES高剂量组微血管密度低于中、小剂量ES组(P<0.01)；ES中剂量组微血管密度也低于ES低剂量组(P<0.01)。
结论
1.内皮抑素能抑制实体肿瘤的血管生成及肿瘤的生长。
2.内皮抑素抑制血管生成和肿瘤生长的作用效果有明显的量效关系：随剂量的增多而作用增强。
第三部分重组人血管内皮抑素联合异环磷酰胺对肉瘤小鼠移植瘤血管生成及肿瘤生长抑制作用的实验研究
目的
探讨内皮抑素(Endostatin. ES)联合异环磷酰胺(ifosfamide. IFO)对小鼠肉瘤皮下移植瘤血管生成和肿瘤生长的抑制作用。
材料和方法
1.建立小鼠肉瘤模型
将S180肉瘤腹水种鼠传代,无菌条件下抽取传代至第4天的S180肉瘤小鼠腹水,1000r/min,离心5min,PBS稀释,反复三次,最后DMEM夜稀释,制成肿瘤细胞悬液,细胞计数为5×107个/ml,将细胞悬液在小鼠右前肢腋下注射0.1ml,观察小鼠皮下移植瘤成瘤情况,S180细胞接种后5天,75只荷瘤鼠全部成瘤,测量肿瘤最大径(a)和最小径(b),计算肿瘤体积：V=ab2/2,将肿瘤体积过大及过小者排除,肿瘤体积约90mm3的60只荷瘤小鼠进入实验。
2.实验分组
将60只荷瘤小鼠随机等分为四组：生理盐水组15只：给予等量生理盐水；ES组15只：给予ES剂量10mg/kg；IFO组15只：给予IFO剂量50mg/kg；ES+IFO组只15只：给予ES剂量10mg/kg+IFO剂量50mg/kg。ES及生理盐水瘤周皮下注射,连续给药14天,IFO组腹腔注射给药,连续给药5天。
3.检测指标
观察肿瘤生长情况,从小鼠药物治疗的第一天算起,每3天用游标卡尺测量肿瘤最大径(a)和最小径(b),直至药物治疗药结束后第二天,计算每次测量肿瘤的体积,取平均值,绘制肿瘤生长曲线。在药物干预结束后第二天处死小鼠,处死前1分钟尾静脉注射DiOC7(3)(1mg/kg),然后迅速将肿瘤组织完整剥离取出,称瘤重,测量肿瘤最大径(a)和最小径(b),计算肿瘤体积及抑瘤率：抑瘤率(%)：(对照组瘤体平均体积一实验组瘤体平均体积)/对照组瘤体平均体积x100%。将剥离的肿瘤组织一半制作冰冻切片：新鲜标本用OCT包埋后,4-5μm连续切片,荧光显微镜观察组织血管标记,摄取DiOC7(3)标记血管图片(绿色荧光),用相关软件将肿瘤血管图片用于定量分析,计算肿瘤灌注血管平均距离,血管平均距离越大,血管密度越低,血管平均距离越小,血管密度越高；另一半肿瘤组织立即用10%福尔马林固定,石蜡包埋、5μm切片、免疫组化染色检测肿瘤微血管密度(MVD)：切片观察先在低倍光镜下(40倍)确定肿瘤内新生血管最密集区作为“热点”,进行血管计数,然后在高倍镜视野(200倍)计数5个视野,每个视野的血管平均数为该切片的MVD值。
4.统计学分析
所有数据采用SPSS13.0软件进行统计学分析。定量数据采用均数±标准差(x±s)表示。组间比较采用单因素方差分析,组间多重比较根据各组间方差齐性检验结果选择不同的方法,按0.05检验水准,若组间方差齐同,则采用LSD-t检验,若组间方差不齐,则采用Tamhane's T2检验。以P≤0.05认为差别有统计学意义。
结果
在整个实验过程中,所有小鼠均生长良好,进食、饮水正常,未出现明显消瘦等表现.小鼠在植瘤5天后,接种部位可触及小结节,以后渐呈类圆形或椭圆形生长,至实验结束时(用药2周后)接种部可见明显膨隆肿瘤瘤结块。在整个实验过程中无小鼠死亡。用药后第3天,各组肿瘤生长平缓,体积比较无统计学差异(P>0.05),第3天后,各组肿瘤生长出现差异,联合用药组生长曲线平缓,但未停滞,对照组生长较快,曲线陡直,第12天,联合用药组与单独用药组比较有统计学差异(P<0.05),实验结束各组体积比较有统计学差异(P<0.01)。接种后第6天,开始给予药物治疗,药物干预结束后第二天处死动物称肿瘤瘤重及计算肿瘤体积,各治疗组平均瘤重及体积与对照组相比差别有显著统计学意义(P<0.01),各治疗组平均瘤重及平均体积小于对照组(P<0.01)；各治疗组间的平均瘤重及体积相比差别也有显著统计学意义(P<0.01),联合治疗组平均瘤种及体积小于单独治疗组(P<0.01),ES组、IFO组及联合用药组肿瘤生长抑制率分别为45.83%和76.57%、84.66%,联合用药组抑瘤作用最强。荧光显微镜观察内皮抑素治疗组及联合治疗组灌注血管减少,血管稀疏。对照组、ES组、IFO组与联合用药组的移植瘤MVD及灌注血管平均距离分别为43.13±9.86、17.87±7.69、29.20±9.28、7.60±3.18和43.33±8.21、84.94±6.99、68.96±10.90、121.08±22.75,在荷瘤鼠各组中灌注血管(DiOC7(3))的平均距离比较,各治疗组灌注血管的平均距离与对照组灌注血管的平均距离相比差别有显著统计学意义(P<0.01),对照组灌注血管平均距离小于各治疗组(P<0.01)；联合治疗组灌注血管平均距离与单独治疗组比较差别有显著统计学意义(P<0.01),联合治疗组灌注血管平均距离大于单独治疗组(P<0.01)。免疫组化检测MVD,发现各治疗组均可降低肿瘤组织微血管密度,各治疗组微血管密度与对照组相比差别有显著统计学意义(P<0.01),各治疗组微血管密度低于对照组微血管密度(P<0.01);联合治疗组微血管密度与单独治疗组比较差别有显著统计学意义(P<0.01),联合治疗组微血管密度低于单独用药组(P<0.01)。
结论
内皮抑素结合异环磷酰胺能明显抑制肿瘤血管生成及肿瘤生长,二者联合治疗肿瘤效果比单一使用效果好,联合治疗抗肿瘤具有协同增效作用。
Part one Background
Tumor is a serious harm to human health,Currently,there are surgery, chemotherapy and radiotherapy, biological therapy etc.Various treatments,but curative effect is not ideal, the mortality rate remains high. in the 21st century totally conquering the disease、looking for more effective treatment methods and new no-side-effects drug treatment, have become a hot spots of general medical researchers.Many studies show that tumor formation and growth, the key is invasion and metastasis of tumor vascularization. Tumor did ont enter the invasion of the avascular, when tumor diameter general less than 2-3mm,cell count in 1 x 107, tumor cells are mainly supplied nutrition by dispersion. When the tumor diameter is more than 2-3mm,Tumor will need new blood vessels to supply nutrition,.discharge metabolites and provide transfer channel. The formation of a tumor blood essels is complicated process, many kinds of cell factors participate the process, and influences the formation of new blood vessels, Herefore, the research development of the antiangiogenesis drugs is to inhibit tumor growth, and prevent tumor metastasis effective strategy. Since the 20th century 70's, Folkman puts forward the growth of tumor relys on new angiogenesis, antiangiogenic can treat the substantive tumor. Many preclinical experimental study attempt to deal with a certain angiogenesis link by antiangiogenic drugs in situ tumor of animal or subcutaneous transplantation tumor, which provide some clinical application of the theory and experiment. Currently, antiangiogenic methods mainly have three directions:The first is to neutralize or inhibit the factor, which can promote the formation of blood vessels; The sceond is to give the factor, which can inhibit the formation of blood vessels; The third is to provide paranormal effect of toxic material or antibody make tumor infarction, Many antiangiogenic drugs already have been into clinical trials stage, and hav already obtained encouraging results, Especially vascular