【作者】 杨家骍；

【导师】 所剑；

【作者基本信息】 吉林大学 ， 外科学， 2014， 博士

【摘要】 半乳糖凝集素（Galectin）是一类专一结合糖蛋白聚糖β-半乳糖残基的凝集素家族，存在于多种生物体内，广泛的分布于细胞核、细胞质和细胞外基质中。Galectin具有多种生物学功能，如细胞粘附、细胞生长调节、细胞凋亡、炎症反应、免疫调节等。Galectin-9是Galectin家族中的一员，具有Galectin家族典型的保守区结构，广泛分布在细胞内外，在调节免疫和炎症反应、细胞凋亡、肿瘤新生血管形成和肿瘤转移等方面发挥重要作用。近年来，Galectin-9与肿瘤的关系受到越来越多的关注，在黑色素瘤、宫颈癌、肝癌等均有相关报道。有研究认为，Galectin-9可能成为新的肿瘤治疗靶点。T细胞免疫球蛋白粘蛋白（Tim）基因家族，主要表达在免疫细胞表面，调节Thl和Th2细胞介导的免疫应答。Tim-3蛋白为T细胞免疫球蛋白粘蛋白家族的一个重要成员，主要表达于活化的Thl细胞表面，与其配体Galectin-9结合后发挥免疫调节作用。目前，有关Galectin-9和Tim-3在胃癌组织中的表达情况及作用鲜有报道，本研究旨在观察胃组织和胃癌细胞系中Galectin-9和Tim-3的表达及分布，并研究其与胃癌临床参数的关系，以及其在胃癌发生发展过程中的角色，探讨其可能的作用途径。我们首先通过免疫组织化学方法观察了在胃组织和胃癌细胞系中Galectin-9和Tim-3的表达情况。发现胃组织和胃癌细胞系中Galecitn-9主要表达在细胞膜和细胞质中，而Tim-3在细胞质和细胞核中均有表达。我们成对收集了自2008年9月至2013年7月间于吉林大学第一医院胃结直肠外科行胃癌根治术病人癌组织、癌旁组织（距肿瘤<2cm）及正常组织（距肿瘤>5cm）标本52例，全部病例术后病理均证实为胃癌。分别采用荧光定量PCR方法和Western blot方法检测Galectin-9mRNA水平及蛋白的表达，同正常组织及癌旁组织相比较，癌组织中的Galectin-9mRNA水平明显降低（P<0.001，P<0.001），差异有统计学意义。为了验证Galectin-9mRNA水平改变是否引起蛋白表达量变化，我们通过Western blot方法检测了样本中Galectin-9蛋白表达。结果显示，癌组织中Galectin-9蛋白表达（0.999±0.435）明显低于正常组织（1.796±0.684），差异有统计学意义（P<0.05）。癌组织中Galectin-9mRNA水平与临床参数分析结果表明，T3和T4期癌组织中的Galectin-9mRNA水平明显低于T1期（P<0.05，P<0.05）；而在T2期与T1期无明显差别，提示Galectin-9可能与肿瘤浸润深度相关。N0—N3各分期癌组织中Galectin-9mRNA水平均无明显差别。而同未发生远处转移组（M0）相比较，远处转移组(M1) Galectin-9mRNA相对表达量明显降低（P<0.05）。我们又分析了Galectin-9mRNA水平与肿瘤临床分期间关系。同Ⅰ期比较，II、III、IV期癌组织中Galectin-9mRNA水平均降低，且II期和IV期病例癌组织中Galectin-9mRNA水平降低明显，差异有统计学意义（P<0.05）。癌组织中Galectin-9mRNA表达量高的患者术后生存时间高于Galectin-9mRNA表达量低组，提示Galectin-9表达与患者术后生存时间相关。综合以上结果可以看出，Galectin-9在胃癌组织中的表达明显下降，并与肿瘤的恶性程度有一定的相关性，而与性别、年龄等因素无明显关系（P>0.05）。同时Tim-3mRNA和蛋白检测显示，同正常组织及癌旁组织相比较，癌组织中的Tim-3mRNA水平和蛋白表达虽有所降低，但差异无统计学意义。另外我们又分别通过荧光定量PCR方法和Western blot方法观察了胃癌细胞系SGC-7901和MGC-803中Galectin-9和Tim-3mRNA和蛋白的表达情况。与胃粘膜正常细胞GES-1相比较，SGC-7901和MGC-803中Galectin-9mRNA水平明显降低，差异有统计学意义（P<0.05，P<0.01）。Western blot结果表明，同正常胃粘膜细胞GES-1相比较，SGC-7901和MGC-803中Galectin-9蛋白表达明显降低，差异有统计学意义（P<0.05，P<0.01）。而SGC-7901和MGC-803细胞中Tim-3mRNA水平和蛋白的表达与GES-1细胞相比较无明显差别。这与Galectin-9和Tim-3在胃组织中的表达趋势是一致的。为进一步探讨Galectin-9在胃癌发生和发展中的作用和作用机制，以及Galectin-9和Tim-3在胃癌组织中的相互关系，我们构建了pcDNA3.1（-）-Galectin-9质粒，瞬时转染MGC-803细胞，建立了瞬时转染表达体系。观察到Galectin-9过表达能明显抑制MGC-803细胞的增殖，DAPI染色显示Galectin-9过表达能促进MGC-803细胞凋亡。Western blot检测凋亡信号通路相关蛋白结果显示，转染Galectin-9后能下调Bcl-2蛋白表达，而Bax蛋白、Caspase-3蛋白的表达上调，提示Galectin-9过表达可能通过线粒体凋亡途径，引起细胞凋亡。检测转染Galectin-9的MGC-803细胞中Tim-3蛋白表达结果显示，Galectin-9过表达不引起Tim-3表达变化，同时免疫共沉淀复合物中只检测到Galectin-9蛋白，而无Tim-3蛋白沉淀，提示二者在MGC-803细胞中无直接作用，Galectin-9对胃癌细胞的作用可能不是通过Galectin-9/Tim-3途径发挥的。综上所述，Galectin-9和Tim-3在胃正常粘膜组织及胃癌组织中均有表达；在胃癌组织和胃癌细胞系中Galectin-9mRNA和蛋白表达水平明显低于正常组织；Galectin-9在胃癌细胞系中过表达，能抑制肿瘤细胞生长、促进肿瘤细胞凋亡；Galectin-9能通过下调Bcl-2蛋白表达，上调Bax蛋白、Caspase-3蛋白的表达发挥促进细胞凋亡作用；在MGC-803细胞中Galectin-9和Tim-3不存在直接相互作用，提示Galectin-9在胃癌细胞中可能不是通过Galectin-9/Tim-3途径发挥作用的。Galectin-9在胃癌发生发展中的作用机制还需在以后的研究中进一步探讨。更多还原

【Abstract】 The Galectins are a family of β-galactoside-binding animal lectins. Galectins are widelydistributed in many animals and localized in the nucleus, cytoplasm and extra cellular matrix.They have many functions, such as cell adhesion, proliferation, cell apoptosis, modulation ofinflammation and immune regulation. Galectin-9, as a member of Galectin family, which hasa typical conservative structure of the Galectin family is widely distributed in the inside andoutside of cells. Galectin-9has been found to regulate