【作者】 肖钟迪；

【导师】 房学东；

【作者基本信息】 吉林大学 ， 外科学， 2010， 博士

【摘要】 目的：探讨TSLC1基因抑制人肝癌细胞HepG2的机理,为肝癌的基因治疗提供有效的靶点。方法：应用基因重组技术构建了真核表达载体pReceiver-M29-TSLC 1重组质粒,采用脂质体Lipofectamine 2000介导重组载体转染到HepG2肝癌细胞中,筛选后建立稳定转染细胞株,采用MTT法检测细胞增值情况、流式细胞仪检测细胞周期、利用酶标仪检测caspase-3和caspase-8酶活性,RT-PCR方法检测DAL-1mRNA的表达,探讨TSLC1真核表达载体对肝癌HepG2细胞的作用及其抗肝癌机制的实验研究。结果：(1)成功的构建了pReceiver-M29-TSLC1重组质粒。(2)重组载体转染到HepG2肝癌细胞后,TSLC1mRNA表达明显增多,TSLC1蛋白表达明显增高；(3)我们成功证实了TSLC1能抑制细胞生长、阻滞细胞周期、提高caspase-3、caspase-8酶活性以及DAL-1的mRNA表达。结论：TSLC1基因的表达可能通过抑制细胞周期、诱导肿瘤细胞凋亡、提高caspase-3、caspase-8酶活性及DAL-1mRNA的表达等方式起到抑制人肝癌细胞HepG2的作用,这为TSLC1作为肝癌基因治疗的有效靶点提供实验基础及理论依据。更多还原

