不同剂量IGF-1对人脐带间充质干细胞增殖与分化的影响

发布时间：2018-07-01 20:58

本文选题：1、人脐带间充质干细胞 + IGF-I　；参考：《兰州大学》2011年硕士论文

【摘要】：目的：1、探讨并优化人脐带间充质干细胞(hunman Umbilical Cord Mesenchymal Stem Cells hUCMSCsi)体外分离.纯化.扩增及培养条件,并对其基本生物学特性进行研究；2、观察不同剂量外源性IGF-1对hUCMSCs体外增殖的影响及其量效关系；3、观察在不同浓度IGF-1处理下,人脐带间充质干细胞(hUCMSCsi)诱导成脂后生长状态的变化,初步探讨用于促进hUCMSCs诱导成脂的最适IGF-1浓度。方法：1、将剔除血管的新鲜新生儿脐带组织切成小块培养,得到贴壁细胞,倒置相差显微镜观察细胞形态,细胞计数绘制细胞生长曲线；流式细胞仪测定细胞表面抗原；化学染色检测其体外成脂和成骨诱导分化能力；2、取第3代hUCMSCs接种于96孔板,分为7组(G1,G2…,G7)。前5组分别加入含不同剂量IGF-I(40,60,80,100,120μg/L)的体积分数为0.02的胎牛血清培养液；第6组加入体积分数为0.05的胎牛血清培养液做为阳性对照,第7组加入体积分数为0.02的胎牛血清培养液做为阴性对照,采用MTT法观察不同剂量的IGF-I对细胞增殖的影响。3、将第3代的hUCMSCs随机分为5组,在成脂诱导24h后,分别加入40 ng/ml,、60 ng/ml、80 ng/ml,、100 ng/ml和120 ng/ml的IGF-1,对5组样本进行四甲基偶氮唑盐(MTT)法检测其光密度值(optical density,OD值)。取第8天的样本进行油红O染色,观察脂滴形成情况,计算红色脂滴数量,并利用多个样本均数的方差分析法比较5组之间的差别结果：1、体外培养4-6天后,有细胞从组织块中游出；细胞传代培养至第10代,无明显的形态和增殖能力改变；细胞表达CD13,CD44,而CD14、D34、HLA-DR表达成阴性。体外诱导实验证实,该细胞成脂和成骨分化的能力；2、①第3、第6天,G1组、G2组和G7组之间差异无显著性(P0.05)；G3组、G4组和G5组之间差异无显著性(P0.05)。②第3天,G6组增殖高于G1组与G2组,低于G3组、G4组和G5组,差异有显著性(P0.000或0.006)；第4天,G6组增殖高于其余各组,差异有显著性(P0.000或0.006)。③第3天、第6天,G1组、G2组和G6组低于G3组,G4组,G5组,差异有显著性(P0.000)；G6组增殖高于G7组,统计分析差异有显著性(P0.000)。3、①各组OD值之间的差异没有统计学意义(P0.05)；②80 ng/ml组、100ng/ml组和120ng/ml组成脂数量明显多于40ng/ml组与60ng/ml组,P值均小于0.01；而80ng/ml组、100ng/ml组和120ng/ml组3组之间在成脂数量上的差异没有显著统计学意义(P0.05).结论：80 ng/ml为IGF-1促进人脐带间充质干细胞诱导成脂的适宜浓度。结论：1、hUCMSCs能在体外培养、纯化及扩增,其基本生物学特性和骨髓间充质干细胞相似；2、外源性IGF-I对hUCMSCs增殖的促进作用,随IGF-I浓度增高而有增高趋势,100μg/L为其较适合浓度,为实现hUCMSCs体外快速扩增提供适宜的条件；3、80 ng/ml为IGF-1促进人脐带间充质干细胞诱导成脂的适宜浓度。
[Abstract]:Objective to investigate and optimize the isolation of human umbilical cord mesenchymal stem cells (hunman UCM SCsi) in vitro. Purification. The effects of different doses of exogenous IGF-1 on the proliferation of hUCMSCs in vitro and the dose-response relationship between IGF-1 and HUCMSCs were observed, and the effects of different concentrations of IGF-1 on the proliferation of hUCMSCs were observed. The changes of growth state of human umbilical cord mesenchymal stem cells (hUCMSCsi) induced by lipids were studied. Methods: 1. Fresh umbilical cord tissue was cut into small pieces and adherent cells were obtained. Cell morphology was observed by inverted phase contrast microscope, cell growth curve was plotted by cell count, and cell surface antigen was measured by flow cytometry. The ability of adipogenic and osteogenic differentiation in vitro was detected by chemical staining. The 3rd generation of hUCMSCs was inoculated on 96 well plate and divided into 7 groups (G1G 2. G7). The first five groups were fed with 0.02 fetal bovine serum culture medium containing different doses of IGF-I (4060 渭 g / L, 80100120 渭 g / L), the sixth group added 0.05 fetal bovine serum culture medium as positive control. In group 7, the effects of different doses of IGF-I on cell proliferation were observed by MTT assay, and the third passage of hUCMSCs were randomly divided into 5 groups. The optical density value (OD value) was measured by MTT method in 5 groups of IGF-1 with the addition of 40 ng / ml ~ (60) ng / ml ~ (-1) ~ (80) ng / ml ~ (-1) and 120 ng/ml of IGF-1, respectively. The samples on the 8th day were stained with oil red O, the formation of lipid droplets was observed, the number of red lipid droplets was calculated, and the difference between the five groups was compared by the variance analysis method of the mean numbers of several samples. The difference between the five groups was compared by using the analysis of variance. After 4-6 days of culture in vitro, the difference between the five groups was compared. There were no obvious changes in morphology and proliferative ability of the cells from tissue mass to the 10th passage, and the expression of CD13, CD44 and HLA-DR in CD14 D34 was negative. In vitro induction experiments confirmed that there was no significant difference between G _ 2 group and G _ 7 group in the ability of adipogenic and osteogenic differentiation between G _ 2 group and G _ 7 group on the 3rd and 6th day (P0.05). There was no significant difference between G3 group G4 group and G5 group (P0.05) on the 3rd day, the proliferation of G6 group was higher than that of G1 group and G2 group, and lower than that of G3 group G4 group and G5 group (P0.000 or 0.006), the proliferation of G6 group was higher than that of other groups on the 4th day. The difference was significant (P0. 000 or 0.006) on the 3rd day. On the 6th day, the proliferation of G6 group was higher than that of G7 group, while that of G6 group was lower than that of G3 group G4 group G5 group, and on the 6th day, the proliferation of G6 group was higher than that of G7 group. There was no significant difference in OD value between the three groups (P0. 000). 3. There was no significant difference in OD value between the three groups (P0.05). The amount of lipid composition of 100ng / ml and 120ng/ml in 280 ng/ml group was significantly higher than that in 40ng/ml group and 60ng/ml group (P < 0.01). However, there was no significant difference in fat production between 80ng/ml group (100ng / ml) and 120ng/ml group (P0.05). Conclusion the optimal concentration of IGF-1 for inducing lipids induced by human umbilical cord mesenchymal stem cells is ng/ml. Conclusion the primary biological characteristics of human UCMSCs cultured, purified and amplified in vitro are similar to those of bone marrow mesenchymal stem cells. The effect of exogenous IGF-I on the proliferation of hUCMSCs is similar to that of bone marrow mesenchymal stem cells. The suitable concentration of IGF-I is 100 渭 g / L with the increase of IGF-I concentration. In order to achieve rapid expansion of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro, the suitable conditions for the rapid expansion of hUCMSCs in vitro were established.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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