人IL-8基因在卵巢上皮性癌浸润转移中作用机制的研究
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摘要
目的:卵巢癌是威胁妇女健康常见的妇科肿瘤,因其早期发现较难,肿瘤病理类型多,组织结构复杂并各具不同的生物学特性,一直是妇科肿瘤领域研究的难点。卵巢上皮性癌其恶性程度比性索间质性肿瘤高,治疗效果也远不如恶性生殖细胞肿瘤,病程发展迅速,而且以盆腹腔种植和直接浸润蔓延为主要特征,不易早期诊断,死亡率极高,是卵巢癌中最有待研究的一种肿瘤,因此一种全新的治疗方法亟待需要探讨。基因的异常表达及其与卵巢肿瘤的发生发展关系的研究已成为分子生物学研究的热点。卵巢癌的发生和发展是一个复杂、多步骤的过程,与许多基因和蛋白的表达密切相关。
白细胞介素8(interleukin 8,IL-8),是Yoshimur于1987年发现的第一个趋化性细胞因子,属于CXC家族,在炎症和免疫过程中起重要的调节作用。近年来IL-8作为恶性肿瘤相关因子越来越多地引起了国内外学者的关注。目前已知多种肿瘤细胞均可分泌IL-8,实体肿瘤组织IL-8过度表达与肿瘤生长侵袭转移密切相关,而其促进肿瘤发生发展的机制尚不完全清楚。
DNA重组技术的建立与发展在理论和实践上为卵巢癌治疗提供了新的策略——基因治疗有望成为卵巢癌治疗的新途径。理想的治疗基因应有效的抑制肿瘤细胞生长,而对正常组织、细胞无明显抑制作用。本研究将人IL-8基因克隆到真核表达载体pcDNA3.1(+)中,在脂质体作用下瞬时和/或稳定转染低表达IL-8基因的上皮性卵巢癌细胞株OVCAR-3中,探讨了其对卵巢癌细胞生长增殖及侵袭转移能力的作用机制,将培养好的各实验组卵巢癌OVCAR-3细胞悬液直接接种于裸鼠背部皮下组织内,观察了裸鼠生长状况和/或出瘤时间及IL-8cDNA对裸鼠皮下移植瘤生长及卵巢癌肺转移的影响,为IL-8基因在上皮性卵巢癌发生发展与治疗及判断预后作用机制的深入研究奠定了实验及理论基础。
方法:
1构建IL-8基因真核表达载体
提取健康志愿者外周血单个核细胞(PBMC)的总RNA,通过RT-PCR扩增IL-8编码序列基因片段,并将其克隆至PMD-18T载体构成PMD-18T/ IL-8,BamHI和XhoI双酶切后,与经相同双酶切后的真核pCDNA3.1(+)表达载体连接,构建成pcDNA3.1(+)/IL-8重组质粒。
2瞬时和/或稳定转染人卵巢癌细胞OVCAR-3
以脂质体介导法将pcDNA3.1(+)/IL-8重组质粒转染低表达该目的基因的OVCAR-3细胞,经RT-PCR、ELISA、Western-blot法检测转染细胞中IL-8的表达情况。
3检测细胞增殖情况
MTT法检测转染pcDNA3.1(+)/IL-8组、pcDNA3.1(+)组与未转染组OVCAR-3细胞的生长增殖能力,绘制生长增殖曲线。
4检测各组OVCAR-3细胞的细胞周期变化
用流式细胞仪(FCM)检测3组细胞,即转染pcDNA3.1(+)/IL-8重组质粒组、转染pcDNA3.1(+)空质粒组和未转染组OVCAR-3细胞周期变化。
5测定各组OVCAR-3细胞的Bax、Bcl-2、VEGF和MMP-9四种蛋白的表达情况
采用FCM检测转染pcDNA3.1(+)/IL-8重组质粒组、转染pcDNA3.1(+)空质粒组和未转染组3组OVCAR-3细胞的Bax、Bcl-2、VEGF和MMP-9四种蛋白的表达变化。
6 Transwell小室法检测卵巢癌OVCAR-3细胞侵袭转移能力
选用Transwell小室和Matrigel胶建立了体外侵袭模型,评价稳定转染pcDNA3.1(+)/IL-8重组质粒的卵巢癌细胞侵袭能力的变化情况。Transwell小室分为上下两室,中间以聚碳酸脂滤膜(微孔直径为8μm)相隔,微孔允许肿瘤细胞穿越,Matrigel是大鼠ESH肉瘤细胞外基质提取物,由Ⅳ型胶原、层粘蛋白、硫酸肝磷脂蛋白多糖及整合素等组成,其成分与组织基底膜相似,可模拟组织基底膜,用以观察细胞同细胞外基质间的相互作用。将Matrigel胶铺在Transwell小室的聚碳酸脂膜上层,计数并比较穿膜细胞数。
7 IL-8cDNA对裸鼠皮下移植瘤生长及卵巢癌肺转移的影响
常规消化培养中的各组OVCAR-3细胞,PBS洗3次,台盼蓝染色证实活细胞数>95%,调整细胞浓度至7.5×107/ml,取细胞悬液0.2 ml接种于裸鼠右后背部皮下组织内,观察裸鼠生长状况和/或出瘤时间。接种细胞9周后,断颈处死全部动物,取出肿瘤组织,测量肿瘤最大直径及相对应的横径,根据公式V=π/6×肿瘤直径(mm)×肿瘤横径(mm),计算肿瘤大小;并将肿瘤组织作成切片,CD34和NF-κB免疫组织化学染色,应用Motic Med 6.0数码医学图像分析系统,在相同的放大倍数(400×)下,以阳性细胞染色的平均光密度值(OD)值来表示NF-κB、CD-34的表达量,每张片子随机选取五个视野,取平均值表示每张片子NF-κB、CD-34的表达量。NikonFXA尼康光镜显微照相机拍照。取裸鼠肺组织,10%甲醛固定,石蜡切片,HE染色,显微镜下观察细胞。
结果:
1经PCR扩增、双酶切及DNA测序鉴定,克隆的目的基因序列正确,证实重组真核质粒pcDNA3.1(+)/IL-8构建成功。
2 RT-PCR法检测到瞬时和稳定转染的OVCAR-3细胞中有大量IL-8的表达,转染pcDNA3.1(+)/ IL-8后IL-8基因的扩增片段灰度值为226.88±5.47,与相应β-actin灰度值之比1.55 ;未转染组的IL-8基因相应位置的灰度值120.38±6.72,与相应β-actin灰度值之比0.82,增殖率为189.02%;
ELISA法检测结果,转染pcDNA3.1(+)/ IL-8重组质粒6h、48h后OVCAR-3细胞上清和细胞裂解液中IL-8表达量分别为3.69±0.37、19.67±0.24、11.10±1.14,而未转染组和转染空质粒组仅检测到极少量,后两者与前者比较有统计学意义(P<0.05);
Western-blot法检测前者有IL-8特异性蛋白条带(8KD),后两者未见特异性条带。
3 MTT法结果显示,转染pcDNA3.1(+)/ IL-8后第1~6天OVCAR-3细胞光吸收值分别为: 0.56±0.08、0.63±0.06、0.93±0.09、1.20±0.06、1.32±0.12、1.39±0.14;未转染组第1~6天为: 0.62±0.07、0.70±0.10、0.84±0.12、0.92±0.10、0.96±0.14、1.01±0.10,第1~3天两两比较均无统计学意义(P>0.05),转染3天后,两组细胞的光吸收值在统计学上具有显著差异(P<0.05),且细胞的增殖率随着时间的延长呈现上升趋势。
