FAK基因与胃癌、肝癌细胞恶性表型关系的研究及其基因敲除细胞系试构建

发布时间：2018-01-13 18:41

本文关键词：FAK基因与胃癌、肝癌细胞恶性表型关系的研究及其基因敲除细胞系试构建　出处：《陕西师范大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景及目的在我国,由癌症引起的死亡高居全国死亡率的第二位。其中,肝癌、胃癌分别位居癌症死亡率的第二、第三位。癌症的发病率在过去几十年中一直呈上升趋势,近年来明显加速,构成对人类生命安全的重大威胁!而且,人类在应对癌症特别是对癌症的有效诊治方面,仍有漫长的路要走。目前,针对胃癌、肝癌最有效的治疗方法仍是手术、化疗、放疗等方法,但这些方法仍有很多不足,如毒副作用大、复发率高、治愈率低。作为新兴的诊治手段,基因诊治在癌症的诊断、治疗及预后中具有显著的优势,有可能是未来癌症诊治的重要辅助手段。因此,对肿瘤相关基因功能的研究显得尤为重要。研究基因功能常用的手段有基因敲低(Inhibition/Knockdown)和基因敲除(Deletion/Knockout)。RNA干扰(RNAi)是最常用的基因敲低技术,已被广泛应用于与基因相关疾病的研究中。RNA干扰虽然对靶基因具有较高的抑制效率,但无法达到100%抑制,在基因功能研究方面存在一定的局限性,但技术简单,成本较低,易于成功,而基因敲除技术可以针对特定基因,使基因功能完全丧失,是一种更为可靠的基因研究手段。黏着斑激酶(Focal adhesion kinase,FAK)在许多肿瘤中都过表达,其异常表达与肿瘤的发生、发展密切相关。本研究利用RNAi技术,抑制FAK在人肝癌SMMC-7721、胃癌SGC-7901细胞内的表达,进而研究了FAK表达对肝癌、胃癌细胞表型及运动相关蛋白的影响。为深入探究FAK功能,以及为后续研究提供更确切的实验平台,本研究尝试了以TALEN技术构建FAK敲除的SGC-7901胃癌细胞系模型。方法一、FAK基因与胃癌、肝癌细胞恶性表型关系的研究1.利用磷酸钙转染法,将靶向FAK的干扰重组质粒转入SGC-7901、SMMC-7721细胞中。2.利用扫描电镜观察细胞形态及运动相关微结构。3.利用免疫荧光法、免疫细胞化学法及考马斯亮蓝染色对细胞骨架相关蛋白(F-actin、β-tubulin、Lamin B1)的表达、分布及重构进行研究。4.利用MitoViewTM 633、Hoechst 33342染色法检测线粒体膜电位、细胞核形态变化,EB染色法检测细胞膜通透性改变。二、利用TALEN构建FAK基因敲除的SGC-7901细胞系1.FAK基因组信息学分析,NCBI database中查找人类FAK基因组序列,找到CDS区,确定TALEN作用靶点位置。’2.根据TALEN设计原则,设计针对FAK的TALEN作用靶点位置识别序列。3.构建识别FAK基因敲除靶点的TALEN左右臂同源重组DNA质粒。4.靶细胞(胃癌细胞SGC-7901)培养及其转染(磷酸钙法)。5.连续稳定转染细胞的Puromycin(嘌呤霉素)抗性筛选。6. Puromycin筛选细胞的单克隆化(有限稀释法)及其扩大培养。7.阳性克隆的初步鉴定(细胞克隆FAK基因TALEN作用靶点序列的测定)。结果一、FAK基因与胃癌、肝癌细胞怒性表型关系的研究1.提取的FAK siRNA质粒浓度、纯度较好,对两种细胞的转染效率均≥80%。2.扫描电镜结果显示,FAK表达抑制后,细胞运动有关形态有所改变,片状、丝状伪足发育明显受到抑制,癌细胞最具特征的恶性表型—接触抑制丢失(Contact inhibition lost)有所恢复。3.FAK表达抑制后,F-actin表达减弱,微丝变细；由β-tubulin所形成的微管结构的Polymerization弱化；Lamin B1表达量降低,并呈时间依赖性。4.线粒体膜电位、细胞核形态有一定的变化,膜通透性改变不明显,FAK表达抑制对细胞凋亡的促进作用不明显。二、利用TALEN构建FAK基因敲除的SGC-7901细胞系1.酶切鉴定结果显示FAK基因敲除靶点的TALEN左右臂同源重组DNA质粒构建成功。2.质粒共转效率≥60%。3. Puromycin抗性筛选后,有限稀释法获得6株细胞克隆。4.对6株细胞克隆进行FAK基因组靶点序列测定,证明无FAK-/-SGC-7901克隆。结论1.FAK表达抑制可有效引起SMMC-7721、SGC-7901细胞运动相关微结构发育的抑制。这一改变对癌细胞侵袭、转移等癌症发展关键环节意义重大。2.FAK表达抑制与细胞凋亡的关系不明显。这与FAK基因的已知基本功能是相符的。3.本课题对FAK在癌细胞恶性表型方面的研究结果再次提示该基因的过表达对胃癌、肝癌发展过程的重要作用,提示FAK基因及其产物在胃癌、肝癌的基因诊治方面具有作为靶标的潜在性。4.以斯丹赛公司提供的TALEN试剂盒构建FAK基因敲除的SGC-7901细胞系未成功。结合行业内有关信息,以及本实验室另一尝试结果,说明该公司的该试剂盒不易于成功敲除靶基因。
[Abstract]:Background and purpose in our country, caused by cancer death in the National Death second. Among them, liver cancer, gastric cancer mortality were ranked second and third. The incidence rate of cancer in the past few decades has been rising in recent years, significantly accelerated, pose a major threat to human life and safety! In response to the human cancer especially effective in diagnosis and treatment of cancer, there is still a long way to go. At present, the most effective treatment for gastric cancer, liver cancer is surgery, chemotherapy, radiotherapy and other methods, but these methods still have many shortcomings, such as high toxicity, high recurrence rate and low cure rate. As a new means of diagnosis and treatment, diagnosis and treatment of genes in cancer diagnosis, treatment and prognosis has significant advantages, there may be an important auxiliary means for future diagnosis and treatment of cancer. Therefore, the research on tumor related gene function is particularly important. Methods to study gene function are used to knockdown gene knockout (Inhibition/Knockdown) and (Deletion/Knockout).RNA interference (RNAi) is the most commonly used gene knockdown technology, has been widely used in the study of.RNA interference and genes related to diseases although has high inhibition efficiency of target genes, but can not reach 100% inhibition and there are some limitations in gene function research, but the technology is simple, low cost, easy success, and gene knockout technology to target specific genes, the gene function completely lost, is a more reliable means of gene research. FAK (Focal adhesion kinase, FAK) in many cancers all over expression, its abnormal expression with tumor occurrence, development is closely related to this study. Using the technology of RNAi, the inhibition of FAK in human hepatocellular carcinoma SMMC-7721, SGC-7901 expression in gastric cancer cells, and then studied the expression of FAK on liver Effect of cancer cell phenotype and motion related proteins in gastric cancer. To explore the function of FAK, to