【作者】 李荣宽；

【导师】 唐建武；

【作者基本信息】 大连医科大学 ， 病理与病理生理学， 2010， 博士

【摘要】 背景：转移潜能是恶性肿瘤最重要的生物学特征之一,也是肿瘤患者死亡率高、预后差的主要原因。对于上皮来源的恶性肿瘤,淋巴道转移是其早期转移的主要方式,但确切机制目前仍不清楚。原发性肝癌(primary carcinoma of the liver)是最常见恶性肿瘤之一,包括肝细胞癌(hepatocellular carcinoma,HCC)、肝内胆管上皮癌(intrahepatic cholangiocarcinoma,IHCC)、肝细胞及胆管上皮细胞混合癌三种细胞类型,其中90%以上为肝细胞癌。全世界每年新发约62.6万例HCC患者,其中的半数以上病例发生在我国。同时,它也是恶性程度最高、致死率最高的肿瘤之一,位居全球恶性肿瘤死因第三位、我国恶性肿瘤死因第二位。即便是经过根治性切除手术治疗,5年的复发转移率仍高达60%,因此,揭示肝癌转移的分子机制及其关键调控环节,进而采取相应的干预措施,将有助于减少、甚至避免病人术后发生复发转移,从而达到降低死亡率的目的。Hca-F和Hca-P是将小鼠肝癌细胞株H22在615小鼠体内反复接种和筛选后得到的一对来源于同一亲本细胞、但是淋巴道转移潜能显著不同的小鼠肝癌腹水型细胞株。其中Hca-F具有高淋巴道转移潜能,接种到615小鼠体内后其淋巴结转移率超过70%,Hca-P是低转移潜能的细胞株,其淋巴结转移率低于30%。通过对比研究二者的差异,可以为我们揭示小鼠肝癌的淋巴道转移机制提供有益的参考。本课题组在前期工作中已经应用不同的方法,对Hca-F和Hca-P这一对具有高低不通淋巴道转移潜能的细胞株进行了一系列系统的研究。首先,利用抑制性消减杂交技术(suppression substractive hybridization, SSH)及基因芯片(Genechip)高通量筛选(high throughput screen, HTS)技术得出了Hca-F细胞和Hca-P细胞之间一系列差异表达基因,继而采用定量蛋白质组学技术建立了高低淋巴道转移潜能小鼠肝癌细胞的荧光差异蛋白表达图谱。在诸多差异基因和蛋白质中,细胞内氯离子通道1 (chloride intracellular channel 1,C LIC 1)在Hca-F细胞中的表达都显著高于Hca-P细胞,其中在mRNA水平(基因芯片)上,CLIC1在Hca-F细胞中的表达是其在Hca-P细胞中表达量的3.48倍,在蛋白水平(双向免疫荧光差异电泳)上,Hca-F中的CLIC1表达量是Hca-P的1.6倍,提示CLIC1可能与小鼠肝癌的淋巴道转移潜能相关。由于人类组织细胞中具有CLIC1同源蛋白,因此,研究小鼠肝癌细胞株CLIC1与淋巴道转移的关系,对人类肝癌淋巴道转移的防治必然具有重要意义。CLIC1是氯离子通道家族中的一员,又称NCC27,是一种氯离子通道蛋白。通常其在细胞中以两种形式存在：溶解于胞浆中的可溶型和结合于胞膜上的膜结合型。研究表明CLIC1的作用不仅局限于细胞内外离子的运输,而且和许多疾病,尤其是肿瘤性疾病有密切的关系。有学者发现CLIC1在乳腺癌、肺癌、肝细胞癌、胆囊癌、胃癌以及大肠癌等多种肿瘤细胞中表达明显增强,同时发现其与胃癌和胆囊癌的淋巴结侵袭能力显著相关,提示CLIC1可能是参与肿瘤发生及淋巴道转移的重要基因之一。本研究以CLIC1为研究对象,探索其在Hca.F细胞株淋巴道转移过程中的作用。经检索,目前国内外尚无CLIC1与肝癌淋巴道转移关系的研究报道。目的：1.在蛋白质水平上比较CLIC1在淋巴道转移潜能不同的小鼠肝癌细胞系Hca-F和Fca-P中的表达,为进一步研究肝癌淋巴道转移的机制奠定基础。2.构建pGPU6/GFP/Neo-shRNA表达载体,稳定转染Hca-F细胞,得到CLIC1的表达被明显下调的Hca-F细胞株。3.对比研究小鼠肝癌细胞株Hca-F在CLIC1表达水平下调前后其细胞增殖能力、细胞周期与凋亡情况、细胞迁移和侵袭能力等方面的变化,探讨CLIC1基因的表达对小鼠肝癌细胞生物学行为的影响。方法：1.采用蛋白免疫印迹(Western Blotting)方法检测Hca-F和Hca-P细胞株中CLIC1的表达差异。2.依据Gene Bank中小鼠CLIC1基因序列设计合成3条shRNA的DNA模板的两条单链,分别命名为shRNA-1.shRNA-2.shRNA-3,同时设计shRNA-无关序列作为阴性对照(negative control,NC).进而与pGPU6/GFP/Neo质粒构建表达载体,分别命名为pGPU6/GFP/Neo-shRNA-1.pGPU6/GFP/Neo-shRNA-2. pGPU6/GFP/Neo-shRNA-3. pGPU6/GFP/Neo- shRNA-NC。对三条pGPU6/GFP/Neo-shRNA表达载体经测序鉴定,结果与我们设计的三条shRNA序列以及Gene Bank中的序列相比对,确定序列无误,将三组pGPU6/GFP/Neo-shRNA表达载体和阴性对照pGPU6/GFP/Neo-shRNA-NC分别应用阳离子聚合物转染试剂梭华-SofastTM稳定转染至Hca-F细胞中,经G418筛选,获得稳定转染表达载体pGPU6/GFP/Neo-shRNA的Hca-F细胞。以空白对照组(未转染的Hca-F细胞株)为参照,应用Real-time RT-PCR和Western Blotting方法在mRNA水平和蛋白质水平上检测三组pGPU6/GFP/Neo-shRNA对CLIC1基因表达的抑制作用,以筛选出RNA干扰效果最好的一组表达载体用于后续实验。3.以三组细胞(分别为Hca-F细胞株、稳定转染pGPU6/GFP/Neo-shRNA-NC的Hca-F细胞株、经筛选稳定转染干扰效率最好的pGPU6/GFP/Neo-shRNA表达载体的Hca-F细胞株)为实验对象。应用CCK-8试剂盒检测三组细胞的增殖能力；应用流式细胞仪检测三组细胞的细胞周期和凋亡情况；应用Transwell小室检测CLIC1的表达被pGPU6/GFP/Neo-shRNA下调后Hca-F细胞的迁移能力和侵袭能力的变化。结果：1.CLIC 1蛋白在小鼠腹水型肝癌细胞株Hca-F和Hca-P中均有表达,CLIC1在Hca-F细胞株中的表达是其在Hca-P中的1.33倍。2.靶向CLIC1 mRNA的三个pGPU6/GFP/Neo-shRNA重组质粒表达载体经测序分析,以及经过限制性内切酶酶切后电泳证实载体构建成功。三组重组质粒表达载体稳定转染Hca-F细胞后,经验证pGPU6/GFP/Neo-shRNA-2对CLIC1表达抑制的效果最好,因而被选择作为后续实验的CLIC1稳定下调的细胞株。