高糖对BMP-2、IGF-Ⅰ基因转染大鼠BMSC增殖的影响

发布时间：2018-12-16 22:58

【摘要】：背景骨折愈合是一个复杂的机体自身修复过程,需要多种生长因子参与。临床上糖尿病合并骨折患者很常见,延迟愈合或不愈合发生率高,是临床医师面临的棘手问题。研究认为糖尿病骨折愈合过程中血清及局部骨痂组织中骨形态发生蛋白-2(Bone morphogenetic protein-2, BMP-2)、胰岛素样生长因子-I(Insulin-like growth factor-1, IGF-I)含量减少是导致骨折延迟愈合、不愈合的原因之一。随着干细胞工程和组织工程的研究进展,骨髓基质干细胞已成为研究和治疗多种骨科疾病及其合并症的一种重要方法。BMP-2、IGF-I均具有促进BMSC增殖及向成骨细胞分化的作用,而关于两者联合应用对BMSC增殖及分化效应尚缺乏系统研究。目的高糖环境下通过测定BMP-2、IGF-I基因转染大鼠BMSC后,目的蛋白表达情况及BMSC的增殖情况,进一步了解基因转染BMSC的生物学特性,为构建新型组织工程骨种子细胞提供思路,从而为临床上应用BMSC移植治疗糖尿病骨折不愈合、延迟愈合提供实验依据。方法用Ad-BMP-2和Ad-IGF-I转染大鼠BMSC,按转染情况分为5组：A组：未转染组；B组：转染空载体组；C组：转染BMP-2组；D组：转染IGF-I组；E组：BMP-2加IGF-I共转染组。分别于转染处理后24h、48h、72h、96h荧光显微镜下观察转染效果；不同时间点观察各组大鼠BMSC的形态变化；利用蛋白印迹法(Western blotting)检测转染后24h、48h、72h各组细胞BMP-2和IGF-I的表达情况。绘制细胞生长曲线、采用噻唑蓝(MTT)比色法及流式细胞仪,观察大鼠BMSC的生长及增殖情况。利用SPSS16.0统计软件进行统计,P0.05表示差异有统计学意义。结果(1)基因转染组(C、D、E组)与未转染组及空载体组比较,转染处理后初期,细胞形态无明显变化；随着培养时间延长,基因转染组多角形细胞增多。(2)荧光显微镜下观察到基因转染组大部分细胞呈现绿色荧光,Ad-BMP-2转染大鼠BMSC后48h时最强,Ad-IGF-I转染大鼠BMSC后72h时最强,Ad-BMP-2-IGF-I转染大鼠BMSC后72h时最强。转染率均高达90%以上。(3)蛋白印迹法检测到转染组细胞中有BMP-2和IGF-I蛋白表达,且随时间延长目的蛋白表达逐渐增强。(4)生长曲线、MTT法及流式细胞仪结果显示第5天各组BMSC增殖能力趋于达到高峰,其中E组增殖能力最强(P0.055)。5d光吸收值：A-E组分别为0.324±0.027,0.319±0.017,0.622±0.028,0.626±0.020,0.778±0.031。流式细胞术结果显示A-E组细胞处于增殖期的比重分别为23.92±3.07,23.51±2.11,34.37±6.85,35.04±1.45,42.56±1.15。结论高糖环境下在腺病毒载体介导下BMP-2、IGF-I基因转染BMSC是可行的,经BMP-2、IGF-I基因转染的BMSC能够稳定表达外源性BMP-2和IGF-I。BMP-2和IGF-I均有促进体外培养的BMSC增殖的作用,联合应用促增殖作用最强,两者对BMSC增殖有协同增强效应。
[Abstract]:Background fracture healing is a complex process of self-repair, which requires a variety of growth factors. Diabetes mellitus complicated with fracture is very common in clinic. Delayed healing or high incidence of nonunion is a difficult problem for clinicians. Bone morphogenetic protein-2 (Bone morphogenetic protein-2, BMP-2 and insulin-like growth factor I (Insulin-like growth factor-1,) in serum and local callus during the healing process of diabetic fracture were considered. Reduction of IGF-I content is one of the causes of delayed union and non-union of fracture. With the development of stem cell engineering and tissue engineering, bone marrow stromal cells have become an important method for the study and treatment of various orthopaedic diseases and their complications. BMP-2, IGF-I can promote the proliferation and differentiation of BMSC into osteoblasts. However, there is no systematic study on the effect of BMSC proliferation and differentiation. Objective to investigate the biological characteristics of BMP-2,IGF-I gene transfection into rat BMSC by measuring the expression of target protein and the proliferation of BMSC after transfection of BMSC gene in high glucose environment. To provide ideas for constructing new tissue engineering bone seed cells and provide experimental basis for clinical application of BMSC transplantation in the treatment of nonunion of diabetic fracture and delayed healing. Methods Rats with Ad-BMP-2 and Ad-IGF-I were divided into five groups according to the transfection conditions: group A: untransfected group, group B: empty vector group, group C: transfected BMP-2 group, group D: transfected IGF-I group, group B: transfected empty vector group, group C: transfected BMP-2 group, group D: transfected IGF-I group; Group E: BMP-2 and IGF-I co-transfection group. The transfection effect was observed under fluorescence microscope at 24 h, 48 h, 72 h and 96 h after transfection, and the morphologic changes of BMSC were observed at different time points. The expression of BMP-2 and IGF-I was detected by Western blot (Western blotting) at 24 h, 48 h and 72 h after transfection. The growth and proliferation of rat BMSC were observed by (MTT) colorimetry and flow cytometry. The use of SPSS16.0 statistical software for statistics, P0.05 means the difference is statistically significant. Results (1) compared with the control group and the empty vector group, the cell morphology in the gene transfection group (CnDX E group) did not change significantly at the beginning of transfection. With the increase of culture time, the number of polygonal cells in the gene transfection group increased. (2) most of the cells in the gene transfection group showed green fluorescence under fluorescence microscope, which was the strongest at 48h after Ad-BMP-2 transfection into BMSC of rats. The strongest was at 72 h after Ad-IGF-I transfection into BMSC and the strongest at 72 h after Ad-BMP-2-IGF-I transfection into BMSC. The transfection efficiency was above 90%. (3) the expression of BMP-2 and IGF-I in transfected cells was detected by Western blotting, and the expression of target protein was gradually increased with time. (4) growth curve. The results of MTT and flow cytometry showed that the proliferative ability of BMSC tended to peak on the 5th day. The value of light absorption in group A was 0.324 卤0.027 卤0.319 卤0.017 卤0.622 卤0.028 卤0.626 卤0.0200.78 卤0.031, respectively. The results of flow cytometry showed that the proportion of cells in proliferation phase in A-E group was 23.92 卤3.07 卤23.51 卤2.1134.37 卤6.85 卤35.04 卤1.45 卤42.56 卤1.15, respectively. Conclusion it is feasible to transfect BMP-2,IGF-I gene into BMSC under high glucose environment mediated by adenovirus vector, and it is feasible to transfect BMSC by BMP-2,. BMSC transfected with IGF-I gene could stably express exogenous BMP-2, IGF-I.BMP-2 and IGF-I, and both of them could promote the proliferation of BMSC cultured in vitro. The combination of IGF-I.BMP-2 and IGF-I had the strongest effect on the proliferation of BMSC, and they had synergistic effect on the proliferation of BMSC.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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