TEAD1基因敲除对糖尿病性ED大鼠阴茎海绵体平滑肌细胞表型转化的影响  

CRISPR/Cas9-mediated TEAD1 knockout induces phenotypic modulation of corpus cavernosum smooth muscle cells in diabetic rats with erectile dysfunction

作　　者：张涛 李维丽[2] 邱晓拂[1] 刘百川[1] 李高远[1] 冯才鑫 廖俊发 林康健 ZHANG Tao;LI Weili;QIU Xiaofu;LIU Baichuan;LI Gaoyuan;FENG Caixin;LIAO Junfa;LIN Kangjian(Department of Urology,Second Guangdong Provincial People's Hospital,Guangzhou 510317,China;Department of Obstetrics and Gynecology,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]广东省第二人民医院泌尿外科,广东广州510317 [2]南方医科大学南方医院妇产科,广东广州510515

出　　处：《南方医科大学学报》2021年第4期567-573,共7页Journal of Southern Medical University

基　　金：国家自然科学基金青年科学基金(81601273);广东省中医药局科研项目(20192004)。

摘　　要：目的利用CRISPR/Cas9技术敲除糖尿病性ED大鼠阴茎海绵体平滑肌细胞(CCSMCs)中TEAD1基因,探讨TEAD1基因对糖尿病性ED大鼠CCSMCs表型转化的影响。方法利用链脲佐菌素建立糖尿病性ED大鼠模型。原代及传代培养CCSMCs,并进行免疫荧光染色鉴定。根据CRISPR/Cas9靶点设计规则设计了3组特异性sgRNA(sgRNA-1、sgRNA-2、sgRNA-3),构建表达载体并转染293T细胞,包装并收集慢病毒,将其感染糖尿病性ED大鼠CCSMCs,嘌呤霉素筛选阳性细胞,Western blot检测TEAD1蛋白的表达水平。然后将实验分3组:敲除TEAD1基因的糖尿病性ED大鼠CCSMCs为实验组(CCSMCs-sgRNA-2)、空载体病毒感染组为阴性对照组(CCSMCs-sgRNA-NC)、不感染病毒组为空白对照组(CCSMCs-CK),分别使用qRT-PCR和Western blot检测各组细胞表型标志物平滑肌肌球蛋白重链(SMMHC)、碱性调宁蛋白(Calponin)和增殖细胞核抗原(PCNA)mRNA和蛋白水平的表达。结果原代培养的糖尿病性ED大鼠CCSMCsα-SMA阳性细胞率达95%以上。成功包装了含有TEAD1-sgRNA的重组慢病毒,筛选得到了稳定低表达TEAD1基因的糖尿病性ED大鼠CCSMCs细胞系。Western blot结果显示感染TEAD1-sgRNA-2的糖尿病性ED大鼠CCSMCs中TEAD1蛋白水平最低(P<0.05)。qRT-PCR和Western blot结果显示,敲除TEAD1基因的糖尿病性ED大鼠CCSMCs,SMMHC和Calponin mRNA和蛋白水平的表达均显著上调(P<0.05),而PCNAmRNA和蛋白水平的表达均显著减少(P<0.05)。结论利用CRISPR/Cas9基因技术成功构建了定向敲除TEAD1基因的慢病毒载体,TEAD1基因的缺失可使糖尿病性ED大鼠CCSMCs表型从合成型向收缩型转化。Objective To construct a corpus cavemosum smooth muscle cell(CCSMCs)line with TEAD1 knockout from diabetic rats with erectile dysfunction(ED)using CRISPR/Cas9 technology and explore the role of TEAD1 in phenotypic modulation of CCSMCs in diabetic rats with ED.Methods Models of diabetic ED were established in male Sprague-Dawley rats by intraperitoneal injection of streptozotocin.CCSMCs from the rat models were primarily cultured and identified with immunofluorescence assay.Three sgRNAs(sgRNA-1,sgRNA-2 and sgRNA-3)were transfected via lentiviral vectors into 293T cells to prepare the sgRNA-Cas9 lentivirus.CCSMCs from diabetic rats with ED were infected by the lentivirus,and the cellular expression of TEAD1 protein was detected using Western blotting.In CCSMCs infected with the sgRNA-Cas9 lentivirus(CCSMCs-sgRNA-2),or the empty lentiviral vector(CCSMCs-sgRNA-NC)and the blank control cells(CCSMCs-CK),the expressions of cellular phenotypic markers SMMHC,calponin and PCNA at the mRNA and protein levels were detected using real-time fluorescence quantitative RT-PCR(qRT-PCR)and Western blotting,respectively.Results The primarily cultured CCSMCs from diabetic rats with ED showed a highα-SMA-positive rate of over 95%.The recombinant lentivirus of TEAD1-sgRNA was successfully packaged,and stable TEAD1-deficient CCSMC lines derived from diabetic rat with ED were obtained.Western blotting confirmed that the protein expression of TEAD1 in TEAD1-sgRNA-2 group was the lowest(P<0.05),and this cell line was used in subsequent experiment.The results of qRT-PCR and Western blotting showed significantly up-regulated expressions of SMMHC and calponin(all P<0.05)and down-regulated expression of PCNA(all P<0.05)at both the mRNA and protein levels in TEAD1-deficient CCSMCs from diabetic rats with ED.Conclusion We successfully constructed a stable CCSMCs line with CRISPR/Cas9-mediated TEAD1 knockout from diabetic rats with ED.TEAD1 gene knockout can induce phenotype transformation of the CCSMCs from diabetic rats with ED from the synthe

关 键 词：TEAD1 勃起功能障碍 糖尿病 平滑肌细胞 表型转化 CRISPR/Cas9 

分 类 号：R587.2[医药卫生—内分泌] R698.1[医药卫生—内科学]