發(fā)布時間：2018-01-13 18:41

  本文關(guān)鍵詞：FAK基因與胃癌、肝癌細(xì)胞惡性表型關(guān)系的研究及其基因敲除細(xì)胞系試構(gòu)建　出處：《陜西師范大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 黏著斑激酶 胃癌 肝癌 RNA干擾 TALEN 基因功能

【摘要】：背景及目的在我國,由癌癥引起的死亡高居全國死亡率的第二位。其中,肝癌、胃癌分別位居癌癥死亡率的第二、第三位。癌癥的發(fā)病率在過去幾十年中一直呈上升趨勢,近年來明顯加速,構(gòu)成對人類生命安全的重大威脅!而且,人類在應(yīng)對癌癥特別是對癌癥的有效診治方面,仍有漫長的路要走。目前,針對胃癌、肝癌最有效的治療方法仍是手術(shù)、化療、放療等方法,但這些方法仍有很多不足,如毒副作用大、復(fù)發(fā)率高、治愈率低。作為新興的診治手段,基因診治在癌癥的診斷、治療及預(yù)后中具有顯著的優(yōu)勢,有可能是未來癌癥診治的重要輔助手段。因此,對腫瘤相關(guān)基因功能的研究顯得尤為重要。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干擾(RNAi)是最常用的基因敲低技術(shù),已被廣泛應(yīng)用于與基因相關(guān)疾病的研究中。RNA干擾雖然對靶基因具有較高的抑制效率,但無法達(dá)到100%抑制,在基因功能研究方面存在一定的局限性,但技術(shù)簡單,成本較低,易于成功,而基因敲除技術(shù)可以針對特定基因,使基因功能完全喪失,是一種更為可靠的基因研究手段。黏著斑激酶(Focal adhesion kinase,FAK)在許多腫瘤中都過表達(dá),其異常表達(dá)與腫瘤的發(fā)生、發(fā)展密切相關(guān)。本研究利用RNAi技術(shù),抑制FAK在人肝癌SMMC-7721、胃癌SGC-7901細(xì)胞內(nèi)的表達(dá),進而研究了FAK表達(dá)對肝癌、胃癌細(xì)胞表型及運動相關(guān)蛋白的影響。為深入探究FAK功能,以及為后續(xù)研究提供更確切的實驗平臺,本研究嘗試了以TALEN技術(shù)構(gòu)建FAK敲除的SGC-7901胃癌細(xì)胞系模型。方法一、FAK基因與胃癌、肝癌細(xì)胞惡性表型關(guān)系的研究1.利用磷酸鈣轉(zhuǎn)染法,將靶向FAK的干擾重組質(zhì)粒轉(zhuǎn)入SGC-7901、SMMC-7721細(xì)胞中。2.利用掃描電鏡觀察細(xì)胞形態(tài)及運動相關(guān)微結(jié)構(gòu)。3.利用免疫熒光法、免疫細(xì)胞化學(xué)法及考馬斯亮藍(lán)染色對細(xì)胞骨架相關(guān)蛋白(F-actin、β-tubulin、Lamin B1)的表達(dá)、分布及重構(gòu)進行研究。4.利用MitoViewTM 633、Hoechst 33342染色法檢測線粒體膜電位、細(xì)胞核形態(tài)變化,EB染色法檢測細(xì)胞膜通透性改變。二、利用TALEN構(gòu)建FAK基因敲除的SGC-7901細(xì)胞系1.FAK基因組信息學(xué)分析,NCBI database中查找人類FAK基因組序列,找到CDS區(qū),確定TALEN作用靶點位置。’2.根據(jù)TALEN設(shè)計原則,設(shè)計針對FAK的TALEN作用靶點位置識別序列。3.構(gòu)建識別FAK基因敲除靶點的TALEN左右臂同源重組DNA質(zhì)粒。4.靶細(xì)胞(胃癌細(xì)胞SGC-7901)培養(yǎng)及其轉(zhuǎn)染(磷酸鈣法)。5.連續(xù)穩(wěn)定轉(zhuǎn)染細(xì)胞的Puromycin(嘌呤霉素)抗性篩選。6. Puromycin篩選細(xì)胞的單克隆化(有限稀釋法)及其擴大培養(yǎng)。7.陽性克隆的初步鑒定(細(xì)胞克隆FAK基因TALEN作用靶點序列的測定)。結(jié)果一、FAK基因與胃癌、肝癌細(xì)胞怒性表型關(guān)系的研究1.提取的FAK siRNA質(zhì)粒濃度、純度較好,對兩種細(xì)胞的轉(zhuǎn)染效率均≥80%。2.掃描電鏡結(jié)果顯示,FAK表達(dá)抑制后,細(xì)胞運動有關(guān)形態(tài)有所改變,片狀、絲狀偽足發(fā)育明顯受到抑制,癌細(xì)胞最具特征的惡性表型—接觸抑制丟失(Contact inhibition lost)有所恢復(fù)。3.FAK表達(dá)抑制后,F-actin表達(dá)減弱,微絲變細(xì)；由β-tubulin所形成的微管結(jié)構(gòu)的Polymerization弱化；Lamin B1表達(dá)量降低,并呈時間依賴性。4.線粒體膜電位、細(xì)胞核形態(tài)有一定的變化,膜通透性改變不明顯,FAK表達(dá)抑制對細(xì)胞凋亡的促進作用不明顯。二、利用TALEN構(gòu)建FAK基因敲除的SGC-7901細(xì)胞系1.酶切鑒定結(jié)果顯示FAK基因敲除靶點的TALEN左右臂同源重組DNA質(zhì)粒構(gòu)建成功。2.質(zhì)粒共轉(zhuǎn)效率≥60%。3. Puromycin抗性篩選后,有限稀釋法獲得6株細(xì)胞克隆。4.對6株細(xì)胞克隆進行FAK基因組靶點序列測定,證明無FAK-/-SGC-7901克隆。結(jié)論1.FAK表達(dá)抑制可有效引起SMMC-7721、SGC-7901細(xì)胞運動相關(guān)微結(jié)構(gòu)發(fā)育的抑制。這一改變對癌細(xì)胞侵襲、轉(zhuǎn)移等癌癥發(fā)展關(guān)鍵環(huán)節(jié)意義重大。2.FAK表達(dá)抑制與細(xì)胞凋亡的關(guān)系不明顯。這與FAK基因的已知基本功能是相符的。3.本課題對FAK在癌細(xì)胞惡性表型方面的研究結(jié)果再次提示該基因的過表達(dá)對胃癌、肝癌發(fā)展過程的重要作用,提示FAK基因及其產(chǎn)物在胃癌、肝癌的基因診治方面具有作為靶標(biāo)的潛在性。4.以斯丹賽公司提供的TALEN試劑盒構(gòu)建FAK基因敲除的SGC-7901細(xì)胞系未成功。結(jié)合行業(yè)內(nèi)有關(guān)信息,以及本實驗室另一嘗試結(jié)果,說明該公司的該試劑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.

【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R730

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Wanqing Chen;Rongshou Zheng;Hongmei Zeng;Siwei Zhang;Jie He;;Annual report on status of cancer in China, 2011[J];Chinese Journal of Cancer Research;2015年01期

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本文編號：1420089