【作者】 韦安慧；

【导师】 颜炜群；

【作者基本信息】 吉林大学 ， 生物医学工程， 2009， 硕士

【摘要】 糖尿病是由多种病因引起的以慢性高血糖为特征的代谢性疾病，严重危害着人类健康。目前糖尿病的治疗手段有胰岛素注射，胰腺和胰岛移植，细胞治疗等。由于长期注射胰岛素存在自体耐受和较高的并发症，胰岛移植面临着供体不足和长期使用免疫抑制剂的问题。因此，干细胞以其多向分化潜能成为糖尿病治疗的一条很好的途径。本实验以人脂肪来源基质细胞（human adipose-derived stromal cells,hADSCs）为研究对象，利用碱性成纤维细胞生长因子、胰高血糖素样肽-1（GLP-1）、肝脑匀浆因子和尼克酰胺等在体外将其诱导分化为胰岛素分泌细胞，通过形态学、RT-PCR、双硫腙染色、免疫细胞化学等方法，对诱导后的细胞进行分泌功能和活性测定，探讨脂肪来源基质细胞分化为胰岛素分泌细胞的潜能及其分化机制。结果显示，体外hADSCs在适当葡萄糖浓度，匀浆因子、GLP-1和尼克酰胺共同作用下，可以分化为胰岛素分泌细胞，双硫腙染色呈阳性，RT-PCR检测到胰岛素基因的表达，而且外环境中葡萄糖浓度变化影响着胰岛素的分泌。利用诱导后的细胞分泌胰岛素的特性，为今后干细胞临床治疗糖尿病提供一定的理论和实践依据。更多还原

【Abstract】 Diabetes is a human hazardous metabolic disease caused by a variety offactors,characterized with chronic hyperglycaemia.Traditionally diabetes are treatedmainly by drug therapy and exogenous insulin administration. Long-term insulininjection posesses many problems,such as autologous tolerance, impreciselyregulating blood sugar concentration and unable to intervene the complicationscaused by diabetes.Recently, the treatment of diabetes have made a great progressionby pancreas transplantation and islet cell transplantation.Treatment by these two waysof therapy can effectively recover endogenous insulin secretion, improve life qualityand bring new hope to the diabetic patience. However, the lack of supply of isletdonor and many side effects caused by long-term immunosuppression restrict theirclinical application.Stem cells are a kind of original cells with self-renewal ability and differentiationpotential, which can be directionally induced to various targeted tissue cell typesunder specific micro-environment.Stem cell is classified into two sub-categories--embryonic stem cell and adultstem cell. It is reported that embryonic stem cell can be induced to insulin-producingcells.However, because of their social ethics, tumorigenicity, potentially pathogenicgene and other problems, embryonic stem cells have been limited to clinicalapplication. Adipose-derived stromal cells, a class of adult stem cells,drew greaterattention in recent years because they show very similar biological characteristicswith bone marrow-derived mesenchymal stem cells and have advantage of adiposetissue such as rich resources, convenience of extraction, autologous transplantationwithout immune rejection.Fat-derived mesenchymal stem cells may substitute orcomplement bone marrow-derived mesenchymal stem cells as seed cells for diabetes cell transplatation treatment.However,the potential of differentiation from fat-derivedmesenchymal stem cells into insulin-secreting cell is unclear and the mechanism ofthe directed differentiation regulation is still unknown.To investigate the in vitro differentiation potential of adipose-derived stromalcells induced to insulin-secreting cells and the underlying possible mechanism ofdifferentiation, we focused on human adipose-derived stromal cells(hADSCs),established and optimized the experimental method and conditions for inducing thefat-derived mesenchymal stem cells to insulin-secreting cells.By means of RTPCR,dithizone staining,immunocytochemistry and so on, we detected and assessedthe induced cells to evaluate the effect of directed differentiation.Our study willprovide experimental basis for the use of fat-derived mesenchymal stem cells as seedcell in the cell transplantation therapy for diabetes.First: adipose tissue-derived stromal cell separation, amplification andidentification.①seperation and amplification of hADSCs: Firstly to obtain the liposuctionobjects by mean of lipo-suction, then digest the erythrocyte with typeⅡcollagenaseand disolve the erythrocyte with 0.16mol/L NH4Cl, and finally get the hADSCs byadherent method. Cells were cultured at 37℃, with 5% CO2 and 95% air. The resultsshowed that part of the separated cells got attached after 24 hours and the adherentcells were round with little protrusions; After 72 hours the cells have cytoplasmicprocesses with spindle shape. Through subculture, the cells grew in parallel or aswhirlpool, similar to the bone marrow-derived MSCs in morphology.②detection of the cell surface markers such as CD29, CD44, CD117, CD105,CD45R, HLA-DR and so on by flow cytometry. Our results demonstrated that theisolated and amplified cells showed positive expression for CD29, CD44, CD105, butdid not express the CD45R, CD117, HLA-DR, confirming that the separated cellswere mesenchymal stem cells.Second, induced differentiation of Adipose tissue-derived stromal cells intoinsulin-secreting cells.Take the P3 cells from logarithmic phase, digest them with separation midium, adjust the cell density to 0.5～1×105/ml, inoculate in collagen coated 6-well plates,and culture them in 37℃with 5% CO2 and 95% air. When the cells adhered andgrew to occupation of 80% of the bottom of the culture dish, add inducing medium toinduce the differentiation.①: cell-conditioned inducing medium: GLP-1, bFGF and nicotinamide.With 10 days of inducement by GLP-1 and bFGF, the hADSCs cells graduallycontracted from a long spindle shape,with some cells polygonal and beginning togather. After one week induced with GLP-1 and nicotinamide, the cells wereobserved to aggregate obviously, although the number of cluster was small.Dithizone staining was positive, immunofluorescence detection showed expressionof insulin, RT-PCR illustrated Pdx-1, insulin and Glucagon gene expression for theinduced cells.②: cell-conditioned inducing medium: homogenate factor, bFGF and nicotinaamide.Preparation of homogenate factor: under sterile conditions adopt the brain tissueand the liver tissue of aborted fetuses, homogenize with high-speed, then centrifugeat 13500rpm for 30min, and reserve the supernatant. The cells showed positive signalfor Dithizone staining, immunofluorescence assay confirmed the expression ofinsulin is positive and RT-PCR detection ascertain ed the Pdx-1, Glut-2, insulin,Glucagon gene expression.After 4-6 days of inducement by the inducing medium, part of the cells wereobserved to display radial aggregation and local small cluster of cells were alsoseen.10 days later, we could find that the aggregates significantly increased in numberand size to form islet-like cell clusters, semi-suspended in culture medium.Third, discussion of the mechanism of differentiation of the hADSCs .In this experiment we used liver and brain homogenate to induce differentiation.After treatment with inducing, the cells expressed the Pdx-1，Pdx-1 to activate theinsulin gene transcription, which led to insulin secretion under high glucosestimulation. These result also indicated the exsitence of the key factors of the liverand brain that influence the pancreas differentiation. To sum up: Our experiment successfully induced the differentiation of hADSCsto insulin-secreting cells, demonstrating the potential of hADSCs in differentiation toinsulin-secreting cells, and explored the mechanisms of differentiation of hADSCs.These results not only provided new experimental evidence for the induceddifferentiation of stem cells, but also offered important theoretical and experimentalbasis for treatment of diabetes.更多还原