provide more precise and experimental platform for subsequent research, this study attempts to construct the FAK TALEN technology on SGC-7901 gastric cancer cell line model. Methods, FAK gene and gastric cancer, liver cancer cell malignant phenotype relationship by 1. calcium phosphate transfection, the targeting of the recombinant plasmid FAK was transfected into SGC-7901 SMMC-7721 cells,.2. cell morphology was observed by scanning electron microscopy and micro motion related structure of.3. by immunofluorescence, immunocytochemistry and Coomassie blue staining of cytoskeleton associated protein (F-actin, beta -tubulin, Lamin B1) expression, distribution and the reconstruction of.4. using MitoViewTM 633, Hoechst 33342 staining was used to detect the mitochondrial membrane potential, morphological changes, cell membrane permeability was detected by EB staining. Two, using TALEN Construction of FAK gene on SGC-7901 cell line 1.FAK in genomic information analysis, NCBI database find FAK human genome sequence, find the CDS area, to determine the location of the target TALEN. "2. according to TALEN design principles, design for TALEN target location recognition sequence.3. FAK construction to identify FAK gene knockout target TALEN left arm homologous target cells of recombinant plasmid DNA.4. (SGC-7901 gastric cancer cell culture and transfection) (calcium phosphate).5. transfected cells Puromycin (puromycin resistant) screening of.6. Puromycin screening cell monoclonal (limited dilution) and expand the preliminary identification of cultured.7. positive clones (determination of target gene TALEN point sequence of FAK cell clones). Results, FAK gene and gastric cancer, FAK concentration of siRNA plasmid on hepatocellular carcinoma cell line of anger phenotype correlation of 1. extracted with good purity and transfection of two cells Efficiency is greater than or equal to 80%.2. scanning electron microscopy showed that the inhibition of FAK gene expression, morphology of cell movement change, flake, filopodia significantly inhibited development of the malignant phenotype of cancer cells, and the main characteristics of the loss of contact inhibition (Contact inhibition lost) has been restored after inhibition of.3.FAK expression, F-actin expression decreased, actin thin microtubules; weakening the structure formed by the beta -tubulin Polymerization; Lamin B1 expression decreased, and time-dependent.4. mitochondrial membrane potential, cell morphology changed with the change of membrane permeability, no significant inhibition of FAK gene expression on the apoptosis promoting effect is not obvious. Two, using TALEN to construct FAK gene knock in SGC-7901 cell line the results showed that FAK enzyme digestion 1. knockout target TALEN arm homologous recombinant DNA plasmid was successfully constructed.2. plasmid co transfection efficiency higher than 60%.3. Puromycin after resistance screening 6 strains,.4. cell clones of FAK genomic target sequences of 6 strains of cell clones obtained by limited dilution method, prove that there is no FAK-/-SGC-7901 clone. Conclusion 1.FAK can effectively inhibit the expression of SMMC-7721 induced SGC-7901 cell movement related micro structure development. This change of inhibition of cancer cell invasion, metastasis of cancer is very important to the development of key links relationship between the expression of.2.FAK and inhibit the cell apoptosis is not obvious. The FAK gene and known basic functions are consistent with the research of.3. FAK in the malignant phenotype of cancer cells the results suggest that the overexpression of this gene in gastric cancer, an important role in HCC development, suggesting that the FAK gene and its product in gastric cancer, gene diagnosis and treatment liver cancer is a TALEN kit of.4. as a potential target to match the company's construction of FAK gene in SGC-7901 cell line in addition to the knock binding within the industry without success. The information, as well as another experiment in our laboratory, showed that the company's kit was not easy to successfully knock off the target gene.

【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R730

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