本文選題：臍帶間充質(zhì)干細(xì)胞 + 原代肝細(xì)胞�。� 參考：《蘇州大學(xué)》2014年碩士論文

【摘要】：目的 探討肝衰竭大鼠血清對(duì)臍帶間充質(zhì)干細(xì)胞（UMSCs）表面免疫學(xué)標(biāo)記CD86的影響。了解UMSCs對(duì)大鼠原代肝細(xì)胞增殖、凋亡與功能的影響。 方法 采用膠原酶和胰酶兩步法分離UMSCs，采用貼壁法培養(yǎng)、純化；觀察細(xì)胞形態(tài)特征及生長(zhǎng)狀態(tài)；流式細(xì)胞儀檢測(cè)UMSCs細(xì)胞表面分子標(biāo)記CD90、CD105；將達(dá)70%～80%融合的第3代UMSCs隨機(jī)分為正常培養(yǎng)基組，正常血清組（含10%正常大鼠血清培養(yǎng)基），肝衰竭血清組（含10%肝衰竭大鼠血清培養(yǎng)基）。以上措施干預(yù)培養(yǎng)24h后用流式細(xì)胞儀定量監(jiān)測(cè)細(xì)胞表面免疫學(xué)標(biāo)記CD86的變化。用膠原酶灌注法分離大鼠肝細(xì)胞，肝細(xì)胞與UMSCs應(yīng)用Transwell共培養(yǎng)。原代大鼠肝細(xì)胞分為三組：UMSCs組、大鼠原代肝細(xì)胞組及UMSCs與大鼠原代肝細(xì)胞共培養(yǎng)組，分別于共培養(yǎng)第1d、3d、5d、7d通過(guò)MTT法檢測(cè)各組細(xì)胞增殖能力；UMSCs與肝細(xì)胞共培養(yǎng)組、肝細(xì)胞組，采用脂多糖（LPS）誘導(dǎo)肝細(xì)胞凋亡，于共培養(yǎng)24h后采用流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率；收集各組細(xì)胞共培養(yǎng)第1d、3d、5d培養(yǎng)上清液，ELISA法檢測(cè)上清液中白蛋白含量。 結(jié)果 1．采用膠原酶和胰酶兩步酶法能獲得大量的單個(gè)UMSCs，在體外可以穩(wěn)定的生長(zhǎng)、擴(kuò)增。流式細(xì)胞儀檢測(cè)結(jié)果顯示，分離得到的細(xì)胞穩(wěn)定表達(dá)相關(guān)抗原標(biāo)記CD90、CD105，表達(dá)率為99.7%、99.5%。2．不同組誘導(dǎo)培養(yǎng)UMSCs24小時(shí)后，正常培養(yǎng)基組、正常血清組，肝衰竭血清組細(xì)胞表面CD86表達(dá)率為0.5%，0.6%，0.4%。相比差異無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.05）。3．（1）UMSCs與原代大鼠肝細(xì)胞共培養(yǎng)組在第1d、3d、5d、7d的OD值大于相應(yīng)時(shí)間點(diǎn)的UMSCs組OD值與原代大鼠肝細(xì)胞組OD值之和（P＜0.05）。（2）誘導(dǎo)肝細(xì)胞凋亡后，流式細(xì)胞儀檢測(cè)陽(yáng)性對(duì)照組、陰性對(duì)照組及UMSCs密度為1*104/ml、1*106/ml與肝細(xì)胞共培養(yǎng)組凋亡率分別為93.3%、54.2%、59.7%、72.9%，相比差異有統(tǒng)計(jì)學(xué)意義（P0.05）。（3）UMSCs無(wú)白蛋白分泌能力，UMSCs與大鼠原代肝細(xì)胞共培養(yǎng)組培養(yǎng)上清液中白蛋白水平第1d、3d、5d均較原代大鼠肝細(xì)胞組明顯升高（P0.05）。 結(jié)論 膠原酶和胰酶兩步消化法是一種簡(jiǎn)單、可靠的分離UMSCs的方法；大鼠UMSCs穩(wěn)定表達(dá)相關(guān)抗原標(biāo)記CD90、CD105。經(jīng)肝衰竭大鼠血清干預(yù)培養(yǎng)UMSCs后CD86仍不表達(dá)。UMSCs在體外可以促進(jìn)肝細(xì)胞增殖，抑制其凋亡，，并具有增強(qiáng)白蛋白分泌的功能。
[Abstract]:PurposeTo investigate the effect of serum from rats with liver failure on CD86 labeled by UMSCs on the surface of umbilical cord mesenchymal stem cells (UMSCs).To investigate the effects of UMSCs on the proliferation, apoptosis and function of rat primary hepatocytes.MethodUMSCs were separated by collagenase and trypsin, cultured and purified by adherent method; morphological characteristics and growth status of UMSCs cells were observed; CD90 CD105, a molecular marker on the surface of UMSCs cells, was detected by flow cytometry; and the third generation of UMSCs, which fused up to 70%, was randomly divided into normal medium group.Normal serum group (containing 10% normal rat serum medium), liver failure serum group (containing 10% liver failure rat serum medium).After 24 hours of culture, flow cytometry was used to quantitatively monitor the changes of immuno-labeled CD86 on cell surface.Rat hepatocytes were isolated by collagenase perfusion and co-cultured with UMSCs with Transwell.The primary rat hepatocytes were divided into three groups: the primary rat hepatocytes group, the primary rat hepatocyte group and the co-culture group of UMSCs and rat primary hepatocytes. The proliferation ability of each group was detected by MTT assay on the 1st day, 3d after incubation, and 5 days after co-culture, respectively.Hepatocyte apoptosis was induced by lipopolysaccharide (LPS), the apoptosis rate was detected by flow cytometry after 24 h co-culture, and the albumin content in supernatant was detected by Elisa.Result1.A large number of single UMCs can be obtained by using collagenase and trypsin two-step enzymatic method, which can grow and amplify stably in vitro.The results of flow cytometry showed that the stable expression of CD90 and CD105 was detected by flow cytometry, and the expression rate of CD90 and CD105was 99.70.2.After induction and culture of UMSCs24 in different groups, the expression rate of CD86 on the cell surface of normal medium group, normal serum group and serum group of liver failure was 0.5% and 0.64% respectively.There was no significant difference between 0.05).3.(1)UMSCs and primary rat hepatocyte coculture group. The OD value of the co-cultured rat hepatocytes on the 1st day was higher than that of the UMSCs group and the primary rat hepatocyte group on the 1st day (P < 0.05), and the positive control group was detected by flow cytometry after the OD value of the UMSCs group was higher than that of the primary rat hepatocyte group (P < 0.05), and the total OD value of the primary rat hepatocyte co-culture group was significantly higher than that of the control group (P < 0.05).The apoptotic rates of the negative control group and the UMSCs density of 1 / 104 / ml / ml and the hepatocyte co-culture group were 93.3% and 54.2%, respectively. The difference was significant compared with the control group (P 0.05 / ml) and the rat primary hepatocyte co-culture group (P < 0.05). The albumin level in the supernatant of the primary rat hepatocyte co-culture group was significantly higher than that in the control group (P < 0.05) and the rat primary hepatocyte co-culture group (P < 0.05), respectively, and the level of albumin in the supernatant was significantly higher than that in the control group (P < 0.05).Compared with the primary rat hepatocyte group, the level of P0. 05 was significantly increased at 1 d, 3 d and 5 d.ConclusionThe two-step digestion of collagenase and trypsin is a simple and reliable method for the separation of UMSCs and the stable expression of CD90 and CD105 in rat UMSCs.In vitro, CD86 could promote the proliferation of hepatocytes, inhibit their apoptosis and enhance the secretion of albumin.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575.3;R457.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 秦愛(ài)蘭,周霞秋;肝臟干細(xì)胞——肝病的細(xì)胞治療[J];國(guó)外醫(yī)學(xué)(內(nèi)科學(xué)分冊(cè));2004年04期