different biological functions, such asimmune regulation, inflammation, cell apoptosis, angiognesis and matastasis of tumors.Recently the relationship of Galectin-9on tumors, such as melanomas, cervical squamous cellcarcinomas and hepatocellular carcinomas, has attracted more attention, and Galectin-9maybe a new therapeutic field of study related to the treatment of tumors.The T-cell immunoglobulin domain and mucin domain (Tim) family, mainly expressed inthe surface of immune cells, regulates the immune response which is mediated by Th1andTh2cells. Tim-3, a Th1-specific type1membrane protein, emerges on the cell surface of fullyactivated Th1cells. Binding to its ligand, Galectin-9, to regulate the immune system.At present, few articles about the expression of Galectin-9and Tim-3in gastric cancerhave been reported. The aim of this study has been to observe the expression and distributionof Galectin-9and Tim-3in gastric tissues and gastric cancer cell lines, and to explore its rolein the genesis and development of gastric cancer and its possible mechanism.In gastric tissues and gastric cancer cell lines, the Galectin-9has been localized in bothcytomembrane and cytoplasm, and Tim-3has been localized in both the nucleus andcytoplasm by the immunohistochemical method. To investigate the involvement of theGalectin-9mRNA and protein expression in the pathogenesis of primary gastric cancer,52clinical gastric cancer tissues and matched adjacent (<2cm away from the tumor) and/ornormal (>5cm away from the tumor) tissues were collected in pairs from September2008upuntil July2013in the Department of Gastrointestinal Surgery of The First Hospital of JilinUniversity. All cases were confirmed to have gastric cancer by postoperative pathology.Galectin-9mRNA and protein expression levels were measured by qPCR and Western blot.Compared to matched normal or adjacent tissues, the mRNA expression of Galectin-9wassignificantly decreased in gastric cancer tissues (P<0.001, P<0.001). To determine whether the reduction of Galectin-9mRNA expression resulted in decreasing of Galectin-9proteinlevels, gastric cancer and corresponding normal or adjacent tissues were analyzed by Westernblotting. Similar to our expected results, significant reduction of Galectin-9protein in gastriccancer (0.999±0.435) compared to those matched normal tissues (1.796±0.684) were detected(P<0.05). To extend above observations and to know the relationship between Galectin-9mRNA levels and clinicopathological parameters, we analyzed qPCR results according to theclinical characteristics of gastric cancer. Compared to the mRNA expression of Galectin-9inT1stage, Galectin-9mRNA levels in T3and T4stage were significantly decreased (P<0.05,P<0.05). However, there were no differences in the T2and T1stages, which suggest thatGalectin-9may be related to the degree of tumor invasion. N0-N3stage cancer Galectin-9mRNA levels were not significantly different. With no distant metastasis group (M0)compared with distant metastasis group (M1), Galectin-9mRNA expression decreasedobviously (P<0.05). Compared to stageⅠ, Galectin-9mRNA expression levels weredecreased in stage II、III、IV gastric tumors, and there was a significant difference in stages IIand IV (P<0.05). The survival time after surgery