3.CCK-8实验结果显示CLIC1基因表达下调后Hca-F细胞增殖情况明显增强；流式细胞仪检测结果显示CLIC1基因表达下调后,停留于G2／M期的Hca-F细胞数目比例明显增多(下调组46.07%*；空白对照组16.72%；阴性对照组19.79%,*P<0.05),凋亡细胞比例显著减少(下调组5.33%*；空白对照组29.78%；阴性对照组28.53%,*P<0.05)；体外迁移实验结果显示CLIC1表达下调后穿膜细胞数明显低于空白对照组和阴性对照组(下调组128.67+33.54*；空白对照组214.53+24.96；阴性对照组197.73+24.48,*P<0.05)；体外侵袭实验结果显示CLIC1表达下调后穿过人工基底膜的细胞数明显低于空白对照组和阴性对照组(下调组50.734-3.89*；空白对照组98.93±5.00；阴性对照组96.27±2.60,*P<0.05)。结论：1.CLIC 1在小鼠腹水型肝癌细胞系Hca-F和Hca-P均有表达,在Hca-F细胞株中的表达量明显高于在Hca-P中的表达。2.利用真核细胞表达质粒pGPU6/GFP/Neo和shRNA成功构建了三组pGPU6/GFP/ Neo-shRNA表达载体,并稳定转染Hca-F细胞株,获得CLIC1表达稳定下调的Hca-F细胞株,经过检验,pGPU6/GFP/Neo-shRNA-2对Hca-F细胞中CLIC1抑制效果最明显,通过其下调CLIC1在Hca-F细胞中的表达,提供了揭示CLIC1基因在小鼠肝癌淋巴道转移潜能中所起作用的可能。3.CLIC 1作为小鼠肝癌细胞株Hca-F和Hca-P的差异表达蛋白质,降低其表达水平可促进小鼠肝癌细胞的增殖,使得停留于细胞周期中G2／M期的细胞数量增多,凋亡细胞数量减少,并且能够降低小鼠肝癌细胞的体外迁移和侵袭能力,提示CLIC1基因具有抑制小鼠肝癌细胞增殖、调节细胞周期、促进肿瘤细胞凋亡、促进肿瘤细胞的迁移和侵袭能力的作用,表明其在Hca-F细胞的淋巴道转移潜能机制中可能起着重要的作用。更多还原

【Abstract】 Background:Metastasis is one of the most important biological characteristics of malignant tumor, at the same time, it is the fundamental cause of high mortality and poor prognosis of the patients who suffer from malignant tumors.Lymphatic metastasis is the early mode in epithelial malignant tumors, but the mechanism of it is unclear. As one of the most common malignant tumor, the primary carcinoma of the liver includes 3 types:hepatocellular carcinoma （HCC）, which accounts more than 90%, and the other types are intrahepatic cholangiocarcinoma （IHCC） and the mixed type carcinoma. There are about 626,000 new cases of HCC and more than half in China. Meanwhile, as one of the most fatal malignant tumor, HCC has a high relapse incidence though the mass has been cut off closely. Therefore, to reveal the mechanism and the key steps in metastasis of HCC is helpful for reducing the mortality of it.Hca-P and Hca-F is a pair of syngenetic mouse hepatocarcinoma ascites cell lines which have different rates of lymphatic metastasis. Hca-F is the cell line whose metastatic rate is more than 70%, compared with it, Hca-P has a low lymphatic metastasis rate, which is less than 30%. They are the ideal models for the research of mechanism of lymphatic metastasis. Our research team has engaged in the study about the mechanism of lymphatic metastasis. We have picked out the lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines by using suppressive subtractive hybridization and high throughput screen of genechips assays respectively and obtained the lymphatic metastasis-associated proteins by using quantitative proteomics technique. The expressing level of CLIC1 was much higher in Hca-F than that in Hca-P cell lines in both mRNA and protein levels which showed that CLIC1 maybe play an important role in lymphatic metastasis of mouse hepatocarcinoma. Because there is the same origin of CLIC1 protein in the tissue