【作者】 胡敬海；

【导师】 王春喜；

【作者基本信息】 吉林大学 ， 外科学， 2009， 博士

【摘要】 在我国肾癌是仅次于膀胱癌,占第二位的泌尿系恶性肿瘤,同时也是成人肾脏最常见的恶性实体瘤,其中肾细胞癌占肾脏原发恶性肿瘤的85﹪左右。由于肾细胞癌的发病机制不完全清楚,因此,治疗手段仍以手术为主。虽然对于丧失手术时机等晚期肾细胞癌近年出现了靶向治疗手段,但效果并不理想。因此,探索新的生物治疗方法是目前研究的热点。有研究显示,抑癌基因TIP30与多种肿瘤的发生、发展及侵袭进程密切相关,然而它与肾细胞癌发生发展的具体相关关系迄今尚不清楚。本研究应用免疫组化及Western blotting方法探讨了肾细胞癌组织中TIP30的表达及其临床意义。并进一步构建了TIP30/CC3的真核表达载体,筛选含有外源性TIP30/CC3的人肾透明细胞腺癌细胞系786-0,探讨TIP30/CC3对786-0的生长抑制作用,寻找肾癌基因治疗的潜在靶点。结果显示TIP30/CC3在人肾细胞癌中表达降低,并与肿瘤的侵润程度、淋巴结转移以及肾静脉癌栓密切相关。重组质粒载体pcDNA3.1-TIP30构建成功,转染pcDNA3.1-TIP30的786-0细胞能够稳定表达外源性TIP30蛋白。各种结果显示TIP30/CC3有抑制786-0细胞增殖的作用,转染TIP30/CC3基因后诱导了786-0细胞的G1期细胞阻滞作用和细胞凋亡的增加。本研究提示:针对TIP30/CC3基因表达产物的检测,将对肾细胞癌的恶性程度及预后提供有意义的客观指标。提高肾癌细胞中TIP30/CC3蛋白的表达,能抑制肿瘤细胞的生长,TIP30/CC3是肾癌基因治疗的潜在靶点。更多还原

