【摘要】 目的:用酶消化法从正常女性松质骨中分离成骨细胞,进行体外培养和功能鉴定,从mRNA水平研究吗啡对体外培养人成骨细胞雌激素受体(estrogen receptor,ER)和孕激素受体(progesterone receptor,PR)表达的影响,探讨吗啡对成骨细胞的直接作用,为进一步寻求减少或阻断吗啡依赖对骨的负面影响提供一定的理论依据。方法:用胰酶-胶原酶消化法从正常人松质骨中分离培养成骨细胞,用茜素红染色法、Gomonri改良钙钴法鉴定成骨细胞;制备不同浓度的吗啡及其拮抗剂纳洛酮,作用于体外培养的成人正常成骨细胞上,通过RT-PCR法半定量分析不同浓度的吗啡及其拮抗剂纳洛酮对成骨细胞ER、PRmRNA及BGP(骨钙素)mRNA表达的影响。结果:用酶消化法分离的人成骨细胞,在体外培养时可合成碱性磷酸酶(ALP)、形成钙化结节。不同浓度吗啡组成骨细胞上ERmRNA表达与对照组比较有显著性下降(P<0.05)且与吗啡浓度负相关(r=-0.996,P<0.001);不同浓度吗啡组成骨细胞上PRmRNA表达与对照组比较均有显著性下性(P<0.05),且与吗啡浓度负相关(r=-0.983,P<0.001);不同浓度吗啡组成骨细胞上BGPmRNA表达与对照组比校均有显著性下(P<0.05),且与吗啡浓度负相关(r=-0.988,P<0.001)。结论:用酶消化法进行人成骨细胞培养,简捷易行,可培养大量高纯度人成骨细胞供研究使用;从mRNA水平证明了吗啡可以引起体外培养的女性成骨细胞内ER、PR及BGP数量下调,且与吗啡浓度呈负相关,可剂量依赖下调人成骨细胞ER、PRmRNA及BGPmRNA的表达。更多还原

【Abstract】 Objective:To evaluate the effect of morphine on expression of ER、PR mRNA of female Osteoblasts in Vitro and investigate the direct effect of morphine on Osteoblasts, so as to provide theoretic basis for reducing or obstructing negative effect of morphine dependence on Osteoblasts. Methods:The human osteoblasts cell (HOBs) were isolated from the cancellous bone by trypsin and collagenase digestion and identified by alizarin red staining and modified Gomonri method. Different concentrations of morphine and naloxone were prepared and acted on HOBs cultured in Vitro. The goal was to analyze effects of different concentrations of morphine and naloxone on expression of ER、PR mRNA 、BGP mRNA of female Osteoblasts by RT-PCR method. Results:The HOBs purified by enzyme digestion in vitro functioned normally as observed in vivo; Alkaline phosphatase(ALP)and Calcified nodules were expressed by HOBs; RT-PCR showed that comparing with the control group, expression of ER、PR mRNA、BGP mRNA of female osteoblasts in different concentration morphine groups decreased significantly(P<0.05),and they had negative correlation with the concentration of morphine(P<0.001). Conclusions :It is easy to purify a large number of HOBs with normal functions by enzyme digestion; In mRNA level, morphine may drop the expression of ER、PR mRNA 、BGP mRNA of female osteoblasts directly and reduce the expression of ER、PR mRNA 、BGP mRNA of female osteoblasts with dose dependent.更多还原