with high Galectin-9mRNA expression waslonger than that with low Galectin-9mRNA expression, which suggests Galectin-9expressionis associated with survival time. In summary, Galectin-9mRNA expression levels have acertain correlation with the degree of malignancy found in the tumor. However, there was nosignificant difference between age and gender (P>0.05). Compared to matched normal tissues,Tim-3mRNA and protein expression were decreased in the tumors. Statistical analysisconfirmed that there was no significant difference between the tumor and matched normaltissues.The expressions of Galectin-9and Tim-3in gastric cancer cell lines (SGC-7901andMGC-803) and the normal gastric mucosa cell line (GES-1) were measured by qPCR andWestern blot. Compared to GES-1, the mRNA expression of Galectin-9was significantlydecreased in SGC-7901and MGC-803（P<0.05, P<0.01）, and significant reduction ofGalectin-9protein in SGC-7901and MGC-803to GES-1were detected (P<0.05, P<0.01) byWestern blot. Expression of Tim-3mRNA and protein in the SGC-7901and MGC-803cellsare not significantly lower than in GES-1cells. The expression trend of Tim-3and Galectin-9in gastric cell lines is consistent with that in gastric tissues.For the further study of Galectin-9in the carcinogenesis, development of gastric cancerand the mechanism, and to further clarify whether there is a correlation regulatory relationship between Galectin-9and Tim-3in cells, we constructed the pcDNA3.1(-)-Galectin-9plasmid,transiently transfected into MGC-803cells and established a transient transfection expressionsystem. We observed that over expression of Galectin-9can inhibit the proliferation ofMGC-803cells, and DAPI staining showed that over expression of Galectin-9can induce theapoptosis of MGC-803cells. The result of Western blot to detect the apoptosis signalingpathway related protein showed that transfection of Galectin-9can down regulate theexpression of Bcl-2protein, but Bax protein and Caspase-3protein expression increase, whichsuggests that over expression of Galectin-9may induce cell apoptosis through themitochondrial pathway. To determine whether the Tim-3expression was regulated byGalectin-9, MGC-803cells were transiently transfected with Galectin-9DNA, the resultshowed that the expression of Tim-3protein had not been changed. The immunoprecipitationcomplexes only detected Galectin-9protein, and no Tim-3protein precipitation, whichsuggests that the two had no direct effect on MGC-803cells, the effects of Galectin-9ongastric cancer cells may not be played by Galectin-9/Tim-3pathway.In summary, the normal stomach mucosa and gastric cancer tissues can show Galectin-9and Tim-3protein expression. In gastric cancer tissues and gastric cancer cell lines, theexpression of Galectin-9mRNA and protein were significantly lower than that of normaltissues. The over expression of Galectin-9in gastric cancer cell lines can inhibit tumor cellgrowth, promote the apoptosis of tumor cells, Galectin-9can down regulate Bcl-2andupregulate the expression of Bax and Caspase-3protein, and then promote apoptosis.Galectin-9may not interact with Tim-3directly in MGC-803cells, but the mechanism ofGalectin-9and Tim-3in the occurrence and development of gastric carcinoma should besubject to further studies.更多还原