本文關(guān)鍵詞：TGF-β1、TIMP-1、TIMP-2 RNAi質(zhì)粒的構(gòu)建及鑒定　出處：《重慶醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： TFG-β1 TIMP-1 TIMP-2 RNA干擾 肝星狀細(xì)胞-T6(HSC-T6)

【摘要】： 目的:利用RNA干擾技術(shù),分別以TGF-β1、TIMP-1和TIMP-2為靶基因,構(gòu)建靶向TGF-β1、TIMP-1和TIMP-2基因的RNA干擾真核表達(dá)載體并進行鑒定分析及體外觀察干擾效率。 方法:針對目的基因TGF-β1、TIMP-1和TIMP-2分別設(shè)計合成編碼目的基因的反向重復(fù)序列,中間間隔9個核苷酸序列,經(jīng)退火形成互補雙鏈,通過定向克隆至質(zhì)粒pGenesil-1,構(gòu)建siRNA真核表達(dá)載體。轉(zhuǎn)化JM109大腸桿菌,提取質(zhì)粒進行酶切鑒定和測序分析。并體外轉(zhuǎn)染培養(yǎng)的HSC-T6細(xì)胞,觀察轉(zhuǎn)染效率及對目的基因的抑制效率。 結(jié)果:酶切證實目的DNA定向克隆至載體上,測序分析結(jié)果與目的序列相同。熒光顯微鏡下觀察,可見大量帶熒光的細(xì)胞。RT-PCR結(jié)果顯示,六個特異性的siRNA表達(dá)載體均有抑制效果,其中psi-TGF-Β1-1、psi-TIMP1-1、psi-TIMP2-2抑制效率較高。 結(jié)論:成功構(gòu)建了針對TGF-β1、TIMP-1和TIMP-2基因的RNA干擾真核表達(dá)載體;將重組質(zhì)粒成功轉(zhuǎn)染入體外培養(yǎng)的HSC-T6細(xì)胞,并且重組質(zhì)粒顯著抑制了目的基因的表達(dá)。
[Abstract]:Objective: to construct TGF- 尾 1 targeting TGF- 尾 1 by using RNA interference technique and targeting TGF- 尾 1 TIMP-1 and TIMP-2 as target genes, respectively. The RNA interference eukaryotic expression vector of TIMP-1 and TIMP-2 gene was identified and analyzed and the interference efficiency was observed in vitro. Methods: the reverse repeats of the target gene TGF- 尾 1 TIMP-1 and TIMP-2 encoding the target gene were designed, with an intermediate interval of 9 nucleotide sequences. SiRNA eukaryotic expression vector was constructed by directional cloning into plasmid pGenesil-1 and transformed into JM109 Escherichia coli. The plasmid was identified by restriction endonuclease digestion and sequenced. The transfection efficiency and inhibition efficiency of the target gene were observed by transfection of HSC-T6 cells in vitro. Results: the target DNA was cloned into the vector by restriction endonuclease digestion, and the sequencing result was the same as the target sequence. Under fluorescence microscope, a large number of fluorescent cells. RT-PCR results showed. Among the six specific siRNA expression vectors, psi-TGF- 尾 1-1psi-TIMP1-1psi-TIMP2-2 showed higher inhibition efficiency. Conclusion: the RNA interference eukaryotic expression vector targeting TIMP-1 and TIMP-2 genes of TGF- 尾 1 was successfully constructed. The recombinant plasmid was successfully transfected into HSC-T6 cells in vitro, and the expression of the target gene was significantly inhibited by the recombinant plasmid.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R346

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