【Abstract】 In China, the renal carcinoma is the second urinary system malignant tumor afer the bladder carcinoma, but also the most common renal malignant solid tumor in the adult ,and the 85﹪-90﹪of renal primary malignant tumor is the renal cell carcinoma .Research shows that, TIP30 was considered to have tumor suppressor activity by inhibiting tumor growth, invasion, and angiogenesis and inducing apoptosis.However, the relationship with occurrence and development of renal cell carcinoma is not reported.This research discussed the expression of TIP30 and their clinical significance in renal cell carcinoma tissues with immunohistochemical and Western blotting method. And further construct the eukaryotic expression vector of TIP30, screen the 786-0 cell line containing the exogenous TIP30, research the growth inhibition function of TIP30 to 786-0, looking for the potential targets of the renal carcinoma gene therapy. The research was divided into three parts:1. Expression of TIP30/CC3 protein in human renal cell carcinoma Objective:To study the expression of TIP30 protein in renal cell carcinoma tissues and adjacent tissues,and to analyze its clinical significance. Meanwhile, TIP30 protein expression in some common cell lines of the human urinary system carcinoma was observed and established the basis for the subsequent research work. Method: Sixty-five patients with renal cell carcinoma treated at the Institute of Urology in the present hospital, from June 2006 to December 2007 were studied respectively. There were 46 male and 19 female patients in the present study, aging between 17-75 years, and the average age is 56.4. The expressions of TIP30 protein were detected in 65 renal cell carcinoma tissues and their adjacent tissues by S-P immunohistochemical method, and Western blot was used to detect the expression of TIP30 in 22 fresh renal cell carcinoma tissues and their adjacent tissues. And TIP30 protein expression in the prostate carcinoma cell line PC-3M, the bladder carcinoma cell line T24 and the renal carcinoma cell line of clear cell was observed with immonohistochemical and Western blotting method. Results:The negative rate of TIP30 expression in renal cell carcinoma (49.2 %) was higher than that in control group(10.8 %) ( P < 0.01) . TIP30 expression was not different between every clinical pathological type ( P > 0.05) . The negative rate of TIP30 expression in real cell carcinoma was related to invasion degree ( P < 0. 05) , lymph node metastasis ( P <0. 05) and tumor thrombus ( P <0. 05). Moreover, Western blotting analysis showed that there was low expression of TIP30 in 77.3% (17/22) of the renal cancer tissues. There was high level TIP30 protein expression in the PC-3M and T24 cells, especially in T24 cells and 786-0 cells showed relative lower level TIP30 protein expression. Conclusion: There was low level TIP30 protein expression in human renal carcinoma tissues. The negative expression of TIP30 protein may be related to the initiation and development of real cell carcinoma; As an tumor suppressor gene product, TIP30 protein may be considered as a new clinical indicator for monitor of real cell carcinoma and provide information for adjuvant therapy.2. Construction and sequence analysis of the eukaryotic expression vector Objective: To construct and sequence the eukaryotic expression vector of TIP30 gene. Methods: The total RNA was extracted from normal renal tissue. TIP30 gene cDNA was amplified by RT-PCR, and then was cloned into eukaryotic expression vector pcDNA3.1(+) and sequenced. Results: After amplification by RT-PCR, the normal renal tissue produced specific bands which were located about 800 bp according to expectation. By BamH-I and EcoR-I digestion, pcDNA3.1-TIP30 produced 2 bands, which agreed with the expected position, and the sequence of obtained TIP30 gene cDNA was homologous (100% ) to the encoding region of TIP30 that was shown in documents(Genbank No.AF039103), and the target gene was correctly cloned into the vector. Conclusions: The sequence of TIP30 gene cDNA encoding region was cloned, and the recombinant eukaryotic expression vector pcDNA3.1-TIP30 was successfully constructed. It lays the foundation for the further research on the function of TIP30 gene.3. TIP30 gene suppresses growth of human renal carcinoma 786-0 cells Objective: To explore the effect of TIP30 gene on the growth inhibition of renal carcinoma cell line 786-0 and look for a potential therapeutic target for RCC. Methods: The pcDNA3.1(+) and pcDNA3.1-TIP30 plasmids were transfected into the 786-0 cells with lipo2000, screening the stable expression cell line with G418. The expression of TIP30 in 786-0 cells was detected by RT-PCR and Western blotting. The changes of cell proliferations and cell cycles were observed by MTT and FCM assay. Scorching method was used to observe the transfected 786-0 cells migration ability and Transwell chamber combining matrigel was used to detect the transfected 786-0 cells invasion ability. Results:The mRNA and protein expression of TIP30 gene in pcDNA3.1-TIP30-transfected 786-0 cells were significantly increased than those in untreated and pcDNA3.1(+)-transfected cells(P <0.05). However, there was no difference in TIP30 expression between untreated and pcDNA3.1(+)-transfected 786-0 cells(P >0.05).The inhibitory rate of pcDNA3.1- TIP30- transfected 786-0 cells was significantly higher than that in untreated and pcDNA3.1(+)- transfected cells(P <0.01); Cell cycle analysis by flow cytometry showed that the number of cells in G0-G1 phase of pcDNA3.1-TIP30-transfected 786-0 cells was significantly increased while cells in phase S and G2-M was decreased compared with the untreated and pcDNA3.1(+)-transfected cells(P <0.01). 786-0 cells transfected with pcDNA3.1-TIP30 showed increased apoptosis percentage, slower migration ability and attenuating invasion ability compared with normal 786-0 cells.786-0 cells transfected with pcDNA3.1-TIP30 showed increasing vacuoles and diminishing villus with transmitting electron microscope. Conclusions:786-0 can stably express exogene TIP30. Transduction of TIP30 gene into lower expression renal cancer cells can restore its suppressive effect on cell growth, suggesting that TIP30 gene may be a new therapeutic target for RCC.更多还原