骨形态蛋白2 siRNA靶向抑制对体外培养人脐动脉平滑肌细胞成骨样分化及钙化的影响

目的研究骨形态蛋白2（BMP2）小分子干扰RNA（siRNA）靶向抑制对人脐动脉平滑肌细胞（HUASMC）由白介素6（IL-6）诱导刺激的成骨样分化和细胞层钙化的影响。方法以体外培养的原代HUASMC作为实验对象。建立RNA干扰（RNAi）实验平台,通过FAM标记阴性对照siRNA优化转染条件;将BMP2基因的4条候选siRNA分别转染原代HUASMC,Real-TimePCR检测BMP2mRNA表达,筛选有效抑制siRNA。将复苏的原代HUASMC分为siRNA转染组（转染有效抑制siRNA）和模拟转染组（对照组,不转染siRNA）,两组均加入重组人IL-6（rhIL-6）（10ng/mL）刺激孵育,Real-TimePCR检测刺激孵育12、48h时点成骨样转化相关基因BMP2、骨特异性碱性磷酸酶（BAP）、骨钙素（OC）和骨桥蛋白（OPN）mRNA表达。将复苏的原代HUASMC分为siRNA转染组、对照组和空白组（不予rhIL-6刺激孵育,其余处理与对照组相同）,siRNA转染组和对照组均加入rhIL-6（50ng/mL）刺激孵育,甲氧-酚酞络合酮法检测rhIL-6刺激孵育前及孵育后2、4d时点各组HUASMC细胞层钙盐含量,以总蛋白含量校正。结果成功建立合适的RNAi实验平台。10ng/mLrhIL-6刺激孵育后12h时点,siRNA转染组HUASMC BMP2和BAPmRNA表达明显低于对照组（P〈0.05）;刺激孵育后48h时点,siRNA转染组OC和OPNmRNA表达显著低于对照组（P〈0.05）。加入50ng/mLrhIL-6刺激孵育前,siRNA转染组、对照组和空白组HUASMC细胞层钙盐含量比较差异无统计学意义（P〉0.05）;在siRNA转染组,50ng/mLrhIL-6刺激孵育后2、4d时点的细胞层钙盐含量均明显低于对照组（P〈0.05）,刺激孵育后2d时点的细胞层钙盐含量明显高于空白组（P〈0.05）。结论体外培养的原代HUASMC能够实现BMP2基因的siRNA靶向抑制,能显著抑制由IL-6诱导刺激的成骨样分化和钙化。

Effect of bone morphogenetic protein 2 siRNA targeted suppression on osteogenic differentiation and calcification of human umbilical arterial smooth muscle cells cultured in vitro

Objective To investigate the effect of bone morphogenetic protein 2 (BMP2) small interfering RNA (siRNA) targeted suppression on osteogenic differentiation and calcification of human umbilical arterial smooth muscle cells (HUASMC) induced by interleukin-6 (IL-6). Methods Primary HUASMC cultured in vitro were served as study objective. RNA interference (RNAi) experiment platform was established, and transfection condition was optimized with FAM negative siRNA. Primary HUASMC were transfected with four candidate siRNA of BMP2 gene, respectively, the expression of BMP2 mRNA was detected by Real-Time PCR, and active suppression siRNA was screened. The thawed primary HUASMC were divided into siRNA transfection group (transfection with active suppression siRNA) and mocking transfection group (control group, without transfection with siRNA), and both group were stimulated with recombinant human IL-6 (10 ng/mL). The expression of BMP2, bone specific alkaline phosphatase (BAP), osteocalcin (OC) and osteopontin (OPN) mRNA was detected by Real-Time PCR after simulation for 12 and 48 h. The thawed primary HUASMC were divided into siRNA transfection group, control group and blank group (without rhIL-6 stimulation, similar to control group for the other treatment). rhIL-6 (50 ng/mL) was added to siRNA group and control group for stimulation. The calcium deposition in cell layers was measured by o-cresolphthalein complexone method before stimulation, 2 d and 4 d after stimulation with rhIL-6, and the results were corrected by total protein content. Results The suitable RNAi experiment platform was successfully established. Twenty hours after stimulation with 10 ng/mL rhIL-6, the expression of BMP2 and BAP mRNA of HUASMC in siRNA transfection group was significantly lower than that in control group (P<0.05). Forty-eight hours after stimulation, the expression of OC and OPN mRNA in siRNA transfection group was significantly lower than that in control group (P<0.05). Before stimulation with 50 ng/mL rhIL-6, there was no significant difference in calcium deposition in HUASMC cell layers among siRNA transfection group, control group and blank group (P>0.05). For siRNA transfection group, calcium deposition in cell layers after stimulation with 50 ng/mL rhIL-6 for 2 d and 4 d was significantly lower than that in control group (P<0.05), and calcium deposition in cell layers after stimulation for 2 d was significantly higher than that in blank group (P<0.05). Conclusion Primary HUASMC cultured in vitro can lead to siRNA targeted suppression of BMP2 gene, and can significantly inhibit the osteogenic differentiation and calcification induced by IL-6.