【作者】 王化丽；

【导师】 张淑兰；

【作者基本信息】 中国医科大学 ， 妇产科学， 2006， 博士

【摘要】 目的 肿瘤的侵袭和转移是肿瘤细胞的恶性生物学行为,是一个多环节、多因素、多基因参与的复杂过程,也是临床绝大多数肿瘤患者致死的原因。侵袭和转移是指肿瘤细胞的直接扩散和肿瘤细胞的不连续播散,并在远隔部位生长。基本过程包括:脱离(detachment)、侵入(intravasation)、循环(circulation)、粘附(adhesion)、血管生成(angiogenesis)和生长(growth)等。细胞外信息通过信号转导通路,传递和调控细胞生命活动,探讨肿瘤细胞信号传递及相关机制,为揭示肿瘤侵袭和转移的本质并为抗肿瘤的治疗提供理论依据。由间质细胞产生的HGF可作用其特异性受体c-met引起一系列信号转导蛋白的酶促反应产生相应的生物学效应。HGF/c-met信号通路在促进肝癌、乳癌细胞的生长、侵袭和转移方面起重要作用,但HGF/c-met在卵巢癌的研究国内外罕见报导。本研究以卵巢癌为研究对象,检测HGF/c-met在卵巢癌相关的表达,观察HGF对卵巢癌细胞的作用及信号转导通路,并应用反义基因治疗技术,构建c-met反义真核表达载体,探讨阻断HGF/c-met信号转导通路的抗侵袭转移的效应。从正反两方面探讨HGF/c-met在卵巢癌侵袭转移中的作用及其分子基础。本论文共分三部分。第一部分,题目:肝细胞生长因子及其受体c-met在卵巢上皮性肿瘤的表达及意义。目的:研究HGF和c-met蛋白在卵巢上皮性肿瘤组织中的表达,探讨其与临床病理特征的关系。第二部分,题目:肝细胞生长因子对卵巢癌细胞生长侵袭作用及信号转导通路的研究。目的:研究HGF在卵巢癌细胞生长侵袭中的作用、可能机制及其NFκB信号传导通路的作用。第三部分,题目:反义c-met基因对人卵巢癌细胞生长侵袭的影响。目的:构建c-met反义真核表达载体,转染卵巢癌细胞,阻断HGF/c-met信号更多还原

