作者：赵倩，陈玉霞，王平，任安经，宋金超

单位：

1.上海理工大学2.上海理工大学附属市东医院麻醉科3.海军军医大学基础医学院病理生理学教研室4.海军军医大学基础医学院基础医学实验教学中心

Units：

1.University of Shanghai for Science and Technology, Shanghai 200093, China 2.Department of Anesthesiology, Shidong Hospital Affiliated to University of Shanghai for Science and Technology, Shanghai 200082, China 3.Department of Pathophysiology, School of Basic Medicine, Naval Medical University, Shanghai 200433, China 4.Experimental Teaching Center of Basic Medicine, School of Basic Medicine, Naval Medical University, Shanghai 200433, China

关键词：

锌指蛋白; ZBTB20; 血管平滑肌细胞; 慢病毒感染;

Keywords：

Zinc finger protein; ZBTB20; Vascular smooth muscle cells; Lentivirus infections

CLC：

DOI：

10.12114/j.issn.1008-5971.2023.00.154

Funds：

国家自然科学基金面上项目（31471090，32071115）

摘要：

目的 构建并鉴定锌指蛋白ZBTB20敲低的血管平滑肌细胞稳转株。方法 本实验时间为2022-04-18至2022-11-29。设计合成针对ZBTB20基因的短发夹RNA(shRNA)，构建ZBTB20干扰慢病毒载体——pHBLVZBTB20 shRNA，包装ZBTB20干扰慢病毒。将人肺动脉平滑肌细胞（HPASMCs）接种于6 cm培养皿中，分别加入含8μg/ml polybrene和0.5 ml ZBTB20干扰慢病毒上清液的无血清培养液3 ml（实验组）和含8μg/ml polybrene和0.5 ml含无意义干扰序列的慢病毒的无血清培养液3 ml（阴性对照组）进行转染，48 h后加入嘌呤霉素（优化剂量为1.5μg/ml）以筛选转染成功的HPASMCs。采用荧光定量PCR检测两组ZBTB20 mRNA相对表达量，采用Western blot法检测两组ZBTB20，采用免疫组化试验检测两组ZBTB20表达情况，并进行细胞增殖实验和细胞迁移实验。结果 实验组ZBTB20mRNA相对表达量、ZBTB20均低于阴性对照组（P<0.05）。免疫组化试验结果显示，ZBTB20在阴性对照组的细胞核内高度表达，而在实验组的细胞核内无明显表达，表明ZBTB20敲低的血管平滑肌细胞稳转株构建成功。感染后第2～5天，实验组细胞计数少于对照组，OD值低于阴性对照组（P<0.05）。感染后48 h，实验组划痕愈合面积百分比低于阴性对照组（P<0.05）。结论 本实验成功构建了锌指蛋白ZBTB20敲低的血管平滑肌细胞稳转株，且ZBTB20敲低后HPASMCs的增殖和迁移能力明显受到抑制。

Abstract：

Objective To construct and identify a stable strain of vascular smooth muscle cell with knockdown zinc finger protein ZBTB20. Methods The experiment was conducted from April 18, 2022 to November 29, 2022. We designed and synthesized a short hairpin RNA (shRNA) targeting ZBTB20 gene, constructed a ZBTB20 interference lentivirus vector, pHBLVZBTB20 shRNA, and packaged ZBTB20 interference lentivirus. Human pulmonary artery smooth muscle cells (HPASMCs) were seeded in a 6 cm culture dish, 3 ml serum-free culture medium containing 8 μg/ml polybrene and 0.5 ml ZBTB20 interfering lentivirus supernatant (experimental group) , or 3 ml serum-free culture medium containing 8 μg/ml polybrene and 0.5 ml lentivirus containing insignificant interfering sequence (negative control group) were added for transfection, respectively. After 48 hours, puromycin (optimized dose of 1.5 μg/ml) was added and the successfully transfected HPASMCs were selected. The relative expression of ZBTB20 mRNA in the two groups was detected by fluorescent quantitative PCR, the ZBTB20 in the two groups was detected by Western blot, and the expression of ZBTB20 was detected by immunohistochemistry, and the cell proliferation test and cell migration test were performed. Results The relative expression of ZBTB20 mRNA and ZBTB20 in the experimental group were lower than those in the negative control group (P < 0.05) . The results of immunohistochemistry showed that ZBTB20 was highly expressed in the nuclei of the negative control group, while there was no significant expression in the nuclei of the experimental group, indicating that stable strain of vascular smooth muscle cell with knockdown ZBTB20 were successfully constructed. On the 2nd to 5th day after infection, the cell count and OD value in the experimental group were lower than those in the control group (P < 0.05) . At 48 h after infection, the percentage of cell scratch healing area in the experimental group was lower than that in the negative control group (P < 0.05) . Conclusion The stable strain of vascular smooth muscle cell with knockdown ZBTB20 is successfully established in this study, and the proliferation and migration of HPASMCs are significantly inhibited after knockdown of ZBTB20.

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