| 摘要: |
| 目的 构建人蛋白激酶R样内质网激酶(protein kinase R-like ER kinase, PERK)慢病毒载体并包装慢病毒颗粒(lentivirus),探讨成肌细胞C2C12中PERK基因慢病毒修饰对内质网应激介导凋亡的作用。方法 设计并合成人PERK基因的上、下游引物,采用PCR方法从真核质粒pcDNA3.l(-)-PERK中扩增目的基因。鉴定后与慢病毒载体pWPT-GFP进行重组,再将重组慢病毒载体pWPT-GFP-PERK与慢病毒包装质粒pMD2G、pSPAX2共转入293T细胞中,进一步包装形成PERK慢病毒(LV-PERK);收集转染后48 h的细胞上清,体外感染成肌细胞C2C12,观察绿色荧光蛋白(GFP)感染效率,并应用流式细胞仪(FCM)检测内质网应激时重组慢病毒PERK对C2C12细胞增殖凋亡的影响,蛋白质印迹法检测凋亡相关基因Cleaved Caspase-3与Chop蛋白的表达。结果 成功构建和包装了滴度为4.2×108 efu /mL的PERK重组慢病毒。FCM检测结果表明,TM+LV-PERK处理组G1期细胞比例为54.37%,低于TM组(77.91 %)和LV-GFP组(66.41%);TM LV-PERK处理组S期细胞比例为31.36 %,高于TM组(12.14%)和LV-GFP组(16.96%),各组差异均具有统计学意义(P<0.05);此外,TM LV-PERK处理组细胞凋亡率为25.91%,高于TM组(11.79%)和Ad-GFP组(11.24 %),各组差异具有统计学意义(P<0.05)。免疫印迹检测Cleaved Caspase-3和Chop蛋白的表达与FCM结果一致。结论 成功构建和包装LV-PERK重组慢病毒颗粒,在内质网应激时,LV-PERK慢病毒可促进C2C12细胞的增殖和凋亡。 |
| 关键词: PERK 慢病毒 成肌细胞 内质网应激 细胞凋亡 |
| DOI：10.3724/SP.J.1008.2014.01183 |
| 投稿时间：2014-03-19修订日期：2014-05-16 |
| 基金项目:国家自然科学基金(81371928,81171697), 教育部新世纪优秀人才支持计划(NCET-12-1090), 人力资源和社会保障部留学回国人员择优资助项目(2011-235). |
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| Influence of lentivirus modification of human PERK gene on endoplasmic reticulum stress-mediated apoptosis |
| ZHANG Peng,XIA Fei,LI Mei-ling,HAN Xiao-feng,GUO Feng-jin* |
(1. Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, China;
2. Platform of Development Biology and Model Animal, Chongqing Medical University, Chongqing 400016, China
*Corresponding authors) |
| Abstract: |
| Objective To construct the lentivirus vector targeting human protein kinase R-like endoplasmic reticulum kinase (PERK) gene, and to study the effect of lentivirus modification of human PERK gene on endoplasmic reticulum(ER) stress-mediated apoptosis in C2C12 cells. Methods Primer pairs of PERK gene were designed and synthesized. Human PERK gene was amplified by PCR from the expression plasmid pCDNA3.l (-)-PERK and was cloned into pWPT-GFP lentivirus vector. Then the recombinant PERK lentivirus were produced in 293T cells following co-transfection between pWPT-GFP-PERK and the lentivirus packaging plasmids pMD2G, pSPAX2. The supernatants were collected 48 h after transfection and myoblast C2C12 were infected in vitro. Infection efficiency of green fluorescent protein (GFP) was observed. The effect of recombinant lentivirus PERK on proliferation and apoptosis in C2C12 cells was detected by flow cytometry (FCM) in ER stress. Western blotting analysis was used to examine the expression of apoptosis-related proteins Cleaved Caspase-3 and Chop. Results The recombinant lentivirus PERK was successfully constructed, with a titer of 4.2×108 efu/mL. FCM analysis showed that under ER stress condition, the ratio of G1 phase C2C12 cells in the TM+LV-PERK group (54.37 %) was significantly lower than those in the TM group (77.91%) and LV-GFP (66.41%, P<0.05); the ratio of S phase C2C12 cells in the TM LV-PERK group (31.36%) was significantly higher than those in the TM group (12.14%) and LV-GFP (16.96%, P<0.05). The apoptosis rate in the TM LV-PERK group(25.91%) was significantly higher than those in the TM group (11.79%) and Ad-GFP group (11.24%, P<0.05). Western blotting analysis showed that the expression of Cleaved Caspase-3 and Chop protein was consistent with the results of FCM. Conclusion We have successfully constructed and packaged recombinant lentivirus PERK. Under ER stress condition, LV-PERK can promote the proliferation and apoptosis in C2C12 cells. |
| Key words: PERK lentivirus myoblasts endoplasmic reticulum apoptosis |