statin and endothelial statin. Molecular biology research shows that ES can by induce endothelial cell apoptosis, block activity of the vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP), thereby inhibit tumor growth and metastasis, ES is currently accepted the strongest inhibit angiogenesis factor, has the very strong antitumor activity, can significantly reduce tumor microvascular density within the tumor, increase tumor cell apoptosis. ES can strongly inhibit tumor endothelial cells, and can effectively restrain the growth of the tumor above 60, can adjust 12% of the human genome, and then cut the express pathological angiogenesis,and no side effects, also no resistance and side effects. Several studies have also reported that ES has the good anti-tumor effect in the liver, thyroid cancer, breast cancer, colorectal, cervical cancer and lung adenocarcinoma, and can prevent the formation of metastases. Currently ES are III period clinical trials, many experiments showed that ES has specific inhibition of vascular endothelial cells, has a stronger inhibitory effect for the clinical treatment of tumor, and break a new path. Recombinant human endothelial statin injection is our country experts and scholars research a new human blood endothelial statin, which can specific strongly inhibit endothelial cell proliferation and growth, is a kind of brand-new inhibit angiogenesis inhibitors, with the chemotherapy drug joint act synergistically. With the deepening of the research of molecular biology, chemotherapy combined molecular target therapy has become a hotspot of tumor treatment research.
Part two The inhibitory effect of recombinant human endostatin on angiogenesis in tumors
Objective
1. To evaluate the inhibitory effect of endostatin on angiogenesis and sarcoma growth in mice.
2. To evaluate the concentration-response relationship of endostatin on antiangiogenesis and inhibit sarcoma growth in mice.
Material and methods
1. Establish animal model
S180 cells was provided form abdomen of mice by the department of biology of Nanjing University. The cells were collected and centrifuged at 1000r/min,and then prepared in DEME into 5×107/ml cell suspension.0.1ml cell suspension was inoculated subcutaneouly into the right axillary of mice with sterile syringe, thus the animal model of sarcomas were set up.Observe mice subcutaneous transplant tumors, 75 mice have tumor after S180 cells inoculation only 5 days, all tumor diameter of biggest (a) and the least(b) are measured, calculation tumor size:V=ab2/2, tumor size too big and too small is excluded, There are 60mice only enter laboratory,tumor size about 90mm3.
2. Grouping and drug treatments
The sixty mice implanted with sarcoma were randomly divided into the control, ES low dose(5mg/kg.d)groups、ES middle dose(10mg/kg.d)groups and ES high dose(15mg/kg.d) groups.ES and NS was injected around the tumor,once a day for 14 days.
3. Testing index
The tumor growth situation are observed, the tumor diameter of the biggest (a) and the least (b) are measured with vernier caliper every 3 days until over drug injection, Calculating volume, Taking average, drawing tumor growth curve, The mice in the 4 groups were sacrificed by cervical dislocation at 15 days after initiation of therapy. This DiOC7 was injected into the tail vein 1 min before the mice were sacrificed, at a concentration of 1.0 mg/kg. the tumors were complete removed, Tumor volume was calculated by the formula:l/2×a×b2.where a and b are the major and minor diameters of the tumor as measured using vernier caliper every 3 days after administration. The tumor inhibition rate (TIR) was calculated by the formula:TIR= [(A-B)/A]×100%, where A and B are mean volume of the tumors of the control and treatment groups, respectively. The tumors were then removed and split. Half of the tissue was made frozen sections (5μm thick) for DiOC7 perfusion staining immediately and then the perfusion vessels was observed using fluorescence microscopy,and the average distance of perfusion blood vessels are calculated, and the other half was fixed in formalin and embedded in paraffin, Then, Paraffin sections (5μm thick) were maked for immunohistochemistry. The MVD was quantified in each of the five 'hotspot' areas on a slide at high at×200 magnification,Tumor MVD was calculated as the mean of vessel counts recorded at 5 representative locations.
