通过RNAi制备不同AFP表达水平的大鼠肝癌细胞

发布时间：2018-06-06 05:39

本文选题：肝癌 + AFP　；参考：《河北医科大学》2016年硕士论文

【摘要】：目的:肝癌是全球发病率第六的恶性肿瘤,每年约有70万人被诊断为肝癌。在癌症引起的死亡中30%为肝癌,近年来其病死率呈逐年上升的趋势,已经对人类健康构成了极大的威胁。肝癌是涉及多基因、多过程的复杂疾病,其致病机理仍不清楚。目前,手术切除治疗仍然是肝癌最有效的治疗方法,然而肝癌切除术或肝移植术后患者预后情况往往并不理想,其主要原因之一就是肿瘤的复发和转移。因此探讨肝癌细胞转移发生、发展的分子生物学机制对肝癌的治疗具有重要意义,也是肝癌研究的热点和难点。另外,建立与人类肝癌临床特征相近的动物模型对于研究肝癌机理寻找新的治疗方法也具有重要的现实意义。甲胎蛋白(alpha—fetoprotein,AFP)是目前已知的与肝癌关系最密切的基因之一,约70%的肝癌患者高表达AFP,AFP作为血清标记物已广泛应用于肝癌的临床诊断、治疗及预后判断。AFP主要在胎儿时期合成,出生后1-2年降至成人水平,健康成年人肝脏失去产生AFP的能力。但是,当成年人患肝癌或者肝切除时,肝脏恢复合成AFP的能力,AFP的合成量与胎儿肝脏或者肝细胞再生的分裂细胞数呈正相关,因此认为AFP是分裂增殖的肝细胞产生,靶向Afp的治疗能同时针对所有的肝癌细胞。近年研究表明,AFP在肝癌细胞的生长、分化、增殖和迁移中发挥重要作用,是肝癌进展过程中一个重要的调节因子。RNA干扰(RNA Interference)通过双链RNA在mRNA水平部分或者完全抑制特异性基因表达。RNAi在生物体内广泛存在能同时抑制多个基因,并且操作简单、特异性高、能快速使靶向基因功能抑制,为肝癌的基因治疗带来了希望。本研究采用DEN和NMOR联合诱导大鼠产生肝癌,通过组织培养、克隆,筛选出具有高、低转移潜能的大鼠肝癌细胞各1株。并以得到的高转移潜能大鼠肝癌细胞为研究对象,构建靶向Afp的shRNA并用脂质体法转染细胞,干扰其AFP表达,分析不同AFP表达水平对细胞迁移率和克隆形成能力的影响。不同AFP表达水平的细胞的获得可为制备相应的大鼠肝癌模型做准备,从而为肝癌的转移、复发机理研究及新药治疗效果评价等提供与临床相一致的动物模型。方法:无菌取诱导20周肝癌大鼠肝脏肿瘤进行原代培养,收取细胞后经Transwell筛选获得AFP高表达的高、低转移潜能的肝癌细胞株Wrh-f2和Wrh-s2,分析细胞生物学特性;将靶向AFP的4个shRNA载体及阴性载体转染AFP高表达的Wrh-f2细胞,实验分为空白对照组、阴性对照组、转染AFP-si RNA组,获得5个稳定转染细胞株。通过Real-time PCR和Western blot方法检测各组细胞Afp mRNA和AFP的表达变化,采用t检验和方差分析进行比较。选择干扰效果最好的8号细胞,检测细胞克隆形成率和迁移率。结果:1 DEN和NMOR联合诱导大鼠于20周末获得诱发性肝癌模型,肝脏有大的肿瘤块;肿块经培养、克隆获得肝癌细胞。2经Transwell筛选获得高、低转移潜能的肝癌细胞株Wrh-f2和Wrh-s2,AFP表达均为阳性。Wrh-f2细胞具有更高的迁移率和克隆形成率,有多核细胞,异常核分裂相多见,其特征与病人肝癌组织相同。经染色体核型分析发现Wrh-s2株细胞为超2倍体,众数为55;Wrh-f2细胞大部分核型为超4倍体,各细胞内部差异较大,f2众数为83。且高转移潜能细胞Wrh-f2易发生肺部转移,这与人体内的肝癌细胞特性相近。3合成针对Afp的shRNA4个分别为5、6、7、8号,另有阴性对照0号,转染Wrh-f2细胞,获得稳转细胞。Real-time PCR和Western blot定量结果显示,转染8号shRNA的细胞mRNA及蛋白表达水平均显著降低,因此选择8号细胞进行下面实验。4经检测Wrh-f2、0号、8号细胞克隆形成率分别为:(85.7±7.2)%、(59±5.8)%、(5.5±0.7)%,差异极显著;细胞迁移率结果显示Wrh-f2、0号、8号平均穿膜细胞数分别为98±12,45±15,30±10,差异显著。结论:1经Transwell筛选获得Wrh-f2和Wrh-s2细胞分别为高、低转移潜能的肝癌细胞株,AFP表达均为阳性。Wrh-f2细胞具有更高的迁移率和克隆形成率,且从形态学及染色体分析发现Wrh-f2分化水平较低,恶化程度较高,且高转移潜能细胞Wrh-f2易发生肺部转移,这与人体内的肝癌细胞特性相近。2合成的4组shRNA均可靶向抑制Afp mRNA的表达量,其中8号可同时显著抑制蛋白的表达,且干扰效果最好。经试验8号可以有效降低肝癌细胞的转移能力。
[Abstract]:Objective: liver cancer is a malignant tumor of sixth in the world. About 700 thousand people are diagnosed as liver cancer every year. In the death of cancer, 30% of them are liver cancer. In recent years, the mortality rate is increasing year by year, which has posed a great threat to human health. Liver cancer is a complex disease involving multi cause and multi process. The pathogenesis is still unclear. At present, surgical resection is still the most effective treatment for liver cancer. However, the prognosis of the patients after hepatectomy or liver transplantation is often not ideal. One of the main reasons is the recurrence and metastasis of the tumor. Therefore, it is important to explore the metastasis of hepatoma cells and the development of the sub biological mechanism for the treatment of liver cancer. It is also a hot and difficult point in the study of liver cancer. In addition, it is of great practical significance to establish an animal model similar to the clinical characteristics of human liver cancer for the study of the mechanism of liver cancer. Alpha fetoprotein (AFP) is one of the most closely related genes associated with liver cancer, and about 70% of the liver cancer patients High expression of AFP, AFP as a serum marker has been widely used in the clinical diagnosis of liver cancer. The treatment and prognosis of.AFP are mainly synthesized in the fetal period, 1-2 years after birth to adult level. The liver of healthy adults loses the ability to produce AFP. However, when adults suffer from liver cancer or hepatectomy, the liver can restore the ability to synthesize AFP and the combination of AFP. There is a positive correlation between the number and the number of split cells regenerated from fetal liver or liver cells. Therefore, AFP is considered to be a proliferating hepatocyte, and the targeted Afp therapy can simultaneously target all liver cancer cells. In recent years, the study shows that AFP plays an important role in the growth, differentiation, proliferation and migration of hepatoma cells, and is one of the progress of liver cancer. The important regulatory factor.RNA interference (RNA Interference) can inhibit multiple