4流式细胞(FCM)周期结果表明转染pcDNA3.1(+)/IL-8与转染pcDNA3.1(+)、未转染组三组OVCAR-3细胞进入S期数分别为55.18±2.98、39.72±4.16、31.36±1.46,前者进入S期数明显增多,且与后两者比较有统计学意义(P<0.05);三者增殖指数分别为58.83±2.85、44.32±4.20、35.31±1.56,转染pcDNA3.1(+)/IL-8组PI值明显增加,且与后两者比较具有统计学意义(P<0.05)。
5 FCM对转染pcDNA3.1(+)/IL-8、转染pcDNA3.1(+)和未转染三组OVCAR-3细胞蛋白分析结果表明,与细胞增殖相关细胞因子VEGF和MMP-9表达量分别为:430.44±21.70、358.70±17.77、335.52±19.68;356.15±22.70、279.37±27.48、283.56±26.67,转染pcDNA3.1(+)/IL-8组与后两者比较有统计学意义(p<0.05);三组凋亡抑制因子Bcl-2结果分别为380.86±15.27、319.15±17.89、305.04±28.90;凋亡因子Bax分别为343.12±26.10、416.27±25.45、420.13±23.65,前者与后两者比较有统计学意义(p<0.05),而未转染组与转染空质粒组比较均无统计学意义(p>0.05)。
6 Transwell小室侵袭实验结果显示,未转染组、空质粒组和IL-8组穿膜细胞数分别为:101.47±13.52、98.07±13.97、163.73±12.98。转染IL-8cDNA组细胞较转染空质粒组和未转染组细胞穿膜细胞数明显增加,(P<0.05)差异有显著性意义,提示转染IL-8基因后增加了卵巢癌细胞的侵袭能力。
7转染IL-8cDNA对裸鼠皮下移植瘤生长的影响
IL-8组成瘤时间为16.00±1.53天,空质粒组成瘤时间为19.90±1.28天,阴性对照组成瘤时间为19.70±1.25天,各组成瘤率均为100%。各组成瘤时间比较有统计学意义(P<0.05)。
接种肿瘤细胞后7、8、9周,IL-8组肿瘤生长明显活跃于空质粒组和阴性对照组。7、8、9周肿瘤体积分别为:IL-8组:1394.795±108.559mm3、1773.860±133.087 mm3、2207.659±209.713 mm3 ;空质粒组:1106.625±146.984 mm3、1402.206±127.053mm3、1657.190±173.368mm3;阴性对照组: 1110.330±186.458 mm3、1355.918±210.983 mm3、1582.372±169.642 mm3。IL-8组肿瘤体积大于空质粒组和阴性对照组,且两两比较有统计学意义(P<0.05)。实验前6周各组肿瘤体积比较无明显差别(P>0.05)。
各动物实验组肺组织均未见癌组织转移。
8 MVD和NF-κB免疫组化结果:
MVD免疫组化结果:组织内有棕黄色粗颗粒分布或棕黄色细腻颗粒弥漫分布者为阳性反应。空质粒和阴性对照组的肿瘤组织内可见少量阳性反应。IL-8组肿瘤组织中阳性表达明显增加,阳性目标平均光密度值为0.1942±0.0175,明显高于空质粒组和阴性对照组肿瘤组织的光密度值0.1330±0.0134、0.1330±0.0134。两两比较结果显示,IL-8组与空质粒组,IL-8组与阴性对照组均有明显差异(P<0.05),空质粒组与阴性对照组之间无明显差异(P>0.05)。
NF-κB免疫组化结果:组织内有棕黄色粗颗粒分布或棕黄色细腻颗粒弥漫分布者为阳性反应。空质粒和阴性对照组的肿瘤组织内可见少量阳性反应。IL-8组肿瘤组织内阳性表达明显增加,阳性目标平均光密度值为0.1072±0.0049 ,明显高于空质粒组和阴性对照组的光密度值0.0812±0.0100、0.0794±0.0098(P<0.05)。两两比较结果显示,IL-8组与空质粒组,IL-8组与阴性对照组均有明显差异(P<0.05),空质粒组与阴性对照组之间无明显差异(P>0.05)。
结论
1、成功构建IL-8基因的重组真核质粒pcDNA3.1(+)/IL-8;并在脂质体介导下转染人卵巢癌细胞株OVCAR-3,经RT-PCR、ELISA、Western-blot法检测从基因和蛋白水平证实转染成功有效;
2、MTT法检测IL-8可明显促进OVCAR-3细胞增长,并随着时间的增长,细胞的增值率呈现上升趋势;
3、FCM法显示转染IL-8的卵巢癌细胞S期数增多,细胞增殖指数相应升高,表明IL-8基因可以使细胞进入分裂期数增加,因此认为,IL-8基因促进细胞周期进入S期很可能是其促进肿瘤发生发展的重要机制之一;
4、ELISA检测结果显示转染空质粒48h后也有大量IL-8分泌,可以认为转染本身也可以作为一种刺激因子而使IL-8作为炎性因子或自分泌因子大量分泌,但现在还没有关于此方面的确切报导,值得我们今后进一步研究。
5、Transwell小室侵袭实验结果显示转染IL-8基因后增加了卵巢癌细胞的侵袭能力。
6、IL-8基因可使VEGF/MMP-9/Bcl-2表达上调,使Bax表达下调,
其机制与促血管生成因子和凋亡相关因子密切相关。7、动物实验研究发现IL-8能够诱导NF-κB的表达影响肿瘤细胞的增殖、侵袭和转移;IL-8同时促进肿瘤组织内毛细血管的生成,为卵巢癌组织生长侵袭和远处转移提供必要条件。
所有这些为进一步研究IL-8在卵巢上皮性癌发生浸润转移和判断预后中的作用机制提供了坚实的理论和实验基础。
Objective: Being one of department of gynecology tumor, ovarian cancer which is seriously attacking women’s health has driven professor’s attention which is hard to search out in early stage. There are varieties of tumor of the ovary whose organism construction are complicated and owe different bionomics. The malignant degree of epithelium tissue cancer is higher than interstitial substance cancer. While its therapeutic efficacy can not reach the germinocarcinoma. And this tumor which grows very fast, can’t be diagnosed easily in the early stage and its death rate is very high is eager to be researched in the region of woman tumor. So a new therapy should be found. Recently, the research that specialists try to find out the cause and developing relationship of some uncommon genes and ovarian cancer is hitting at the district of boss molecular biology research. Ovarian cancer’s cause and developing relationship is a complex and mixed-step process which involves quantities genes and proteins’expression.
