CAJ | 학술논문

目的观察骨形态发生蛋白-2(BMP-2)基因转染人脐带间充质干细胞(hUCMSC)对其成骨分化和体内异位成骨的影响.方法实验分为3组,空白组:10％胎牛血清+ DMEM培养基,对照组:10％胎牛血清+DMEM培养基+空慢病毒载体转染,实验组:10％胎牛血清+DMEM培养基+BMP-2+增强型绿色荧光蛋白(EGFP)基因转染,检测3组不同培养基培养后人脐带间充质细胞的碱性磷酸酶(ALP)活性变化,蛋白印迹法检测骨桥蛋白(OPN)、Ⅰ型胶原蛋白(COL1)、BMP-2的蛋白表达变化.yon kossa染色观察hUCMSC钙结节形成.大鼠肌袋内植入BMP-2转染hUCMSC/COL1,通过CT三维重建及苏木素-伊红(HE)染色观察其体内成骨能力.结果 hUCMSC BMP-2转染良好,转染率可达到(90.95±4.35)％,实验组细胞在第5天,ALP活性就由(26.492 ±7.105) U/g·蛋白增加至(38.625±11.592) U/g·蛋白,在第14天,实验组的ALP的活性可以高达(49.732±13.068) U/g·蛋白,对比空白组和对照组都有明显增加(P＜0.05).对比空白组和对照组,实验组COL1、BMP-2和OPN蛋白呈现高表达(P＜0.05).转染BMP-2基因后,hUCMSC培养28 d可形成大量的钙结节.BMP-2转染hUCMSC/COL1在大鼠肌袋内可异位成骨.结论转染BMP2基因可促进hUCMSC细胞成骨能力.
목적 관찰골형태발생단백-2(BMP-2)기인전염인제대간충질간세포(hUCMSC)대기성골분화화체내이위성골적영향.방법 실험분위3조,공백조:10％태우혈청+ DMEM배양기,대조조:10％태우혈청+DMEM배양기+공만병독재체전염,실험조:10％태우혈청+DMEM배양기+BMP-2+증강형록색형광단백(EGFP)기인전염,검측3조불동배양기배양후인제대간충질세포적감성린산매(ALP)활성변화,단백인적법검측골교단백(OPN)、Ⅰ형효원단백(COL1)、BMP-2적단백표체변화.yon kossa염색관찰hUCMSC개결절형성.대서기대내식입BMP-2전염hUCMSC/COL1,통과CT삼유중건급소목소-이홍(HE)염색관찰기체내성골능력.결과 hUCMSC BMP-2전염량호,전염솔가체도(90.95±4.35)％,실험조세포재제5천,ALP활성취유(26.492 ±7.105) U/g·단백증가지(38.625±11.592) U/g·단백,재제14천,실험조적ALP적활성가이고체(49.732±13.068) U/g·단백,대비공백조화대조조도유명현증가(P＜0.05).대비공백조화대조조,실험조COL1、BMP-2화OPN단백정현고표체(P＜0.05).전염BMP-2기인후,hUCMSC배양28 d가형성대량적개결절.BMP-2전염hUCMSC/COL1재대서기대내가이위성골.결론 전염BMP2기인가촉진hUCMSC세포성골능력.
Objective To investigate the biological characteristics of human umbilical cord mesenchymal stem cells (hUCMSC) transfected with.bone morphogenetic protein-2 (BMP-2) gene in vitro and in vivo.Methods hUCMSC were cultured in vitro.Based on the different culture media added,three groups were designed:blank group (10％ FCS/DMEM),control group (10％ FCS/DMEM + empty lentiviral particles),experimental group (10％ FCS/DMEM + BMP-2 lentiviral particles).Cell differentiation was examined by alkaline phosphatase (ALP) measurement kit.The expression of osteopontin ( OPN ),type 1 collagen ( COL1 ) and BMP-2 was detected by using Western boltting.Von kossa staining method was used to study the calcification effects. In the rat muscle bag, the complex containing BMP-2-transfected hUCMSCs/COL1 was transplanted.The ectopic bone in the implanted area was observed by the CT 3D reconstruction and hematoxylin and eosin (HE) staining.Results The positive rate of hUCMSC transfected with BMP-2 gene was (90.95 ± 4.35 ) ％.As compared with the blank group and control group,the proliferation in experimental group was promoted at 5th and 7th day after culture (P ＜ 0.05 ).The ALP activity was increased from (26.492 ±7.105) U/g·prot at 3rd day to (38.625 ± 11.592) U/g·prot at 5th day,even to (49.732 ± 13.068) U/g·prot at 14th day in experimental group as compared with other two groups (P ＜ 0.05).The COL1,OPN and BMP-2 proteins were overexpressed in experimental group,but weakly expressed in the blank group and control group ( P ＜ 0.05 ).Many mineralized nodes were observed 18 days after transfection of hUCMSC with BMP-2 gene.The ectopic bone formed in the rat muscle bag implanted with BMP-2-transfected hUCMSCs/COL1.Conclusion Transfection of BMP-2 gene can promote osteogenic potential of hUCMSCs.