慢病毒介导siRNA hsa-circ-0000885转染BMSCs与破骨细胞共培养体系对细胞分化、增殖和凋亡相关研究

Lentivirus-mediated siRNA hsa-circ-0000885 transfection of BMSCs and osteoclast co-culture system on cell differentiation,proliferation and apoptosis

目的:探究将siRNA hsa-circ-0000885修饰的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)后与破骨细胞共培养对BMSCs的成骨分化、细胞增殖和凋亡的影响,为临床上骨质疏松症(osteoporosis,OP)的治疗提供新的思路和方法。方法:选择自2018年9月至2020年2月收治的13例骨质疏松患者为研究对象,其中女11例,男2例,年龄(65.45±10.77)岁;取得患者知情同意后,抽取患者外周血组织。然后用circ RNA芯片检测外周血单个核细胞(peripheral blood mononuclear cell,PBMC)的circRNA的表达水平,用siRNA技术沉默circ RNA的表达,通过慢病毒转染BMSCs,根据是否转染hsa-circ-0000885的siRNA干扰质粒,将细胞分成空白组,空载体组和siRNA干扰组。各组细胞处理72 h后,用流式细胞仪检测细胞的周期,用AV-PI试剂盒检测细胞的凋亡水平,用ALP染色检测BMSCs的成骨分化能力。结果:OP患者外周血PBMC中hsa-circ-0000885的表达量明显高于健康对照组患者(t=2.119,P<0.05)。ALP染色结果表明,siRNA hsa-circ-0000885质粒可以促进BMSCs的成骨分化,明显高于空白组和空白质粒组(F=9.132,q=2.995,2.897;P=0.009,0.012<0.05)。CCK-8试剂盒检测结果显示,siRNA hsa-circ-0000885干扰组BMSCs的细胞增殖率明显高于空白组和空白质粒组(F=9.881,q=2.457,2.904;P=0.032,0.016<0.05)。而AV-PI试剂盒检测结果显示,siRNA干扰组细胞的凋亡率明显低于空白组和空白质粒组(F=10.208;q=2.885,3.001;P=0.019,0.011<0.05)。结论:慢病毒介导siRNA hsa-circ-0000885质粒转染BMSCs与破骨细胞共培养体系可以促进细胞增殖,抑制细胞凋亡,促进BMSCs的成骨分化,可以作为OP患者潜在的治疗靶点。

Objective:To explore the effects of siRNA hsa-circ-0000885 modified bone marrow mesenchymal stem cells(BMSCs)on osteogenic differentiation,cell proliferation and apoptosis in order to provide new ideas and methods for the clinical treatment of osteoporosis(OP).Methods:From September 2018 to February 2020,13 patients with osteoporosis admitted to our hospital were selected as the research objects,including 11 females and 2 males,with an age of(65.45±10.77)years old.After obtaining the informed consent of patients,peripheral blood tissues were extracted.Then the expression level of circRNA in peripheral blood mononuclear cells(PBMC)was detected by circ RNA chip.The expression of circ RNA was silenced by siRNA technology.The BMSCs were transfected with lentivirus.According to the siRNA interference plasmid hsa-circ-0000885,the cells were divided into the blank group,the empty vector group and the siRNA interference group.After 72 hours of treatment,the cell cycle was detected by flow cytometry,the apoptosis level was detected by AV-PI kit,and the osteogenic differentiation ability of BMSCs was detected by ALP staining.Results:The expression of hsa-circ-0000885 in PBMC of patients with osteoporosis was significantly higher than that of healthy controls(t=2.119,P<0.05).ALP staining showed that siRNA hsa-circ-0000885 could promote the osteogenic differentiation of BMSCs,which was obviously too much in the blank group and blank plasmid group(F=9.132,q=2.995,2.897;P=0.009,0.012<0.05).The results of CCK-8 showed that siRNA hsa-circ-0000885 could promote the proliferation of BMSCs,which was significantly higher than that of the blank group and blank plasmid group(F=9.881,q=2.457,2.904;P=0.032,0.016<0.05).The results of AV-PI showed that the apoptosis rate of siRNA interference group was significantly lower than that of blank group and blank plasmid group(F=10.208;q=2.885,3.001;P=0.019,0.011<0.05).Conclusion The lentivirus mediated siRNA hsa-circ-0000885 plasmid transfected into BMSCs and osteoclast co culture system can promote cell proliferation,inhibit apoptosis and promote osteogenic differentiation of BMSCs,which can be used as a potential therapeutic target for OP patients.