IL-8 which is found by Yoshimur at 1987 is the first chematropism cell factor belonging to CXC family that plays an important role in the contribution of inflammation and immunologic process. Recently, it has drawn more and more attention of researchers as malignant tumor correlative molecule. Nowadays, it is known that many tumor cell can secrete IL-8, IL-8 overexpression relates to tumorous growth and metastasis though it is not clear that the mechanism of encouraging tumor appearance and development.
Recent years, the construction and development of recombination technology can provide new strategy for ovarian cancer in theory and practice——gene therapy can be expected to become new channel of ovarian cancer therapy. Ideal therapeutic gene can inhibit tumor cell’s growth effectively without inhibitory action to normal tissue and cell. In this research, to explore the intereffecting mechanism of IL-8 and epithelial ovarian cancer growth, invasion and metastasis , the eukaryotic expression vector of hIL-8 was constructed and expressed in OVCAR-3 cells after transient and stable transfection. Meanwhile pcDNA3.1(+)/IL-8, pcDNA3.1(+) and non-tranfected OVCAR-3 cells were inoculated subcutaneously into every nude mouse respectively according to three divided groups, Mice’s general condition and time of tumour growth were observed. The volume of transplanted tumor and pulmonary metastasis were compared with different groups. The foundation was settled for further investigation of tumorigenesis and tumor growth.
Method:
1 Construct the eukaryotic expression vector of IL-8 gene. Total RNA was isolated from PBMC of healthy volunteers, IL-8 gene encoding fragment was amplified by RT-PCR and cloned into the vector PMD-18T. BamHI/XhoI double digested product of PMD-18T/IL-8 was connected with the vector pcDNA3.1(+) which was digested by BamHI/XhoI .
2 Transient and stable transfect recombinant plasmid into OVCAR-3 cells. The recombinant plasmid pcDNA3.1(+)/IL-8 was transfected into OVCAR-3 cells which express IL-8 a little with Lipofectamine2000. The expression of IL-8 was tested in OVCAR-3 cells by RT-PCR ,ELISA and Western blot assay.
3 Detect cell proliferation. Detect these cells’reproductive activities by MTT and draw growth curve.
4 The cell cycle was detected by flow cytometry (FCM) on three group cells. pcDNA3.1(+)/IL-8 transfected group, pcDNA3.1(+) group and OVCAR-3 none transfected group.
5 Measure Bax,Bcl-2,VEGF and MMP-9 expression analysis.The expression of Bax,Bcl-2,VEGF and MMP-9 were detected by FCM on three group cells the same as method 4.
