CAJ | 학술논문

背景:人胰岛素样生长因子1基因对脂肪源性干细胞的增殖和分化也可能会产生有效作用.目的:验证人胰岛素样生长因子1基因转染对体外培养的脂肪源性干细胞的效应.方法:构建含人胰岛素样生长因子1基因的双顺反子真核表达载体 pIRES2-EGFP-hIGF-1,利用阳离子脂质体 Lipofectamine 2000介导转染体外培养的人脂肪源性干细胞.观察基因转染后细胞增殖及形态的变化,倒置荧光显微镜观测标记基因增强绿色荧光蛋白的表达并计算转染效率,酶联免疫吸附试验检测培养上清中人胰岛素样生长因子1的浓度,免疫组织化学染色及 RT-PCR 检测人胰岛素样生长因子1的表达,流式细胞仪检测转染前后细胞周期的变化.结果与结论:测序及酶切证实真核表达载体 pIRES2-EGFP-hIGF-1构建正确.体外培养的脂肪源性干细胞为多种形态并存,转染后6 h 检测到有 EGFP 的表达,至60 h 达到高峰,转染效率为(16±3)%.细胞上清中人胰岛素样生长因子1的浓度在60 h 达到22.65μg/L.免疫组织化学染色及 RT-PCR 均检测到人胰岛素样生长因子1的表达.转染后的细胞分裂增殖加快,细胞群体倍增时间缩短,S 期细胞比例增多.证实人胰岛素样生长因子1基因可有效转染脂肪源性干细胞并表达人胰岛素样生长因子1蛋白质,同时可促进细胞增殖.
배경:인이도소양생장인자1기인대지방원성간세포적증식화분화야가능회산생유효작용.목적:험증인이도소양생장인자1기인전염대체외배양적지방원성간세포적효응.방법:구건함인이도소양생장인자1기인적쌍순반자진핵표체재체 pIRES2-EGFP-hIGF-1,이용양리자지질체 Lipofectamine 2000개도전염체외배양적인지방원성간세포.관찰기인전염후세포증식급형태적변화,도치형광현미경관측표기기인증강록색형광단백적표체병계산전염효솔,매련면역흡부시험검측배양상청중인이도소양생장인자1적농도,면역조직화학염색급 RT-PCR 검측인이도소양생장인자1적표체,류식세포의검측전염전후세포주기적변화.결과여결론:측서급매절증실진핵표체재체 pIRES2-EGFP-hIGF-1구건정학.체외배양적지방원성간세포위다충형태병존,전염후6 h 검측도유 EGFP 적표체,지60 h 체도고봉,전염효솔위(16±3)%.세포상청중인이도소양생장인자1적농도재60 h 체도22.65μg/L.면역조직화학염색급 RT-PCR 균검측도인이도소양생장인자1적표체.전염후적세포분렬증식가쾌,세포군체배증시간축단,S 기세포비례증다.증실인이도소양생장인자1기인가유효전염지방원성간세포병표체인이도소양생장인자1단백질,동시가촉진세포증식.
BACKGROUND: Human insulin-like growth factor 1 gene produces effects on the proliferation and differentiation of adipose-derived stem cells. OBJECTIVE: To investigate the possible effects of human insulin-like growth factor 1 gene transfection on the adipose-derived stem cells cultured in vitro. METHODS: The cDNA for human insulin-like growth factor 1 was cloned into the recombinant eukaryotic expression plasmid pIRES2-EGFP-hIGF-1. The adipose-derived stem cells were derived from adipose tissue and cultured in vitro, and then the plasmid was transfected into cells by Lipofectamine 2000. After gene transfection, changes in cel proliferation and morphology were observed. Through the use of inverted fluorescent microscopy, the marker gene coding enhanced green fluorescent protein was observed and the transfection efficiency was determined. The human insulin growth factor 1 concentration in the supernatant fluids was measured by ELISA. The expression of human insulin-like growth factor 1 was detected by immunohistochemical staining and RT-PCR. The changes in cel cycle before and after transfection were assessed by flow cytometry. RESULTS AND CONCLUSION: The recombinant eukaryotic expression plasmid pIRES2-EGFP-hIGF-1 was confirmed by gene sequencing and enzyme digestion. The adipose-derived stem cells cultured in vitro were in multiple shapes. The expression of enhanced green fluorescent protein was detected for the first time at 6 hours after transfection and peaked at 60 hours, with transfection efficacy of 16±3%. The concentration of insulin-like growth factor 1 in the supernatant fluid was 22.65 μg/L at 60 hours after transfection. Human insulin growth factor 1 expression was detected by immunohistochemical staining and RT-PCR. Division and proliferation of the cells were accelerated after gene transfection, leading to the decrease of the population doubling time, and the increase of the percentage of stem cells in the S stage. These results indicate that human insulin growth factor 1 gene can be transfected efficiently into the adipose-derived stem cells by liposome and human insulin-like growth factor 1 protein can be secreted into the supernatant fluid, therefore accelerating the proliferation of the cells.