摘要目的:探讨Ras超家族亚群A(RhoA)/Rho关联含卷曲螺旋结合蛋白激酶1(Rock-1)在无机磷酸盐中诱导大鼠瓣膜间质细胞(VICs)成骨分化的作用机制。方法:取8只雄性大鼠(购自上海西普尔必凯动物实验有限公司)的主动脉瓣膜消化培养至第3代后免疫荧光鉴定。小干扰RNA(siRNA)-RhoA转染VICs后用无机磷酸盐成骨培养基(IP-OIM)分别诱导转染组(RNAi)，阴性对照组(NC)及空白对照组(CON)的成骨分化，并通过蛋白质印迹法(Western blot)与聚合酶链反应(PCR)评估转染后RhoA、Rock-1、Runt相关转录因子2(Runx-2)及骨钙素(OST)的变化；7 d行碱性磷酸酶(ALP)活性相关检测，14 d行钙含量相关检测评估其早期与晚期成骨分化趋势。用IP-OIM刺激转染后的VICs 60 min，并通过Western blot检测SMAD1/5/9的磷酸化变化。采用单因素方差分析。结果:鉴定后的VICs转染siRNA-RhoA并用IP-OIM培养，经过Western blot及PCR检测后可知RNAi的RhoA(Western blot：0.330±0.020；PCR：0.357±0.060)及Rock-1(Western blot：0.366±0.020；PCR：0.383±0.040)比CON均出现表达下调(RhoA Western blot： t＝36.680， P<0.05 PCR： t＝10.610， P<0.05；Rock-1 Western blot： t＝31.240， P<0.05 PCR： t＝13.210， P<0.05)，差异有统计学意义，RNAi的OST(Western blot：0.520±0.030；PCR：0.510±0.070)和Runx-2(Western blot：0.573±0.020；PCR：0.57±0.060)比CON也出现了下降(OST Western blot： t＝16.630， P<0.05；PCR： t＝6.970， P<0.05；Runx-2 Western blot： t＝21.040， P<0.05；PCR： t＝6.710， P<0.05)，差异有统计学意义。在7 d的ALP染色中RNAi的颜色最浅，ALP活性检测中RNAi [(1.005±0.101) U/mg]活性比较NC[(2.042±0.116) U/mg]和CON[(1.931±0.136) U/mg]明显下降( t＝10.570， P<0.05； t＝9.400， P<0.05)，差异有统计学意义。在14 d的相对钙含量测定中RNAi[(22.290±1.981) ng/L]钙含量比较NC[(41.100±2.440) ng/L]和CON[(41.760±1.215) ng/L]明显下降( t＝10.530， P<0.05； t＝14.510， P<0.05)，差异有统计学意义；同样茜素红钙沉积染色中，也是RNAi颜色最浅。经IP-OIM分别刺激60 min，RNAi的SMAD1/5/9的磷酸化水平(0.337±0.030)比CON明显下降( t＝25.480， P<0.05)，差异有统计学意义。 结论:无机磷酸盐可通过RhoA/Rock-1引起瓣膜间质细胞的成骨分化，SMAD1/5/9的磷酸化在此过程中起重要作用。

abstractsObjective:To investigate the action mechanism of RhoA/Rock-1 in inorganic phosphate-induced osteogenic differentiation of rat valvular interstitial cells (VICs).Methods:The rat aortic valve (n=8, male, Shanghai Sppir BK) was digested and cultured to the third generation, and immunofluorescence identification was performed. Then VICs were transfected with small interfering RNA (siRNA)-RhoA, and in the transfection group (RNAi), the negative control group (NC) and blank control group (CON) the osteogenic differentiation was induced by inorganic phosphate-osteogenic inducing medium (IP-OIM). Western blotting and polymerase chain reaction (PCR) were used to evaluate whether there were significant changes in RhoA, Rock-1, Runx-2 and Osteocalcin (OST). Changes in alkaline phosphatase (ALP) activity on the 7th day, and calcium assay on the 14th day were used to assess osteogenic differentiation trend. Transfected VICs were stimulated by IP-OIM for 60 min, and the SMAD1/5/9 level of phosphorylation was detected by Western blotting.Results:Identified VICs were transfected with siRNA-RhoA and then cultured with IP-OIM. Western blotting and PCR showed that RhoA (Western blotting: 0.330±0.020; PCR: 0.357±0.060) and Rock-1 (Western blotting: 0.366±0.020; PCR: 0.383±0.040) in the RNAi decreased significantly compared to CON (RhoA, for Western blotting: t=36.680, P<0.05; for PCR: t=10.610, P<0.05. Rock-1, for Western blotting: t=31.240, P<0.05; for PCR: t=13.210, P<0.05), while OST (Western blotting: 0.520±0.030; PCR: 0.510±0.070) and Runx-2 (Western blotting: 0.573±0.020; PCR: 0.570±0.060) in the RNAi also decreased (OST, for Western blotting: t=16.630, P<0.05; for PCR: t=6.970, P<0.05. Runx-2, for Western blotting: t=21.040, P<0.05; for PCR: t=6.710, P<0.05). The RNAi was the lightest in ALP staining on day 7, while compared with NC [(2.042±0.116) U/mg] and CON [(1.931±0.136) U/mg], the activity of RNAi [(1.005±0.101) U/mg] in ALP activity test was significantly decreased ( t=10.570, P<0.05; t=9.400, P<0.05). On the 14th day, the calcium content assay revealed that the RNAi [(22.290±1.981) ng/L] was significantly lower than that in the NC [(41.100±2.440) ng/L] and the CON [(41.760±1.215) ng/L] ( t=10.530, P<0.05; t=14.510, P<0.05). The alizarin red calcium deposition staining showed also the lightest color in the RNAi. After stimulation with IP-OIM 60 min later, the SMAD1/5/9 phosphorylation level in the RNAi (0.337±0.030) was significantly lower than that in the CON ( t=25.480, P<0.05). Conclusion:Inorganic phosphate can cause osteogenic differentiation of VICs through RhoA/Rock-1, and SMAD1/5/9 phosphorylation plays an important role in this process.