慢病毒介导SiRNA沉默H22肝癌细胞TLR4基因对肝癌细胞生物特性的影响

发布时间：2018-07-06 07:52

本文选题：TLR4 + 慢病毒载体　；参考：《昆明医科大学》2016年硕士论文

【摘要】：[目的]构建TLR4基因沉默siRNA慢病毒载体并鉴定；培养H22肝癌细胞后用慢病毒载体进行转染,实时荧光定量PCR和Western blot检测肝癌细胞目的基因的表达。采用MTT法检测H122细胞增殖情况。从而研究H22肝癌细胞生物学特性的改变。[方法](一)TLR4基因沉默siRNA慢病毒载体构建：应用siRNA设计软件按照siRNA设计原则设计靶序列,分别与u6载体连接,构建重组慢病毒载体,(二)转染感受态细胞,提取质粒,酶切鉴定,测序,再进行慢病毒颗粒的包装。(三)培养H122肝癌细胞并感染慢病毒,收集感染肝癌细胞进行实时荧光定量PCR分析,筛选出具有较高效的干扰率的设计序列。(四)West-blotting检测：选取RNAi组、空载体转染组及对照组细胞,分别提取总蛋白,经电泳分离,显影,灰度值计算比较等步骤,检测TLR4、MyD88及NF-kB的表达并比较各组差异。(五)将RNAi组和对照组细胞按一定浓度铺板,设置空白孔,连续3天使用MTT法测量其OD值,最终利用各组每天平均OD减去空白孔OD所得值绘制生产曲线,并比较其差异。[结果](一)通过shRNA设计软件设计TLR4基因沉默shRNA序列,并插入慢病毒载体中,构建了3条TLR4基因沉默慢病毒序列和1条含空载体序列的对照慢病毒载体,将重组慢病毒干扰载体进行质粒小量提取,然后酶切、电泳、测序,将二者逐一比对,可发现设计基因序列与测序序列完全相符,慢病毒载体构建成功。(二)采用不同滴度的慢病毒颗粒分别转染H22肝癌细胞,可观察到不同滴度转染后细胞的生长情况,MOI=10时通过光学显微镜观察,H22肝癌细胞形态良好,荧光显微镜下观察,存在大量荧光,即荧光率较高。(三)通过实时荧光定量PCR检测3条shRNA分别感染H22肝癌细胞所表达的mRNA水平,可检测到shRNA#1具有最高的干扰效率。(四)经West-biotting检测提示各蛋白各组表达量具有差异,差异有统计学意义,且RNAi组表达最少。(五)RNAi组和对照组增殖有统计学差异,且RNAi组增殖减弱。[结论](一)通过TLR4基因与u6慢病毒载体连接并包装生产成功构建了TLR4基因沉默siRNA慢病毒载体。(二)测定了最适MOI值,并感染H22肝癌细胞。(三)经实时荧光定量PCR测定显示shRNA#1能高效沉默TLR4基因。(四)siRNA'慢病毒载体能干扰肝癌细胞TLR4基因使TLR4、MyD88及NF-kB表达降低。(五)沉默肝癌细胞的TLR4基因后,可以抑制其增殖。
[Abstract]:[objective] to construct and identify the lentivirus vector silencing siRNA of TLR4 gene, culture H22 hepatoma cell line and transfect it with lentivirus vector, and detect the expression of the target gene by real-time fluorescence quantitative PCR and Western blot. The proliferation of H122 cells was detected by MTT assay. To study the changes of biological characteristics of H 22 hepatoma cells. [methods] (1) TLR4 gene silencing siRNA lentivirus vector construction: siRNA design software was used to design target sequence according to the principle of siRNA design, ligated with U6 vector respectively, constructed recombinant lentivirus vector, (2) transfected into receptive cells and extracted plasmid. Enzyme digestion, sequencing and packaging of lentivirus particles. (3) H122 hepatoma cells were cultured and infected with lentivirus. The infected hepatoma cells were collected and analyzed by real-time fluorescence quantitative PCR, and the design sequence with high interference rate was screened. (4) West-blotting: the total protein was extracted from RNAi group, empty vector transfection group and control group, respectively. The expression of TLR4, MyD88 and NF-kB was detected by electrophoresis separation, development and comparison of gray value. (5) the cells of RNAi group and control group were set up blank hole according to a certain concentration, and the OD value was measured by MTT method for 3 days. Finally, the production curve was drawn with the average OD value of each group minus the OD value of blank hole, and the difference was compared. [results] (1) the silencing shRNA sequence of TLR4 gene was designed by shRNA design software, and inserted into lentivirus vector, and three LLR4 gene silencing lentivirus sequences and one control lentivirus vector containing empty vector sequence were constructed. The recombinant lentivirus interference vector was extracted in a small amount, then digested by enzyme, electrophoretic, sequenced, and compared one by one. It was found that the designed gene sequence was completely consistent with the sequencing sequence, and the lentivirus vector was successfully constructed. (2) different titers of lentivirus particles were used to transfect H22 hepatoma cells, and the growth of H22 hepatoma cells after transfection with different titers was observed. The morphology of H22 hepatoma cells was observed by optical microscope at 10:00 and observed under fluorescence microscope. There is a large amount of fluorescence, that is, high fluorescence rate. (3) the expression of mRNA in H22 hepatoma cells infected with three shRNAs was detected by real-time fluorescent quantitative PCR, and #shRNA1 had the highest interference efficiency. (4) the results of West-biotting analysis showed that the protein expression in each group was different, the difference was statistically significant, and the expression of RNAi group was the least. (5) the proliferation of RNAi group was significantly different from that of control group, and the proliferation of RNAi group was weakened. [conclusion] (1) TLR4 gene silencing siRNA lentivirus vector was successfully constructed by linking TLR4 gene with U6 lentivirus vector and packaging production. (2) the optimal moi was measured and infected with H 22 hepatoma cells. (3) real-time fluorescent quantitative PCR assay showed that shRNA #1 could effectively silence TLR4 gene. (4) siRNA' lentivirus could interfere with the expression of TLR4 gene and decrease the expression of TLR4 MyD88 and NF-kB. (5) silencing TLR 4 gene can inhibit the proliferation of hepatoma cells.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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