發(fā)布時(shí)間：2018-07-06 07:52

  本文選題：TLR4 + 慢病毒載體��； 參考：《昆明醫(yī)科大學(xué)》2016年碩士論文

【摘要】：[目的]構(gòu)建TLR4基因沉默siRNA慢病毒載體并鑒定；培養(yǎng)H22肝癌細(xì)胞后用慢病毒載體進(jìn)行轉(zhuǎn)染,實(shí)時(shí)熒光定量PCR和Western blot檢測(cè)肝癌細(xì)胞目的基因的表達(dá)。采用MTT法檢測(cè)H122細(xì)胞增殖情況。從而研究H22肝癌細(xì)胞生物學(xué)特性的改變。[方法](一)TLR4基因沉默siRNA慢病毒載體構(gòu)建：應(yīng)用siRNA設(shè)計(jì)軟件按照siRNA設(shè)計(jì)原則設(shè)計(jì)靶序列,分別與u6載體連接,構(gòu)建重組慢病毒載體,(二)轉(zhuǎn)染感受態(tài)細(xì)胞,提取質(zhì)粒,酶切鑒定,測(cè)序,再進(jìn)行慢病毒顆粒的包裝。(三)培養(yǎng)H122肝癌細(xì)胞并感染慢病毒,收集感染肝癌細(xì)胞進(jìn)行實(shí)時(shí)熒光定量PCR分析,篩選出具有較高效的干擾率的設(shè)計(jì)序列。(四)West-blotting檢測(cè)：選取RNAi組、空載體轉(zhuǎn)染組及對(duì)照組細(xì)胞,分別提取總蛋白,經(jīng)電泳分離,顯影,灰度值計(jì)算比較等步驟,檢測(cè)TLR4、MyD88及NF-kB的表達(dá)并比較各組差異。(五)將RNAi組和對(duì)照組細(xì)胞按一定濃度鋪板,設(shè)置空白孔,連續(xù)3天使用MTT法測(cè)量其OD值,最終利用各組每天平均OD減去空白孔OD所得值繪制生產(chǎn)曲線,并比較其差異。[結(jié)果](一)通過shRNA設(shè)計(jì)軟件設(shè)計(jì)TLR4基因沉默shRNA序列,并插入慢病毒載體中,構(gòu)建了3條TLR4基因沉默慢病毒序列和1條含空載體序列的對(duì)照慢病毒載體,將重組慢病毒干擾載體進(jìn)行質(zhì)粒小量提取,然后酶切、電泳、測(cè)序,將二者逐一比對(duì),可發(fā)現(xiàn)設(shè)計(jì)基因序列與測(cè)序序列完全相符,慢病毒載體構(gòu)建成功。(二)采用不同滴度的慢病毒顆粒分別轉(zhuǎn)染H22肝癌細(xì)胞,可觀察到不同滴度轉(zhuǎn)染后細(xì)胞的生長(zhǎng)情況,MOI=10時(shí)通過光學(xué)顯微鏡觀察,H22肝癌細(xì)胞形態(tài)良好,熒光顯微鏡下觀察,存在大量熒光,即熒光率較高。(三)通過實(shí)時(shí)熒光定量PCR檢測(cè)3條shRNA分別感染H22肝癌細(xì)胞所表達(dá)的mRNA水平,可檢測(cè)到shRNA#1具有最高的干擾效率。(四)經(jīng)West-biotting檢測(cè)提示各蛋白各組表達(dá)量具有差異,差異有統(tǒng)計(jì)學(xué)意義,且RNAi組表達(dá)最少。(五)RNAi組和對(duì)照組增殖有統(tǒng)計(jì)學(xué)差異,且RNAi組增殖減弱。[結(jié)論](一)通過TLR4基因與u6慢病毒載體連接并包裝生產(chǎn)成功構(gòu)建了TLR4基因沉默siRNA慢病毒載體。(二)測(cè)定了最適MOI值,并感染H22肝癌細(xì)胞。(三)經(jīng)實(shí)時(shí)熒光定量PCR測(cè)定顯示shRNA#1能高效沉默TLR4基因。(四)siRNA'慢病毒載體能干擾肝癌細(xì)胞TLR4基因使TLR4、MyD88及NF-kB表達(dá)降低。(五)沉默肝癌細(xì)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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.7

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