4. Data and Statistical
All the data were processed by Spss 13.0.All the data were noted in mean±standard deviation (mean±SD); To determine the statistically significant difference in parameters among in the four groups, the one-way ANOVA test was employed, and the LSD or Dunnett's was used in multiple comparison. Statistical significance was set at P< 0.05.
Results
No one mice in the 4 groups died of their tumor burdens before scheduled sacrifice, All mice were killed at 15 days after initiation of therapy. All animals in each group developed tumors (100% tumorigenicity). Five days later, subcutaneous tumor nodules can be seen in 60 mice. The curves showed that tumors size increased gradually in the four groups. All treatment groups decreased tumor volume and weight relative to the control group (P< 0.05), Tumor growth inhibition was greater. Tumor growth was significantly inhibited in the 3 treatment groups(P<0.05), The TIR in the low dose、the middle dose and the high dose groups were 24.91%、45.83%and65.3%,respectively,and showed a marked inhibition of Tumor angiogenesis, The value of MVD and perfusion vessel average distance in Control groups、ES the low dose groups、ES the middle dose groups and ES the high dose groups were 43.13±9.86、30.27±9.95、17.87±7.69、9.60±4.31 and 43.33±8.21、66.06±14.98、84.94±6.99、108.16±18.10, respectively; while Compared with the control group, and among ES significantly (P<0.01).The effect of endostatin on tumor perfused vessels were evaluated using DiOC7 staining.endostatin-treated tumors had markedly increase in mean distances to the nearest perfused vessel compared with control group (P< 0.01),The numbers of perfused vessels was decrease. Mean distance to the perfused vessel was markedly lower in control group.in high dose therapy group, the distance to the perfused vessel was markedly increased than the low dose group and the middle dose group(P< 0.01), indicating a pronounced vascular reduce. The MVD was significantly less in tumors treated with high dose therapy compared to control tumors and tumors treated with low dose and the middle dose esdostatin (P< 0.01).
Conclusion
1.The results show that ES markedly inhibits tumor angiogenesis and tumor growth.
2.The results show that ES have the obvious curative effect depends on the doses.
Part three The inhibitory effect of endostatin combined with Ifosfamide chemoththerapy in treatment of transplant sarcoma in mice
Objective
To evaluate the inhibitory effect of recombinant human endostatin combined with ifosfamide on angiogenesis and transplant sarcoma growth in mice.
Material and methods
1. Establish animal model
S180 cells was provided form abdomen of mice by the department of biology of Nanjing University. The cells were collected and centrifuged at 1000r/min,and then prepared in DEME into 5×107/ml cell suspension.0.1ml cell suspension was inoculated subcutaneouly into the right axillary of mice with sterile syringe, thus the animal model of sarcomas were set up.Observe mice subcutaneous transplant tumors, 75 mice have tumor after S180 cells inoculation only 5 days, all tumor diameter of biggest (a) and the least (b) are measured, calculation tumor size:V=ab2/2, tumor size too big and too small is excluded, There are 60 mices only enter laboratory,tumor size about 90mm3.
2. Grouping and drug treatments
60 nodules with unanimous size were randomized into one control group, two single agent groups and one combination group, fifteen for each group. In the ifosfamide (IFO) group, ifosfamide, 50mg.kg-1, was given by intraperitoneal injection, once a day for 5 days. In the endostatin (ES) group, endostatin,10mg.kg-1, was injected around the tumors, once a day for 14 days. In the combination(ES+IFO) group, endostatin, 10mg.kg-1, was injected around the tumor, once a day for 14 days, meanwhile the ifosfamide,50mg.kg-1,was given by intraperitoneal injection, once a day for 5 days. In the control (NS) group, the same volume of normal saline was given by intraperitoneal injection.
3. Testing index
The tumor growth situation are observed, the tumor diameter of the biggest (a) and the least (b) are measured with vernier caliper every 3 days until over drug injection, Calculating volume, Taking average, drawing tumor growth curve, The mice in the 4 groups were sacrificed by cervical dislocation at 15 days after initiation of therapy. This DiOC? was injected into the tail vein 1 min before the mice were sacrificed, at a concentration of 1.0 mg/kg. the tumors were complete removed, Tumor volume was calculated by the formula:1/2×a×b2.where a and b are the major and minor diameters of the tumor as measured using vernier caliper every 3 days after administration. The tumor inhibition rate (TIR) was calculated by the formula:TIR= [(A-B)/A]×100%, where A and B are mean volume of the tumors of the control and treatment groups, respectively. The tumors were then removed and split. Half of the tissue was made frozen sections (5μn thick) for DiOC7 perfusion staining immediately and then the perfusion vessels was observed using fluorescence microscopy,and the average distance of perfusion blood vessels are calculated, and the other half was fixed in formalin and embedded in paraffin, Then, Paraffin sections (5μm thick) were maked for immunohistochemistry. The MVD was quantified in each of the five 'hotspot' areas on a slide at high at×200 magnification,Tumor MVD was calculated as the mean of vessel counts recorded at 5 representative locations.
4. Data and Statistical
All the data were processed by Spss 13.0.All the data were noted in mean±standard deviation (mean±SD); To determine the statistically significant difference in parameters among in the three groups, the one-way ANOVA test was employed, and the LSD or Dunnett's was used in multiple comparison. Statistical significance was set at P< 0.05.
