中性粒细胞弹性蛋白酶的过表达促进K562细胞增殖并抑制其凋亡  

Over-expression of neutrophil elastase promotes proliferation and inhibits apoptosis in K562 cells

作　　者：蒋开玲[1,2] 马鹏鹏[2] 阳小群[2] 钟梁[2] 王慧[2] 朱新瑜[2] 刘北忠[1,2] 

机构地区：[1]重庆医科大学附属永川医院中心实验室,重庆402160 [2]重庆医科大学临床检验诊断学教育部重点实验室,重庆400016

出　　处：《细胞与分子免疫学杂志》2015年第2期159-162,167,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81171658);重庆市自然科学基金计划重点项目(2011BA5037)

摘　　要：目的利用Ad Easy系统构建携带人中性粒细胞弹性蛋白酶(NE)基因的重组腺病毒表达质粒,并感染K562细胞,观察其对K562细胞增殖和凋亡的影响。方法以急性早幼粒细胞白血病(APL)细胞株HL-60的RNA为模板,经反转录PCR得到NE基因的编码区并克隆入穿梭载体p Ad Track-CMV,得到p Ad-NE,经HindⅢ、EcoRⅤ酶切鉴定和测序正确后,将其转化入p Ad Easy-1-BJ5183进行同源重组,得到重组子Ad-NE,经PacⅠ酶切线性化后转染AD293细胞进行包装。14 d之后收获原代病毒,经5轮扩增后,测定病毒滴度,进行PCR鉴定。感染K562细胞后,荧光显微镜初步观察感染效率,同时用流式细胞术检测感染效率;Western blot法进一步鉴定NE是否表达上调;CCK-8法测定细胞增殖情况;流式细胞术测定细胞凋亡和细胞周期变化。结果酶切鉴定及测序结果表明重组穿梭质粒p Ad-NE构建成功;酶切鉴定显示同源重组成功,得到重组子Ad-NE;荧光显微镜表明病毒包装成功;经过5轮扩增后,病毒滴度达到1.64×1012pfu/m L;感染K562细胞后,荧光显微镜观察感染效率达80%左右;Western blot法检测到NE表达上调;CCK-8实验显示NE表达上调后K562细胞增殖能力增强;流式细胞术显示S期细胞明显增多并且细胞凋亡减少。结论过表达NE可促进K562细胞增殖并抑制其凋亡。Objective To establish recombinant adenovirus carrying human neutrophil elastase （NE） gene using AdEasy system, over-express NE in 1（562 cell line and observe the effects of NE on K562 cell proliferation and apoptosis. Methods NE gene was amplified with RNA extracted from acute premyelocytic leukemia （APL） HL-60 cells as a template using reverse transcription-PCR. The coding sequence was cloned into shuttle plasmid pAdTrack-CMV to obtain the recombinant plasmid named pAd-NE. After digested with HindⅢ and EcoRⅤ and sequenced, the pAd-NE was transformed to competent E. co//BJ5183 containing adenovirus backbone plasmid pAdEasy-1. The obtained recombinant adenovirus plasmid Ad-NE was digested with Pac Ⅰ and transfected into AD293 cells for packaging. Fourteen days later, primary recombinant adenovirus Ad-NE was harvested, and then subjected to five cycles of amplification, titer determination and PCR identification. K562 cells were infected by the recombinant adenovirus. The infection efficiency was observed under a fluorescence microscope and detected by flow cytometry. Western blotting was used to detect NE expression. The proliferation of K562 cells was detected by CCK-8 assay. Cell cycle and apoptosis was measured by annexin V/PI accompanied by flow cytometry. Results HindⅢ and EcoRⅤ digestion and sequencing suggested that the recombinant vector Ad-NE was successfully constructed. The recombinant plasmid Ad-NE was packaged in AD293 cells as expected. Following five-cycle amplification, the viral titer was up to 1.64 × 10^12 pfu/mL. GFP expression observed by fluorescence microscopy and flow cytometry implied that the infection efficiency of Ad-NE in K562 cells reached about 80%. Western blotting showed that NE expression was up-regulated in K562 cells. CCK-8 assay revealed that the proliferation of K562 cells over-expressing NE was enhanced. Meanwhile, flow cytometry indicated that the K562 cells were arrested in S phase and the apoptosis rate was highly reduced. Conclusion Over-expressed NE

关 键 词：中性粒细胞弹性蛋白酶 重组腺病毒 增殖 凋亡 K562细胞 

分 类 号：R392-33[医药卫生—免疫学] Q279[医药卫生—基础医学]