of both human being and mouse, it is very helpful to study the function of CLIC1 in the lymphatic metastasis of mouse hepatocarcinoma.CLIC1, also known as NCC27, is a member of the family of chloride intracellular ion channels. As a chloride channel, which play an important role in the cells.CLIC1 usually exists in both soluble and memberane-assosiated forms. Studies have shown that the role of CLIC1 is not limited to intra and extra cellular ion transport, but also, and many diseases, especially neoplastic diseases are closely related to. Some scholars have found that CLIC1 markedly enhanced expression in breast cancer, lung cancer, gallbladder cancer, hepatocellular carcinoma, colorectal cancer, gastric cancer, as well as a variety of tumor cells, and found it was significantly correlated with lymph node invasion in stomach and gallbladder carcinoma, suggesting that CLIC1 may be one of the important genes involved in tumor occurrence and lymphatic metastasis. In this study, CLIC1 as the research object to explore in Hca-F cell line. There was not the same reports home and abroad by retrieval.Objective:1. To observe the different expression levels of CLIC1 in mouse hepatocarcinoma ascites cell lines Hca-F and Hca-P by Westen Blottin for further study of its function and mechanism in lymphatic metastasis of hepatocarcinoma.2.To build expression vector of pGPU6/GFP/ Neo-shRNA and get cell line Hca-F which expression of CLIC1 has been markedly decreased after stable transfection.3. To study the influence on proliferation, cell cycle, apoptosis, migration and invasion of mouse hepatocellular carcinoma cell line Hca-F after down regulation of CLIC1 expression by shRNA and discuss the correlation between expressing level of CLIC1 and lymphatic metastasis of mouse hepatocarcinoma.Methods:1. The expression of CLIC1 protein was detected in mouse hepatocarcinoma ascites cell lines of Hca-F and Hca-P by Western Blotting. 2. Three shRNAs （shRNA-1, shRNA-2, shRNA-3） and unrelated sequence were designed according to the gene sequence of CLIC1 （NM₀033444） in Gene Bank. pGPU6/GFP/Neo vector was obtained and restriction enzymes were used to digest. The annealing of shRNA compositive sequence was performed and T4 DNA ligase was used to connect the pGPU6/GFP/Neo vector with three shRNAs and unrelated sequence（negative control, NC） after annealing. The connecting products were named pGPU6/GFP/Neo-shRNA-1, pGPU6/GFP/Neo-shRNA-2, pGPU6/GFP/Neo-shRNA-3 and pGPU6/GFP/Neo-shRNA-NC. The expressing vector was obtained by extraction of vectors. The sequence of expressing vectors was identified by sequencing and the result was compared to that in Gene Bank. The three pGPU6/GFP/Neo-shRNA expressing vectors were transfected into Hca-F cells respectively by SofastTM reagent and the most effective pGPU6/ GFP/Neo-shRNA