6 Using transwell cabin to detect invasive ability of different OVCAR-3 cell lines. Establish vitro invasive model by transwell cabin and matrigel to evaluate the invasive ability of OVCAR-3 cells which stable transfected recombinant plasmid IL-8. Transwell cabin divided into two cabin by polycarbonate fliter membrance(micropore diamerer is 8μm). Micropore allow cell to pass through. Matrigel is composed by typeⅣcollagen、laminin and integrin, similar with basal membrane. The number of cells which pass through the micropore was counted and compared with each other.
7 Effect of IL-8cDNA on transplanted tumour growth and lung metastasis. Mice are divided into three group, and OVCAR-3, OVCAR-3/pcDNA3.1(+) and OVCAR-3/pcDNA3.1(+)/IL-8 cells were subcutaneously inoculated into nude mice according to three divided groups. Mice were killed after 9 weeks, the volume of tumour in each group was compared in different time and the expression of CD34 and NF-κB of tumours from different group were detected by immunohistochemical method. The tissue slice of lung was observed.
RESULTS:
1 After PCR amplification, BamHI/XhoI digestion and DNA sequencing confirmation, the eukaryotic expression vector pcDNA3.1(+)/IL-8 was constructed successfully.
2 The IL-8’s expressions in quantity were founded in the cells of transient transfection when detected by RT-PCR. The gray scale value of pcDNA3.1(+)/ IL-8 transfected group which was 226.88±5.47 correspondingly againstβ-actin was 1.55, while OVCAR-3 none transfected group which was 120.38±6.72 correspondingly againstβ-actin was 0.82. The growth rate was 189.02%;The IL-8’s expressions in cell sap and the cells after transfected pcDNA3.1(+)/ IL-8 6h and 48h were 3.69±0.37, 19.67±0.24, 11.10±1.14 respectively by ELISA, while the others were a little, P<0.05; the specific protein strap was detected only in the former by Western-blot.
3 The results by MTT indicated that the light absorption value of OVCAR-3 cells transfected IL-8/pcDNA3.1 were 0.56±0.08, 0.63±0.06, 0.93±0.09, 1.20±0.06, 1.32±0.12, 1.39±0.14 respectively, while OVCAR-3 none transfected group were 0.62±0.07, 0.70±0.10, 0.84±0.12, 0.92±0.10, 0.96±0.14, 1.01±0.10 respectively. There was no statistical significance compared pcDNA3.1(+)/ IL-8 transfected group with the ones not transfected within 3days(P>0.05), while pcDNA3.1(+)/ IL-8 transfected group was obviously more active after 3 days(P<0.05), and the former’s increasing rate presents an evident tendency of going up along with the time’s extension.
4 The cellular S stage in pcDNA3.1(+)/IL-8, pcDNA3.1(+) and OVCAR-3 cells were 55.18±2.98, 39.72±4.16, 31.36±1.46 respectively by FCM. The former was much higher than the others, P<0.05. These proliferation index were 58.83±2.85, 44.32±4.20, 35.31±1.56.The former PI was much higher and the comparison between them has statistical significance(P<0.05).
5 The expression of VEGF and MMP-9 in pcDNA3.1(+)/IL-8, pcDNA3.1(+) and OVCAR-3 cells were 430.44±21.70, 358.70±17.77, 335.52±19.68 and 356.15±22.70, 279.37±27.48, 283.56±26.67 respectively; while Bcl-2 and Bax in these cells were 380.86±15.27, 319.15±17.89, 305.04±28.90 and 343.12±26.10, 416.27±25.45, 420.13±23.65 respectively. There was statistical significance against pcDNA3.1(+) transfected group and OVCAR-3 none transfected group (p<0.05), while the comparison between them has no statistical significance.
6 Cell number that pass through micropore of OVCAR-3 group, empty plasmid group and IL-8 group is 101.47±13.52, 98.07±13.97, 163.73±12.98. There was statistical significance against OVCAR-3 group, empty plasmid group and IL-8 group. This confirm that IL-8 maybe increase invasive ability of OVCAR-3.
7 Effect of transplanted tumor growth in nude mice by IL-8. Days of tumour growth of OVCAR-3 group, empty plasmid group and IL-8 group is 19.70±1.25, 19.90±1.28, 16.00±1.53. Rate of tumor growth is 100%. Tumor volume of IL-8 group is much bigger than other two groups in 7, 8, 9 week . There have no difference in other times. Cancer cells couldn’t found in lung in each group.
8 Immunohistochemical result of MVD and NF-κB. The MVD and NF-κB in mice treated with IL-8 was much higher than other two groups. Light absorption value of CD34 of OVCAR-3 group, empty plasmid group and IL-8 group are 0.1330±0.0134, 0.1330±0.0134, 0.1942±0.0175. There was statistical significance. Light absorption value of NF-κB of OVCAR-3 group, empty plasmid group and IL-8 group are 0.0794±0.0098, 0.0812±0.0100, 0.1072±0.0049. There was statistical significance.
Conclusions: The IL-8 gene is transfected into OVCAR-3 cells effectively by liposome. Moreover, IL-8 also promotes the proliferation of cell by increasing the number of cell S stage, that the mechanism is connected with angiogenesis factor and correlation factor about apoptosis, which provides a foundation for researching the mechanism of IL-8 on ovary cancer. Result of ELISA showed that cell secretes much more IL-8 after empty plasmid transfection, we consider that transfection maybe stimulate the secretion of IL-8, but we could not find any report about this respect, we should study it more deeply in the future. Invasive experiment showed that IL-8 could increases the invasive ability of OVCAR-3. IL-8 enhances the express of VEGF/MMP-9/Bcl-2 and inhibits the espress of Bax. This mechanism is intimate correlated with angiogenesis factors and apoptosis factors. The animal experiment showed that IL-8 couses the growth, invasion and metastasis of tumor by induce the express of NF-κB and increase the value of MVD. All of this provide the theoretical and experimental basis for us to study the mechanism of growth, invasion ,metastasis and prognosis in epithelial ovarian cancer.
