非病毒载体Lip2000介导BMP2基因转染BMSCs促进大鼠骨折愈合

Effects of non viral vector Lip2000 mediated-BMP2 transfection of BMSCs on promoting fracture healing in rats

目的探讨非病毒载体LipofectamineTM2000(Lip2000)介导人骨形态发生蛋白2(BMP2)基因体内转染骨髓间充质干细胞(BMSCs)促进大鼠骨折愈合的效果。方法 20只大鼠建立股骨骨折模型,根据随机抽签原则分为观察组与对照组大鼠各10只。观察组大鼠股骨骨折断端间隙局部注射Lip2000转染BMP2:注射Lip2000后取大鼠红骨髓,将传代培养后生长旺盛的红骨髓细胞与BMP2转染试剂的混合物(聚乙烯亚胺和BMP2基因质粒的混合液)在细胞培养液中培养以转染BMSCs,转染后再植入骨折部位。对照组骨折断端注射相同体积生理盐水。观察骨折愈合情况,回滴定法测定骨密度,Western blot法检测BMP2在骨折间软组织中的相对表达水平。结果大体标本观察发现观察组骨痂生成量多于对照组。X线显示观察组基因转染处的骨折线较对照组明显模糊。观察组大鼠的骨密度随基因转染的时间增加(转染后1周→转染后8周)而增加[(0.32±0.09)mg/cm^3vs(0.39±0.11)mg/cm^3,P<0.05],但在转染后1周[(0.32±0.09)mg/cm^3vs(0.26±0.11)mg/cm^3,P<0.01]和转染后8周[(0.39±0.11)mg/cm^3vs(0.22±0.21)mg/cm^3,P<0.01]观察组的骨密度均高于对照组。Western blot检测分析显示基因转染1周与8周后,观察组的BMP2相对表达水平均明显高于对照组。结论 Lip2000介导BMP2基因进入大鼠的BMSCs中可诱导其向成骨细胞分化,促进骨折后骨小梁形成和缩短骨折愈合时间。

Objective To investigate the effects of human bone morphogenetic protein 2 （ BMP＇2 ） transfection of bone marrow mesenchymal stem cells （BMSCs） mediated by non viral vector Lip2000 on promoting fracture healing of rats. Methods Establishing rat femoral fracture model,the rats were divided into observation group and control group （ n = 10 each） according to the principle of drawing. In observation group, Lip2000 was injected into the local space between femoral fractured ends to transfect BMP2：taking rat bone marrow after injecting Lip2000, the eugonic red bone marrow cells after subculture were cultured with the mixture of BMP＇2 transfection reagent contenting polyethyleneimine and BMP2 gene plas-mid in cell culture medium to transfect BMSCs and transplanted back to the fractured site after transfection. In control group, the normal saline of same volume was injected into the space between femoral fractured ends. Fracture healing status was observed. Back titration method was used to detect bone density. Western blot method was used to detect the relative expression level of BMP2 in the soft tissues between fractured ends. Results Gross specimen observation showed that the amount of callus formation in observation group was more than that in control group. X-ray displayed that the fracture line in transfection site in observation group was obvious indistinct than that of control group. Bone density values in observation group increased with the increase of transfection time [ （ 0.32±0.09 ） mg/cm^3 （ at 1 w） vs （0. 39±0.11 ） mg/cm3 （ at 8w） ,P 〈0.05 ] and the bone density values at 1- and 8- week after transfection in observation group were significantly higher than those at 1 - and 8- week after modeling in control group [ （0.32±0. 09 ） mg/cm^3 vs （0.26±0. 11 ） mg/cm3 , P 〈0. 01; （0.39±0.11 ） mg/cm^3 vs （0.22±0.21 ） mg/cm^3 ,P 〈0.01 ] ）. Western blot result showed that the relative ex-pression levels of BMP2 one week and eight weeks after transfection in observation group were significantly higher than those in control group （ all P 〈 0.05 ）. Conclusion BMSCs trasfected by Lip2000 mediated-BMP2 can be induced to differentiate into osteoblasts, promote the formation of bone trabecula and shorten the healing time of fracture.