vector was selected according to the results of Real-time RT-PCR and Western Blotting. The unrelated shRNA transfected Hca-F cell line and normal Hca-F cell line were negative control group and blank control group respectively.3. The cell viability was evaluated by CCK-8. The cell cycle and apoptosis were evaluated by flow cytometry. The cell migration and invasion capability were evaluated by transwell assays.Results:1. The expression of CLIC1 in cell line Hca-F was 1.33 times more than that in Hca-P.2. The expression vector of pGPU6/GFP/Neo-shRNA was built and transfected into Hca-F cells successfully. The sequence of three shRNAs was identified by sequencing and the result was identical to that in Gene Bank. At the same time, all expression vectors were verified by restriction enzyme cutting to be built successfully. The expression vector pGPU6/GFP/Neo-shRNA-2 was the most effective one. The expressions of mRNA and protein of CLIC1 in Hca-F cells after transfection with pGPU6/GFP/Neo-shRNA-2 were markedly decreased compared with the other groups. pGPU6/GFP/Neo-shRNA-2 group was chosen as the cell line for further study.3. The results of CCK-8 showed an obvious increase of cell proliferation was detected in pGPU6/GFP/Neo-shRNA-2 group cells after the expression of CLIC1 has been decreased. The results detected by flow cytometer showed more cells were arrested in G2/M phase and the cell percentage of apoptosis significantly decreased after the expression of CLIC1 was decreased compared with the blank group and negative control group cells （P<0.05）. Transwell assays showed that both migration and invasion capability were decreased after knock-down of CLIC1 expression in Hca-F cell line （128.67±33.54* vs 214.53±24.96, 197.73±24.48,*P<0.05; 50.73±3.89* vs98.93±5.00,96.27±2.60,*P <0.05）.Conclusions:1. The expreesion of CLIC1 in Hca-F was greatly higher than that in Hca-P.2. The expression vectors pGPU6/GFP/Neo-shRNAs were constrcted successfully with plasmid pGPU6/GFP/Neo and shRNAs. After the expression vectors were stable transfected into Hca-F cell line, pGPU6/GFP/Neo-shRNA-2 was proved by Real-time RT-PCR and Western Blotting to be the most effective one, which can decreased the expression of CLIC1 remarkably. And pGPU6/GFP/Neo-shRNA-2 was chosen as the cell line for further research which provided a solid foundation for further studies about the relationship between CLIC1 and lymphatic metastasis of hepatocellular carcinoma.3. As one of the proteins expressed differently in Hca-F and Hca-P cell lines, CLIC1 could inhibit proliferation, promote apoptosis, promote cells to pass G2/M phase, increase migration and invasion capability of mouse hepatocellular carcinoma cells. Results above pointed out that CLIC1 may play an important role in lymphatic metastasis of hepatocellular carcinoma.更多还原