摘要
目的探讨绝经后骨质疏松症病人骨髓间充质干细胞(BMSCs)基质交感分子1(STIM1)与成骨分化关键因子碱性磷酸酶(ALP)、骨桥蛋白(OCN)、骨钙素(OPN)的表达变化情况。方法通过临床取材获得并培养绝经后骨质疏松症病人BMSCs,加入成骨诱导液孵育14天,采用RT-PCR检测ALP、OPN、OCN的转录水平及ALP的活性,并检测STIM1的表达情况。采用ALP染色法检测ALP活性;采用Alizarin Red染色法检测钙结节形成数量;采用Fluo荧光法检测钙离子内流变化。结果通过无菌培养,获得高纯度绝经后骨质疏松症(观察组)病人的BMSCs,经成骨诱导14天后,观察组病人BMSCs中成骨分化关键因子ALP、OPN、OCN的转录水平以及ALP的活性均低于对照组(P<0.05),同时STIM1 mRNA的表达水平也相应下降,钙离子内流较对照组减少(P<0.05)。另外,观察组病人BMSCs钙结节形成数量少于对照组。结论绝经后骨质疏松的发病机制可能与由STIM1参与调控的钙释放激活钙离子通道开放情况密切相关,可作为绝经后骨质疏松病人预防和治疗的潜在靶点。
Objective To observe the expression of stromal interaction molecule-1(STIM1)and other osteogenetic differentiation related factors(such as ALP,OCN,OPN) in bone marrow stromal stem cells(BMSCs)of postmenopausal osteoporosis patients.Methods BMSCs were cultured in the presence of osteogenic induction for 14 d after obtained from postmenopausal osteoporosis patients.We analyzed the Ca~(2+) concentration and Ca~(2+) release activated Ca~(2+) channel(CRAC)by Fluo-3 staining before and after osteogenic induction respectively.The expressions of STIM1 and other osteogenic differentiation related factors(ALP,OPN and OCN)were further measured and evaluated by quantitative real time PCR.Mineralization was evaluated by the calcium chelating dye alizarin red at the start of osteogenic induction and after 7 and 14 days.BMSCs were stained for alkaline phosphatase(ALP)at 14 d.Results BMSCs were successfully cultured after osteogenic induction for 14 d.The expressions of STIM1 and other osteogenic differentiation related factors(ALP,OPN,and OCN)in observation group were lower than the control group by RT-PCR(P<0.05).Similarly,ALP staining was significantly reduced in observation group compared with the normal control cells(P<0.05).Besides,the expression of STIM1 in observation group after osteogenic induction for 14 d was reducing,but less than control group.Ca~(2+) concentration was reduced in observation group than control group(P<0.05).The numbers of calcium chelating dye alizarin red in observation group were less than control group.Conclusion There are significant evidences that STIM1 in BMSCs would be contributed to the postmenopausal osteoporosis,which could provide a new research direction and therapeutic strategy for osteoporosis.
引文
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