本文選題：巨噬細胞集落刺激因子(M-CSF)　切入點：核內(nèi)定位載體pCMV/myc/nuc　出處：《南華大學》2006年碩士論文

【摘要】：目的：構(gòu)建GFP-M-CSF融和蛋白真核表達載體pEGFP／M-CSF，分析M-CSF在NIH3T3細胞內(nèi)的定位分布；構(gòu)建M-CSF細胞核內(nèi)定位表達載體pCMV／M-CSF，，建立M-CSF核內(nèi)穩(wěn)定表達細胞系，探討核內(nèi)M-CSF對NIH3T3細胞運動和細胞骨架的影響。 方法：采取PCR的方法擴增人M-CSF活性片段，將其插入真核表達載體pEGFP-C1中，PCR、酶切分析及測序鑒定篩選陽性重組體pEGFP／M-CSF，經(jīng)脂質(zhì)體瞬時轉(zhuǎn)染NIH3T3細胞，用熒光顯微鏡觀察GFP-M-CSF融和蛋白在NIH3T3細胞內(nèi)的表達分布，以確定在未加信號肽的條件下M-CSF在NIH3T3細胞中的定位。然后將M-CSF片段插入核內(nèi)真核表達載體pCMV／myc／nuc，強制性把M-CSF引入細胞核內(nèi)，通過PCR、酶切分析及測序鑒定篩選陽性重組體pCMV／M-CSF，脂質(zhì)體介導轉(zhuǎn)染NIH3T3細胞，經(jīng)G418篩選并擴增陽性克隆后，用RT-PCR、免疫細胞化學及western blot鑒定其在真核細胞中的表達及定位分布，用細胞劃痕實驗和考馬斯亮藍染色細胞微絲測定M-CSF進入細胞核后對細胞運動能力及細胞骨架的影響。 結(jié)果：PCR擴增和限制性雙酶切重組體pEGFP／M-CSF和pCMV／M-CSF后，瓊脂糖電泳結(jié)果顯示插入片段為1400bp左右，與預期M-CSF分子大小相當；DNA測序分析表明插入質(zhì)粒的M-CSF無讀碼框移位，并與來源序列一致。熒光顯微鏡下見到GFP-M-CSF融和蛋白表達定位于NIH3T3細胞質(zhì)和細胞核。RT-PCR和Westorn blot結(jié)果顯示轉(zhuǎn)染pCMV／M-CSF的NIH3T3細胞能穩(wěn)定表達M-CSF mRNA與M-CSF蛋白；免疫細胞化學結(jié)果顯示表達的M-CSF定位于NIH3T3細胞核。細胞劃痕實驗顯示轉(zhuǎn)染pCMV／M-CSF的NIH3T3細胞有較強的運動能力，考馬斯亮藍染色結(jié)果顯示轉(zhuǎn)染pCMV／M-CSF的NIH3T3細
[Abstract]:Aim: to construct eukaryotic expression vector pEGFP / M-CSF of GFP-M-CSF fusion protein, analyze the localization and distribution of M-CSF in NIH3T3 cells, construct M-CSF expression vector pCMV / M-CSF in nucleus, and establish a stable expression cell line of M-CSF nucleus. To investigate the effects of nuclear M-CSF on NIH3T3 cell motility and cytoskeleton. Methods: the active fragment of human M-CSF was amplified by PCR and inserted into the eukaryotic expression vector pEGFP-C1. The positive recombinant pEGFP / M-CSF was screened by restriction endonuclease analysis and sequencing. NIH3T3 cells were transiently transfected with liposome. The expression and distribution of GFP-M-CSF fusion protein in NIH3T3 cells were observed by fluorescence microscope to determine the location of M-CSF in NIH3T3 cells without signal peptide. Then the M-CSF fragment was inserted into the eukaryotic expression vector pCMV / myc / nuclear, and M-CSF was forcibly introduced into the nucleus. NIH3T3 cells were transfected by liposome mediated by pCMV / M-CSF by PCR, restriction endonuclease analysis and sequencing. After G418 screening and amplification of positive clones, the expression and localization of pCMV / M-CSF in eukaryotic cells were identified by RT-PCR, immunocytochemistry and western blot. The effects of M-CSF on cell motility and cytoskeleton were measured by cell scratch test and Coomassie brilliant blue staining. Results after PCR amplification and restriction digesting the recombinant pEGFP/M-CSF and pCMV/M-CSF, the agarose electrophoresis results showed that the inserted fragment was about 1400bp, and the sequence analysis showed that the M-CSF of the inserted plasmid was translocated without reading frame, which was equivalent to the expected size of M-CSF. The results of RT-PCR and Westorn blot showed that the NIH3T3 cells transfected with pCMV/M-CSF could stably express M-CSF mRNA and M-CSF protein. The results of immunocytochemistry showed that the expressed M-CSF was located in the nucleus of NIH3T3. The cell scratch assay showed that the NIH3T3 cells transfected with pCMV/M-CSF had strong motor ability. Coomassie brilliant blue staining showed that the NIH3T3 transfected with pCMV/M-CSF was fine.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R346

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1 張曉紅;M-CSF在NIH3T3細胞核內(nèi)的表達及其對細胞運動的影響[D];南華大學;2006年

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