双基因重组载体对乙醇作用下兔干细胞增殖与成骨活性的影响

Effect of double gene recombinant vector on the proliferation and osteogenic activity of rabbit stem cells induced by ethanol

目的 观察双基因重组载体对乙醇作用下兔干细胞增殖与成骨活性的影响.方法 取第3代兔骨髓间充质干细胞（BMSCs）,随机分为6组：（1）正常组：正常培养BMSCs,无基因转染;（2）模型组：无基因转染BMSCs;（3）无关序列组：10μl无关序列载体转染BMSCs;（4）沉默过氧化物酶体增殖子活化受体γ （siPPARγ）组：10μl siPPARγ基因载体转染BMSCs;（5）表达降钙素基因相关肽（exCGRP）组：10μl CGRP基因载体转染BMSCs;（6）双基因组：10μl双基因重组载体转染BMSCs.除正常组外,其余各组细胞给予终质量浓度0.09 mol/L乙醇.采用噻唑蓝（MTT）法检测BMSCs增殖,第14天检测各组细胞内碱性磷酸酶活性、层粘连蛋白、Ⅰ型胶原、培养基中骨钙素含量,并作碱性磷酸酶（ALP）染色.结果 双基因组细胞增殖明显,增殖曲线接近于正常组.细胞中ALP活性、层粘连蛋白、Ⅰ型胶原、培养基中骨钙素含量：双基因组数值最高,分别为（18.593±2.350） U/L、（24.422 ±3.110） ng/ml、（5.951 ±0.650） μg/L、（5.836 ±0.630） ng/ml,显著高于模型组[（6.528 ±0.830） U/L、（9.422±1.250） ng/ml、（1.733±0.230） μg/L、（2.016 ±0.240） ng/ml]、无关序列组[（7.011±0.850） U/L、（9.693±1.260） ng/ml、（1.663 ±0.220） μg/L、（1.913±0.230） ng/ml],明显高于siPPARγ组[（13.536 ±1.710） U/L、（17.103±2.230） ng/ml、（3.486±0.410） μg/L、（3.686±0.420） ng/ml]、exCGRP组[（13.692 ±1.760） U/L、（17.185 ±2.240） ng/ml、（3.525±0.420） μg/L、（3.833±0.430） ng/ml]、正常组[（15.056±1.910） U/L、（18.528±2.430） ng/ml、（4.035±0.460）μg/L、（4.012±0.460） ng/ml],差异均有统计学意义（均P=0.000）.模型组、无关序列组数值显著低于正常组,差异均有统计学意义（均P=0.000）.siPPARγ组、exCGRP组数值略低于正常组,差异均无统计学意义：ALP活性（P=0.222、0.274）,层粘连蛋白（P =0.362、0.390）,Ⅰ型胶原（P=0.082、0.105）,骨钙素（P =0.276、0.543）.双基因组数值显著高于正常组,差异有统计学意义：ALP活性（P=0.031）,层粘连蛋白（P=0.010）,Ⅰ型胶原、骨钙素（均P =0.001）;且明显高于siPPARγ组、exCGRP组,差异均有统计学意义：ALP活性（P =0.005、0.006）,层粘连蛋白（P=0.003、0.003）,Ⅰ型胶原、骨钙素（均P=0.000）.ALP染色显示：双基因组中大量细胞胞质染色为深蓝色,且较正常组多.结论 双基因重组载体可以保持乙醇作用下兔干细胞正常增殖,显著促进成骨活性,优于单一基因作用.

Objective To investigate the effects of double gene recombinant vector on the prolifer-ation and osteogenesis of alcohol-induced rabbit stem cells.Methods The rabbit bone marrow mesenchy-mal stem cells （BMSCs） at the third-generation were randomly divided into 6 groups：（1） normal group[bone marrow stem cells （BMSCs） without special treatment];（2） model group （BMSCs without gene transfection）;（3） unrelated sequence group （10 μl unrelated sequence transfected BMSCs）;（4） siPPARγ group （10 μ l siPPARγ gene vector transfected BMSCs）;（5） exCGRP group （10 μl CGRP gene vector transfected BMSCs）;（6） double gene group （10 μl double gene recombinant vector transfected BMSCs）.The final mass concentration of 0.09 mol/L alcohol was added to all groups except the normal group.BMSCs proliferation was detected by methyl thiazol tetrazolium （MTT） method.Alkaline phosphatase （ALP） activity,laminin content,collagen type Ⅰ content,and osteocalcin content in medium and ALP staining were detected at 14th day later.Results The proliferation in double gene group was obvious,and the proliferation curve was close to the normal group.The ALP activity,laminin content,collagen type Ⅰ coutent and osteocalcin content in medium of the cells in the double gene group were the highest [（18.593 ±2.350） U/L,（24.422 ±3.110） ng/ml,（5.951 ±0.650） μg/L,（5.836 ±0.630） ng/ml respectively],which were significantly higher than those in model group [（6.528 ±0.830） U/L,（9.422 ±1.250） ng/ml,（1.733 ± 0.230） μg/L,（2.016 ± 0.240） ng/ml],unrelated sequence group [（7.011 ±0.850） U/L,（9.693 ± 1.260） ng/ml,（1.663 ±0.220） μg/L,（1.913 ±0.230） ng/ml],obviously higher than those in siPPARγ group [（13.536 ± 1.710） U/L,（17.103 ± 2.230） ng/ml,（3.486 ± 0.410） μg/L,（3.686 ± 0.420） ng/ml],exCGRP group [（13.692 ± 1.760） U/L,（17.185± 2.240） ng/ml,（3.525 ± 0.420） μg/L,（3.833 ± 0.430） ng/ml],normal group [（15.056±1.910） U/L,（18.528±2.430） ng/ml,（4.035 ±0.460） μg/L,（4.012±0.460） ng/ml],and the difference was statistically significant （all P =0.000）.The contents in model group and unrelated sequence group were significantly lower than those in normal group （all P =0.000）.The contents in siPPARγ and exCGRP groups were slightly lower than those in normal group：for ALP activity （P =0.222,0.274）,for laminin （P =0.362,0.390）,for collagen type Ⅰ （P =0.082,0.105）,and for osteocalcin （P =0.276,0.543）.The contents in double gene group were significantly higher than those in normal group：for ALP activity （P =0.031）,for laminin （P =0.010）,for collagen type Ⅰ and osteocalcin （all P =0.001）.The contents in double gene group were significantly higher than those in siPPARγ and exCGRP groups：for ALP activity （P =0.005,0.006）,for laminin （all P =0.003）,for collagen type Ⅰ and osteocalcin （all P =0.000）.ALP staining showed that the cytoplasm in a large number of cells in double gene group was dyed dark blue and more than that in normal group.Conclusion The double gene recombinant vector can maintain the normal proliferation of rabbit stem cells induced by ethanol,and significantly promote osteogenic activity,which is superior to the single gene effect.