不同浓度HGF及EGF体外诱导兔BMSCs向肝细胞分化的研究

发布时间：2018-04-23 07:49

本文选题：骨髓间充质干细胞 + 肝细胞　；参考：《中南大学》2009年硕士论文

【摘要】： 目的通过不同浓度肝细胞生长因子(hepatocyte growth factor,HGF)和表皮细胞生长因子(epidermal cell growth factor,EGF)体外诱导兔骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)向肝细胞分化,以探讨HGF和EGF的最适浓度组合。方法密度梯度离心法分离兔BMSCs,体外培养,传代纯化。建立以HGF(30.0ug/L)+EGF(1.5mg/L)、HGF(40.0ug/L)+EGF(2.5mg/L)、HGF(50.0ug/L)+EGF(3.5mg/L)、HGF(60.0ug/L)+EGF(4.5mg/L)为诱导的培养体系。观察诱导细胞形态学变化,于7、14、21、28d采用免疫细胞化学及免疫荧光分别检测肝细胞表面标志甲胎蛋白(alphafetoprotein AFP)及白蛋白(albumin ALB),留取不同时段培养上清液,用溴甲酚绿法(bromcresol green BCG)检测上清液中ALB含量,用尿素合成试验检测尿素含量。结果14d开始可观察到多极性的肝细胞样细胞,呈短梭形和多角形,随诱导时间延长(21d)形成多细胞集落,各组形态学比较无明显差异。免疫细胞化学检测,7d时AFP表达阳性,14d达最大值后即开始下降(P＜0.05),各浓度组间表达无差别(P＞0.05)。免疫荧光检测,14d开始ALB表达阳性,1组、4组各时间点比较无差别(P＞0.05),2组及3组随诱导时间延长,阳性表达细胞数增加(P＜0.05)。纵向比较:总体上各组之间有显著性差异,且浓度越高,阳性细胞数越高(P＜0.05)。两两比较,在14d时1组与2组无差别,在21d时2组与3组表达无差别,28d时3组与4组表达无差别(p＞0.05)。溴甲酚绿法检测,12d细胞培养上清液中可检测到ALB,ALB含量在早期(9-15d)表现为浓度越高含量越高((P＜0.05),18或21d达高峰后下降,此时各浓度组间含量无差别(p＞0.05),最高峰值在第四组(HGF60ug/L、EGF4.5ug/L)。9d时可检测到尿素含量,各浓度组间表达无差别(p＞0.05)。结论HGF及EGF能诱导兔BMSCs分化为肝细胞,诱导时间约2-3周。诱导剂浓度与诱导分化率之间在诱导早期呈剂量依赖关系,高浓度的HGF及EGF可提高BMSCs向肝细胞的分化率。诱导剂最适组合为HGF60ug/L、EGF4.5mg/L。
[Abstract]:Objective to induce the differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) into hepatocytes by different concentrations of hepatocyte growth factor (HGF) and epidermal cell growth factor (EGF), and to explore the optimal concentration combination of HGF and EGF. Methods BMSCs were isolated by density gradient centrifugation and cultured in vitro. We set up a culture system that was induced by HGF 30.0ugr / L) EGF1.5mg / L) HGFF 40.0ugP / L) EGF2.5mg / L HGF50.0ugP / L) EGF3.5mg / L) EGF3.5mg / L) EGF4.5mg / L). The morphological changes of induced cells were observed. The hepatocyte surface marker alphafetoprotein AFPand albumin albumin albumin were detected by immunocytochemistry and immunofluorescence for 28 days. The content of ALB in supernatant was determined by bromcresol green method and urea content was detected by urea synthesis test. Results Multi-polarity hepatocyte-like cells were observed on the 14th day, which were spindle-shaped and polygonal, and formed multicellular colonies with the prolongation of induction time (21 d). There was no significant difference in morphology among the groups. Immunocytochemistry showed that the expression of AFP reached the maximum at day 14 and then decreased (P < 0.05). There was no difference in the expression of AFP between different concentration groups (P > 0.05). Immunofluorescence assay showed that there was no difference at each time point between group 1 and group 1 (P > 0.05) and group 3 (P > 0.05). The number of positive cells increased with the time of induction (P < 0.05). Longitudinal comparison: there was significant difference among the groups, and the higher the concentration, the higher the number of positive cells (P < 0.05). There was no difference between group 1 and group 2 at day 14, and group 2 and group 3 on day 21. There was no difference in expression between group 3 and group 4 on day 28 at day 2 and group 4 (P > 0.05). The concentration of ALB in the supernatant of cell culture for 12 days was detected by bromocresol green method. The results showed that the higher the concentration of ALB was, the higher the concentration was, the higher the concentration was, the higher the concentration was, P < 0.05, P < 0.05, or 21 days after reaching the peak. At the same time, there was no difference in the content of urea between the different concentration groups. The highest peak value of urea was detected at day 9 of HGF 60ugr / L, EGF 4.5ugP / L, and there was no difference in the expression of urea among the different concentration groups (P > 0.05). Conclusion HGF and EGF can induce rabbit BMSCs to differentiate into hepatocytes for about 2-3 weeks. There was a dose-dependent relationship between the concentration of inducer and the rate of induced differentiation at the early stage of induction. High concentrations of HGF and EGF could increase the differentiation rate of BMSCs into hepatocytes. The optimal combination of inducers was HGF 60ugr LnEGF4.5 mg / L 路L ~ (-1) 路L ~ (-1) 路L ~ (-1).
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

【参考文献】