【作者】 陆琰；

【导师】 张洹；

【作者基本信息】 暨南大学 ， 内科学， 2009， 博士

【摘要】 目的:从人胎盘中分离、培养和纯化间充质干细胞(Mesenchymal stem cells,MSCs),探索四种诱导方案使人胎盘间充质干细胞(Human placenta mesenchymalstem cells,hPL-MSCs)分化为胰岛β样细胞。方法:1.胎盘间充质干细胞的分离、培养和鉴定经胶原酶Ⅱ消化和贴壁培养法,从胎盘组织中获取MSCs。采用流式细胞仪检测细胞表面标志以及测定细胞周期。RT-PCR检测SSEA-4的表达。诱导细胞向脂肪细胞、成骨细胞分化,并以油红O染色、碱性磷酸酶染色和Von Kossa染色进行鉴定。2.诱导胎盘间充质干细胞向胰岛β样细胞分化方案1:①IMDM(含5%FBS)添加β-巯基乙醇(β-Me)诱导2d;②经消化后,以添加了激活素A、全反式维甲酸和2%B27的IMDM诱导2d;③用含100ng/mL表皮生长因子(EGF)、碱性成纤维生长因子(bFGF)和纤连蛋白(FN)的IMDM(含3%FBS)诱导5d;④在DMEM/F12(含3%～5%FBS)培养基中加入β细胞调节素(BTC)、胰高血糖素样肽1(GLP-1)和尼克酰胺(NIC)诱导7～9d。方案2:在方案1的基础上降低EGF用量(20ng/mL),不添加FN,未经过消化过程。方案3:前3步和方案1相同,但最后1步采用高糖(添加相同的细胞因子)诱导。方案4:构建含pdx-1基因的重组腺病毒载体,并于293细胞中包装出完整的腺病毒(pAdxsi-CMV-Pdxl)。以pAdxsi-CMV-Pdxl病毒感染hPL-MSCs,经消化后再以EGF、bFGF、肝细胞生长因子(HGF)、BTC、GLP和NIC进行诱导。经诱导后的细胞用化学发光免疫法检测培养上清中胰岛素和C肽的分泌量,并进行胰岛素释放实验;RT-PCR检测胰岛相关基因的表达;免疫细胞化学染色检测诱导后的细胞是否表达PDX-1,Insulin和C肽;Western blot检测细胞内是否含有PDX-1和Insulin。3.诱导后的细胞在1型糖尿病小鼠体内的作用采用链脲霉素一次性腹腔注射法制备1型糖尿病小鼠模型,将诱导后的细胞注射到小鼠右侧肾被膜下,观察小鼠血糖变化;移植术后第24d,摘除其右肾以观察血糖是否反弹,最后取小鼠的肾脏和胰腺进行免疫组化检测。结果:1.胎盘间充质干细胞的分离、培养和鉴定胎盘MSCs表达CD44、CD29,不表达CD34、CD45、CDl06、CDl4、CD40和HLA-DR。细胞周期分析显示92.6%的细胞处在G0/G1期。向脂肪细胞诱导10d后,油红O染色为阳性。向成骨诱导3w,碱性磷酸酶染色和Von Kossa染色也均为阳性。而且hPL-MSCs能表达SSEA-4。2.胰岛β样细胞分化的鉴定经过诱导后的细胞聚集形成胰岛样细胞团。RT-PCR显示诱导后的细胞表达胰岛相关基因。免疫细胞化学染色表明诱导后的细胞表达PDX-1,胰岛素和C肽。Western blot也证实诱导后的细胞含有PDX-1和胰岛素。经方案1诱导后的细胞分泌胰岛素的浓度为(222.00±584.97)mU/L,与方案2(72.00:±27.75)mU/L、方案4(722.00±51 79.36)mU/L、MSC组(0.08±50.03)mU/L和空载体组(1.40±0.57)mU/L相比,差异有统计学意义(P<0.05);方案3(226.00:±73.35)mU/L与MSC组(0.08±0.03)mU/L相比,差异也有统计学意义(P<0.05)。方案1、方案2、方案3、方案4诱导的细胞以5.5mmol/L和25mmmol/L葡萄糖分别刺激1h后,刺激指数分别为4.69±2.18,2.40±1.04,3.49±1.70,4.91±1.80。而经方案4诱导后的细胞分泌的C肽为(3.380±52.3lO)ng/mL,与方案1(0.447±0.381)ng/mL、MSC组(0ng/mL)和空载体组(0ng/mL)相比,差异有统计学意义(P<0.05)。3.诱导后的细胞在1型糖尿病小鼠体内的作用诱导后的细胞注射到1型糖尿病小鼠肾被膜后,血糖逐渐下降,但是在下降过程中,部分小鼠的血糖会出现波动。直到实验结束,实验组小鼠的血糖降至6-8mmol/L(方案4转基因组)或10mmol/L(方案1细胞因子组)。而对照组的小鼠血糖则持续处于高水平。实验组的小鼠被摘除右肾后,血糖迅速反弹。胰岛素免疫组织化学染色显示,注射诱导细胞的小鼠肾被膜下有大量染成棕色的胰岛素阳性细胞;同时取出的小鼠胰腺显示胰岛数量仍然明显减少,胰岛内胰岛素阳性细胞极少。结论:1.胎盘中能分离出MSCs,而且能在体外扩增培养;胎盘MSCs表达CD44、CD29,不表达CD34、CD45、CDl06、CDl4、CD40和HLA-DR;还能表达胚胎干细胞特异性标志之一的SSEA-4。2.胎盘MSCs在体外能向脂肪细胞、成骨细胞分化。3.四种诱导方案都能诱导胎盘间充质干细胞向胰岛β样细胞分化;其中以pAdxsi-CMV-Pdxl联合细胞因子诱导的方案最佳,能提高分化效率,稳定分泌胰岛素,并使糖尿病小鼠血糖降至正常水平。更多还原

【Abstract】 Objectives:To isolate mesenchymal stem cells from human placenta and induce them to differentiate into isletβ-like cells by four protocols. Methods:1. Isolation, culture and identification of human placenta mesenchymal stem cells.Mesenchymal stem cells were isolated from human placenta by digestion of collagenaseⅡand adhesive screening method. Cell cycle and the expressions of cell