【作者】 赵娟；

【导师】 程建新； 李万胜；

【作者基本信息】 河北医科大学 ， 妇产科学， 2008， 硕士

【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热点。上皮性癌其恶性程度比间质性肿瘤高,治疗效果远不如生殖细胞癌,此种肿瘤发展迅速,不易早期诊断,死亡率高,是该领域中最有待研究的一种肿瘤,因此一种新的治疗方法亟待需要。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。近来作为恶性肿瘤相关分子越来越多地引起了研究者的关注。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。近年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转染低表达IL-8基因的上皮性卵巢癌细胞株OVCAR-3中,探讨该基因诱导的上皮性卵巢癌细胞功能的改变及其生物学机制,为进一步研究IL-8基因治疗上皮性卵巢癌的提供了可行性。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Xho I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组与未转染组细胞的生长增殖能力,绘制生长增殖曲线。4检测各组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(+)空载体组和未转染组OVCAR-3细胞周期变化。5测定各组细胞的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(+)空载体组和未转染组3组细胞的Bax、Bcl-2、VEGF和MMP-9四种蛋白的表达变化。结果: 1经PCR扩增、双酶切及DNA测序鉴定,证实pcDNA3.1(+)/IL-8构建成功。2 RT-PCR法检测到瞬时转染的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质粒6h、48h后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,而未转染组和转空载体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,两种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流式细胞周期结果表明转染pcDNA3.1(+)/IL-8与转染pcDNA3.1(+)、未转染组三组细胞进入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)。结论:目的基因IL-8可以在脂质体介导下有效的转染人卵巢癌细胞株OVCAR-3,其通过提高进入细胞S期数明显促进卵巢癌细胞的生长,且其机制与促血管生成因子和凋亡相关因子密切相关,这为进一步研究IL-8在卵巢肿瘤中的作用机制提供了基础。更多还原

【Abstract】 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 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 but it is not clear that the mechanism of encouraging tumor appearance and development.Recent years, the construction and development of recombi- nation 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, we clone the human IL-8 gene into eukaryotic expression vector pcDNA3.1(+), with the act of liposome, transient transfection OVCAR-3 which is epithelial ovarian cancer cells expressing little IL-8. The functional change and biology mechanism are investigated induced by IL-8, and provide a feasibility to IL-8 gene therapy ovarian cancer.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 transfect 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 Detect the cell cycle The cell cycle was detected by Using flow cytometry (FCM) on three group cells: pcDNA3.1(+)/IL-8 transfection group、pcDNA3.1(+) group and OVCAR-3 none transfection 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 using FCM on three group cells the same asmethod 4. 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 the way of RT-PCR. The gray scale value of pcDNA3.1(+)/ IL-8 transfection group which was 226.88±5.47 correspondingly againstβ-actin was 1.55, while OVCAR-3 none transfection 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 transfecting pcDNA3.1(+)/ IL-8 6h and 48h were respectively 3.69±0.37、19.67±0.24、11.10± 1.14 by using ELISA, while the others were a little, P<0.05; the specific protein strap was detected only in the former by the way of Western-blot.3 The results by MTT indicated that the light absorption value of OVCAR-3 cells transfected IL-8/pcDNA3.1 were respectively 0.56±0.08、0.63±0.06、0.93±0.09、1.20±0.06、1.32±0.12、1.39±0.14, while OVCAR-3 none transfection group were respectively 0.62±0.07、0.70±0.10、0.84±0.12、0.92±0.10、0.96±0.14、1.01±0.10. There was no statistical significance compared pcDNA3.1(+)/ IL-8 transfection group with the ones not transfected within 3days(P>0.05), while pcDNA3.1(+)/ IL-8 transfection 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 respectively 55.18±2.98、39.72±4.16、31.36±1.46 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 respectively 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; while Bcl-2 and Bax in these cells were respectively 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. There was statistical significance against pcDNA3.1(+) transfection group and OVCAR-3 none transfection group (p<0.05), while the comparison between them has no statistical significance.Conclusions: IL-8 could transfect OVCAR-3 cells effectively by liposome. Moreover, IL-8 can also promote cell’proliferation function by increasing the number of cell S stage, that the mechanism was connected with angiogenesis factor and correlation factor about apoptosis, which provides a foundation for researching the mechanism of IL-8 on ovary cancer.更多还原