白细胞介素8(interleukin 8,IL-8),是Yoshimur于1987年发现的第一个趋化性细胞因子,属于CXC家族,在炎症和免疫过程中起重要的调节作用。近年来IL-8作为恶性肿瘤相关因子越来越多地引起了国内外学者的关注。目前已知多种肿瘤细胞均可分泌IL-8,实体肿瘤组织IL-8过度表达与肿瘤生长侵袭转移密切相关,而其促进肿瘤发生发展的机制尚不完全清楚。
DNA重组技术的建立与发展在理论和实践上为卵巢癌治疗提供了新的策略——基因治疗有望成为卵巢癌治疗的新途径。理想的治疗基因应有效的抑制肿瘤细胞生长,而对正常组织、细胞无明显抑制作用。本研究将人IL-8基因克隆到真核表达载体pcDNA3.1(+)中,在脂质体作用下瞬时和/或稳定转染低表达IL-8基因的上皮性卵巢癌细胞株OVCAR-3中,探讨了其对卵巢癌细胞生长增殖及侵袭转移能力的作用机制,将培养好的各实验组卵巢癌OVCAR-3细胞悬液直接接种于裸鼠背部皮下组织内,观察了裸鼠生长状况和/或出瘤时间及IL-8cDNA对裸鼠皮下移植瘤生长及卵巢癌肺转移的影响,为IL-8基因在上皮性卵巢癌发生发展与治疗及判断预后作用机制的深入研究奠定了实验及理论基础。
方法:
1构建IL-8基因真核表达载体
提取健康志愿者外周血单个核细胞(PBMC)的总RNA,通过RT-PCR扩增IL-8编码序列基因片段,并将其克隆至PMD-18T载体构成PMD-18T/ IL-8,BamHI和XhoI双酶切后,与经相同双酶切后的真核pCDNA3.1(+)表达载体连接,构建成pcDNA3.1(+)/IL-8重组质粒。
2瞬时和/或稳定转染人卵巢癌细胞OVCAR-3
以脂质体介导法将pcDNA3.1(+)/IL-8重组质粒转染低表达该目的基因的OVCAR-3细胞,经RT-PCR、ELISA、Western-blot法检测转染细胞中IL-8的表达情况。
3检测细胞增殖情况
MTT法检测转染pcDNA3.1(+)/IL-8组、pcDNA3.1(+)组与未转染组OVCAR-3细胞的生长增殖能力,绘制生长增殖曲线。
4检测各组OVCAR-3细胞的细胞周期变化
用流式细胞仪(FCM)检测3组细胞,即转染pcDNA3.1(+)/IL-8重组质粒组、转染pcDNA3.1(+)空质粒组和未转染组OVCAR-3细胞周期变化。
5测定各组OVCAR-3细胞的Bax、Bcl-2、VEGF和MMP-9四种蛋白的表达情况
采用FCM检测转染pcDNA3.1(+)/IL-8重组质粒组、转染pcDNA3.1(+)空质粒组和未转染组3组OVCAR-3细胞的Bax、Bcl-2、VEGF和MMP-9四种蛋白的表达变化。
6 Transwell小室法检测卵巢癌OVCAR-3细胞侵袭转移能力
选用Transwell小室和Matrigel胶建立了体外侵袭模型,评价稳定转染pcDNA3.1(+)/IL-8重组质粒的卵巢癌细胞侵袭能力的变化情况。Transwell小室分为上下两室,中间以聚碳酸脂滤膜(微孔直径为8μm)相隔,微孔允许肿瘤细胞穿越,Matrigel是大鼠ESH肉瘤细胞外基质提取物,由Ⅳ型胶原、层粘蛋白、硫酸肝磷脂蛋白多糖及整合素等组成,其成分与组织基底膜相似,可模拟组织基底膜,用以观察细胞同细胞外基质间的相互作用。将Matrigel胶铺在Transwell小室的聚碳酸脂膜上层,计数并比较穿膜细胞数。
7 IL-8cDNA对裸鼠皮下移植瘤生长及卵巢癌肺转移的影响
常规消化培养中的各组OVCAR-3细胞,PBS洗3次,台盼蓝染色证实活细胞数>95%,调整细胞浓度至7.5×107/ml,取细胞悬液0.2 ml接种于裸鼠右后背部皮下组织内,观察裸鼠生长状况和/或出瘤时间。接种细胞9周后,断颈处死全部动物,取出肿瘤组织,测量肿瘤最大直径及相对应的横径,根据公式V=π/6×肿瘤直径(mm)×肿瘤横径(mm),计算肿瘤大小;并将肿瘤组织作成切片,CD34和NF-κB免疫组织化学染色,应用Motic Med 6.0数码医学图像分析系统,在相同的放大倍数(400×)下,以阳性细胞染色的平均光密度值(OD)值来表示NF-κB、CD-34的表达量,每张片子随机选取五个视野,取平均值表示每张片子NF-κB、CD-34的表达量。NikonFXA尼康光镜显微照相机拍照。取裸鼠肺组织,10%甲醛固定,石蜡切片,HE染色,显微镜下观察细胞。
结果:
1经PCR扩增、双酶切及DNA测序鉴定,克隆的目的基因序列正确,证实重组真核质粒pcDNA3.1(+)/IL-8构建成功。
2 RT-PCR法检测到瞬时和稳定转染的OVCAR-3细胞中有大量IL-8的表达,转染pcDNA3.1(+)/ IL-8后IL-8基因的扩增片段灰度值为226.88±5.47,与相应β-actin灰度值之比1.55 ;未转染组的IL-8基因相应位置的灰度值120.38±6.72,与相应β-actin灰度值之比0.82,增殖率为189.02%;
ELISA法检测结果,转染pcDNA3.1(+)/ IL-8重组质粒6h、48h后OVCAR-3细胞上清和细胞裂解液中IL-8表达量分别为3.69±0.37、19.67±0.24、11.10±1.14,而未转染组和转染空质粒组仅检测到极少量,后两者与前者比较有统计学意义(P<0.05);
Western-blot法检测前者有IL-8特异性蛋白条带(8KD),后两者未见特异性条带。
3 MTT法结果显示,转染pcDNA3.1(+)/ IL-8后第1~6天OVCAR-3细胞光吸收值分别为: 0.56±0.08、0.63±0.06、0.93±0.09、1.20±0.06、1.32±0.12、1.39±0.14;未转染组第1~6天为: 0.62±0.07、0.70±0.10、0.84±0.12、0.92±0.10、0.96±0.14、1.01±0.10,第1~3天两两比较均无统计学意义(P>0.05),转染3天后,两组细胞的光吸收值在统计学上具有显著差异(P<0.05),且细胞的增殖率随着时间的延长呈现上升趋势。
4流式细胞(FCM)周期结果表明转染pcDNA3.1(+)/IL-8与转染pcDNA3.1(+)、未转染组三组OVCAR-3细胞进入S期数分别为55.18±2.98、39.72±4.16、31.36±1.46,前者进入S期数明显增多,且与后两者比较有统计学意义(P<0.05);三者增殖指数分别为58.83±2.85、44.32±4.20、35.31±1.56,转染pcDNA3.1(+)/IL-8组PI值明显增加,且与后两者比较具有统计学意义(P<0.05)。
5 FCM对转染pcDNA3.1(+)/IL-8、转染pcDNA3.1(+)和未转染三组OVCAR-3细胞蛋白分析结果表明,与细胞增殖相关细胞因子VEGF和MMP-9表达量分别为:430.44±21.70、358.70±17.77、335.52±19.68;356.15±22.70、279.37±27.48、283.56±26.67,转染pcDNA3.1(+)/IL-8组与后两者比较有统计学意义(p<0.05);三组凋亡抑制因子Bcl-2结果分别为380.86±15.27、319.15±17.89、305.04±28.90;凋亡因子Bax分别为343.12±26.10、416.27±25.45、420.13±23.65,前者与后两者比较有统计学意义(p<0.05),而未转染组与转染空质粒组比较均无统计学意义(p>0.05)。