surface antigens were detected by flow cytometry. RT-PCR detected the expression of SSEA-4. The cells were cultured in an adipogenic medium or in an osteogenic medium. After induction, the cells were observed by oil red O staining, alkaline phosphatase staining and Von Kossa staining.2. Differentiation of placenta mesenchymal stem cells into islet P-like cells.Protocol 1: MSCs from placenta were cultured in IMDM medium supplemented with p-mercaptoethanol (β-Me) and 5%FBS for 2 days. After digestion, cells were cultured in IMDM medium supplemented with Activin A, ATRA and 2%B27 for 2 days. Then cells were switch to IMDM medium supplemented with EGF, bFGF, FN and 3%FBS for 5 days. Finally, cells were cultured in DMEM/F12 medium supplemented with BTC、GLP-1, NIC and 3%-5%FBS for 7-9 days.Protocol 2: The procedures were similar to Protocol 1 .But Protocol 2 reduced the dose of EGF, and without FN and digestion.Protocol 3: The procedures were similar to Protocol 1.But Protocol 3 applied IMDM medium not DMEM/F12 medium in the final procedure.Protocol 4: Recombinant adenovirus vector carrying pdx-1 gene was constructed and transfected into 293 cell line to generate recombinant adenovirus (pAdxsi-CMV-Pdx1). Then hPL-MSCs were infected with pAdxsi-CMV-Pdx1.After digestion, cells were induced by EGF, bFGF, hepatocyte growth factor (HGF), BTC, GLP and NIC.After induction, the levels of insulin secretion, C peptide secretion and glucose-simulated insulin release test were examined by chemiluminescence immunoassay. The genes’ expression related to isletβcells were detected by RT-PCR. PDX-1, insulin and C peptide in the treated cells were examined by immunocytochemistry and immumofluorescence method. The expressions of PDX-1 and insulin were examined by Western blot. 3. The effect of induced cells in type 1 diabetic mice.Type 1 diabetic mice were made by intraperitoneal injection of streptozocin. The induced cells were implanted into the right renal subcapsular space of diabetic mice. Blood glucose levels were monitored every 3 days. The right kidneys were removed at the 24th day after implantation and blood glucose levels were still monitored. At last, the pancreata and the grafts in the right kidneys were detected for immunohi stochemi stry. Results:1. Isolation, culture and identification of human placenta mesenchymal stem cells.Human placenta mesenchymal stem cells expressed CD44 and CD29, but didn’t express CD34, CD45, CD106, CD14, CD40 and HLA-DR. 92.6% of cells was in G0/G1 phase. After induction, the cells were positive for oil red O staining, alkaline