【Abstract】 Hepatocellular carcirioma(HCC)is reported to be the sixth most common cancer,and over 626,000 people newly issued in patients each year in the world, and about 50% of the patients occurred in china。Moreover,the mortality of HCC is the third highest among all cancers,only behind lung and colon cancer. Since 1990s and is now the second highest among all cancers.Hepatocellular carcinoma deaths increased to the second place in China. Liver resection is best way to treatment.of HCC,and could be supplemented by radiotherapy and chemotherapy.But about 15% of patients only could benefited from liver resection. Hepatocellular carcinoma cell are not sensitive to radiotherapy and chemotherapy. Five years survival rate is low, only 3%-6%,in hepatocellular carcinoma patients. At present,for the minimally invasive treatment of patients with hepatocellularcarcinoma is TACE, EIT, MTC. electric Chemotherapy treatment, RFA treatment, Immunotherapy, Brachytherapy. The efficient still reached less than 20%, the prognosis remains poor.So,we are realized that we should find new ways to treatment of liver cancer based on mechanism of liver cancer. Studies have shown that the pathogenesis of liver cancer related to a variety of oncogene activation and inactivation of tumor suppressor genes. Choosen the key genes of oncogene or tumor suppressor genes of liver cancer, is expected to reach the purpose of treatment of liver cancer through gene therapy.TSLC1, a tumor suppressor gene few studies,1993, Satoh speculated that the human chromosome 11 that there is a tumor suppressor gene. After years of study, 2002, Murakami et al transfected normal long arm of chromosome 11 DNA fragments into the NSCLC cell line A549 by functional complementation method,and then the transfected cells Injectied into the backs of nude mice,observed the DNA fragment can inhibit the tumor formation and confirmed human chromosome 11q23.2 has a new tumor suppressor gene, due to significantly inhibit the growth of lung cancer cell lines, so named for the TSLC1 (tumor suppressor in lung cancer 1, TSLC1).TSLC1 gene mapping to human chromosome 11q23.2, the total length is more than 300kb, a total of 12 exons. TSLC1 gene 5’, CpG islands, CpG island about the size of 900bp, is located in exon 1 and intron 1 upstream. In which exon 8a,8b and 8c in brain, there are many types of splicing isoforms, normal epithelial cells express only the exon 8a. TSLC1 gene transcripts of 4.4kb or 1.6kbmRNA precursors, translation 442 amino acid residues of the transmembrane glycoprotein. TSLC1 protein structure contains the extracellular domain, a transmembrane region and cytoplasmic region; these three regions may be related to TSLC1 protein has an important function of the relationship.It is inactivated in 44% of NSCLC and 30-60% of various cancers,including liver, pancreatic, and prostate cancers, especially in those with invasion or metastasis. Inactivation occurs by two hits:through promoter methylation, and through loss of heterozygosity at the gene locus. Now that promoter methylation is the main reason. The later stage cancer, the higher the degree of malignancy, TSLC1 promoter methylation frequency of the higher.A great deal of research about TSLC1 anti-tumor mechanism of action At home and abroad, and its anti-tumor mechanism of action includes the following aspects: (1)TSLC1 associates with an actin-binding protein, DAL-1, and members of the membrane-associated guanylate kinase homologue (MAGuK)group, providing a novel tumor suppressor cascade that is inactivated in tumors.(2) TSLC1 as necl-2 induced tumor cells were phagocytosis by NK cell.(3) TSLC1, as antigen presented cells to CD8+T cells inhibit the occurrence of cancer.(4) TSLC1 may be activating apoptosis factor caspase-3, and raise its expression, promoting tumor cell apoptosis.(5) TSCL1 can inhibit tumor cell from the early DNA synthesis (G0/G1) phase to the DNA synthesis phase (S phase). And then inhibit tumor cell growth.The TSLC1 research focused on lung cancer, leukemia, breast cancer, prostate cancer, breast cancer, but the research of TSLC1 in liver disease is still rare. at home and abroad.Although many studies have discovered that the expression of TSLC1 downregulate in patient of hepatoma carcinoma. But the study on mechanism of TSLC1 effect HepG2 hepatoma cell is lacking. This experiment was successfully constructed the eukaryotic expression vector pReceiver-M29-TSLC1, and the vector was transfected into HepG2 hepatoma cells line for research the mechanism of anti-tumor of TSLC1.and provide the theoretical and experimental basis for gene therapy. In this study, the research results obtained are as follows.1. The construction of eukaryotic expression vector pReceiver-M29-TSLC1 The full length of TSLC1 gene was amplificated using specific primers for RT-PCR reaction by Genetic engineering technology,and linked with eukaryotic vector pReceiver-M29, and transformed into E.coli JM109. We identified this recombinant vector through application of restriction enzyme digestion, PCR and sequencing and confirmed that the TSLC1 gene fragment was correctly inserted into this eukaryotic expression vector.The results show that the eukaryotic expression vector pReceiver-M29-TSLC1 was constructed successfully, and established the experimental basis for further study of the mechanism in the inhibition of tumor.2. Establish a stable TSLC1 gene transfected HepG2 cell line.Eukaryotic expression vector pReceiver-M29-TSLC1 transfected into HepG2 hepatoma cells mediated through Lipofectamine 2000 liposome.G418 selection to establish a stable transfected cell lines.We detected that pReceiver-M29-TSLC1 has been transfected into HepG2 hepatoma cells by RT-PCR, immunohistochemical and westernblot methods.The experiment provides a cytological basis about TSLC1 and investigates the inhibition mechanism of TSLC1 in hepatoma cells HepG2.3. TSLC1 can inhibit cell proliferation of hepatoma cells HepG2.We detected pReceiver-M29-TSLC1-HepG2 group, pReceiver-M29-HepG2 and normal cell proliferation in HepG2 group by MTT methods. From the 3rd day,pReceiver-M29-TSLC1-HepG2 cells group growed slowly and proliferation were inhibited in a row of 7 days.We compared the MTT values between TSLC1-HepG2 cells and normal HepG2 cell group and found the significantly different of the value(P<0.05). Results showed that TSLC1 could inhibit hepatoma cells HepG24. TSLC1 could inhibit hepatoma cells HepG2 DNA synthesis from the early (GO/Gl phase) to the DNA synthesis phase (S phase) transformation.We cultured pReceiver-M29-TSLC1-HepG2 cells and pReceiver-M29-HepG2 cells 3 days,and detected the change of cell cycle by the flow cytometry.We found early DNA synthesis cells (GO/G1 phase) were significantly increased and the DNA synthesis phase (S phase) cells decreased significantly in pReceiver-M29-TSLC1-HepG2 cells(P<0.05).Results show that the TSLC1 can inhibit DNA synthesis of hepatoma cells HepG2 in interphase of cell cycle, and then inhibit the growth of hepatoma cells HepG2.5. TSLC1 can increase the caspase-3 significantly We cultured the pReceiver-M29-TSLC1-HepG2 cells and pReceiver-M29-HepG2 cells 3 days,then detected the content of caspase-3. We compare the pReceiver-M29-TSLC1-HepG2 cells with pReceiver-M29-HepG2 cells found that the content of caspase-3 increased significantly in pReceiver-M29-TSLC1-HepG2 cells. The results showed that TSLC1 can increase the content of caspase-3 and caspase-8, leading to tumor cells apoptosis,then inhibit the growth of hepatoma cells HepG2.6. TSLC1 can increase the caspase-8 significantlyWe cultured the pReceiver-M29-TSLC1-HepG2 cells and pReceiver-M29-HepG2 cells 3 days,then detected the content of caspase-8. We compare the pReceiver-M29-TSLC1-HepG2 cells with pReceiver-M29-HepG2 cells found that the content of caspase-8 increased significantly in pReceiver-M29-TSLC1-HepG2 cells. The results showed that TSLC1 can increase the content of caspase-8, leading to tumor cells apoptosis,then inhibit the growth of hepatoma cells HepG2.7. TSLC1 can increase the expression of the DAL-1 mRNAWe cultured the pReceiver-M29-TSLC1-HepG2 cells and pReceiver-M29-HepG2 cells 3 days,then detected the expression of DAL-1 mRNA.We compare the pReceiver-M29-TSLC1-HepG2 cells with pReceiver-M29-HepG2 cells found that the increased significantly expression of DAL-1 mRNA in pReceiver-M29-TSLCl-HepG2 cells. The results showed that TSLC1 can increase the expression of DAL-1 mRNA leading to inhibit the growth of hepatoma cells HepG2.更多还原