【Abstract】 ObjectivesMetastasis is the most lethal attribute of cancer, which several affects the effectiveness and prognosis of cancer patients. Tumor metastasis is an extremely complex process involving multi - genes, multi - factors and multi - steps, in which neoplastic cells must escape from the primary tumor, degrade the extracellular matrix, migrate to distant sites, arrest in the capillaries, migrate through the basement membrane and underlying connective tissue to the meta-static site. The essential events of the process are detachment, intravasation, circulation, arrest, extravasation, angiogenesis and proliferation, which come down to interaction of neoplastic cells with host cells and extracellular matrix --------- governed by adhesion, migration, invasion, proteaselysis, and the complicated signal transduction.. In view of signaling play the leading roles in the cell function, signal transduction system and its mechanisms in the tumor metastasis are the key to understanding of essence of the process and exploring new anti - metastasis techniques. HGF, a multifunction cell growth factor yielded by interstitial cell, has been proved to promote adhesiveness, invasiveness, motion and metastasis for cancer cells, c - met is a receptor tyrosine kinase shown to be over - expressed and mutated in a variety of malignancies. HGF - c - met signal pathway leads to a plethora of biological and biochemical effect in the cell. It has been proved that HGF specially binding to its receptor c - met induces phos-phorylation of the intracellular kinase domain resulting in activation of a complex set of intracellular pathway that lead to cell growth, differentiation, invasion and angiogenesis in liver cancer and breast cancer. However, little is known about the role of HGF - c - met signal pathway in ovarian cancer. This paper aims toinvestigate the relationship between HGF - c - met and ovarian cancer, the role of HGF in ovarian cancer and signal pathway, and blockage of HGF - c - met signal pathway by antisense gene technology to probe into anti - metastasis effect and mechanisms and to find a new way for antisense gene therapy. It includes three parts in our report. The first part.Title: Clinical significance of HGF and c - met expression in patients with epithelial ovarian cancer . Objective: The aim of this study is to detect the expression of HGF and c - met in epithelial ovarian tumor tissues to probe the relationship between HGF or c - met and clinical characteristics of patients with epithelial ovarian tumor and correlation of them. The second partTitle: Role of HGF in ovarian cancer invasion and its signal transduction pathway. Objective;This study is designed to investigate the role and associated of HGF in ovarian cancer cells invasion and its signal transduction pathway. The third part.Title: Effect of transferred c - met antisense gene on biological characteristics of human ovarian cancer cell. Objective: To construct eukaryotic expressing vector of c — met antisense gene and transfer it into human ovarian cancer cell line SKOV3. To observe the effect of transfected c — met antisense gene on the biological behaviors of SK0V3 cells.Methods1. Immunohistochemistry was used to detect the expression of HGF and c -met protein in 45 cases of epithelial ovarian cancer and 10 ovarian benign tumors. Western blot analysis was used to examine HGF and c - met protein in these tumors.2. HGF - induced proliferation and invasion activity of SKOV3 cell were analyzed with cells growth curve and Boy den chamber invasion assay. The expression changes of c - met and MMP9 in SKOV3 cells before and after treatment of HGF and NF kB inhibitor PDTC were measured by real time RT - PCR, Western blot and flow cytometric analysis, respectively. The expression of NFkB induced by HGF in ovarian cancer cell was evaluated by immune flouorescencetechnique and western blot.3. Anti - c - met gene is inserted into eukaryotic expressing vector PcD-NA3.1 to construct PcDNA3. 1 - c - met, in which restriction enzyme analysis was used to confirm the vector. The recombinant of PcDNA3. 1 - c - met anti-sense plasmid was transfected into human ovarian cancer cell SK0V3 by lipo-fectamine and positive clone was screened by G418. Real time RT - PCR and flow cytometric analysis were used to detect the expression of c - met and MMP9 mRNA and protein levels, respectively. The proliferation and invasion capabilities of the transfected cells were evaluated by growth curves and Boyden chamber, respectively.ResultsIn ovarian cancer tissues , HGF protein was expressed more strongly on the epithelial cells than that on the stromal cells, c - met protein was positive expression on the epithelial cells and negative expression on the stromal cells . The positive rates of HGF and c - met protein in malignant tissues were 60.0% (27/ 45) and 3. 0% (33/45) respectively which were significantly higher than these in benign ones, 20. 0% (2/10) and 50. 0% (5/10) ( p < 0. 01). In epithelial ovarian cancer, the positive rates of HGF and c - met in stage III ~ IV and lymph node metastasis groups were significantly higher than these in stage I ~ II and no lymph node metastasis groups (p <0. 01). There was no relationship between the histologic grade and the expression of both HGF and c - met (p > 0. 05). The relative intensity of HGF and c - met protein expression in epithelial ovarian cancer were 1. 24 0. 98 andl. 69 0. 90, respectively, which were also significantly higher than these in benign ones, 0. 17 0.34 and 0.44 0.46 (p < 0. 01). Expression of HGF protein was positively correlated with expression of c -met protein ( p <0.01) .The invasion cells were significantly more in HGF group than in control group and PDTC group (140.. 2 3.3 vs. 64.2 1.9 and 93.4 2.9, P <0.01 ). The proliferation of SKOV3 cells were more in HGF group than in control group and PDTC group. HGF promoted the expression of MMP9 mRNA by 5.66 0.10folds, but had no effect on c - met mRNA expression;PDTC suppressed the HGF - driven expression of MMP9 mRNA by 2. 75 0. 80 folds. The positive rates of c - met and MMP9 protein were significantly higher in HGF - treated cells than in control cells [ (96. 6 ± 2. 0) % vs. (73. 3 ± 2. 4) % , P < 0. 01;(74. 6 ±4.4) % vs. (16. 0 ±2. 9) % , P < 0.01 ]. The protein levels of c - met and MMP9 protein were significantly higher in HGF - treated cells than in control cells (2. 84 ±0.18 vs. 1.65 ±0.19, P<0.01;2.94 ±0.13 vs. 0.54 ±0. 18, P <0. 01 ). PDTC significantly suppressed the HGF - driven expression of MMP9 protein;the positive rate and protein level of MMP9 were (25. 8 ±3. 7 ) % and 0. 87 ± 0. 14 (P < 0. 05). HGF promoted the expression of NFKB protein in a time - dependant manner. The expression peak appeared 1 h after treatment of HGF (from 0.42 ± 0. 11 to 1. 16 ± 0. 21, P < 0. 01). PDTC significantly inhibited the HGF - driven expression of NFkB protein (0. 38 ±0. 12, P<0. 01).The PcDNA3. 1 - c - met vector was obtained . Antisense c - met gene was stably transfected into human ovarian cancer cell SKOV3. Antisense c - met RNA blocked c - met and MMP9 mRNA expression of ovarian cancer cell SK-OV3 by 0. 24 ± 0. 03 folds ( p < 0. 01) and 0. 70 ± 0. 03 folds ( p < 0. 01 ). The positive rates of c - met and MMP9 protein were significantly decreased in SK-OV3 cells [from (98.92 ±0. 62) % to(27.17 ±1. 23 ) % ,P<0.01;from(36. 47 ±2. 94) % to( 17. 32 ± 0. 46) % , P < 0. 01 ] after transfected. The protein levels of c - met and MMP9 protein were significantly less in SKOV3 cells (from 3.00 ±0.12 to 0.85 ±0.08 , p<0.01;from 1.06 0.10 to 0.48 0.06, p<0. 01). The invasion cells were significantly less after transfected (from 61.4 3.0 to 42.2 1.9, P<0.01). The proliferation of SKOV3 cells were less after transfected.Conclusions1. HGF and c - met protein are generally expressed in epithelial ovarian cancer. HGF and c - met protein are prognostic indicators in patients with epithelial ovarian cancer. These findings suggest that HGF and c - met may play acrucial role in epithelial ovarian cancer invasion.2. HGF induces the expression of c - met protein and increases the expression of MMP9 mRNA and protein. HGF up - regualtes the expression of nucleus NFkB protein. HGF stimulates proliferation and invasion activity of ovarian cancer SKOV3 cells. HGF stimulates invasion activity of ovarian cancer cell by up - regulating the expression of MMP9. MMP9 is a key component of HGF induced ovarian cancer cell invasion . HGF modulates the expression of MMP9 in ovarian cancer cell via NFkB pathway. HGF - c - met could be a useful target for therapeutic intervention.3. The way using lipofect to transfect anti - c - met into SKOV3 cells is safe, high - efficiency and low toxicity. The expression of c - met and MMP9 are decreased significantly in the transfected cell lines. The proliferation and invasion ability of the transfected cell lines decrease significantly. HGF - c - met signal pathway plays an important role in proliferation, metastasis and invasion of ovarian cancer cells. Blockage HGF - c - met signal pathway can inhibit the growth and invasion of ovarian cancer SKOV3 cells and it is of significance for o-varian cancer specific antisense gene therapy.更多还原