【作者】 王娟；

【导师】 何冬梅； 张洹；

【作者基本信息】 暨南大学 ， 内科学， 2010， 硕士

【摘要】 目的：探讨胰十二指肠同源框-1基因(pancreatic and duodenal homeobox factor 1, Pdx1)联合细胞因子体外诱导人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUC-MSCs)分化为胰岛β样细胞。方法：1.脐带间充质干细胞的分离、培养和鉴定采用胶原酶消化法体外分离脐带MSCs;用MTT检测细胞活性,并绘制生长曲线；用流式细胞术检测MSCs表面标志和测定细胞周期；RT-PCR检测OCT-3的表达；用地塞米松、胰岛素诱导MSCs向脂肪细胞分化,用地塞米松溶液、β-甘油磷酸、维生素C诱导MSCs向成骨细胞分化。2.PDX1联合细胞因子诱导脐带间充质干细胞向胰岛p样细胞分化用携带PDX1基因的重组腺病毒(Adxsi-CMV-PDX1,为本所构建包装并保存)感染MSCs7d后,联合以下细胞因子------人表皮生长因子(epidermai growth factor,EGF)、B27、胰高血糖素样肽-1(glucagons-like peptide-1, GLP-1)、人β细胞调节素(betacelluin)、肝细胞生长因子(hepatocye growth factor, HGF)、烟酰胺(nicotinamide,NIC)、β-巯基乙醇(β-Mercaptoethanol)诱导分化。RT-PCR检测诱导后细胞PDX1、神经元素3(Neurogenin, NGN3),胰岛素(insulin),葡萄糖转运体2(glucose transporter-2, Glut2)和NK转录因子6.1(NK6 transcription factor related, locus 1, NKX6.1)基因表达的变化；Western blot、免疫细胞化学染色、免疫荧光染色等检测诱导后PDX1、NKX6.1和insulin蛋白表达变化；化学发光法检测诱导后细胞培养液上清中胰岛素和C肽的分泌水平；再用25mmol/L葡萄糖刺激1h后,检测胰岛素与C-肽的分泌水平变化。用流式细胞术检测诱导后细胞insulin(+)细胞百分率。结果：1.脐带间充质干细胞的分离、培养和鉴定分离培养所得到的原代细胞24-48h贴壁,细胞呈梭形。传到第4代时,细胞形态均一,呈长梭形,成漩涡状排列生长。脐带MSCs强表达CD44、CD29,不表达CD106,CD34、CD45、、CD14、CD31和HLA-DR。细胞周期分析显示处在G0／G1期的细胞比例占89.3%。向脂肪细胞诱导14d后,油红O染色见胞浆中有桔红色脂滴形成。向成骨诱导3w,碱性磷酸酶染色见细胞呈立方状,细胞被染成褐色,为阳性反应。而且脐带MSCs能表达OCT-3。2.胰岛p样细胞鉴定经Adxsi-CMV-Pdxl感染脐带MSCs7d并联合细胞因子诱导3d,梭形细胞聚集形成岛样细胞团,用双硫腙(Dithizone,DTZ)染色胞浆呈亮红色,为阳性反应。诱导10-17d,RT-PCR检测显示诱导后的细胞表达PDX1、ngn3、NKX6.1、insulin和glut-2胰岛相关基因,免疫细胞化学染色、免疫荧光染色和Western blot均表明诱导后的细胞表达PDX-1、NKX6.1和胰岛素蛋白。在诱导第17d,培养液上清中胰岛素分泌量为(473.11±51.52)mU/L, C肽分泌量为(1.61±0.41) ng/mL,在25mmol/L高糖协同作用1h后,胰岛素含量高达(964.42±68.19)mU/L, C肽含量高达(3.72±1.52) ng/mL。实验组不同阶段间差异具有统计学意义(P<0.05)。用流式细胞术检测诱导后insulin(+)细胞百分率为(11.61±4.83)%。结论：1.脐带中能分离出MSCs,而且能在体外培养、扩增。脐带MSCs体外具有向脂肪细胞、成骨细胞分化的能力。2.PDX1联合细胞因子诱导,在体外能分化为胰岛β样细胞,分化的胰岛β样细胞能分泌胰岛素和C肽,且能够响应高糖的刺激。更多还原