phosphatase staining and Von Kossa staining. And hPL-MSCs could express SSEA-4.2. Identification of islet (3-like cells.After induction, islet-like cell clusters formed. The genes’ expression related to isletβcells were found by RT-PCR. The differentiated cells expressed PDX-1, insulin and C peptide, which were confirmed by immunocytochemistry, immumofluorescence method and Western blot.The chemiluminescence immunoassay demonstrated that the cells induced by Protocol 1 secreted much more insulin than that of Protocol 2 group (222.00±84.97mU/L vs. 72.00±27.75mU/L, P<0.05) , pAdxsi-CMV-EGFP group (222.00±84.97mU/L vs. 1.40±0.57mU/L, ,P<0.05) and MSC group (222.00±84.97mU/L vs. 0.08±3.03mU/L, P<0.05) . And protocol 3 secreted much more insulin than that of MSC group (226.00±73.35mU/L vs. 0.08±3.03mU/L, P<0.05) . The clusters induced by Protocol 4 secreted much more insulin than that of Protocol 1 group (722.00±179.36mU/L vs. 222.00±84.97mU/L, P<0.05) . The isletβ-like cells aquired by the four protocols stimulated index under 5.5mmol/L and 25mmol/L glucose for one hour was 4.69±2.18, 2.40±1.04, 3.49±1.70, 4.91±1.80, respectively.The clusters induced by Protocol 4 secreted much more C peptide than that of Protocol 1 group ( 3.380±2.310ng/mL vs. 0.447±0.381ng/mL, P < 0.05 ) , pAdxsi-CMV-EGFP group (0ng/mL, P<0.05) and MSC group (Ong/mL, P<0.05). 3. The effect of induced cells in type 1 diabetic mice.After differentiated cells transplantation, the blood glucose levels began to decrease. At the end of the experiment, the blood glucose levels reached to 6-8 mmol/L (Protocol 4 group) or 10mmol/L (Protocol 1 group). But the blood glucose levels of some mice emerged undulation during the declining course. The control groups remained hyperglycemia. When the kidneys that contain the differentiated cells were removed, the hyperglycemia reappeared. Immunohistochemistry showed the differentiated cells were positive for insulin. Conclusions:1. Mesenchymal stem cells can be isolated from human placenta. Human placenta MSCs express CD29 and CD44, but no CD34, CD45, CD106, CD14, CD40 and HLA-DR. Moreover, hPL-MSCs express SSEA-4, which is one of the specific markers of embryonic stem cells.2. They have the ability to differentiate into adipocyte and osteoblast in vitro.3. Four protocols all can induce hPL-MSCs to differentiate into isletβ-like cells. The protocol that pAdxsi-CMV-Pdxl combined with cytokines is more effective than other protocols. The differentiated cells induced by this protocol express insulin stably, and normalize the blood levels of diabetic mice.更多还原