發(fā)布時間：2018-06-16 02:22

  本文選題：SARI + 病毒感染��； 參考：《中國實驗血液學雜志》2015年03期

【摘要】：目的:通過構(gòu)建SARI慢病毒表達載體,探討SARI基因過表達對K562細胞生物學功能的影響。方法:采用RT-PCR方法獲得目的基因并重組p LOV.CMV.GFP質(zhì)粒,構(gòu)建pLOV.CMV.GFP-SARI表達載體。通過測序鑒定、病毒包裝和滴度測定后,獲得陽性病毒顆粒,并以病毒顆粒感染K562細胞系。采用Q-PCR及Western blot方法驗證感染后K562細胞SARI基因轉(zhuǎn)錄和蛋白水平的表達情況,同時采用CCK-8法檢測K562細胞的增殖情況,以流式細胞術(shù)分析細胞凋亡和細胞周期變化。結(jié)果:利用RT-PCR方法獲得SARI基因并成功構(gòu)建了含SARI基因的慢病毒表達載體,從分子及蛋白水平驗證了其在感染后K562細胞中的高表達;與空白組和感染空載體的Mock組相比,過表達SARI載體組的細胞增殖顯著受抑、細胞凋亡率增加,而細胞周期無顯著變化。結(jié)論:SARI基因過表達可抑制K562細胞的增殖并促進其凋亡,提示誘導SARI基因過表達可能對于抑制CML發(fā)生發(fā)展具有重要的作用。
[Abstract]:Aim: to investigate the effect of sari gene overexpression on the biological function of K562 cells by constructing the expression vector of sali lentivirus. Methods: the target gene was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and the pLOV.CMV.GFP plasmid was recombined to construct the expression vector pLOV.CMV.GFP-SARI. Positive viral particles were obtained by sequencing, packaging and titer determination. K562 cell line was infected with viral particles. Q-PCR and Western blot were used to detect the expression of SARI gene and protein in K562 cells. The proliferation of K562 cells was detected by CCK-8 method. Apoptosis and cell cycle were analyzed by flow cytometry. Results: Sari gene was obtained by RT-PCR and the lentivirus expression vector containing sari gene was successfully constructed. The high expression of SARI gene in K562 cells was verified at molecular and protein levels, which was compared with that of blank group and Mock group infected with empty vector. In the overexpression of sari vector, the cell proliferation was significantly inhibited, the apoptosis rate was increased, but the cell cycle was not changed significantly. Conclusion the overexpression of the Sari gene can inhibit the proliferation and promote the apoptosis of K562 cells, suggesting that inducing the overexpression of the sari gene may play an important role in inhibiting the occurrence and development of CML.
【作者單位】： 華中科技大學同濟醫(yī)學院附屬協(xié)和醫(yī)院檢驗科;華中科技大學同濟醫(yī)學院附屬協(xié)和醫(yī)院干細胞中心;
【基金】：國家自然科學基金(81100356) 湖北省自然科學基金(2014CFB466)
【分類號】：R329.2

【參考文獻】

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本文編號：2024788