【Abstract】 Objectives:To explore PDX1 combined with cytokines induce the human umblical cord mesenchymal stem cells (MSCs) to differentiate into insulin-producing cells in vitro.Methods:1. Isolation, culture and identification of human umbilical cord mesenchymal stem cells.MSCs were isolated from human umbilical cord by digestion of collagenase.Cell activity was detected by MTT. Cell cycle and the expressions of cell surface antigens were detected by flow cytometry. RT-PCR detected the expression of OCT-3. Dexamethasone solution and insulin induced MSCs to differentiate into adipogenic.Dexamethasone solution,β-glycerol phosphate and vitamin C induced MSCs to differentiate into osteoblasts. After induction, the cells were observed by oil red O staining, alkaline phosphatase staining.2. PDX1 combined with cytokines induced MSCs to differentiate into isletβ-like cells in vitro.Recombined adenovirus vectors inserted with PDX1 (Adxsi-CMV-Pdxl, constructed, package and saved in our institute) transfected MSCs for 7 days. After infected, cells were induced by cytokines----epidermai growth factor(EGF), B27, glucagons-like peptide-1(GLP-1),betacelluin,hepatocye growth factor (HGF), nicotinamide (NIC) and P-Mercaptoethanol (β-Me). The genes’expression related to isletβcells such as pancreatic and duodenal homeobox factor 1(Pdx1), Neurogenin3 (ngn3), insulin, glucose transporter-2, (Glut2) and NKX6.1 were detected by RT-PCR. PDX-1, insulin and C peptide in the induced cells were examined by immunocytochemistry and immumofluorescence method. The expressions of PDX-1, NKX6.1and insulin were examined by Western blot. The levels of insulin secretion and C peptide secretion were examined by chemiluminescence immunoassay. The levels of insulin secretion and C peptide secretiont were examined with 25mmol/L glucose stimulation after one hour. Insulin(+) cell rate was detected by flow cytometry.Results:1. Isolation, culture and identification of human umbilical cord mesenchymal stem cells.It took about 24-72 hours for the primary MSCs attachmen. After passage 4, cells were highly homogeneous, spindle and growing whirlpool-like. MSCs expressed C44 and CD29, but didn’t express CD34, CD45, CD106, CD14, CD31 and HLA-DR.89.3% of cells was in G0/G1 phase. After induction, the cells were positive for oil red O staining, lkaline phosphatase staining. And MSCs could express OCT-3.2. Identification of isletβ-like cells.After infected by recombined adenovirus Adxsi-CMV-Pdxl 7 days and combined with cytokines induced 3 days, MSCs were aggregated and islet-like cell clusters formed. Dithizone staining of these cells was positive. After induction 10-17 days, the genes’ expression related to isletβcells,such as Pdxl, ngn3, insulin, Glut2 and NKX6.1 could be detected by RT-PCR. PDX-1, insulin and C peptide in the inducted cells could be detected by immunocytochemistry and immumofluorescence method. The expressions of PDX-1, NKX6.1 and insulin could be detected by Western blot. After induction 17 days,the levels of insulin secretion and C peptide secretion were(473.11±51.52)mU/L, (1.61±0.41) ng/mL respectively. The levels of insulin secretion and C peptide secretion were (964.42±68.19)mU/L, (3.72±1.52) ng/mL respectively with 25mmol/L glucose stimulation after one hour. Insulin(+) cell rate was detected by flow cytometry is (11.61±4.83)%。Conclusions:1. Mesenchymal stem cells can be isolated, cultured and expanded from human human umbilical cord and they have the ability to differentiate into adipocyte and osteoblast in vitro.2 Adxsi-CMV-Pdxl combined with cytokines can induce MSCs from human human umbilical cord to differentiate into isletβ-like cells. They can secret insulin and c-p, and have the sensitivity to the stimulation of glucose.更多还原