本文選題：臍帶間充質(zhì)干細胞　切入點：培養(yǎng)　出處：《蘇州大學》2013年碩士論文　論文類型：學位論文

【摘要】：目的 建立小鼠臍帶間充質(zhì)干細胞UMSCs的分離、培養(yǎng)和擴增的方法；確定分離、培養(yǎng)的細胞能夠穩(wěn)定傳代，有很強的增殖活性，為以后的細胞移植創(chuàng)造了較好的前提條件。采用四氯化碳建立小鼠急性肝衰竭模型，并觀察小鼠的一般狀況；了解UMSCs移植對急性肝衰竭的療效，包括肝功能和肝組織病理變化；觀察移植前后血清中IL-4和IFN-含量變化；檢測不同時間點肝組織中PD-1mRNA的水平變化。 方法 應(yīng)用雙酶消化法消化C57BL/6孕鼠臍帶組織，行貼壁和傳代培養(yǎng)，，繪制細胞生長曲線和觀察細胞形態(tài)。用四氯化碳(CCl4)溶于橄欖油,配成濃度為50%的四氯化碳，予3ml/kg分組腹腔注射給藥。小鼠分為三組，實驗組、移植對照組和空白對照組，分別在7d、14d、21d檢測肝功能。血清IL-4和IFN-含量采用酶聯(lián)免疫吸附實驗(ELISA)測定。取不同時間點的肝組織行石蠟切片和HE染色，觀察肝臟病理變化情況。RT-PCR檢測不同時間點肝組織中PD-1mRNA的水平表達變化。 結(jié)果 1.以膠原酶和胰酶兩步消化法對小鼠UMSCs進行了分離培養(yǎng)，獲得了成功。兩周之后，主要為長梭形或扁平形的成纖維樣細胞，細胞呈束狀密集平行排列。經(jīng)過多次消化、傳代后，細胞形態(tài)仍為長梭狀，且形態(tài)更加均勻，有很強的增殖活性。群體倍增時間為24h左右。2.成功建立小鼠急性肝衰竭模型。3.移植組小鼠肝功能和造模組比較差異有統(tǒng)計學差異（P＜0.05）。4.移植UMSCs1w、2w、3w后各時間點IL-4和IFN-濃度和造模組比較，IL-4呈上升趨勢，而IFN-呈下降趨勢。5.UMSCs可改善急性肝衰竭小鼠的炎癥浸潤，有利于肝小葉結(jié)構(gòu)恢復正常。6.在正常的小鼠肝臟組織中，PD-1有一定的表達，造模組12h、24h、48h PD-1和正常組比較，差異有統(tǒng)計學意義（P＜0.05）。移植UMSCs后24h、48h PD-1和正常組比較，差異無統(tǒng)計學意義(P＞0.05)，和造模組比較，差異有統(tǒng)計學意義（P＜0.05）。 結(jié)論 成功建立了小鼠UMSCs的分離及培養(yǎng)體系，傳代培養(yǎng)后仍有較強的增殖能力。UMSCs移植可改善肝衰竭小鼠的存活率、肝功能和肝組織病理變化。UMSCs移植可以反映IL-4和IFN-的抗炎/促炎的平衡關(guān)系。PD-1可以抑制正向免疫，可能在急性肝衰竭小鼠和UMSCs移植早期肝臟炎癥損傷中發(fā)揮重要免疫調(diào)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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R575.3

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