HSP70-2基因慢病毒载体的构建及其对鼻咽癌6-10B细胞增殖的影响
孙伟伟1，唐发清2，李名薇1，邹飞雁1
基金项目：国家自然科学基金（81071718） 作者简介：孙伟伟（1988年），女，发育调控与肿瘤发生 通信联系人：邹飞雁（1963年），女，副教授，发育调控与肿瘤发生.

（1. 暨南大学生命科学技术学院发育与再生生物学系，广东广州 510632；2. 暨南大学附属珠海医院检验科和实验中心，广东珠海 519000）
摘要：背景与目的 HSP70-2在鼻咽癌6-10B细胞中过表达及功能研究国内外未见报道。本研究构建HSP70-2基因慢病毒载体及其转染6-10B细胞，观察HSP70-2基因对6-10B细胞生长增殖的影响。方法 利用慢病毒技术，使目的基因HSP70-2与pLenti/V5-DEST载体连接，经转化筛选鉴定后得到pLV-HSP70-2-IRES/eGFP-Neo慢病毒载体，包装慢病毒并测定病毒滴度，感染6-10B细胞，经G418筛选后得到高表达HSP70-2的6-10B稳转细胞株，即6-10B-HSP70-2/eGFP细胞。同时设两对照组，空载对照组为6-10B空载慢病毒稳转细胞株，即6-10B-blank/eGFP细胞；空白对照组为6-10B未感染细胞株，即6-10B细胞。应用Western blotting检测HSP70-2的蛋白表达水平；CCK8检测细胞增殖情况；流式细胞仪分析细胞增殖指数（PI）、S期细胞比例（SPF）和凋亡百分比。结果 HSP70-2蛋白表达，培养24h，6-10B-HSP70-2/eGFP细胞与6-10B-blank/eGFP细胞或6-10B细胞比较，升高4.15±0.68%（P<0.01）或4.12±0.65%（P<0.01）。细胞增殖率，培养24h、48h和72h，6-10B-HSP70-2/eGFP细胞与6-10B-blank/eGFP细胞比较，分别升高29.18±0.014%（P<0.01）、81.72±0.124%（P<0.001）和41.84±0.004%（P<0.001）；与6-10B细胞比较，分别升高25.69±0.031%20 （P<0.01）、84.53±0.060%（P<0.001）和45.28±0.044%（P<0.001）。PI和SPF，培养24h，6-10B-HSP70-2/eGFP细胞与6-10B-blank/eGFP细胞比较，分别增加73.31±5.882%（P<0.001）和71.09±7.495%（P<0.01）；与6-10B细胞比较，分别增加72.60±1.300%（P<0.001）和69.29±13.42%（P<0.01）。细胞凋亡百分比，各组间比较无明显变化（P>0.05）。结论 成功构建pLV-HSP70-2-IRES/eGFP-Neo慢病毒载体以及6-10B-HSP70-2/eGFP细胞和25 6-10B-blank/eGFP细胞，初步观察到HSP70-2基因可能有促进鼻咽癌6-10B细胞生长与增殖作用。
关键词：慢病毒；HSP70-2；6-10B细胞
中图分类号：R739.62

Construction of HSP70-2 gene lentiviral vector and Its Effects on 6-10B Cells’ Growth
SUN weiwei1, TANG faqing2, LI mingwei1, ZOU feiyan1
(1. Department of Developmental and Regenerative Biology,College of Life Science and Technology, Jinan University,Guangdong Guangzhou 510632; 35 2. Clinical Laboratory and Medical Research Center,the Affiliated Zhuhai Hospital,Jinan University,Guangzhou Zhuhai 519000)
Abstract: Background and Objective Over expression and function study of HSP70-2 in nasopharyngeal carcinoma cell lines 6-10B has not been reported at home and abroad. In this study, HSP70-2 gene lentiviral vector and high expression of HSP70-2 in the stably transfected  cell lines 6-10B were constructed. The effect of HSP70-2 gene on 6-10B cell growth and proliferation was observed. Methods The target gene of HSP70-2 was connected to the pLenti/V5-DEST vector by lentiviral technology. After the screening and identification, the product was transformed into pLV-HSP70-2-IRES/eGFP-Neo lentiviral vector. The lentivirus was packed and measured virus titer, and then the lentivirus infected the 6-10B cells. It could get high expression of HSP70-2 in the stably transfected cell lines 6-10B by G418 screening, i.e.6-10-HSP70-2/eGFP cells. While the experiment set two groups: the blank control group was the stably transfected 6-10B cell lines with the empty vector infected, i.e.6-10B-blank/eGFP cells and the no-treatment control group was the uninfected 6-10B cell lines, i.e.6-10B cells. HSP70-2 protein expression of the three groups was analyzed by western blotting; cell proliferation of the three groups was  detected by CCK8; cell proliferation index (PI), S-phase fraction (SPF) and cell apoptosis percentage of the three groups were analyzed by flow cytometry. Results Western blotting assay showed that 6-10B-HSP70-2/eGFP cells compared with 6-10B-blank/eGFP cells or 6-10B cells, HSP70-2 protein expression was respectively increased by 4.15 ± 0.65% (P<0.01) or 4.12 ± 0.68% (P <0.01) after 24h of culture. 6-10B-HSP70-2/eGFP cells compared with 6-10B-blank/eGFP cells,  the proliferation rates were respectively increased by 29.18±0.014% (P<0.01), 81.72±0.124% (P <0.001) and 41.84 ± 0.004% (P <0.001) after 24h, 48h or 72h of culture; or compared with 6-10B cells, the proliferation rates were respectively increased by 25.69 ± 0.031% (P <0.01), 84.53 ± 0.060% (P <0.001) and 45.28 ± 0.044% (P <0.001) after 24h, 48h or 72h of culture. 6-10B- HSP70-2/eGFP cells compared with 6-10B-blank/eGFP cells, PI and SPF were respectively  increased by 73.31 ± 5.882% (P <0.001) and 71.09 ± 7.495% (P <0.01) after 24h of culture; or compared with 6-10B cells, PI and SPF were respectively increased by 72.60 ± 1.300% (P <0.001) and 69.29 ± 13.42% (P <0.01) after 24h of culture. Cell apoptosis percentage was no significantly changed among the three groups (P> 0.05). Conclusion pLV-HSP70-2-IRES/eGFP-Neo lentiviral vector, 6-10B-HSP70-2/eGFP cells and 6-10B-blank/eGFP cells were successfully constructed.  There might be initially observed that HSP70-2 gene could promote the growth and proliferation of nasopharyngeal carcinoma cell lines 6-10B.
Key words: HSP70-2; Lentivirus; 6-10B cells