NF-кB、P38MAPK在骨髓间充质干细胞肝向分化过程中的机制研究

发布时间：2018-05-20 17:56

本文选题：骨髓间充质干细胞 + 肝细胞　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:探究信号传导分子核因子-кB(NF-кB)和P38MAPK在参与调控肝细胞生长因子(Hepatocyte Growth Factor,HGF)为诱导剂的条件下体外培养骨髓间充质干细胞(bone mesenchymal stem cell,BMSC)向肝样细胞定向分化的进程中,NF-кB信号通路和P38MAPK信号通路对分化的调控是否存在着某种联系。P38MAPK作为NF-кB的上游通路,在分化的过程中是参与了NF-кB的激活。方法:本实验中所用到的骨髓间充质干细胞来源于四周龄的雄性SD大鼠。细胞的获取通过采用全骨髓贴壁法,利用不同细胞贴壁时间的差异,筛选出所需要的目标细胞。分离后的细胞在含10%胎牛血清的DMEM低糖培养液中进行培养,第一次换液为全换液,可以使得培养瓶中的大部分还没有贴壁或者贴壁不牢的非目的细胞或状态不佳的细胞冲出瓶外。首次换液时间为取材24小时后,之后每隔3天换一次培养液,当培养瓶中细胞融合率达到70%至80%时,使用胶原蛋白酶消化,1:2传代至新的培养瓶中。实验使用传代至第三代的骨髓间充质干细胞作为实验对象,使用肝细胞生长因子诱导细胞向肝样细胞定向分化。实验分为四大组:诱导组、NF-кB抑制组、P38MAPK抑制组以及阴性对照组。诱导组细胞的诱导环境为含有肝细胞生长因子的完全培养液,抑制组分为NF-кB抑制组和P38抑制组,这两组细胞是在HGF诱导组的基础上分别添加了NF-кB信号分子的抑制剂BAY 11-7082和P38MAPK信号分子的抑制剂SB203580,其他培养环境和培养时间相同。阴性对照组细胞培养环境为含有10%胎牛血清的DMEM低糖培养基。取培养20天后的细胞进行靛青绿(ICG)摄取实验检测已分化的肝细胞,蛋白质印迹检测NF-кB、p-P38和α1-抗胰蛋白酶(α1-antitrypsin,AAT)的表达,取培养第7天和第20天的细胞进行免疫组化染色检测NF-кB蛋白。结果:诱导20天后,HGF诱导组中的大部分细胞呈圆形或卵圆形肝样细胞状生长,靛氰绿实验发现这些细胞可摄取ICG,并在一定时间内将ICG排出胞体外,免疫印迹实验结果显示有特异的肝细胞标志物α1-抗胰蛋白酶AAT的表达,免疫组化结果显示,细胞培养7天时和20天时,细胞核内都有NF-кB的聚集,NF-кB在此过程中激活转移至细胞核内;NF-кB抑制组和P38抑制组中细胞与对诱导组比,NF-кB抑制剂BAY 11-7082和P38抑制剂SB203580的抑制效果明显,细胞对ICG的摄取和表达的α1-抗胰蛋白酶蛋白表和诱导组相比来看都有比较明显的降低。免疫印迹结果显示,P38抑制组中NF-кB蛋白表达量降低。这两组抑制组细胞的免疫组化结果显示NF-кB在细胞核中只有少量存在,NF-кB的核转移被抑制。结论:在肝细胞生长因子诱导骨髓间充质干细胞向肝细胞分化的过程中,NF-κB信号通路和P38MAPK信号通路均参与了肝样细胞定向分化的调控。P38MAPK通路可能通过激活下游NF-κB通路发挥作用。
[Abstract]:Objective: to explore the process of differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro under the condition of signal transduction molecular nuclear factor-BNF-NF-B) and P38MAPK participating in the regulation of Hepatocyte Growth FactorHGF (HGFs) as inducers, during the process of differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro. Whether there is some connection between signaling pathway and P38MAPK signaling pathway in the regulation of differentiation. P38 MAPK is the upstream pathway of NF- FB. In the process of differentiation is involved in the activation of NF- B. Methods: the bone marrow mesenchymal stem cells (BMSCs) used in this study were derived from four-week old male SD rats. The target cells were selected by using the whole bone marrow adherent method and the different adherence time of different cells. The isolated cells were cultured in DMEM low glucose medium containing 10% fetal bovine serum. Most of the cells in the culture bottle that have not adhered or are not adherent to the wall can be rushed out of the bottle by non-target cells or cells in poor condition. When the cell fusion rate in the culture bottle reached 70% to 80%, 1: 2 was digested with collagenase to be passed to the new culture bottle. Bone marrow mesenchymal stem cells (BMSCs) from passage to generation 3 were used in the experiment. Hepatocyte growth factor (HGF) was used to induce the differentiation of cells into hepatoid cells. The experiment was divided into four groups: induction group and negative control group. The inducing environment of the cells in the induction group was a complete medium containing hepatocyte growth factor, and the inhibitory group was divided into NF- and P38 inhibition groups. BAY 11-7082 and SB203580 were added to the two groups on the basis of HGF induction, respectively. The other culture environment and the culture time were the same. The culture environment of negative control group was DMEM low glucose medium containing 10% fetal bovine serum. After 20 days of culture, the differentiated hepatocytes were detected by ICG uptake assay, the expression of 伪 1-antitrypsin (AATA) and the expression of NF-BP38 were detected by Western blot, and the NF-B protein was detected by immunohistochemical staining on the 7th and 20th day of culture. Results: after 20 days of induction, most of the cells in the HGF-induced group showed round or oval hepatocyte-like growth. It was found by indigo green experiment that these cells could absorb ICG and expel ICG out of the cell body within a certain period of time. The results of immunoblotting showed that there was a specific hepatocyte marker 伪 1-antitrypsin AAT expression. The immunohistochemical results showed that the cells were cultured at 7 days and 20 days after culture. During this process, NF-NF-B was activated and transferred to the cells in the NF-NF-B inhibition group and the P38 inhibitory group, and the inhibitory effect of NF-B inhibitor BAY 11-7082 and P38 inhibitor SB203580 was more obvious than that of the induced group, and the inhibitory effect of NF-B inhibitor BAY 11-7082 and P38 inhibitor SB203580 was significantly higher than that of the induction group. The uptake and expression of 伪 1-antitrypsin protein in cells were significantly lower than those in the induction group. The results of Western blot showed that the expression of NF- FB protein was decreased in P38 inhibition group. The immunohistochemical results of these two groups showed that only a small number of nuclear metastases of NFNF-B were inhibited in the nuclei of these two groups. Conclusion: both NF- 魏 B signaling pathway and P38MAPK signaling pathway are involved in the regulation of hepatocyte-like differentiation by activating downstream NF- 魏 B pathway in the process of hepatocyte growth factor inducing mesenchymal stem cells to differentiate into hepatocytes.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2

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