UMSCs对急性肝衰竭小鼠的免疫调节作用的研究

发布时间：2018-03-12 09:45

本文选题：脐带间充质干细胞　切入点：培养　出处：《苏州大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的建立小鼠脐带间充质干细胞UMSCs的分离、培养和扩增的方法；确定分离、培养的细胞能够稳定传代，有很强的增殖活性，为以后的细胞移植创造了较好的前提条件。采用四氯化碳建立小鼠急性肝衰竭模型，并观察小鼠的一般状况；了解UMSCs移植对急性肝衰竭的疗效，包括肝功能和肝组织病理变化；观察移植前后血清中IL-4和IFN-含量变化；检测不同时间点肝组织中PD-1mRNA的水平变化。方法应用双酶消化法消化C57BL/6孕鼠脐带组织，行贴壁和传代培养，，绘制细胞生长曲线和观察细胞形态。用四氯化碳(CCl4)溶于橄榄油,配成浓度为50%的四氯化碳，予3ml/kg分组腹腔注射给药。小鼠分为三组，实验组、移植对照组和空白对照组，分别在7d、14d、21d检测肝功能。血清IL-4和IFN-含量采用酶联免疫吸附实验(ELISA)测定。取不同时间点的肝组织行石蜡切片和HE染色，观察肝脏病理变化情况。RT-PCR检测不同时间点肝组织中PD-1mRNA的水平表达变化。结果 1.以胶原酶和胰酶两步消化法对小鼠UMSCs进行了分离培养，获得了成功。两周之后，主要为长梭形或扁平形的成纤维样细胞，细胞呈束状密集平行排列。经过多次消化、传代后，细胞形态仍为长梭状，且形态更加均匀，有很强的增殖活性。群体倍增时间为24h左右。2.成功建立小鼠急性肝衰竭模型。3.移植组小鼠肝功能和造模组比较差异有统计学差异（P＜0.05）。4.移植UMSCs1w、2w、3w后各时间点IL-4和IFN-浓度和造模组比较，IL-4呈上升趋势，而IFN-呈下降趋势。5.UMSCs可改善急性肝衰竭小鼠的炎症浸润，有利于肝小叶结构恢复正常。6.在正常的小鼠肝脏组织中，PD-1有一定的表达，造模组12h、24h、48h PD-1和正常组比较，差异有统计学意义（P＜0.05）。移植UMSCs后24h、48h PD-1和正常组比较，差异无统计学意义(P＞0.05)，和造模组比较，差异有统计学意义（P＜0.05）。结论成功建立了小鼠UMSCs的分离及培养体系，传代培养后仍有较强的增殖能力。UMSCs移植可改善肝衰竭小鼠的存活率、肝功能和肝组织病理变化。UMSCs移植可以反映IL-4和IFN-的抗炎/促炎的平衡关系。PD-1可以抑制正向免疫，可能在急性肝衰竭小鼠和UMSCs移植早期肝脏炎症损伤中发挥重要免疫调控作用。
[Abstract]:Purpose. Methods of isolation, culture and amplification of UMSCs from mouse umbilical cord mesenchymal stem cells were established. The model of acute liver failure in mice was established by carbon tetrachloride, and the general condition of mice was observed, and the curative effect of UMSCs transplantation on acute liver failure, including liver function and pathological changes of liver tissue, was investigated. The changes of serum IL-4 and IFN- were observed before and after transplantation, and the levels of PD-1mRNA in liver tissue were measured at different time points. Method. The umbilical cord tissue of C57BL / 6 pregnant mice was digested by double enzyme digestion, followed by adherent and passage culture, cell growth curve was drawn and cell morphology was observed. CCL _ 4 was dissolved in olive oil and was mixed with carbon tetrachloride at concentration of 50%. The mice were divided into three groups: experimental group, transplant control group and blank control group. The serum IL-4 and IFN- contents were measured by Elisa. The liver tissues at different time points were stained with HE and paraffin sections. To observe the pathological changes of liver. RT-PCR was used to detect the expression of PD-1mRNA in liver tissue at different time points. Results. 1. The mouse UMSCs was isolated and cultured successfully by collagenase and trypsin digestion. After two weeks, the cells were mainly long fusiform or flattened fibroblast cells arranged in bundles. The shape of the cells is still long fusiform, and the shape is more uniform. It has very strong proliferative activity. The population doubling time is about 24 hours. 2. The mouse model of acute liver failure has been established successfully. 3. There is significant difference in liver function and model group between transplantation group and model group (P < 0. 05. 4). The IL-4 and IFN- concentration at each time point 3 weeks after transplanting UMSCs for 1 wk and 2 wW after transplantation have significant difference (P < 0. 05, P < 0. 05, P < 0. 05). Compared with the model group, IL-4 showed an increasing trend. But IFN- decreased. 5. UMSCs could improve the inflammatory infiltration of mice with acute liver failure, which was beneficial to the recovery of hepatic lobule structure to normal .6.There was a certain expression of PD-1 in normal liver tissue of mice. Compared with the normal group, the model group had the same expression of PD-1 at 24 h and 48 h, compared with the normal group, and the expression of PD-1 in the model group was significantly higher than that in the control group. The difference was statistically significant (P < 0.05). There was no significant difference between the PD-1 group and the normal group at 24 h after UMSCs transplantation (P > 0.05), and there was significant difference between the model group and the control group (P < 0.05). Conclusion. The isolation and culture system of mouse UMSCs was successfully established. Liver function and liver histopathological changes. UMSCs transplantation can reflect the anti-inflammatory / pro-inflammatory balance of IL-4 and IFN-. PD-1 can inhibit positive immunity, which may play an important role in regulating liver inflammation injury in mice with acute liver failure and early UMSCs transplantation.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R575.3

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