6 Transwell小室侵袭实验结果显示,未转染组、空质粒组和IL-8组穿膜细胞数分别为:101.47±13.52、98.07±13.97、163.73±12.98。转染IL-8cDNA组细胞较转染空质粒组和未转染组细胞穿膜细胞数明显增加,(P<0.05)差异有显著性意义,提示转染IL-8基因后增加了卵巢癌细胞的侵袭能力。
7转染IL-8cDNA对裸鼠皮下移植瘤生长的影响
IL-8组成瘤时间为16.00±1.53天,空质粒组成瘤时间为19.90±1.28天,阴性对照组成瘤时间为19.70±1.25天,各组成瘤率均为100%。各组成瘤时间比较有统计学意义(P<0.05)。
接种肿瘤细胞后7、8、9周,IL-8组肿瘤生长明显活跃于空质粒组和阴性对照组。7、8、9周肿瘤体积分别为:IL-8组:1394.795±108.559mm3、1773.860±133.087 mm3、2207.659±209.713 mm3 ;空质粒组:1106.625±146.984 mm3、1402.206±127.053mm3、1657.190±173.368mm3;阴性对照组: 1110.330±186.458 mm3、1355.918±210.983 mm3、1582.372±169.642 mm3。IL-8组肿瘤体积大于空质粒组和阴性对照组,且两两比较有统计学意义(P<0.05)。实验前6周各组肿瘤体积比较无明显差别(P>0.05)。
各动物实验组肺组织均未见癌组织转移。
8 MVD和NF-κB免疫组化结果:
MVD免疫组化结果:组织内有棕黄色粗颗粒分布或棕黄色细腻颗粒弥漫分布者为阳性反应。空质粒和阴性对照组的肿瘤组织内可见少量阳性反应。IL-8组肿瘤组织中阳性表达明显增加,阳性目标平均光密度值为0.1942±0.0175,明显高于空质粒组和阴性对照组肿瘤组织的光密度值0.1330±0.0134、0.1330±0.0134。两两比较结果显示,IL-8组与空质粒组,IL-8组与阴性对照组均有明显差异(P<0.05),空质粒组与阴性对照组之间无明显差异(P>0.05)。
NF-κB免疫组化结果:组织内有棕黄色粗颗粒分布或棕黄色细腻颗粒弥漫分布者为阳性反应。空质粒和阴性对照组的肿瘤组织内可见少量阳性反应。IL-8组肿瘤组织内阳性表达明显增加,阳性目标平均光密度值为0.1072±0.0049 ,明显高于空质粒组和阴性对照组的光密度值0.0812±0.0100、0.0794±0.0098(P<0.05)。两两比较结果显示,IL-8组与空质粒组,IL-8组与阴性对照组均有明显差异(P<0.05),空质粒组与阴性对照组之间无明显差异(P>0.05)。
结论
1、成功构建IL-8基因的重组真核质粒pcDNA3.1(+)/IL-8;并在脂质体介导下转染人卵巢癌细胞株OVCAR-3,经RT-PCR、ELISA、Western-blot法检测从基因和蛋白水平证实转染成功有效;
2、MTT法检测IL-8可明显促进OVCAR-3细胞增长,并随着时间的增长,细胞的增值率呈现上升趋势;
3、FCM法显示转染IL-8的卵巢癌细胞S期数增多,细胞增殖指数相应升高,表明IL-8基因可以使细胞进入分裂期数增加,因此认为,IL-8基因促进细胞周期进入S期很可能是其促进肿瘤发生发展的重要机制之一;
4、ELISA检测结果显示转染空质粒48h后也有大量IL-8分泌,可以认为转染本身也可以作为一种刺激因子而使IL-8作为炎性因子或自分泌因子大量分泌,但现在还没有关于此方面的确切报导,值得我们今后进一步研究。
5、Transwell小室侵袭实验结果显示转染IL-8基因后增加了卵巢癌细胞的侵袭能力。
6、IL-8基因可使VEGF/MMP-9/Bcl-2表达上调,使Bax表达下调,
其机制与促血管生成因子和凋亡相关因子密切相关。7、动物实验研究发现IL-8能够诱导NF-κB的表达影响肿瘤细胞的增殖、侵袭和转移;IL-8同时促进肿瘤组织内毛细血管的生成,为卵巢癌组织生长侵袭和远处转移提供必要条件。
所有这些为进一步研究IL-8在卵巢上皮性癌发生浸润转移和判断预后中的作用机制提供了坚实的理论和实验基础。
Objective: Being one of department of gynecology tumor, ovarian cancer which is seriously attacking women’s health has driven professor’s attention which is hard to search out in early stage. There are varieties of tumor of the ovary whose organism construction are complicated and owe different bionomics. The malignant degree of epithelium tissue cancer is higher than interstitial substance cancer. While its therapeutic efficacy can not reach the germinocarcinoma. And this tumor which grows very fast, can’t be diagnosed easily in the early stage and its death rate is very high is eager to be researched in the region of woman tumor. So a new therapy should be found. Recently, the research that specialists try to find out the cause and developing relationship of some uncommon genes and ovarian cancer is hitting at the district of boss molecular biology research. Ovarian cancer’s cause and developing relationship is a complex and mixed-step process which involves quantities genes and proteins’expression.