Results
No one mice in the 4 groups died of their tumor burdens before scheduled sacrifice, All mice were killed at 5 days after initiation of therapy All animals in each group developed tumors (100% tumorigenicity),. Five days later, subcutaneous tumor nodules can be seen in 60 mice. The curves showed that tumors size increased gradually in the four groups.All treatment groups decreased tumor volume and weight relative to the control group (P< 0.01), Tumor growth inhibition was greater. Two single agent groups resulted in a significant reduction of the mean tumor volume and weight compared to control group(P< 0.01), The combined treatment group showed the smallest mean tumor volume and weight relative to the single treatment groups(P<0.01).the growth rate of tumor in combination group was significantly lower than that in other three groups. The tumor-inhibiting rate was 45.83%、76.57% and 84.66% respectively in endostatin group、ifosfamide group and combination group in comparison to control group. The value of MVD and perfusion vessel average distance in Control groups、ES groups、IFO groups and ES+IFO groups were 43.13±9.86、17.87±7.69、29.20±9.28、7.60±3.18 and 43.33±8.21、84.94±6.99、68.96±10.90、121.08±22.75, respectively.The effects of endostatin combined with ifosfamide on tumor perfused vessels were evaluated using DiOC7 staining.endostatin-treated tumors and combination-treated tumors had markedly increase in mean distances to the nearest perfused vessel compared with control group (P<0.01),The numbers of perfused vessels was decrease. Mean distance to the perfused vessel was markedly lower in control group.in combination therapy group, the distance to the perfused vessel was markedly increased (P<0.01) than endostatin group, indicating a pronounced vascular reduce. In summary, in therapy groups The distance to the perfused vessel remained significantly increased compared to control group (P<0.01).The MVD were markedly differences among the four groups.The MVD had a significant reduction in three treatment groups. The MVD was significantly less in tumors treated with endostatin or combination therapy compared to control tumors and tumors treated with ifosfamide alone (P<0.01).In addition, there was a further significant decrease in MVD in tumors treated with endostatin plus ifosfamide compared to tumors treated with either agent alone (P<0.01).
Conclusion
Recombinant human endostatin combined with ifosfamide has synergistic effect on inhibitory activity against the growth of transplanted sarcoma in mice and reduce the formation of capillary than either endostatin or ifosfamide used alone.
肿瘤是一种严重危害人体健康的疾病,目前临床上有手术治疗、化疗、放疗、生物治疗等多种治疗方法,但疗效并不理想,死亡率仍然较高。如何在21世纪彻底攻克这一疾病,寻找更为有效的治疗方法和新的无毒副作用的治疗药物,已成为广大医务科研人员研究的热点之一。许多研究表明肿瘤的形成、生长、浸润和转移的关键是肿瘤的血管化,肿瘤形成后即进入无血管的浸润前期,此时肿瘤的直径一般小于2-3mm,细胞数一般小于1×107,肿瘤细胞主要靠弥散供给营养,当肿瘤直径超过2-3mm时,肿瘤就需要新生的血管维持营养供给、排泄代谢产物和提供转移的通道,而肿瘤血管的形成是一个及其复杂过程,这一过程有许多种相关的血管细胞因子参与,共同影响新生血管的形成。因此,研究开发抑制肿瘤血管生成的抗肿瘤药物是抑制肿瘤生长、防止肿瘤转移的有效策略。自20世纪70年代Folkman提出肿瘤的生长依赖新血管生成,抗血管生成可治疗实体性肿瘤以来,许多临床前的实验研究都想将针对某一血管生成环节的抗血管生成药物应用于动物原位瘤模型或皮下移植肿瘤的治疗,为抗肿瘤血管生成的临床应用提供一定的理论和实验依据,而目前抗肿瘤血管生成的治疗方法主要有3大方向：一是中和或者抑制促进血管形成的因子分泌,例如血管内皮生长因子和纤维细胞生长因子等；二是给予抑制血管形成的因子,例如血管抑制索和内皮抑制素等；三是用对肿瘤血管系统有特异作用的有毒物质或者抗体使肿瘤梗死,而目前对抑制血管生成的相关因子的研究进展最快,多种抗血管生成药物都已进人临床试验阶段,已取得了令人鼓舞的结果,其中又以血管抑素和内皮抑素抗血管生成作用最引人瞩目。分子生物学研究表示：内皮抑素(Endostatin. ES)能通过诱导血管内皮细胞凋亡、阻断血管内皮细胞生长因子(Vascular endothelial cell growth factor. VEGF)和基质金属蛋白酶(Matrix metalloproteinase. MMP)的活性等多种途径抑制新生血管生长,从而抑制肿瘤的生长和转移,是目前公认的最好最强的血管生成抑制因子,具有很强烈的抗肿瘤血管生成活性,能够明显降低肿瘤组织内的微血管密度,增加肿瘤细胞凋亡。内皮抑素对肿瘤血管内皮细胞生长具有强烈的抑制作用,并且能够有效抑制60种以上的肿瘤,能够调节12%的人类基因组表达,进而下调病理性的血管发生,而且没有毒副作用,也无耐药性；多项研究也报道了ES在甲状腺癌、肝癌、乳腺癌、直肠癌、宫颈癌及肺腺癌等多种肿瘤中均显示了良好的抗肿瘤效果,并且能够阻止肿瘤转移的形成。目前内皮抑素正在Ⅲ期临床试验,多项实验都表明内皮抑素具有特异性抑制血管内皮细胞作用,对多种依赖血管生成的实体瘤都有较强的抑制作用,为肿瘤的临床治疗开辟了一条新路。重组人血管内皮抑素注射液是我国专家学者自主创新研发的新型人血管内皮抑素,能够特异性的强烈抑制血管内皮细胞的增殖和生长,是一种全新的抑制血管形成的抑制剂,与化疗药物联合有协同作用,随着分子生物学研究的深入,化疗联合分子靶向治疗已成为肿瘤治疗研究的热点。
第二部分重组人血管内皮抑素对肿瘤血管生成抑制作用的实验研究
目的
1.探讨内皮抑素(Endostatin. ES)对小鼠肉瘤皮下移植瘤血管生成及肿瘤生长的抑制作用。
2.探讨内皮抑素(Endostatin. ES)抑制肿瘤血管生成及肿瘤生长作用的量效关系。
材料和方法。
1.建立小鼠肉瘤模型
将S180肉瘤腹水种鼠传代,无菌条件下抽取传代至第4天的S180肉瘤小鼠腹水,1000r/min,离心5min,PBS稀释,反复三次,最后DMEM液稀释,制成肿瘤细胞悬液,细胞计数为5×107个/ml,将细胞悬液在小鼠右前肢腋下注射0.1ml,然后观察小鼠皮下移植瘤成瘤情况,S180细胞接种后5天,75只荷瘤小鼠全部成瘤,测量肿瘤最大径(a)和最小径(b),计算肿瘤体积：V=ab2/2,将肿瘤体积过大及过小者排除,肿瘤体积约90mm3的60只荷瘤小鼠进入实验。
