大鼠主动脉中膜平滑肌细胞向成骨样细胞转化中骨保护素和核转录因子κB受体配体的变化及意义

Change and significance of osteoprotegerin/receptor activator of NF-κB ligand during differentiation of rat arterial smooth muscle cells into the osteoblast-like cells

目的研究大鼠主动脉中膜平滑肌细胞（SMC）向成骨样细胞转化过程中骨保护素（OPG）／核转录因子WB（NFκB）受体的配体（RANKL）的变化及意义。方法体外培养大鼠主动脉SMC，随机分为成骨样细胞组、阿托伐他汀组和SMC组，每组再分为早、晚2个亚组。成骨样细胞组在正常SMC培养液中加入口磷酸甘油和维生素C，他汀组在加入以上物质的同时再加入阿托伐他汀。早期组在第7天，晚期组在第14天进行检测。采用VonKossa染色、钙离子含量测定、碱性磷酸酶（ALP）活性检测、骨钙素的Westernblot检测来判定钙化情况；采用实时定量PCR检测OPG和RANKI。的mRNA表达情况。结果成骨样细胞组中钙离子含量、Vonkossa染色、ALP活性、骨钙素均高于他汀组（F=0．27、2．48、6．14、10．21，均P〈0．05）。sMC组无论早晚期均有大量的OPG表达（早期2．71土0．08，晚期2．69±0．02），但无RANKL表达；他汀组（早期3．52±0．05，晚期2．50±0．03）和成骨样细胞组（早期4．18±0．10，晚期2．30±0．11）的早期OPG表达上升而晚期下降，而两组RANKI。表达在钙化中一直呈升高趋势（分别由1.01±0．19升高至2．40±0．10及1．70±0．07升至3．22±0．11）。SMC组、他汀组、成骨样细胞组随钙化程度的加重，OPG／RANKL比值呈逐步下降趋势（F=52．93，2．33，均P〈0．05）；同一组中，晚期亚组随着钙化的加重，OPG／RANKL比值也较早期明显下降（F=38．71，1．74，均P〈0．05）。结论OPG／RANKL的比值与SMC向成骨样细胞的转化程度呈负相关，且阿托伐他汀具有抑制钙化的作用。

Objective To study the change and significance of osteoprotegerin（OPG）/receptor activator of NF-kB ligand（RANKL）during the process of rat arterial smooth muscle ceils （SMC） to differentiate subgroups.. 7 d （early） subgroup and 14 d （late） subgroup, into osteoblast-like cells. Methods The rat arterial SMCs were divided randomly into the SMC group, atorvastatin group, and osteoblast-like group. Each group was also divided into 2. The osteoblast-like group was given ]3- g｜ycerophosphate and vitamin C in culture medium, the atorvastatin group was given both - glycerophosphate, vitamin C and atorvastatin. Von Kossa staining, Ca2＋ content assay, ALP activity assay and osteocalcin assayed with Western blot were used to check the ]eve[ of calcification. Real-time PCR was used to check the mRNA expressions of OPG and RANKL. Results There was high expression of OPG （early 2.71±0.08, late 2.69±0.02） but no RANKL in the SMC group all the time. The expressions of OPG were increased in the early subgroup and decreased in late subgroup in the atorvastatin group （early 3.52±0.05,1ate 2.50±0.03） and osteoblast-like group （early 4. 18± 0. 10, late 2.30 ± 0. 11）. And the expressions of RANKL were both increased in the atorvastatin group and osteoblast-like group during the calcification process （from 1. 01±0. 19 to 2. 40±0. 10,and from 1. 70±0.07 to 3.22±0. 11, respectively）. Among the three groups, the ratio of OPG/RANKL was decreased with the increasing calcification level （F= 52.93,2.33,both P〈0.05）. And compared with the early subgroups, the ratio of OPG/RANKL in the late subgroups was also decreased along with the increase of calcification level in each group（F= 38.71,1.74, both P〈0.05）. Conclusions The ratio of OPG/RANKL has a negative correlation with the level of the osteoblast-like cells differentiation, and atorvastatin could inhibit the calcification.