建立β_3肾上腺素能受体过表达的心肌成纤维细胞模型方法的比较  

Comparison of Two Methods of Building Cardiac Fibroblasts Models With β_3-AR Overexpression

作　　者：张源[1] 王丽[1] 张慧[1] 马依彤[2] 杨毅宁[2] 马苗苗[1] 朱晓丽[1] 

机构地区：[1]石河子大学医学院第一附属医院心内科,新疆石河子市832008 [2]新疆医科大学第一附属医院心脏中心

出　　处：《中国全科医学》2016年第15期1774-1780,共7页Chinese General Practice

基　　金：国家自然科学基金资助项目(81260028);石河子大学科学技术研究发展计划"自然科学与技术创新"团队创新项目(2011ZRKXTD-07)

摘　　要：目的用两种不同的方法构建β3肾上腺素能受体(β3-AR)过表达的SD乳鼠心肌成纤维细胞(CFBs)模型,并对两种方法的效果进行比较,为今后在细胞水平观察β_3-AR对心功能,尤其是对慢性心力衰竭(CHF)的影响提供新的实验方法。方法 2015年3—6月,选取出生1~3 d的SFP级SD乳鼠8只。分离SD乳鼠CFBs并体外培养,随机分为3个感染复数(MOI)组,分别为MOI 50组、MOI 100组、MOI 200组,采用β_3-AR基因过表达的慢病毒载体感染CFBs,利用流式细胞仪检测感染率,确定最佳MOI。将SD乳鼠CFBs随机分为4个β_3-AR激动剂干预组,分别为正常对照组、10^(-6)mol/L组、10^(-5)mol/L组、10^(-4)mol/L组,采用不同浓度β_3-AR激动剂干预CFBs,利用Western blotting法检测β_3-AR表达水平,挑选出β_3-AR激动剂干预CFBs的最适浓度。结果普通光学显微镜下观察CFBs,无搏动性,大多数呈梭形,其细胞核较大,胞质透明。倒置荧光显微镜下观察感染24、48、72、96h时CFBs状态及荧光表达情况,感染72 h时,各MOI组CFBs生长状态较好,细胞大多数呈梭形,无搏动性,细胞核较大,且肉眼观察荧光表达最佳。MOI 100组、MOI 200组感染率高于MOI 50组(P<0.05);MOI 200组感染率低于MOI 100组(P<0.05)。10-6mol/L组、10^(-5)mol/L组、10-4mol/L组β_3-AR表达水平高于正常对照组(P<0.05);10^(-5)mol/L组β_3-AR表达水平高于10^(-6)mol/L组、10^(-4)mol/L组(P<0.05)。10^(-5)mol/L组、MOI 100组β_3-AR表达水平高于正常对照组(P<0.05);MOI 100组β_3-AR表达水平高于10^(-5)mol/L组(P<0.05)。结论使用β_3-AR过表达的慢病毒载体感染CFBs与β_3-AR激动剂干预CFBs均可使其β_3-AR过表达,且前者效果优于后者。Objective To build neonatal SD rat cardiac fibroblasts（CFBs）models with β3-AR overexpression by two different methods and compare the two methods so as to provide new experimental method for exploring the effect of β3-AR on heart function,especially on chronic cardiac failure. Methods From March to June in 2015,8 SPF neonatal SD rats born 1 to 3 days ago were selected. CFBs of the rats were cultured in vitro and were randomly divided into three MOI groups which were MOI 50 group,MOI 100 group and MOI 200 group. These CFBs were infected by lentiviral vector with β3-AR overexpression,and infection rate was detected by flow cytometry to determine the optimal MOI. CFBs were randomly divided into four β3-AR agonist intervention groups which were normal control group,10 - 6 mol/ L group,10 - 5 mol/ L group,and 10 - 4 mol/ L group. Intervention was conducted on these CFBs by different concentrations of β3-AR agonist,and β3-AR expression level was detected by Western blotting method to find out the optimal concentration of β3-AR agonist in the intervention on CFBs. Results Observed under bspnormal optics microscope,CFBs showed no pulsation and mostly took on fusiform shape,with large cell nucleus and transparent cytoplasm. The status of CFBs and fluorescent expression were observed under inverted fluorescence microscope at 24,48,72 and 96 h after infection. At 72 h,CFBs of each MOI group were in good growth status,they were mostly in fusiform shape and had no pulsation,with large cell nucleus,and the fluorescent expression observed by naked eye was optimal. MOI 100 group and MOI 200 group were higher than MOI 50 group in infection rate（P 〈 0. 05）;MOI 200 group was lower than MOI 100 group in infection rate（P 〈 0. 05）. 10 - 6 mol/ L group,10 - 5 mol/ L group and 10 - 4 mol/ L group were higher than normal control group in the expression level of β3-AR（P 〈 0. 05）;10 - 5 mol/ L group was higher than 10 - 6 mol/ L group and 10 - 4 mol/ L group in the expression level of β3-AR（P 〈 0. 05）.

关 键 词：受体 肾上腺素能B 成纤维细胞 心肌 慢病毒载体 

分 类 号：R542.2[医药卫生—心血管疾病]