2.实验分组
采用随机数字表法将60只荷瘤小鼠随机等分为四组：生理盐水组15只：给予等量生理盐水；ES1组15只：给予ES剂量5mg/kg；ES2组15只：给予ES剂量10mg/kg；ES3组15只：给予ES剂量15mg/kg。ES及生理盐水瘤周皮下注射,连续给药14天。
3.检测指标
观察肿瘤生长情况,从内皮抑素用药的第一天算起,每3天用游标卡尺测量肿瘤最大径(a)和最小径(b),直至药物干预结束后第二天,计算每次测量肿瘤的体积,取平均值,绘制肿瘤生长曲线。在药物干预结束后第二天处死小鼠,处死前1分钟尾静脉注射DiOC7(3)(1mg/kg),然后迅速将肿瘤组织完整剥离取出,称瘤重,测量肿瘤最大径(a)和最小径(b),计算肿瘤体积和抑瘤率：抑瘤率(%)=(对照组瘤体平均体积-实验组瘤体平均体积)/对照组瘤体平均体积x100%.将剥离的肿瘤组织一半制作冰冻切片：新鲜标本用OCT包埋后,4-5μm连续切片,荧光显微镜下观察组织的血管标记,摄取DiOC7(3)标记血管图片(绿色荧光),将肿瘤血管图片用于定量分析,经相关软件计算灌注血管平均距离；另一半肿瘤组织立即用10%福尔马林固定,石蜡包埋、5μm切片、免疫组化染色检测肿瘤微血管密度(microvessel density. MVD):切片观察先在低倍光镜下(40倍)确定肿瘤内新生血管最密集区作为“热点”,进行血管计数,然后在高倍镜视野(200倍)计数5个视野,每个视野的血管平均数值为该切片的MVD值。
4.统计学分析
所有数据采用SPSS13.0软件进行统计学分析。定量数据采用均数±标准差(x±s)表示。组间比较采用单因素方差分析,组间多重比较根据各组间方差齐性检验结果选择不同的方法,按0.05检验水准,若组间方差齐同,则采用LSD-t检验,若组间方差不齐,则采用Tamhane's T2检验。以P≤0.05认为差别有统计学意义。
结果
在整个实验过程中,所有荷瘤小鼠均生长良好,进食、饮水正常,未出现明显消瘦等表现。小鼠在植瘤5天后,接种部位可触及小结节,以后渐呈类圆形或椭圆形生长,至实验结束时(用药2周后)接种部可见明显膨隆肿瘤瘤结块。在整个实验过程中无小鼠死亡,小鼠于接种第6天开始给药并记录肿瘤大小,各组肿瘤生长曲线见对照组肿瘤生长曲线陡直,各给药组生长曲线均位于对照组曲线下方,相对对照组略微平缓,且ES中、高剂量组比ES低剂量组小鼠肿瘤生长曲线平缓。接种后第6天,开始给予内皮抑素药物治疗,药物干预结束后第二天处死小鼠称瘤重及计算肿瘤体积。各治疗组平均瘤重及体积与对照组比较差别有统计学意义(P<0.05),各治疗组平均瘤重及体积小于对照组(P<0.05)；各治疗组之间平均瘤重及体积比较差别也有统计学意义(P<0.05),ES高剂量治疗组平均瘤重及体积小于ES中、低治疗组(P<0.05)；ES中剂量治疗组平均瘤种及体积也小于ES低治疗组(P<0.05),ES低、中、高治疗组肿瘤生长抑制率分别为24.91%和45.83%、65.30%。对照组、ES低剂量组、中剂量组、高剂量组MVD及灌注血管平均距离分别为43.13±9.86、30.27±9.95、17.87±7.69、9.60±4.31和43.33±8.21、66.06±14.98、84.94±6.99、108.16±18.10,在荷瘤鼠各组中灌注血管(Di0C7(3))的距离比较中,各治疗组灌注血管距离与对照组相比差别有显著统计学(P<0.01),对照组灌注血管平均距离小于各治疗组(P<0.01)；各治疗组间灌注血管平均距离比较差别也有显著统计学意义(P<0.01),ES高剂量组灌注血管平均距离大于中、低剂量组；ES中剂量组灌注血管平均距离明大于低剂量组(P<0.01)。免疫组化检测微血管密度,经内皮抑素治疗的肿瘤血管生成减少,各治疗组微血管密度与对照组比较差别有显著统计学意义(P<0.01),ES高、中、低剂量组的微血管密度低于对照组(P<0.01)；各治疗组之间微血管密度比较差别也有显著统计学意义(P<0.01),ES高剂量组微血管密度低于中、小剂量ES组(P<0.01)；ES中剂量组微血管密度也低于ES低剂量组(P<0.01)。
结论
1.内皮抑素能抑制实体肿瘤的血管生成及肿瘤的生长。
2.内皮抑素抑制血管生成和肿瘤生长的作用效果有明显的量效关系：随剂量的增多而作用增强。
第三部分重组人血管内皮抑素联合异环磷酰胺对肉瘤小鼠移植瘤血管生成及肿瘤生长抑制作用的实验研究
目的
探讨内皮抑素(Endostatin. ES)联合异环磷酰胺(ifosfamide. IFO)对小鼠肉瘤皮下移植瘤血管生成和肿瘤生长的抑制作用。
材料和方法
1.建立小鼠肉瘤模型
将S180肉瘤腹水种鼠传代,无菌条件下抽取传代至第4天的S180肉瘤小鼠腹水,1000r/min,离心5min,PBS稀释,反复三次,最后DMEM夜稀释,制成肿瘤细胞悬液,细胞计数为5×107个/ml,将细胞悬液在小鼠右前肢腋下注射0.1ml,观察小鼠皮下移植瘤成瘤情况,S180细胞接种后5天,75只荷瘤鼠全部成瘤,测量肿瘤最大径(a)和最小径(b),计算肿瘤体积：V=ab2/2,将肿瘤体积过大及过小者排除,肿瘤体积约90mm3的60只荷瘤小鼠进入实验。
2.实验分组
将60只荷瘤小鼠随机等分为四组：生理盐水组15只：给予等量生理盐水；ES组15只：给予ES剂量10mg/kg；IFO组15只：给予IFO剂量50mg/kg；ES+IFO组只15只：给予ES剂量10mg/kg+IFO剂量50mg/kg。ES及生理盐水瘤周皮下注射,连续给药14天,IFO组腹腔注射给药,连续给药5天。
3.检测指标
观察肿瘤生长情况,从小鼠药物治疗的第一天算起,每3天用游标卡尺测量肿瘤最大径(a)和最小径(b),直至药物治疗药结束后第二天,计算每次测量肿瘤的体积,取平均值,绘制肿瘤生长曲线。在药物干预结束后第二天处死小鼠,处死前1分钟尾静脉注射DiOC7(3)(1mg/kg),然后迅速将肿瘤组织完整剥离取出,称瘤重,测量肿瘤最大径(a)和最小径(b),计算肿瘤体积及抑瘤率：抑瘤率(%)：(对照组瘤体平均体积一实验组瘤体平均体积)/对照组瘤体平均体积x100%。将剥离的肿瘤组织一半制作冰冻切片：新鲜标本用OCT包埋后,4-5μm连续切片,荧光显微镜观察组织血管标记,摄取DiOC7(3)标记血管图片(绿色荧光),用相关软件将肿瘤血管图片用于定量分析,计算肿瘤灌注血管平均距离,血管平均距离越大,血管密度越低,血管平均距离越小,血管密度越高；另一半肿瘤组织立即用10%福尔马林固定,石蜡包埋、5μm切片、免疫组化染色检测肿瘤微血管密度(MVD)：切片观察先在低倍光镜下(40倍)确定肿瘤内新生血管最密集区作为“热点”,进行血管计数,然后在高倍镜视野(200倍)计数5个视野,每个视野的血管平均数为该切片的MVD值。
4.统计学分析
所有数据采用SPSS13.0软件进行统计学分析。定量数据采用均数±标准差(x±s)表示。组间比较采用单因素方差分析,组间多重比较根据各组间方差齐性检验结果选择不同的方法,按0.05检验水准,若组间方差齐同,则采用LSD-t检验,若组间方差不齐,则采用Tamhane's T2检验。以P≤0.05认为差别有统计学意义。
结果
在整个实验过程中,所有小鼠均生长良好,进食、饮水正常,未出现明显消瘦等表现.小鼠在植瘤5天后,接种部位可触及小结节,以后渐呈类圆形或椭圆形生长,至实验结束时(用药2周后)接种部可见明显膨隆肿瘤瘤结块。在整个实验过程中无小鼠死亡。用药后第3天,各组肿瘤生长平缓,体积比较无统计学差异(P>0.05),第3天后,各组肿瘤生长出现差异,联合用药组生长曲线平缓,但未停滞,对照组生长较快,曲线陡直,第12天,联合用药组与单独用药组比较有统计学差异(P<0.05),实验结束各组体积比较有统计学差异(P<0.01)。接种后第6天,开始给予药物治疗,药物干预结束后第二天处死动物称肿瘤瘤重及计算肿瘤体积,各治疗组平均瘤重及体积与对照组相比差别有显著统计学意义(P<0.01),各治疗组平均瘤重及平均体积小于对照组(P<0.01)；各治疗组间的平均瘤重及体积相比差别也有显著统计学意义(P<0.01),联合治疗组平均瘤种及体积小于单独治疗组(P<0.01),ES组、IFO组及联合用药组肿瘤生长抑制率分别为45.83%和76.57%、84.66%,联合用药组抑瘤作用最强。荧光显微镜观察内皮抑素治疗组及联合治疗组灌注血管减少,血管稀疏。对照组、ES组、IFO组与联合用药组的移植瘤MVD及灌注血管平均距离分别为43.13±9.86、17.87±7.69、29.20±9.28、7.60±3.18和43.33±8.21、84.94±6.99、68.96±10.90、121.08±22.75,在荷瘤鼠各组中灌注血管(DiOC7(3))的平均距离比较,各治疗组灌注血管的平均距离与对照组灌注血管的平均距离相比差别有显著统计学意义(P<0.01),对照组灌注血管平均距离小于各治疗组(P<0.01)；联合治疗组灌注血管平均距离与单独治疗组比较差别有显著统计学意义(P<0.01),联合治疗组灌注血管平均距离大于单独治疗组(P<0.01)。免疫组化检测MVD,发现各治疗组均可降低肿瘤组织微血管密度,各治疗组微血管密度与对照组相比差别有显著统计学意义(P<0.01),各治疗组微血管密度低于对照组微血管密度(P<0.01);联合治疗组微血管密度与单独治疗组比较差别有显著统计学意义(P<0.01),联合治疗组微血管密度低于单独用药组(P<0.01)。
结论
内皮抑素结合异环磷酰胺能明显抑制肿瘤血管生成及肿瘤生长,二者联合治疗肿瘤效果比单一使用效果好,联合治疗抗肿瘤具有协同增效作用。
Part one Background