genes at the same time in the mRNA horizontal part or completely inhibit the specific gene expression of.RNAi in the organism. It has a simple and high specificity, which can quickly inhibit the target gene function and bring hope for the gene therapy of liver cancer. DEN and NMOR were used to induce liver cancer in rats. Through tissue culture and cloning, 1 rat hepatoma cells with high and low metastatic potential were screened, and the high metastatic potential rat hepatoma cells were selected as the research object. The target Afp shRNA was constructed and transfected with liposome method. The expression of AFP was interfered with the expression of AFP, and the expression of different AFP expression was analyzed. The effect of level on cell mobility and clone formation ability. The acquisition of cells with different AFP expression levels can prepare the corresponding rat model of liver cancer, thus providing a consistent animal model for the metastasis of liver cancer, the study of the mechanism of recurrence and the evaluation of the effect of new drug treatment. Method: aseptic induction of liver cancer of 20 weeks in rat liver After primary culture, after collecting cells, the tumor cells were screened by Transwell to obtain high, low metastatic potential liver cancer cell lines, Wrh-f2 and Wrh-s2, to analyze the cell biological characteristics, and transfect AFP highly expressed Wrh-f2 cells with 4 shRNA carriers and negative vectors targeting AFP, which were divided into blank control group, negative control group and AFP-si transfected AFP-si. In group RNA, 5 stable transfected cell lines were obtained. The expression of Afp mRNA and AFP in each cell was detected by Real-time PCR and Western blot. T test and variance analysis were used to compare. Select the 8 cells with the best interference effect and detect the cell clone formation rate and mobility. Fruit: 1 DEN and NMOR combined rats were obtained at the end of the 20 week. The liver has a large tumor block, the liver has a large tumor block, and the tumor is cloned to obtain the liver cancer cell.2, which is screened by Transwell to obtain high, low metastatic potential liver cancer cell lines Wrh-f2 and Wrh-s2. The expression of AFP is the positive.Wrh-f2 cells with higher mobility and clone formation rate. The liver cancer tissues of the patients were the same. By karyotype analysis, Wrh-s2 cells were found to be ultra 2 fold and 55, and most of the karyotypes of Wrh-f2 cells were super 4 ploidy, the internal difference between each cell was large, the number of F2 was 83. and the high metastatic potential cell Wrh-f2 was easily transferred to the lung, which was similar to the.3 synthesis of Afp shRNA4 Wrh-f2 cells were transfected with.Real-time PCR and Western blot, respectively. The transfection of.Real-time PCR and Western blot showed that the expression of mRNA and protein in the cells transfected to shRNA was significantly reduced, so 8 cells were selected to test the Wrh-f2,0 number of.4, and the clone formation rate of No. 8 cells was respectively: (85.7) 7.2%, (59 + 5.8)%, (5.5 + 0.7)%, and the difference was very significant. The cell migration rate showed that the number of Wrh-f2,0 and 8 average membrane cells was 98 + 12,45 + 15,30 + 10, and the difference was significant. Conclusion: 1 was screened by Transwell to obtain Wrh-f2 and Wrh-s2 cells with high and low metastatic potential respectively. The expression of AFP was more than that of positive.Wrh-f2 cells. The high mobility and clone formation rate, and from the morphological and chromosome analysis, the Wrh-f2 differentiation level is low, the deterioration degree is high, and the high metastatic potential cell Wrh-f2 is prone to pulmonary metastasis, which can target the expression of Afp mRNA in the 4 groups of shRNA, which are similar to the human hepatoma cells in the human body, and the number of Afp mRNA can be inhibited at the same time. The expression of protein is the best and the interference effect is the best. Test 8 can effectively reduce the metastatic ability of liver cancer cells.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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