腺病毒介导BMP-2联合低氧诱导因子1α突变型与单基因BMP-2分别转染BMSCs后成骨效应的比较研究

COMPARATIVE STUDY ON OSTEOGENIC EFFECT OF BONE MARROW MESENCHYMAL STEM CELLS TRANSFECTED BY ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 2-INTERNAL RIBOSOME ENTRYSITE-HYPOXIA INDUCIBLE FACTOR 1α^(mu) AND BY BONE MORPHOGENETIC PROTEIN 2 SINGLE GENE

目的比较腺病毒载体Ad-BMP-2-内部核糖体进入位点(internal ribosome entry site,IRES)-低氧诱导因子1α突变型(hypoxia inducible factor 1αmu,HIF-1αmu)与Ad-巨细胞病毒(cytomegalovirus,CMV)-BMP-2-IRES-人源化海肾绿色荧光蛋白1(human renilla reniformis green fluorescent protein 1,hrGFP-1)分别转染BMSCs后的成骨效应,优化成骨细胞种子来源。方法取1月龄新西兰大白兔骨髓分离培养BMSCs。取第3代BMSCs进行病毒液转染,根据转染病毒液不同将实验分为4组,A、B、C组分别采用感染复数(multiplicity of infection,MOI)为50、100、150和200的Ad-BMP-2-IRES-HIF-1αmu、Ad-CMV-BMP-2-IRES-hrGFP-1、Ad-CMV-IRES-hrGFP-1(空腺病毒载体)转染细胞,D组为未被转染的BMSCs。选择最佳MOI值进行观察。转染后采用免疫组织化学染色检测BMP-2表达,Western blot检测BMP-2和HIF-1α蛋白表达,ALP活性测定和钙结节茜素红染色检测细胞成骨分化能力。结果 A、B、C组最佳MOI值分别为200、150和100。免疫组织化学染色示,A、B组BMP-2染色呈阳性,C、D组呈阴性;A组阳性细胞数明显多于B组(P<0.05)。Western blot检测示,A、B组BMP-2蛋白表达明显高于C、D组(P<0.05),A组高于B组(P<0.05);A组HIF-1α蛋白表达明显高于其余3组(P<0.05),其余3组间差异无统计学意义(P>0.05)。A、B组ALP活性明显高于C、D组(P<0.05),A组高于B组(P<0.05)。茜素红染色示,A、B组可见明显钙结节,C、D组未见钙结节形成;且A组钙结节数量多于B组(P<0.05)。结论单载体双基因Ad-BMP-2-IRES-HIF-1αmu与单载体单基因Ad-CMV-BMP-2-IRES-hrGFP-1分别转染兔BMSCs后,前者BMP-2和成骨效应表达均高于后者。

Objective To compare the osteogenic effect of bone marrow mesenchymal stem cells（BMSCs） transfectedby adenovirus-bone morphogenetic protein 2-internal ribosome entry site-hypoxia inducible factor 1αmu（Ad-BMP-2-IRESHIF1αmu） and by Ad-cytomegalovirus（CMV）-BMP-2-IRES-human renilla reniformis green fluorescent protein 1（hrGFP-1）single gene so as to optimize the source of osteoblasts.Methods BMSCs were separated and cultured from 1-month-old NewZealand white rabbit.The BMSCs at passage 3 were transfected by virus.The experiment was divided into 4 groups（groups A,B,C,and D） according to diff erent virus： BMSCs were transfected by Ad-BMP-2-IRES-HIF-1αmu in group A,by Ad-CMV-BMP-2IRES-hrGFP-1 in group B,by Ad-CMV-IRES-hrGFP-1 in group C,and BMSCs were not transfected in group D.The optimummultiplicity of infection（MOI）（50,100,150,and 200） was calculated and then the cells were transfected by the optimum MOI,respectively.The expression of BMP-2 gene was detected by immunohistochemistry staining after transfected,the expressionsof BMP-2 protein and HIF-1α protein were detected by Western blot method.The osteogenic diff erentiation potential wasdetected by alkaline phosphatase（ALP） activity and Alizarin red staining.Results The optimum MOI of groups A,B,andC was 200,150,and 100,respectively.The expression of BMP-2 was positive in groups A and B,and was negative in groups Cand D by immunohistochemistry staining;the number of positive cells in group A was more than that in group B（P 0.05）.The expression of BMP-2 protein in groups A and B was significantly higher than that in groups C and D（P 0.05）,group A washigher than group B（P 0.05）.The expression of HIF-1α protein in group A was significantly higher than those in the other 3groups（P 0.05）,no significant diff erence was found among the other 3 groups（P 0.05）.ALP activity in groups A and B wassignificantly higher than that in groups C and D（P 0.05）,group A was higher than group B（P 0.05）.Calcium nodules could beseen in groups A and B,but not in groups C and D;the number of calcium nodules in group A was higher than that in group B（P 0.05）.Conclusion The expression of BMP-2 and osteogenic effect of BMSCs transfected by Ad-BMP-2-IRES-HIF-1αmu（doublegenes in single carrier） are higher than those of BMSCs transfected by Ad-CMV-BMP-2-IRES-hrGFP-1（one gene in single carrier）.