Tumor is a serious harm to human health,Currently,there are surgery, chemotherapy and radiotherapy, biological therapy etc.Various treatments,but curative effect is not ideal, the mortality rate remains high. in the 21st century totally conquering the disease、looking for more effective treatment methods and new no-side-effects drug treatment, have become a hot spots of general medical researchers.Many studies show that tumor formation and growth, the key is invasion and metastasis of tumor vascularization. Tumor did ont enter the invasion of the avascular, when tumor diameter general less than 2-3mm,cell count in 1 x 107, tumor cells are mainly supplied nutrition by dispersion. When the tumor diameter is more than 2-3mm,Tumor will need new blood vessels to supply nutrition,.discharge metabolites and provide transfer channel. The formation of a tumor blood essels is complicated process, many kinds of cell factors participate the process, and influences the formation of new blood vessels, Herefore, the research development of the antiangiogenesis drugs is to inhibit tumor growth, and prevent tumor metastasis effective strategy. Since the 20th century 70's, Folkman puts forward the growth of tumor relys on new angiogenesis, antiangiogenic can treat the substantive tumor. Many preclinical experimental study attempt to deal with a certain angiogenesis link by antiangiogenic drugs in situ tumor of animal or subcutaneous transplantation tumor, which provide some clinical application of the theory and experiment. Currently, antiangiogenic methods mainly have three directions:The first is to neutralize or inhibit the factor, which can promote the formation of blood vessels; The sceond is to give the factor, which can inhibit the formation of blood vessels; The third is to provide paranormal effect of toxic material or antibody make tumor infarction, Many antiangiogenic drugs already have been into clinical trials stage, and hav already obtained encouraging results, Especially vascular statin and endothelial statin. Molecular biology research shows that ES can by induce endothelial cell apoptosis, block activity of the vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP), thereby inhibit tumor growth and metastasis, ES is currently accepted the strongest inhibit angiogenesis factor, has the very strong antitumor activity, can significantly reduce tumor microvascular density within the tumor, increase tumor cell apoptosis. ES can strongly inhibit tumor endothelial cells, and can effectively restrain the growth of the tumor above 60, can adjust 12% of the human genome, and then cut the express pathological angiogenesis,and no side effects, also no resistance and side effects. Several studies have also reported that ES has the good anti-tumor effect in the liver, thyroid cancer, breast cancer, colorectal, cervical cancer and lung adenocarcinoma, and can prevent the formation of metastases. Currently ES are III period clinical trials, many experiments showed that ES has specific inhibition of vascular endothelial cells, has a stronger inhibitory effect for the clinical treatment of tumor, and break a new path. Recombinant human endothelial statin injection is our country experts and scholars research a new human blood endothelial statin, which can specific strongly inhibit endothelial cell proliferation and growth, is a kind of brand-new inhibit angiogenesis inhibitors, with the chemotherapy drug joint act synergistically. With the deepening of the research of molecular biology, chemotherapy combined molecular target therapy has become a hotspot of tumor treatment research.
Part two The inhibitory effect of recombinant human endostatin on angiogenesis in tumors
Objective
1. To evaluate the inhibitory effect of endostatin on angiogenesis and sarcoma growth in mice.
2. To evaluate the concentration-response relationship of endostatin on antiangiogenesis and inhibit sarcoma growth in mice.
Material and methods
1. Establish animal model
