高糖對BMP-2、IGF-Ⅰ基因轉(zhuǎn)染大鼠BMSC增殖的影響
發(fā)布時間：2018-12-16 22:58
【摘要】：背景骨折愈合是一個復(fù)雜的機(jī)體自身修復(fù)過程,需要多種生長因子參與。臨床上糖尿病合并骨折患者很常見,延遲愈合或不愈合發(fā)生率高,是臨床醫(yī)師面臨的棘手問題。研究認(rèn)為糖尿病骨折愈合過程中血清及局部骨痂組織中骨形態(tài)發(fā)生蛋白-2(Bone morphogenetic protein-2, BMP-2)、胰島素樣生長因子-I(Insulin-like growth factor-1, IGF-I)含量減少是導(dǎo)致骨折延遲愈合、不愈合的原因之一。隨著干細(xì)胞工程和組織工程的研究進(jìn)展,骨髓基質(zhì)干細(xì)胞已成為研究和治療多種骨科疾病及其合并癥的一種重要方法。BMP-2、IGF-I均具有促進(jìn)BMSC增殖及向成骨細(xì)胞分化的作用,而關(guān)于兩者聯(lián)合應(yīng)用對BMSC增殖及分化效應(yīng)尚缺乏系統(tǒng)研究。 目的高糖環(huán)境下通過測定BMP-2、IGF-I基因轉(zhuǎn)染大鼠BMSC后,目的蛋白表達(dá)情況及BMSC的增殖情況,進(jìn)一步了解基因轉(zhuǎn)染BMSC的生物學(xué)特性,為構(gòu)建新型組織工程骨種子細(xì)胞提供思路,從而為臨床上應(yīng)用BMSC移植治療糖尿病骨折不愈合、延遲愈合提供實驗依據(jù)。 方法用Ad-BMP-2和Ad-IGF-I轉(zhuǎn)染大鼠BMSC,按轉(zhuǎn)染情況分為5組：A組：未轉(zhuǎn)染組；B組：轉(zhuǎn)染空載體組；C組：轉(zhuǎn)染BMP-2組；D組：轉(zhuǎn)染IGF-I組；E組：BMP-2加IGF-I共轉(zhuǎn)染組。分別于轉(zhuǎn)染處理后24h、48h、72h、96h熒光顯微鏡下觀察轉(zhuǎn)染效果；不同時間點觀察各組大鼠BMSC的形態(tài)變化；利用蛋白印跡法(Western blotting)檢測轉(zhuǎn)染后24h、48h、72h各組細(xì)胞BMP-2和IGF-I的表達(dá)情況。繪制細(xì)胞生長曲線、采用噻唑藍(lán)(MTT)比色法及流式細(xì)胞儀,觀察大鼠BMSC的生長及增殖情況。利用SPSS16.0統(tǒng)計軟件進(jìn)行統(tǒng)計,P0.05表示差異有統(tǒng)計學(xué)意義。 結(jié)果(1)基因轉(zhuǎn)染組(C、D、E組)與未轉(zhuǎn)染組及空載體組比較,轉(zhuǎn)染處理后初期,細(xì)胞形態(tài)無明顯變化；隨著培養(yǎng)時間延長,基因轉(zhuǎn)染組多角形細(xì)胞增多。(2)熒光顯微鏡下觀察到基因轉(zhuǎn)染組大部分細(xì)胞呈現(xiàn)綠色熒光,Ad-BMP-2轉(zhuǎn)染大鼠BMSC后48h時最強,Ad-IGF-I轉(zhuǎn)染大鼠BMSC后72h時最強,Ad-BMP-2-IGF-I轉(zhuǎn)染大鼠BMSC后72h時最強。轉(zhuǎn)染率均高達(dá)90%以上。(3)蛋白印跡法檢測到轉(zhuǎn)染組細(xì)胞中有BMP-2和IGF-I蛋白表達(dá),且隨時間延長目的蛋白表達(dá)逐漸增強。(4)生長曲線、MTT法及流式細(xì)胞儀結(jié)果顯示第5天各組BMSC增殖能力趨于達(dá)到高峰,其中E組增殖能力最強(P0.055)。5d光吸收值：A-E組分別為0.324±0.027,0.319±0.017,0.622±0.028,0.626±0.020,0.778±0.031。流式細(xì)胞術(shù)結(jié)果顯示A-E組細(xì)胞處于增殖期的比重分別為23.92±3.07,23.51±2.11,34.37±6.85,35.04±1.45,42.56±1.15。 結(jié)論高糖環(huán)境下在腺病毒載體介導(dǎo)下BMP-2、IGF-I基因轉(zhuǎn)染BMSC是可行的,經(jīng)BMP-2、IGF-I基因轉(zhuǎn)染的BMSC能夠穩(wěn)定表達(dá)外源性BMP-2和IGF-I。BMP-2和IGF-I均有促進(jìn)體外培養(yǎng)的BMSC增殖的作用,聯(lián)合應(yīng)用促增殖作用最強,兩者對BMSC增殖有協(xié)同增強效應(yīng)。
[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 鹵0.027 鹵0.319 鹵0.017 鹵0.622 鹵0.028 鹵0.626 鹵0.0200.78 鹵0.031, respectively. The results of flow cytometry showed that the proportion of cells in proliferation phase in A-E group was 23.92 鹵3.07 鹵23.51 鹵2.1134.37 鹵6.85 鹵35.04 鹵1.45 鹵42.56 鹵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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363
本文編號：2383187
[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 鹵0.027 鹵0.319 鹵0.017 鹵0.622 鹵0.028 鹵0.626 鹵0.0200.78 鹵0.031, respectively. The results of flow cytometry showed that the proportion of cells in proliferation phase in A-E group was 23.92 鹵3.07 鹵23.51 鹵2.1134.37 鹵6.85 鹵35.04 鹵1.45 鹵42.56 鹵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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363
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