UMSCs对急性肝衰竭小鼠的免疫调节作用的研究
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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。
Objective
To explore isolation，cultivation and proliferation of mice umbilical mesenchymalstem cells(UMSCs)；To prove isolated,cultured cells can be stably passage, and have astrong proliferative activility, they can create a better prerequisite for future celltransplantation. To establish acute liver failure mode with carbon tetrachloride(CCl4) andobserve the general status of the mice；To get straight the therapeutic effectiveness ofUMSCs on Acute Liver Failure，including liver function and liver pathology.To observeserum IL-4and IFN-content before and after transplantation.To detect PD-1mRNA levelchanges at different time points in liver tissue.
Methods
UMSCs were isolated from C57BL/6pregnant mice umbilical cord bytrypsin-collagenase digestion. The cells were adherent to culture flask and subculture.Thecell growth curve was plotted and observe cell morphology. With carbontetrachloride(CCl4) dissolved in olive oil,dubbed the concenttaton of50%,and packetintraperitoneal injection. The mice were divided into three groups,including theexperimental group, the transplant control group and blank control group, to detect theliver function in7,14and21days. IL-4and IFN-content were determined byenzyme-linked immunosorbent assay(ELISA). Paraffin section and HE staining were takenat different time points in hepatic tissue samples.And after this,to observe liverpathological changes. Expression of PD-1mRNA in hepatic tissue samples were detectedby reverse transcription-polymerase chain reaction(RT-PCR) at different time points.
Results
1.UMSCs were harvested successfully from UC by trypsin-collagenasedigestion.After two weeks, cells were long spindle or flat-shaped fibroblast-like,and they arranged in bundle-intensive parallel. After several digestions and subculture,they werestill long spindle,had more unifom shape,had a strong proliferative activity.Populationdoubling time of about24hours.2.To establish acute liver failure mode successfully.3.Liver function of the transplanted group and model group mice was statistically significantdifference （P＜0.05）.4. Comparison at different time of IL-4and IFN-content aftertransplantation UMSCs in1,2,and3weeks, IL-4was an upward trend, and IFN-was andownward trend.5. UMSCs can improve inflammatory infiltration of acute liver failure inmice, and hepatic lobule structure was conducive to return to normal.6. In normal miceliver tissue,PD-1had a low expression; after model in12,24and48hours, comparedwith blank control group, the expression of PD-1was statistically significant difference（P＜0.05）. After transplantation in24and48hours, compared with blank controlgroup, the expression of PD-1was not statistically significant （P＜0.05）, comparedwith the model group, the difference was statistically significant （P＜0.05）.
Conclusions
We was successful to isolate and establish of culture system of umbilical cordmesenchymal stem cells from mice. After several subculture,they still had a strongproliferative activity. UMSCs transplantation can improve the survival rate of mice withliver falure,liver function and liver pathology. UMSCs transplantation can balance therelations of IL-4and IFN-. PD-1can be suppressed in forward immune and may play animportant role in mice of acute liver failure and early transplantation liver inflammatoryinjury on immunomodulatory effects.
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。
Objective
To explore isolation，cultivation and proliferation of mice umbilical mesenchymalstem cells(UMSCs)；To prove isolated,cultured cells can be stably passage, and have astrong proliferative activility, they can create a better prerequisite for future celltransplantation. To establish acute liver failure mode with carbon tetrachloride(CCl4) andobserve the general status of the mice；To get straight the therapeutic effectiveness ofUMSCs on Acute Liver Failure，including liver function and liver pathology.To observeserum IL-4and IFN-content before and after transplantation.To detect PD-1mRNA levelchanges at different time points in liver tissue.
Methods
UMSCs were isolated from C57BL/6pregnant mice umbilical cord bytrypsin-collagenase digestion. The cells were adherent to culture flask and subculture.Thecell growth curve was plotted and observe cell morphology. With carbontetrachloride(CCl4) dissolved in olive oil,dubbed the concenttaton of50%,and packetintraperitoneal injection. The mice were divided into three groups,including theexperimental group, the transplant control group and blank control group, to detect theliver function in7,14and21days. IL-4and IFN-content were determined byenzyme-linked immunosorbent assay(ELISA). Paraffin section and HE staining were takenat different time points in hepatic tissue samples.And after this,to observe liverpathological changes. Expression of PD-1mRNA in hepatic tissue samples were detectedby reverse transcription-polymerase chain reaction(RT-PCR) at different time points.
Results
1.UMSCs were harvested successfully from UC by trypsin-collagenasedigestion.After two weeks, cells were long spindle or flat-shaped fibroblast-like,and they arranged in bundle-intensive parallel. After several digestions and subculture,they werestill long spindle,had more unifom shape,had a strong proliferative activity.Populationdoubling time of about24hours.2.To establish acute liver failure mode successfully.3.Liver function of the transplanted group and model group mice was statistically significantdifference （P＜0.05）.4. Comparison at different time of IL-4and IFN-content aftertransplantation UMSCs in1,2,and3weeks, IL-4was an upward trend, and IFN-was andownward trend.5. UMSCs can improve inflammatory infiltration of acute liver failure inmice, and hepatic lobule structure was conducive to return to normal.6. In normal miceliver tissue,PD-1had a low expression; after model in12,24and48hours, comparedwith blank control group, the expression of PD-1was statistically significant difference（P＜0.05）. After transplantation in24and48hours, compared with blank controlgroup, the expression of PD-1was not statistically significant （P＜0.05）, comparedwith the model group, the difference was statistically significant （P＜0.05）.
Conclusions
We was successful to isolate and establish of culture system of umbilical cordmesenchymal stem cells from mice. After several subculture,they still had a strongproliferative activity. UMSCs transplantation can improve the survival rate of mice withliver falure,liver function and liver pathology. UMSCs transplantation can balance therelations of IL-4and IFN-. PD-1can be suppressed in forward immune and may play animportant role in mice of acute liver failure and early transplantation liver inflammatoryinjury on immunomodulatory effects.
引文
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