S180 cells was provided form abdomen of mice by the department of biology of Nanjing University. The cells were collected and centrifuged at 1000r/min,and then prepared in DEME into 5×107/ml cell suspension.0.1ml cell suspension was inoculated subcutaneouly into the right axillary of mice with sterile syringe, thus the animal model of sarcomas were set up.Observe mice subcutaneous transplant tumors, 75 mice have tumor after S180 cells inoculation only 5 days, all tumor diameter of biggest (a) and the least(b) are measured, calculation tumor size:V=ab2/2, tumor size too big and too small is excluded, There are 60mice only enter laboratory,tumor size about 90mm3.
2. Grouping and drug treatments
The sixty mice implanted with sarcoma were randomly divided into the control, ES low dose(5mg/kg.d)groups、ES middle dose(10mg/kg.d)groups and ES high dose(15mg/kg.d) groups.ES and NS was injected around the tumor,once a day for 14 days.
3. Testing index
The tumor growth situation are observed, the tumor diameter of the biggest (a) and the least (b) are measured with vernier caliper every 3 days until over drug injection, Calculating volume, Taking average, drawing tumor growth curve, The mice in the 4 groups were sacrificed by cervical dislocation at 15 days after initiation of therapy. This DiOC7 was injected into the tail vein 1 min before the mice were sacrificed, at a concentration of 1.0 mg/kg. the tumors were complete removed, Tumor volume was calculated by the formula:l/2×a×b2.where a and b are the major and minor diameters of the tumor as measured using vernier caliper every 3 days after administration. The tumor inhibition rate (TIR) was calculated by the formula:TIR= [(A-B)/A]×100%, where A and B are mean volume of the tumors of the control and treatment groups, respectively. The tumors were then removed and split. Half of the tissue was made frozen sections (5μm thick) for DiOC7 perfusion staining immediately and then the perfusion vessels was observed using fluorescence microscopy,and the average distance of perfusion blood vessels are calculated, and the other half was fixed in formalin and embedded in paraffin, Then, Paraffin sections (5μm thick) were maked for immunohistochemistry. The MVD was quantified in each of the five 'hotspot' areas on a slide at high at×200 magnification,Tumor MVD was calculated as the mean of vessel counts recorded at 5 representative locations.
4. Data and Statistical
All the data were processed by Spss 13.0.All the data were noted in mean±standard deviation (mean±SD); To determine the statistically significant difference in parameters among in the four groups, the one-way ANOVA test was employed, and the LSD or Dunnett's was used in multiple comparison. Statistical significance was set at P< 0.05.
Results
No one mice in the 4 groups died of their tumor burdens before scheduled sacrifice, All mice were killed at 15 days after initiation of therapy. All animals in each group developed tumors (100% tumorigenicity). Five days later, subcutaneous tumor nodules can be seen in 60 mice. The curves showed that tumors size increased gradually in the four groups. All treatment groups decreased tumor volume and weight relative to the control group (P< 0.05), Tumor growth inhibition was greater. Tumor growth was significantly inhibited in the 3 treatment groups(P<0.05), The TIR in the low dose、the middle dose and the high dose groups were 24.91%、45.83%and65.3%,respectively,and showed a marked inhibition of Tumor angiogenesis, The value of MVD and perfusion vessel average distance in Control groups、ES the low dose groups、ES the middle dose groups and ES the high dose groups were 43.13±9.86、30.27±9.95、17.87±7.69、9.60±4.31 and 43.33±8.21、66.06±14.98、84.94±6.99、108.16±18.10, respectively; while Compared with the control group, and among ES significantly (P<0.01).The effect of endostatin on tumor perfused vessels were evaluated using DiOC7 staining.endostatin-treated tumors had markedly increase in mean distances to the nearest perfused vessel compared with control group (P< 0.01),The numbers of perfused vessels was decrease. Mean distance to the perfused vessel was markedly lower in control group.in high dose therapy group, the distance to the perfused vessel was markedly increased than the low dose group and the middle dose group(P< 0.01), indicating a pronounced vascular reduce. The MVD was significantly less in tumors treated with high dose therapy compared to control tumors and tumors treated with low dose and the middle dose esdostatin (P< 0.01).
