骨髓基质细胞分化状态对腺病毒介导骨形态发生蛋白2表达效应的影响

Differentiation state of bone marrow stromal cells influences the expression of adenovirus mediated bone morphogenic protein

目的:利用骨形态发生蛋白2腺病毒转染向成骨细胞分化的骨髓基质细胞,在体外观察骨形态发生蛋白2的表达量和反应成骨细胞特性的指标变化。方法:实验于2002-04/12在北京大学第三医院中心实验室进行。6周龄雄性BALB/c小鼠60只用于骨髓基质细胞提取,小鼠骨髓基质细胞分别用Dulbecco改良的Eagle培养基液和骨诱导培养基(含地塞米松10-8mol/L、L-抗坏血酸50mg/L、β-甘油磷酸钠10mmol/LDulbecco改良的Eagle培养基液)培养。①不同量骨形态发生蛋白2腺病毒转染骨髓基质细胞的碱性磷酸酶比活性的比较:取原代培养的骨髓基质细胞,分别加入转染指数25,50,100,200骨形态发生蛋白2腺病毒,以不加骨形态发生蛋白2腺病毒的骨髓基质细胞作为对照。6d后收集细胞,测定碱性磷酸酶活性。②骨髓基质细胞诱导分化后转染骨形态发生蛋白2腺病毒的碱性磷酸酶比活性的比较:实验分5组:骨髓基质细胞组,用Dulbecco改良的Eagle培养基培养9d;骨诱导培养3d组,骨诱导培养3d改用Dulbecco改良的Eagle培养基培养6d;骨诱导培养9d组,骨诱导培养9d;骨诱导培养3d+骨形态发生蛋白2腺病毒组,骨诱导培养3d,加入转染指数50的骨形态发生蛋白2腺病毒作用6d;骨形态发生蛋白2腺病毒组,用Dulbecco改良的Eagle培养基培养3d,加入同量骨形态发生蛋白2腺病毒作用6d。收集细胞,测定碱性磷酸酶比活性。③骨形态发生蛋白2与骨桥蛋白检测:采用免疫组织化学染色与蛋白印迹方法。实验采用拉丁方设计。结果:应用骨诱导培养基或转染骨形态发生蛋白2腺病毒均使细胞碱性磷酸酶比活性明显增加(P<0.05)。①不同量骨形态发生蛋白2腺病毒转染骨髓基质细胞碱性磷酸酶比活性的比较:骨形态发生蛋白2腺病毒(转染指数=50)组碱性磷酸酶比活性显著高于骨髓基质细胞组和骨形态发生蛋白2腺病毒(转染指数=25,100,200)组犤(7658.53±1600.32)比(1932.89±665.30),(4311.53±1108.89),(5360.90±1374.61),划内(2661.86±653.46)nkat/(g·L),P<0.01犦。②骨髓基质细胞诱导分化后转染骨形态发生蛋白2腺病毒的碱性磷酸酶比活性的比较:骨诱导培养3d+骨形态发生蛋白2腺病毒(转染指数=50)组与骨形态发生蛋白2腺病毒(转染指数=50)组差异无显著性(P>0.05),显著高于骨诱导培养3d组和骨诱导培养9d组(P<0.05)。③骨形态发生蛋白2和骨桥蛋白检测结果:骨诱导培养3d+骨形态发生蛋白2腺病毒组两指标表达量均最高。结论:单纯用诱导Dulbecco改良的Eagle培养基以及先诱导培养再加入骨形态发生蛋白2腺病毒均可促进骨髓基质细胞向成骨细胞方向分化,但是骨髓基质细胞用骨诱导培养基作用3d再加入骨形态发生蛋白2腺病毒能分泌表达更多的骨形态发生蛋白2,从而提高了目的基因表达的效应。

AIM： To transfect the bone marrow stromal cells （BMSCs） that have the trend to differentiate into the osteoblasts by bone morphogenic protein-2 （BMP2） mediated with adenovirus（AdBMP-2） and investigate the expression of BMP2 and the changes of indicators which reflect the properties of osteoblasts. METHODS： The experiment was conducted in the central laboratory of Peking University Third Hospital from April to December 2002. Stromal cells were derived from the bone marrow of 60 male BALB/C mice at the age of 6 weeks, and then were cultured in Dulbecco＇s modified Eagle＇ s medium（DMEM） or in bone-induced medium（DMEM consisting of 10^-8 mol/L dexamethasone, 50 mg/L L-aseorbie acid and 10 mmol/L β- glycerophosphate）. ① Comparison of specific activity of alkaline phosphatase（ALP） in BMSCs transfected by various contents of AdBMP-2： The BMSCs cultured primarily were added with AdBMP-2 of transfection exponents of 25, 50, 100 and 200 respectively, and those not transfected with AdBMP-2 were used as controls. The BMSCs were collected 6 days later to measure the activity of ALP. ②Specific activity of ALP in BMSCs transfected by AdBMP-2 after induced to differentiate： Our experiment was conducted in 5 groups： BMSC group, in which the BMSCs were cultured in DMEM for 9 days; 3-day osteoinductive culture group in which the BMSCs were cultured in DMEM for 6 days after a 3-day osteoinductive culture; 9-day osteoinductive culture group in which the BMSCs were cultured in DMEM for 9 days; 3-day osteoinductive culture plus AdBMP-2 group in which the BMSCs were added with the AdBMP-2 （transfection exponent： 50） and cultured for 6 days after a 3-day osteoinductive culture; AdBMP-2 group in with the BMSCs were cultured in DMEM for three days, then simply transduced with AdBMP-2 of the same volume and cultured for next six days. The cultured BMSCs were collected for the detection of specific activity of ALP. ③The changes of BMP2 and osteopontin were detected by immunohistoehemistry and Western blotting. The experiment was performed by latin square design. RESULTS： The specific activity of ALP was promoted by either osteoinductive culture medium or AdBMP-2. ①In the comparison of ALP specific activity in BMSCs transfected by AdBMP-2 of different transfection exponents： The specific activity of ALP was significantly higher in tile BMSCs transfected by AdBMP-2 （transfection exponent： 50） than in the BMSC controls and in the BMSCs transfected AdBMP-2 （transfection exponent： 25, 100, 200）[（7 658.53±1 600.32） nkat/（g.L） vs （1 932.89±665.30）, （4 311.53±1 108.89）, （5 360.90±1 374.61）, （2 661.86±653.46） nkat/（g· L）, P 〈 0.01].②In the comparison of specific activity of ALP in BMSCs transfected by AdBMP-2 after induced to differentiate： The specific activity of ALP was insignificantly different between the 3-day osteoinductive culture plus AdBMP-2（transfection exponent： 50） group and AdBMP-2（transfection exponent： 50） group（P 〉 0.05）, both of which were significantly higher than that in the 3-day osteoinductive culture group and in the 9-day osteoinductive culture group （P 〈 0.05）. ③However, the expressions of BMP2 and osteopontin in BMSCs were the higher in the 3-day osteoinductive culture plus AdBMP-2 group than in the other groups. CONCLUSION： The differentiation in of BMSCs into osteoblasts can be promoted by culturing in DMEM or by osteoinductive medium plus next AdBMP-2, but if induced with osteoinductive medium for 3 days followed by AdBMP-2, the BMSCs can secret more BMP2, so the efficiency of expression of target genes is improved.