丙型肝炎病毒刺激小鼠小胶质细胞后Toll样受体9mRNA的表达及肿瘤坏死因子α、白细胞介素6的分泌  

TLR9 mRNA expression and tumor necrosis factor-alpha/interleukin-6 secretion in murine microglia by hepatitis C virus stimulation

作　　者：胡坤[1] 王国玮[1] 王振海[2] 

机构地区：[1]宁夏医科大学,银川750004 [2]宁夏医科大学附属医院神经内科,宁夏颅脑疾病重点实验室-省部共建国家级重点实验室培育基地

出　　处：《中华医学杂志》2011年第15期1070-1074,共5页National Medical Journal of China

基　　金：国家自然科学基金面上项目（30760067）;教育部春晖计划（Z2008-1-75002）;宁夏医科大学＂博士学位建设学科＂开放课题（KF2010-34）

摘　　要：了解在体外培养条件下丙型肝炎病毒（HCV）刺激小鼠小胶质细胞（BV2）后Toll样受体9 mRNA（TLR9 mRNA）的表达变化及肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）分泌的变化,为进一步探讨TLR9参与小胶质细胞抗HCV的免疫机制提供理论基础.方法 体外培养BV2细胞,随机分为HCV阳性血清组、正常人血清组、空白对照组,每组各20份细胞样本.分别在4、12、20、36 h收集细胞和上清液,以逆转录PCR（RT-PCR）法观察不同时间点TLR9 mRNA的表达,ELISA法检测不同时间点培养上清中TNF-α、IL-6的含量,并用SPSS 13.0统计软件包进行统计学分析.结果 （1）RT-PCR结果显示各组BV2细胞均有TLR9 mRNA的表达,各时间点HCV阳性血清组中TLR9mRNA的表达（0.85±0.11、0.55±0.08、0.58±0.09、0.61±0.07）高于正常人血清组、空白对照组,差异有统计学意义（F=17.258,P＜0.05＝,且HCV阳性血清刺激后4 h的TLR9 mRNA表达量（0.85±0.11）最高,较其他时间点差异有统计学意义（F=7.427,P＜0.05＝;正常人血清组和空白对照组差异无统计学意义（P＞0.05）;12、20、36 h之间差异无统计学意义（P＞0.05）.（2）各时间点HCV阳性血清组TNF-α[（926±133）pg/ml、（327±59）pg/ml、（367±69）pg/ml、（354±71）pg/ml]、IL-6[（125±34）pg/ml、（84±20）pg/ml、（82±24）pg/ml、（87±18）pg/ml]含量高于正常人血清组、空白对照组（F=18.263～27.469,P＜0.05＝;且HCV阳性血清组TLR9 mRNA的表达量与TNF-α（r= 0.425,P〈0.01）、IL-6（r=0.489,P〈0.01）含量呈正相关.结论HCV阳性血清刺激可使体外培养的BV2细胞表达TLR9mRNA增高,4 h表达最高,且可能诱导了TNF-α、IL-6的分泌.提示TLR9可能增加了Th1和Th2细胞因子的分泌,并参与了HCV在中枢神经系统感染中的早期固有免疫应答.Objective To analyze the expression of toll-like receptor 9 mRNA, TNF （tumor necrosis factor）-α and IL （interleukin）-6 after the stimulation of murine microglia （BV2） by hepatitis C virus （HCV） so as to elucidate the immune pathogenesis of HCV in the injury of central nervous system （CNS）. Methods BV2 cells were cultured in vitro and divided into 3 groups： HCV-positive serum group,normal serum group （negative control） and blank control group. Each group had 20 samples. The cells and supernatant were collected at 4, 12, 20, 36 h. And TLR9 mRNA was detected by RT-PCR （reverse transcription-polymerase chain reaction） and the levels of TNF-α and IL-6 were detected by ELISA （enzyme-linked immunosorbent assay）. Comparison analyses were performed by SPSS13. 0 statistical package.Results （1） RT-PCR showed that there was the expression of TLR9 mRNA in BV2 cells. And the level of TLR9 mRNA in HCV-positive serum group （0. 85 ±0. 11, 0. 55 ±0. 08, 0. 58 ±0. 09, 0. 61 ±-0. 07） was higher than that in the other groups. The difference had statistical significance（ F = 17. 258, P ＜ 0. 05 ）. It was the highest at 4 h （0. 85 ± 0. 11 ）. And the difference had statistical significance （ F = 7. 427, P ＜0. 05）; but there were no statistical significance between normal serum group and control group（ P ＞ 0. 05 ）;no significant difference existed among 12, 20 and 36 h （P ＞ 0. 05 ）. （2） The levels of TNF-α[（926 ±133）, （327 ±59）, （367 ±69）, （354 ±71） pg/ml] and IL-6 [（125 ±34）, （84 ±20）, （82 ±24）,（87 ± 18） pg/ml] in the HCV-positive serum group were higher than those in the other groups（ F = 18. 263 -27. 469, P ＜ 0. 05 ）; in HCV-positive serum group, the levels of TLR9 mRNA and TNF-α（r = 0. 425, P ＜0. 01 ）/IL-6（ r =0. 489, P ＜0. 01 ）were positively correlated. Conclusion The expression of TLR9 mRNA is elevated after the stimulation of BV2 cells by HCV-positive serun. It peak comes at 4 h. And it may i

关 键 词：小神经胶质细胞 丙型肝炎病毒 TOLL样受体9 肿瘤坏死因子Α 白细胞介素6 

分 类 号：R51[医药卫生—内科学]