摘要
目的探讨长链非编码RNA胰岛素样生长因子2反义转录物(lncRNA IGF2-AS)调控胰岛素样生长因子2(IGF2)对骨髓间充质干细胞(BMSCs)心肌样分化的影响。方法分离培养SD大鼠的BMSCs,倒置显微镜观察原代细胞、第3代细胞的形态;流式细胞仪检测BMSCs表面抗原CD29、CD90、CD45的表达。将第3代生长良好的BMSCs分为对照组、空载体组(转染p LVX-IRES-Zs Green1载体)、lncRNA IGF2-AS组(转染pLVX-IRES-Zs Green1-IGF2-AS)、5-氮杂胞嘧啶(5-AZA)组(8μmol/L 5-AZA处理)、si-NC组、si-IGF2-AS组、si-IGF2组、lncRNA IGF2-AS+siNC组及lncRNA IGF2-AS+si-IGF2组。实时荧光定量PCR(q RT-PCR)检测IGF2-AS表达;MTT法检测细胞活力;Western blot检测IGF2、间隙连接蛋白43(Cx43)、心肌肌钙蛋白T(cTnT)、心肌肌钙蛋白I(cTnI)蛋白表达;RNA下拉和RNA免疫沉淀(RIP)检测IGF2-AS与IGF2蛋白结合情况。结果原代细胞在培养初期呈悬浮状态,大多数呈圆形,培养48 h后开始贴壁生长;第3代细胞呈长梭形,排列不整齐,相邻细胞间紧密连接,呈成纤维细胞样。第3代BMSCs高表达CD29(98.21%)、CD90(92.54%),低表达CD45(3.67%)。过表达IGF2-AS或5-AZA处理均可促进BMSCs中Cx43、c TnT、c TnI蛋白表达,并降低细胞活力,且5-AZA组相应指标明显低于lncRNA IGF2-AS组(P<0.05)。沉默IGF2-AS或抑制IGF2表达均可降低BMSCs中Cx43、cTnT、cTnI蛋白表达,并降低细胞活力(P<0.05);lncRNA IGF2-AS可靶向上调IGF2蛋白的表达(P<0.05);沉默IGF2逆转了过表达IGF2-AS对BMSCs心肌样分化的促进作用。结论过表达IGF2-AS通过上调IGF2促进BMSCs心肌样分化。
Objective transcript(lncRNA IGF2-AS)on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cells(BMSCs)by regulating insulin-like growth factor 2(IGF2).MethodsBMSCs were isolated and cultured from SD rats.The morphology of primary and third-generation cells was observed under inverted microscope.Flow cytometry was used to detect the expressions of CD29,CD90 and CD45 on the surface of BMSCs.BMSCs with good growth in the third generation were divided into the control group,the empty vector group(transfected with pLVX-IRES-Zs Green1 vector),the lncRNA IGF2-AS group(transfected with pLVX-IRES-Zs Green1-IGF2-AS),the 5-azacytosine group(8μmol/L 5-AZA treatment),the si-NC group,the si-IGF2-AS group,the si-IGF2 group,lncRNA IGF2-AS+si-NC group and the lncRNA IGF2-AS+siIGF2 group.Quantificational real-time polymerase chain reaction(qRT-PCR)was used to detect the expression of IGF2-AS.MTT method was used to detect cell viability.Western blot assay was used to detect the IGF2,gap junction protein 43(Cx43),cardiac troponin T(cTnT)and cardiac troponin I(cTnI)protein expression.RNA pull-down and RNA immunoprecipitation(RIP)were used to detect the IGF2-AS and IGF2 protein binding.ResultsThe primary cells were in suspension at the beginning of the culture flask,most of them were round,and they began to grow adherently after 48 h of culture.The third-generation cells were long spindle-shaped,irregularly arranged,closely connected between adjacent cells,and fibroblast-like.The third-generation BMSCs showed high expression of CD29(98.21%),CD90(92.54%),and low expression of CD45(3.67%).The overexpression of IGF2-AS or 5-AZA could promote protein expressions of Cx43,c TnT and cTnI in BMSCs,and reduce cell viability,and the corresponding indicators were significantly lower in the 5-AZA group than those in the lncRNA IGF2-AS group(P<0.05).Silencing IGF2-AS or inhibiting the expression of IGF2 could reduce the protein expressions of Cx43,cTnT,and c TnI in BMSCs,and reduce cell viability(P<0.05).lncRNA IGF2-AS could target up-regulate the expression of IGF2 protein(P<0.05).Silencing IGF2 reversed the promotion effect of overexpression of IGF2-AS on cardiomyocyte-like differentiation of BMSCs.Conclusion cardiomyocyte-like differentiation of BMSCs by up-regulating IGF2.
作者
钟晓鸣
刘洪洋
张蕾
姚新亮
鲁雪莉
许瑾瑾
程冠昌
ZHONG Xiaoming;LIU Hongyang;ZHANG Lei;YAO Xinliang;LU Xueli;XU Jinjin;CHENG Guanchang(Department of Cardiology,Huaihe Hospital of Henan University,Kaifeng 475000,China)
出处
《天津医药》
CAS
北大核心
2022年第4期350-356,共7页
Tianjin Medical Journal
基金
国家自然科学基金资助项目(81770296)。
