發(fā)布時間：2018-01-11 12:00

  本文關(guān)鍵詞：核內(nèi)M-CSF對Cos7細(xì)胞增殖的影響及其靶分子的鑒定　出處：《南華大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 巨噬細(xì)胞集落刺激因子 核定位信號 細(xì)胞增殖 微小染色體維持蛋白7 Cos7細(xì)胞系

【摘要】：目的：建立M-CSF核內(nèi)穩(wěn)定表達(dá)細(xì)胞系，探討核內(nèi)M-CSF對Cos7細(xì)胞增殖的影響，鑒定M-CSF核內(nèi)相互作用靶分子。 方法：用PCR技術(shù)擴(kuò)增M-CSF活性區(qū)，，并插入真核表達(dá)質(zhì)粒pCMV／myc／nuc，構(gòu)建M-CSF核內(nèi)定位重組表達(dá)質(zhì)粒pCMV／nuc／M-CSF。經(jīng)瓊脂糖凝膠電泳、PCR、限制性酶切和測序鑒定重組子后，用脂質(zhì)體分別將pCMV／nuc／M-CSF、pCMV／myc／nuc和pCMV／nuc／GFP轉(zhuǎn)染Cos7細(xì)胞，經(jīng)G418篩選并擴(kuò)增陽性克隆后，用RT-PCR、免疫細(xì)胞化學(xué)和Western blot證實(shí)陽性克隆細(xì)胞是否穩(wěn)定表達(dá)M-CSF。用細(xì)胞計(jì)數(shù)和MTT法觀察核內(nèi)M-CSF對細(xì)胞增殖的影響，用免疫共沉淀鑒定M-CSF的核內(nèi)相互作用分子。 結(jié)果：瓊脂糖凝膠電泳與限制性酶切分析顯示插入質(zhì)粒的片段大小與M-CSF分子大小相當(dāng)，DAN測序分析表明插入質(zhì)粒的M-CSF無讀碼框移位和突變。熒光倒置顯微鏡觀察綠色熒光只定位于細(xì)胞核內(nèi)，RT-PCR分析顯示pCMV／nuc／M-CSF轉(zhuǎn)染細(xì)胞表達(dá)M-CSF mRNA，免疫細(xì)胞化學(xué)與Western blot顯示M-CSF準(zhǔn)確定位于pCMV／nuc／M-CSF轉(zhuǎn)染細(xì)胞核內(nèi)。細(xì)胞計(jì)數(shù)顯示pCMV／nuc／M-CSF轉(zhuǎn)染細(xì)胞、未轉(zhuǎn)染的Cos7細(xì)胞和pCMV／myc／nuc轉(zhuǎn)染細(xì)胞的倍增時間分別為20.73±0.22h、28.22±0.25h和27.88±0.24h。細(xì)胞計(jì)數(shù)與MTT顯示pCMV／nuc／M-CSF轉(zhuǎn)染細(xì)胞的生長速率比未轉(zhuǎn)染的Cos7細(xì)胞和pCMV／myc／nuc轉(zhuǎn)染細(xì)胞更快。用免疫共沉淀分析表明MCM7能被抗M-CSF抗體沉淀。
[Abstract]:Objective: to establish a stable expression cell line in M-CSF nucleus, to explore the effect of M-CSF on the proliferation of Cos7 cells, and to identify the interaction target molecules in the M-CSF nucleus.
Methods: the amplification of M-CSF activity by PCR, and inserted into the eukaryotic expression plasmid pCMV / myc / NUC, construction of M-CSF nuclear localization of recombinant expression plasmid pCMV / NUC / M-CSF. by agarose gel electrophoresis, PCR, restriction enzyme digestion and sequencing after recombinant plasmid by liposome, respectively pCMV / NUC / M-CSF. PCMV / myc / NUC and pCMV / NUC / GFP was transfected into Cos7 cells, RT-PCR were screened by G418 and amplified after positive clones, immunocytochemistry and Western blot confirmed positive clone cells with stable expression of M-CSF. by cell counting and MTT method to observe the effect of nuclear M-CSF on cell proliferation, nuclear interactions, molecular identification of M-CSF by co immunoprecipitation.
Results: agarose gel electrophoresis and restriction analysis showed that a fragment size and the molecular size of the M-CSF is inserted into the plasmid DAN and inserted into the plasmid M-CSF sequencing analysis showed that the open reading frame shift mutation. And the fluorescence microscope observation of green fluorescence only localized in the nucleus, RT-PCR analysis showed that pCMV / NUC / M-CSF transfected cells expressing M-CSF mRNA Western, immunocytochemistry and blot showed that M-CSF is accurately located in the pCMV / NUC / M-CSF was transfected into cell nucleus. Cells show that pCMV / NUC / M-CSF transfected cells, the doubling time of untransfected Cos7 cells and pCMV / myc / NUC transfected cells were 20.73 + 0.22h, 28.22 + 0.25h and 27.88 + 0.24h. cell count and MTT showed that the growth rate of pCMV / NUC / M-CSF transfected cells faster than non transfected Cos7 cells and pCMV / myc / NUC transfected cells. The results show that MCM7 can be used in CO immunoprecipitation Anti M-CSF antibody precipitation.

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 李劍敏,雷小勇,孫文清,張曉紅,唐圣松;用于酵母雙雜交的M-CSF誘餌載體的構(gòu)建和鑒定[J];南華大學(xué)學(xué)報(醫(yī)學(xué)版);2005年02期

2 唐圣松,劉漢芝,陳桂彬,饒青,鄭國光,耿以琪,鄭德先,吳克復(fù);膜結(jié)合型巨噬細(xì)胞集落刺激因子介導(dǎo)的內(nèi)吞及其半衰期[J];科學(xué)通報;2000年06期

3 吳克復(fù);M-CSF及其受體的生物多樣性和復(fù)雜性觀[J];科學(xué)通報;2000年07期

4 唐圣松,杜喜平,陳桂彬,饒青,耿以琪,吳克復(fù);異型巨噬細(xì)胞集落刺激因子在人白血病細(xì)胞系中的表達(dá)及性質(zhì)[J];中華病理學(xué)雜志;2000年02期

本文編號：1409410