【摘要】： 目的:重癥肝病尚無特效療法,細(xì)胞移植治療肝損傷疾病是一種值得探索的新途徑。羊膜上皮和羊膜間充質(zhì)細(xì)胞具有干細(xì)胞特性,在體外適當(dāng)?shù)恼T導(dǎo)條件下均可分化為肝細(xì)胞,其體內(nèi)是否可以作為肝細(xì)胞供體治療肝損傷疾病尚未探明。本研究的目的是尋找人羊膜細(xì)胞在大鼠肝損傷原位分化為肝細(xì)胞的證據(jù),為其作為細(xì)胞供體臨床治療肝損傷疾病提供實(shí)驗(yàn)依據(jù)。 方法:采用二酶消化法從人羊膜組織中分離hAECs和EAMCs,采用流式細(xì)胞術(shù)(FCM)和免疫熒光染色進(jìn)行表型分析和細(xì)胞鑒定。采用腹腔注射D-氨基半乳糖,按400mg/kg建立大鼠肝損傷模型,隨機(jī)分為hAECs移植組、hAMCs移植組和對照組,每組20只。造模后24h用微量注射器于肝左、中、右葉3點(diǎn)分別注射L-DMEM懸浮的hAECs或hAMCs懸液50μl(約1×10~6個細(xì)胞),對照組注射等量L-DMEM。于移植后48h、1w、2w、3w或4w處死各實(shí)驗(yàn)組3～4大鼠,取移植部位行常規(guī)病理學(xué)檢查和行免疫熒光染色或免疫組化染色以觀察肝組織病理變化、hAECs和hAMCs在受損肝原位的植活與分布及其甲胎蛋白(AFP)、CK18、CK19和白蛋白(Alb)的表達(dá)情況。 結(jié)果:①FCM分析結(jié)果顯示,所分離的hAECs幾乎不表達(dá)CD44,而hAMCs中則部分表達(dá)CD44:免疫熒光染色顯示hAECs只表達(dá)CK19,而hAMCs只表達(dá)波形蛋白。②與對照組比較,hAECs和hAMCs移植組于移植后48h肝門管區(qū)嗜堿性小細(xì)胞增多,肝細(xì)胞壞死較輕;移植后7d肝小葉結(jié)構(gòu)較為完整。③免疫熒光染色結(jié)果顯示,hAECs肝損傷原位移植后48h主要分散于肝血竇,移植后1w表達(dá)AFP,2w表達(dá)CK18,2w和4w表達(dá)Alb;免疫組化和免疫熒光染色結(jié)果顯示,hAMCs移植后48h主要分散于肝小葉,1w表達(dá)CK19,2w表達(dá)CK18,3w仍可檢測到人細(xì)胞核抗原和Alb。 結(jié)論:hAECs和hAMCs在大鼠受損肝組織中能被植活,且可分化為肝細(xì)胞,提示這兩種人羊膜細(xì)胞在臨床治療重癥肝損傷疾病方面可能具有重要的應(yīng)用價值。
[Abstract]:Objective: there is no special effect therapy for severe liver disease. Cell transplantation is a new way worthy of exploration in the treatment of liver injury. Amniotic epithelial cells and amniotic mesenchymal cells have the characteristics of stem cells, which can differentiate into hepatocytes under appropriate induction conditions in vitro. Whether amniotic epithelial cells can be used as liver cell donors in the treatment of liver injury has not been found out. The purpose of this study was to find out the evidence of in situ differentiation of human amniotic cells into hepatocytes in rats with liver injury, and to provide experimental basis for the clinical treatment of liver injury with human amniotic cells as cell donors. Methods: hAECs and EAMCs, were isolated from human amniotic membrane by two enzyme digestion. Flow cytometry (FCM) and immunofluorescence staining were used for phenotypic analysis and cell identification. The rat model of liver injury was established by intraabdominal injection of D-galactosamine according to 400mg/kg. The rats were randomly divided into three groups: hAECs transplantation group, hAMCs transplantation group and control group with 20 rats in each group. 24 hours after modeling, hAECs or hAMCs suspension suspended by L-DMEM (about 1 脳 10 ~ 6 cells) was injected into the left, middle and right lobe of the liver by microsyringe at 3 o'clock, respectively, while the control group was injected with the same amount of L 鈮，

本文編號：2507441