IL-8 which is found by Yoshimur at 1987 is the first chematropism cell factor belonging to CXC family that plays an important role in the contribution of inflammation and immunologic process. Recently, it has drawn more and more attention of researchers as malignant tumor correlative molecule. Nowadays, it is known that many tumor cell can secrete IL-8, IL-8 overexpression relates to tumorous growth and metastasis though it is not clear that the mechanism of encouraging tumor appearance and development.
Recent years, the construction and development of recombination technology can provide new strategy for ovarian cancer in theory and practice——gene therapy can be expected to become new channel of ovarian cancer therapy. Ideal therapeutic gene can inhibit tumor cell’s growth effectively without inhibitory action to normal tissue and cell. In this research, to explore the intereffecting mechanism of IL-8 and epithelial ovarian cancer growth, invasion and metastasis , the eukaryotic expression vector of hIL-8 was constructed and expressed in OVCAR-3 cells after transient and stable transfection. Meanwhile pcDNA3.1(+)/IL-8, pcDNA3.1(+) and non-tranfected OVCAR-3 cells were inoculated subcutaneously into every nude mouse respectively according to three divided groups, Mice’s general condition and time of tumour growth were observed. The volume of transplanted tumor and pulmonary metastasis were compared with different groups. The foundation was settled for further investigation of tumorigenesis and tumor growth.
Method:
1 Construct the eukaryotic expression vector of IL-8 gene. Total RNA was isolated from PBMC of healthy volunteers, IL-8 gene encoding fragment was amplified by RT-PCR and cloned into the vector PMD-18T. BamHI/XhoI double digested product of PMD-18T/IL-8 was connected with the vector pcDNA3.1(+) which was digested by BamHI/XhoI .
2 Transient and stable transfect recombinant plasmid into OVCAR-3 cells. The recombinant plasmid pcDNA3.1(+)/IL-8 was transfected into OVCAR-3 cells which express IL-8 a little with Lipofectamine2000. The expression of IL-8 was tested in OVCAR-3 cells by RT-PCR ,ELISA and Western blot assay.
3 Detect cell proliferation. Detect these cells’reproductive activities by MTT and draw growth curve.
4 The cell cycle was detected by flow cytometry (FCM) on three group cells. pcDNA3.1(+)/IL-8 transfected group, pcDNA3.1(+) group and OVCAR-3 none transfected group.
5 Measure Bax,Bcl-2,VEGF and MMP-9 expression analysis.The expression of Bax,Bcl-2,VEGF and MMP-9 were detected by FCM on three group cells the same as method 4.
6 Using transwell cabin to detect invasive ability of different OVCAR-3 cell lines. Establish vitro invasive model by transwell cabin and matrigel to evaluate the invasive ability of OVCAR-3 cells which stable transfected recombinant plasmid IL-8. Transwell cabin divided into two cabin by polycarbonate fliter membrance(micropore diamerer is 8μm). Micropore allow cell to pass through. Matrigel is composed by typeⅣcollagen、laminin and integrin, similar with basal membrane. The number of cells which pass through the micropore was counted and compared with each other.
