本文關鍵詞：核內M-CSF對Cos7細胞增殖的影響及其靶分子的鑒定　出處：《南華大學》2006年碩士論文　論文類型：學位論文

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

【摘要】：目的：建立M-CSF核內穩(wěn)定表達細胞系，探討核內M-CSF對Cos7細胞增殖的影響，鑒定M-CSF核內相互作用靶分子。 方法：用PCR技術擴增M-CSF活性區(qū)，，并插入真核表達質粒pCMV／myc／nuc，構建M-CSF核內定位重組表達質粒pCMV／nuc／M-CSF。經瓊脂糖凝膠電泳、PCR、限制性酶切和測序鑒定重組子后，用脂質體分別將pCMV／nuc／M-CSF、pCMV／myc／nuc和pCMV／nuc／GFP轉染Cos7細胞，經G418篩選并擴增陽性克隆后，用RT-PCR、免疫細胞化學和Western blot證實陽性克隆細胞是否穩(wěn)定表達M-CSF。用細胞計數和MTT法觀察核內M-CSF對細胞增殖的影響，用免疫共沉淀鑒定M-CSF的核內相互作用分子。 結果：瓊脂糖凝膠電泳與限制性酶切分析顯示插入質粒的片段大小與M-CSF分子大小相當，DAN測序分析表明插入質粒的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胞核內，RT-PCR分析顯示pCMV／nuc／M-CSF轉染細胞表達M-CSF mRNA，免疫細胞化學與Western blot顯示M-CSF準確定位于pCMV／nuc／M-CSF轉染細胞核內。細胞計數顯示pCMV／nuc／M-CSF轉染細胞、未轉染的Cos7細胞和pCMV／myc／nuc轉染細胞的倍增時間分別為20.73±0.22h、28.22±0.25h和27.88±0.24h。細胞計數與MTT顯示pCMV／nuc／M-CSF轉染細胞的生長速率比未轉染的Cos7細胞和pCMV／myc／nuc轉染細胞更快。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】：R392

【參考文獻】

相關期刊論文 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誘餌載體的構建和鑒定[J];南華大學學報(醫(yī)學版);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;膜結合型巨噬細胞集落刺激因子介導的內吞及其半衰期[J];科學通報;2000年06期

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

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

本文編號：1409410