Conclusion
1.The results show that ES markedly inhibits tumor angiogenesis and tumor growth.
2.The results show that ES have the obvious curative effect depends on the doses.
Part three The inhibitory effect of endostatin combined with Ifosfamide chemoththerapy in treatment of transplant sarcoma in mice
Objective
To evaluate the inhibitory effect of recombinant human endostatin combined with ifosfamide on angiogenesis and transplant sarcoma growth in mice.
Material and methods
1. Establish animal model
S180 cells was provided form abdomen of mice by the department of biology of Nanjing University. The cells were collected and centrifuged at 1000r/min,and then prepared in DEME into 5×107/ml cell suspension.0.1ml cell suspension was inoculated subcutaneouly into the right axillary of mice with sterile syringe, thus the animal model of sarcomas were set up.Observe mice subcutaneous transplant tumors, 75 mice have tumor after S180 cells inoculation only 5 days, all tumor diameter of biggest (a) and the least (b) are measured, calculation tumor size:V=ab2/2, tumor size too big and too small is excluded, There are 60 mices only enter laboratory,tumor size about 90mm3.
2. Grouping and drug treatments
60 nodules with unanimous size were randomized into one control group, two single agent groups and one combination group, fifteen for each group. In the ifosfamide (IFO) group, ifosfamide, 50mg.kg-1, was given by intraperitoneal injection, once a day for 5 days. In the endostatin (ES) group, endostatin,10mg.kg-1, was injected around the tumors, once a day for 14 days. In the combination(ES+IFO) group, endostatin, 10mg.kg-1, was injected around the tumor, once a day for 14 days, meanwhile the ifosfamide,50mg.kg-1,was given by intraperitoneal injection, once a day for 5 days. In the control (NS) group, the same volume of normal saline was given by intraperitoneal injection.
3. Testing index
The tumor growth situation are observed, the tumor diameter of the biggest (a) and the least (b) are measured with vernier caliper every 3 days until over drug injection, Calculating volume, Taking average, drawing tumor growth curve, The mice in the 4 groups were sacrificed by cervical dislocation at 15 days after initiation of therapy. This DiOC? was injected into the tail vein 1 min before the mice were sacrificed, at a concentration of 1.0 mg/kg. the tumors were complete removed, Tumor volume was calculated by the formula:1/2×a×b2.where a and b are the major and minor diameters of the tumor as measured using vernier caliper every 3 days after administration. The tumor inhibition rate (TIR) was calculated by the formula:TIR= [(A-B)/A]×100%, where A and B are mean volume of the tumors of the control and treatment groups, respectively. The tumors were then removed and split. Half of the tissue was made frozen sections (5μn thick) for DiOC7 perfusion staining immediately and then the perfusion vessels was observed using fluorescence microscopy,and the average distance of perfusion blood vessels are calculated, and the other half was fixed in formalin and embedded in paraffin, Then, Paraffin sections (5μm thick) were maked for immunohistochemistry. The MVD was quantified in each of the five 'hotspot' areas on a slide at high at×200 magnification,Tumor MVD was calculated as the mean of vessel counts recorded at 5 representative locations.
4. Data and Statistical
All the data were processed by Spss 13.0.All the data were noted in mean±standard deviation (mean±SD); To determine the statistically significant difference in parameters among in the three groups, the one-way ANOVA test was employed, and the LSD or Dunnett's was used in multiple comparison. Statistical significance was set at P< 0.05.
Results
No one mice in the 4 groups died of their tumor burdens before scheduled sacrifice, All mice were killed at 5 days after initiation of therapy All animals in each group developed tumors (100% tumorigenicity),. Five days later, subcutaneous tumor nodules can be seen in 60 mice. The curves showed that tumors size increased gradually in the four groups.All treatment groups decreased tumor volume and weight relative to the control group (P< 0.01), Tumor growth inhibition was greater. Two single agent groups resulted in a significant reduction of the mean tumor volume and weight compared to control group(P< 0.01), The combined treatment group showed the smallest mean tumor volume and weight relative to the single treatment groups(P<0.01).the growth rate of tumor in combination group was significantly lower than that in other three groups. The tumor-inhibiting rate was 45.83%、76.57% and 84.66% respectively in endostatin group、ifosfamide group and combination group in comparison to control group. The value of MVD and perfusion vessel average distance in Control groups、ES groups、IFO groups and ES+IFO groups were 43.13±9.86、17.87±7.69、29.20±9.28、7.60±3.18 and 43.33±8.21、84.94±6.99、68.96±10.90、121.08±22.75, respectively.The effects of endostatin combined with ifosfamide on tumor perfused vessels were evaluated using DiOC7 staining.endostatin-treated tumors and combination-treated tumors had markedly increase in mean distances to the nearest perfused vessel compared with control group (P<0.01),The numbers of perfused vessels was decrease. Mean distance to the perfused vessel was markedly lower in control group.in combination therapy group, the distance to the perfused vessel was markedly increased (P<0.01) than endostatin group, indicating a pronounced vascular reduce. In summary, in therapy groups The distance to the perfused vessel remained significantly increased compared to control group (P<0.01).The MVD were markedly differences among the four groups.The MVD had a significant reduction in three treatment groups. The MVD was significantly less in tumors treated with endostatin or combination therapy compared to control tumors and tumors treated with ifosfamide alone (P<0.01).In addition, there was a further significant decrease in MVD in tumors treated with endostatin plus ifosfamide compared to tumors treated with either agent alone (P<0.01).
Conclusion
Recombinant human endostatin combined with ifosfamide has synergistic effect on inhibitory activity against the growth of transplanted sarcoma in mice and reduce the formation of capillary than either endostatin or ifosfamide used alone.
引文
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