南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (4): 567-573.doi: 10.12122/j.issn.1673-4254.2021.04.13

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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

张 涛，李维丽，邱晓拂，刘百川，李高远，冯才鑫，廖俊发，林康健   

  • 出版日期:2021-04-20 发布日期:2021-04-30

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

  • Online:2021-04-20 Published:2021-04-30

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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CCSMCs α-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PCNA mRNA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。

关键词: TEAD1；勃起功能障碍；糖尿病；平滑肌细胞；表型转化；CRISPR/Cas9

Abstract: 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 high α- 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 synthetic to the contractile type.

Key words: transcription enhancer factor- 1; erectile dysfunction; diabetes mellitus; smooth muscle cells; phenotype modulation; CRISPR/Cas9