7 Effect of IL-8cDNA on transplanted tumour growth and lung metastasis. Mice are divided into three group, and OVCAR-3, OVCAR-3/pcDNA3.1(+) and OVCAR-3/pcDNA3.1(+)/IL-8 cells were subcutaneously inoculated into nude mice according to three divided groups. Mice were killed after 9 weeks, the volume of tumour in each group was compared in different time and the expression of CD34 and NF-κB of tumours from different group were detected by immunohistochemical method. The tissue slice of lung was observed.
RESULTS:
1 After PCR amplification, BamHI/XhoI digestion and DNA sequencing confirmation, the eukaryotic expression vector pcDNA3.1(+)/IL-8 was constructed successfully.
2 The IL-8’s expressions in quantity were founded in the cells of transient transfection when detected by RT-PCR. The gray scale value of pcDNA3.1(+)/ IL-8 transfected group which was 226.88±5.47 correspondingly againstβ-actin was 1.55, while OVCAR-3 none transfected group which was 120.38±6.72 correspondingly againstβ-actin was 0.82. The growth rate was 189.02%;The IL-8’s expressions in cell sap and the cells after transfected pcDNA3.1(+)/ IL-8 6h and 48h were 3.69±0.37, 19.67±0.24, 11.10±1.14 respectively by ELISA, while the others were a little, P<0.05; the specific protein strap was detected only in the former by Western-blot.
3 The results by MTT indicated that the light absorption value of OVCAR-3 cells transfected IL-8/pcDNA3.1 were 0.56±0.08, 0.63±0.06, 0.93±0.09, 1.20±0.06, 1.32±0.12, 1.39±0.14 respectively, while OVCAR-3 none transfected group were 0.62±0.07, 0.70±0.10, 0.84±0.12, 0.92±0.10, 0.96±0.14, 1.01±0.10 respectively. There was no statistical significance compared pcDNA3.1(+)/ IL-8 transfected group with the ones not transfected within 3days(P>0.05), while pcDNA3.1(+)/ IL-8 transfected group was obviously more active after 3 days(P<0.05), and the former’s increasing rate presents an evident tendency of going up along with the time’s extension.
4 The cellular S stage in pcDNA3.1(+)/IL-8, pcDNA3.1(+) and OVCAR-3 cells were 55.18±2.98, 39.72±4.16, 31.36±1.46 respectively by FCM. The former was much higher than the others, P<0.05. These proliferation index were 58.83±2.85, 44.32±4.20, 35.31±1.56.The former PI was much higher and the comparison between them has statistical significance(P<0.05).
5 The expression of VEGF and MMP-9 in pcDNA3.1(+)/IL-8, pcDNA3.1(+) and OVCAR-3 cells were 430.44±21.70, 358.70±17.77, 335.52±19.68 and 356.15±22.70, 279.37±27.48, 283.56±26.67 respectively; while Bcl-2 and Bax in these cells were 380.86±15.27, 319.15±17.89, 305.04±28.90 and 343.12±26.10, 416.27±25.45, 420.13±23.65 respectively. There was statistical significance against pcDNA3.1(+) transfected group and OVCAR-3 none transfected group (p<0.05), while the comparison between them has no statistical significance.
6 Cell number that pass through micropore of OVCAR-3 group, empty plasmid group and IL-8 group is 101.47±13.52, 98.07±13.97, 163.73±12.98. There was statistical significance against OVCAR-3 group, empty plasmid group and IL-8 group. This confirm that IL-8 maybe increase invasive ability of OVCAR-3.
7 Effect of transplanted tumor growth in nude mice by IL-8. Days of tumour growth of OVCAR-3 group, empty plasmid group and IL-8 group is 19.70±1.25, 19.90±1.28, 16.00±1.53. Rate of tumor growth is 100%. Tumor volume of IL-8 group is much bigger than other two groups in 7, 8, 9 week . There have no difference in other times. Cancer cells couldn’t found in lung in each group.
8 Immunohistochemical result of MVD and NF-κB. The MVD and NF-κB in mice treated with IL-8 was much higher than other two groups. Light absorption value of CD34 of OVCAR-3 group, empty plasmid group and IL-8 group are 0.1330±0.0134, 0.1330±0.0134, 0.1942±0.0175. There was statistical significance. Light absorption value of NF-κB of OVCAR-3 group, empty plasmid group and IL-8 group are 0.0794±0.0098, 0.0812±0.0100, 0.1072±0.0049. There was statistical significance.
Conclusions: The IL-8 gene is transfected into OVCAR-3 cells effectively by liposome. Moreover, IL-8 also promotes the proliferation of cell by increasing the number of cell S stage, that the mechanism is connected with angiogenesis factor and correlation factor about apoptosis, which provides a foundation for researching the mechanism of IL-8 on ovary cancer. Result of ELISA showed that cell secretes much more IL-8 after empty plasmid transfection, we consider that transfection maybe stimulate the secretion of IL-8, but we could not find any report about this respect, we should study it more deeply in the future. Invasive experiment showed that IL-8 could increases the invasive ability of OVCAR-3. IL-8 enhances the express of VEGF/MMP-9/Bcl-2 and inhibits the espress of Bax. This mechanism is intimate correlated with angiogenesis factors and apoptosis factors. The animal experiment showed that IL-8 couses the growth, invasion and metastasis of tumor by induce the express of NF-κB and increase the value of MVD. All of this provide the theoretical and experimental basis for us to study the mechanism of growth, invasion ,metastasis and prognosis in epithelial ovarian cancer.
引文
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