2 甘建和,秦愛(ài)蘭;C-kit~+細(xì)胞體外增殖、分化的研究[J];生物醫(yī)學(xué)工程學(xué)雜志;2005年05期

3 胡中杰,郎振為,宋晨朝,張世杰;重型肝炎肝組織中肝前體細(xì)胞增殖分化特點(diǎn)研究[J];中華傳染病雜志;2004年01期

4 袁源;楊樹(shù)源;韓忠朝;張曉輝;;人臍帶間充質(zhì)干細(xì)胞分離純化及基本生物學(xué)特性研究[J];中華實(shí)驗(yàn)外科雜志;2006年01期

5 古彥錚;薛群;王泳;於葛華;沈宇;王明元;張學(xué)光;;人胎盤源與人骨髓源間充質(zhì)干細(xì)胞的生物學(xué)特性的比較及負(fù)性協(xié)同刺激分子PD-L1的表達(dá)[J];中國(guó)免疫學(xué)雜志;2009年05期

6 ;Characterization and enrichment of hepatic progenitor cells in adult rat liver[J];World Journal of Gastroenterology;2004年10期

7 Kei Moriya;Masahide Yoshikawa;Ko Saito;Yukiteru Ouji;Mariko Nishiofuku;Noriko Hayashi;Shigeaki Ishizaka;Hiroshi Fukui;;Embryonic stem cells develop into hepatocytes after intrasplenic transplantation in CCl_4-treated mice[J];World Journal of Gastroenterology;2007年06期

8 張輝挺;勞學(xué)軍;;臍血間充質(zhì)干細(xì)胞治療肝衰竭的研究進(jìn)展[J];醫(yī)學(xué)綜述;2012年15期

9 霍大浪;寧家輝;胡文君;劉明穎;張?jiān)?王娜;馮成軍;王英杰;;BMSC-肝細(xì)胞共培養(yǎng)上清治療大鼠急性肝衰竭療效觀察[J];延安大學(xué)學(xué)報(bào)(醫(yī)學(xué)科學(xué)版);2013年03期

10 張曉培;秦愛(ài)蘭;鄧長(zhǎng)卿;甘建和;石翠翠;劉濤;;大鼠臍帶間充質(zhì)干細(xì)胞對(duì)肝細(xì)胞及卵圓細(xì)胞體外增殖與功能的影響[J];肝臟;2014年05期

本文編號(hào)：1757558