中华创伤杂志
中華創傷雜誌
중화창상잡지
Chinese Journal of Traumatology
2009年
2期
169-172
,共4页
胰岛素样生长因子Ⅰ%间质干细胞%转染%细胞周期%软骨细胞
胰島素樣生長因子Ⅰ%間質榦細胞%轉染%細胞週期%軟骨細胞
이도소양생장인자Ⅰ%간질간세포%전염%세포주기%연골세포
Insulin-like growth factor-Ⅰ%Mesenchymal stem cells%Transfection%Cell cycle%Chondrocyte
目的 探讨人胰岛素样生长因子Ⅰ(hIGF-Ⅰ)转染脂肪间质干细胞(ADSCs)的效果及表达情况,探寻转染后ADSCs向软骨细胞分化的可能性. 方法 从兔颈后脂肪分离培养ADSCs,检测其生长特性.用Lipofectamine 2000介导将质粒pcDNA3.1+hlGF-Ⅰ转染ADSCs,转染后4～72 h、传代后和稳定转染后,分别用倒置显微镜观察细胞的生长情况,计算转染效率.ELISA检测上清液中hlGF-Ⅰ的浓度变化.免疫组化检测Ⅱ型胶原的表达.RT-PCR检测hIGF-Ⅰ mRNA和Western blot检测hIGF-Ⅰ蛋白表达情况.流式细胞仪检测细胞周期分布情况.结果细胞接种后4 h开始贴壁,呈长梭形,24 h时增多,呈集簇状分布.转染后增生活跃,倍增时间缩短.转染72 h后分泌hIGF-Ⅰ达高峰,为32.45 ng/ml,稳定转染后hIGF-Ⅰ保持恒定为28.36 ng/ml.转染后细胞出现多种形态共存.免疫组化证实有大量Ⅱ型胶原形成.hIGF-ⅠmRNA和hIGF-Ⅰ蛋白呈阳性表达.细胞周期中s期增加,与未转染组比较,差异有统计学意义(P<0.05).结论稳定转染后ADSCs可较长时间分泌较高浓度的hIGF-Ⅰ,能促进ADSCs的增殖、分化,并可形成软骨样物质.
目的 探討人胰島素樣生長因子Ⅰ(hIGF-Ⅰ)轉染脂肪間質榦細胞(ADSCs)的效果及錶達情況,探尋轉染後ADSCs嚮軟骨細胞分化的可能性. 方法 從兔頸後脂肪分離培養ADSCs,檢測其生長特性.用Lipofectamine 2000介導將質粒pcDNA3.1+hlGF-Ⅰ轉染ADSCs,轉染後4～72 h、傳代後和穩定轉染後,分彆用倒置顯微鏡觀察細胞的生長情況,計算轉染效率.ELISA檢測上清液中hlGF-Ⅰ的濃度變化.免疫組化檢測Ⅱ型膠原的錶達.RT-PCR檢測hIGF-Ⅰ mRNA和Western blot檢測hIGF-Ⅰ蛋白錶達情況.流式細胞儀檢測細胞週期分佈情況.結果細胞接種後4 h開始貼壁,呈長梭形,24 h時增多,呈集簇狀分佈.轉染後增生活躍,倍增時間縮短.轉染72 h後分泌hIGF-Ⅰ達高峰,為32.45 ng/ml,穩定轉染後hIGF-Ⅰ保持恆定為28.36 ng/ml.轉染後細胞齣現多種形態共存.免疫組化證實有大量Ⅱ型膠原形成.hIGF-ⅠmRNA和hIGF-Ⅰ蛋白呈暘性錶達.細胞週期中s期增加,與未轉染組比較,差異有統計學意義(P<0.05).結論穩定轉染後ADSCs可較長時間分泌較高濃度的hIGF-Ⅰ,能促進ADSCs的增殖、分化,併可形成軟骨樣物質.
목적 탐토인이도소양생장인자Ⅰ(hIGF-Ⅰ)전염지방간질간세포(ADSCs)적효과급표체정황,탐심전염후ADSCs향연골세포분화적가능성. 방법 종토경후지방분리배양ADSCs,검측기생장특성.용Lipofectamine 2000개도장질립pcDNA3.1+hlGF-Ⅰ전염ADSCs,전염후4～72 h、전대후화은정전염후,분별용도치현미경관찰세포적생장정황,계산전염효솔.ELISA검측상청액중hlGF-Ⅰ적농도변화.면역조화검측Ⅱ형효원적표체.RT-PCR검측hIGF-Ⅰ mRNA화Western blot검측hIGF-Ⅰ단백표체정황.류식세포의검측세포주기분포정황.결과세포접충후4 h개시첩벽,정장사형,24 h시증다,정집족상분포.전염후증생활약,배증시간축단.전염72 h후분비hIGF-Ⅰ체고봉,위32.45 ng/ml,은정전염후hIGF-Ⅰ보지항정위28.36 ng/ml.전염후세포출현다충형태공존.면역조화증실유대량Ⅱ형효원형성.hIGF-ⅠmRNA화hIGF-Ⅰ단백정양성표체.세포주기중s기증가,여미전염조비교,차이유통계학의의(P<0.05).결론은정전염후ADSCs가교장시간분비교고농도적hIGF-Ⅰ,능촉진ADSCs적증식、분화,병가형성연골양물질.
Objective To investigate the effectiveness of human insulin-like growth factor-Ⅰ(hIGF-Ⅰ) in transfecting adipose-derived mesenchymal stem cells (AdMSCs) and the expression of the transfected genes and find the possibility of differentiation of ADSCs to chondrocytes after hIGF-Ⅰ transfection. Methods AdMSCs were extracted from the fat tissue of the back of rabbits' neck and cultured in vitro by monolayer. Then, the recombinant eukaryotic expression plasmid poDNA3.1 + hIGF-Ⅰwas transfected into AdMSCs by Lipofectamine 2000. The growth of the ADSCs was observed by inverted microscope at 4-72 hours after transfection, at the end of passage and after stable transfection respectively, and the transfection efficiency was calculated. The hIGF-Ⅰ concentration in the supernatant was detected by enzyme-linked immnnosorbent assay (ELISA), the expression of collagen-Ⅱ by immunohistochemistry, the expression of hIGF-Ⅰ mRNA by reverse transcription polymerase chain reaction (RTPCR), the expression of hIGF-Ⅰ protein by Western blot and the cell cycle by flow cytometry. Results AdMSCs were shaped long shuttle and adhered to the disk at the 4th hour. AdMSCs were increased and distributed in cluster at 24th hour. After tranafection, AdMSCs were proliferated actively, with shortened doubling generation time. The concentration of hlGF-I in the supematant increased slowly after transfection and reached peak at 32.45 ng/ml at the 72nd hour. The hIGF-Ⅰ concentration remained at 28.36 ng/ml after stable transfectian. Immunohistochemistry affirmed a large number of collagen-Ⅱ in the endochylema and positive expression of hIGF-Ⅰ mRNA and hIGF-Ⅰ protein. The percentage of AdMSCs at stage S increased after transfection, which was significantly higher than that of AdMSCs without transfection, with statistical difference (P < 0.05). Conclusion After stable transfection, AdMSCs can secrete high concentration of hIGF-Ⅰ that can promote cell proliferation and differentiation of AdMSCs to chondrocytes.
