【作者】 姜铧；

【导师】 张洹；

【作者基本信息】 暨南大学 ， 内科血液学， 2009， 博士

【摘要】 目的:探讨人胎肝来源的间充质干细胞(mesenchymal stem cells derived fromhuman fetal liver,hFLMSCs)的分离和特性;用转录子TAT-PDX1(trans activitortranscription-pancreatic duodenal homeoboxl)蛋白诱导其分化为胰岛β样细胞能力,以及分化细胞表达胰岛素定性和治疗NOD/SCID鼠糖尿病的效果。方法:取12-20周流产胎儿肝脏,剪切、吹打胎肝组织获取肝细胞悬液,通过二步离心收集肝非实质细胞,再用羟乙基淀粉沉淀红细胞,从而使有核细胞得到富集。接种、培养上述细胞,利用细胞差速贴壁生长的特性纯化hFLMSCs。流式细胞仪检测hFLMSCs的细胞周期和表面标志,对P10代及冻存半年后复苏的hFLMSCs进行染色体分析,用经典方法诱导hFLMSCs向脂肪细胞、成骨细胞分化。用TAT-PDX1蛋白为主的方案体外诱导P3 hFLMSCs分化为胰岛β细胞,观察细胞形态变化,用RT-PCR方法检测基因的表达情况,用双硫腙染色,免疫荧光染色检测胰岛素、C-肽的表达。用化学发光法测定胰岛素累积分泌量并用胰岛素释放实验检测分化的细胞对葡萄糖的反应性;并用Western blot的方法检测胰岛素的表达情况。用STZ腹腔注射制作NOD/SCID鼠糖尿病模型,并将诱导后的细胞移植到糖尿病鼠左肾包膜下,动态观察血糖变化及糖尿病症状变化情况,测定移植前后小鼠胰岛素、C-肽水平,并行腹腔糖耐量试验检测移植细胞对血糖变化的反应情况。对血糖降至正常的小鼠,摘除其左肾以观察血糖是否反弹,对摘除的小鼠的肾脏进行免疫组化检测,以及用RT-PCR的方法检测胰岛素表达相关基因的情况。结果:从人胎儿肝脏中分离、纯化得到梭形贴壁的MSCs;P3代hFLMSCs均有90%以上处于GO/GI期,表达CD90、CD105和CD166,不表达CD14、CD34、CD45以及AFP、HLA-DR等;在多次传代(P10代)及冻存半年后复苏仍保持正常核型。在常规诱导条件下,hFLMSCs可分化为成脂细胞、成骨细胞等。用TAT-PDX1蛋白为主的方案体外诱导后,细胞快速变成圆形或椭圆形,并聚集形成越来越多的胰岛样细胞团;RT-PCR结果显示胰岛样细胞团表达胰岛相关基因,如Neuro D、Nkx2.2、Nkx6.1、Pax4、Pax6、Isl-1和PDX1等;经双硫腙染色后呈现棕红色阳性反应;激光共聚焦显微镜检测细胞团中胰岛素、C-肽等胰岛特异性蛋白表达阳性;化学发光法测得培养液上清中胰岛素累积分泌量逐渐增加,而且随着时间延长分泌量维持在较高水平;胰岛素释放实验表明胰岛样细胞团具有一定的糖反应性;Western blot证实诱导分化的胰岛样细胞团蛋白提取物中有胰岛素的表达。用STZ腹腔注射制作NOD/SCID鼠糖尿病模型成模率高,移植胰岛样细胞团到糖尿病小鼠的肾包膜下,可观察到小鼠的血糖逐渐下降,而非移植组糖尿病小鼠则持续高血糖;移植前后小鼠的胰岛素、C-肽水平有显著性差异,腹腔糖耐量试验检测到移植细胞对血糖变化有反应。摘除血糖降至正常的小鼠左肾,可见血糖出现反弹,再次升高;该鼠肾被膜下移植物的免疫组织化学染色可以看到胰岛素阳性细胞。取小鼠左肾,用RT-PCR的方法检测到人胰岛素相关基因(Nkx2.2、Nkx6.1、Pax6、Isl-1 and PDX1)的表达。结论:人12-20周流产胎儿肝脏含有MSCs,能在体外培养和扩增,hFLMSCs强表达CD90、CD105和CD166,不表达CD14、CD34、CD45以及AFP、HLA-DR等;hFLMSCs具有很强的多向分化能力,且免疫原性弱,能长时间保持正常核型,可以作为人类组织工程和再生医学研究的种子细胞。用TAT-PDX1蛋白为主的方案体外诱导hFLMSCs能分化为胰岛β样细胞并稳定表达胰岛素;移植到糖尿病鼠肾包膜下,能快速降低血糖水平;取出移植物,血糖水平再次升高。更多还原

【Abstract】 ObjectivesTo study the basic biological characteristics of the mesenchymal stem cells derived from human fetal liver(hFLMSCs),the ability of TAT-PDX1 protein to induce hFLMSC_s to differentiate into isletβ-like cells in vitro,and the therapeutic effects of these cells when transplanted into the renal subcapsular space of NOD/SCID diabetic mice.MethodsCells were isolated from the aborted fetuses of 12-20 week’s gestation.The hepatic tissue was cut,and then pipetted to obtain the hepatocyte suspension.The non-parenchyma mesenchymal cells were collected through two-step centrifugation, and then the red blood cells were removed by hydroxyethyl starch sedimentation to enrich the nuclear cells.The cells in suspension were inoculated and cultured,and MSC_s purification was done by adhesive screening method(because different kinds of cells have the different adherence to the wall of culture flask).The cell cycle and surface markers of hFLMSC_s were identified using immunochemistry and flow cytometry.Karyotype analysis of hFL-MSC_s was made at passage 8 after recovery from cryopreservation for 6 months.The hFLMSC_s were induced by routine inducing protocols to differentiate toward adipocytes and osteoblastes.The hFLMSC_s were induced to differentiate toward isletβlike cells by TAT-PDX1 protein primarily in virto.Morphological change was observed under microscope.When the figures of the islet-like clusters changed,the expression of islet cells related genes were detected by RT-PCR,and the islet-specific proteins were tested by immumofluorescent staining.Dithizone(DTZ) staining was performed to identify the Zinc in the islet-like clusters.In addition,the quantity of insulin secretion and intracellular insulin were examined by chemiluminescence immunoassay.The test of glucose-stimulated insulin release was made to evaluate the function of the islet-like clusters.Western blot was conducted to identify the secretion of insulin.A diabetic model in NOD/SCID mice was established by intraperitoneal injection of streptozotocin.Finally,the islet-like clusters were transplanted into the left renal subcapsular space of diabetic mice.Blood glucose levels were dynamic monitored after transplantation.The changes of diabetes symptoms were observed.The level of insulin and C-peptide was examineded before and after transplantation.The test of intraperitoneal glucose tolerance test was made to evaluate the function of the cells.The grafts from the left kidney of the diabetic mice were detected the expression of isletβcells related genes with immunohistochemistry and RT-PCR.ResultsThe hFLMSC_s can be isolated and purified from human fetal liver.There is over 90%hFL-MSC_s of passage 3 in G0/G1 phase,hFLMSC_s expressed adhesion molecules of CD90、CD105and CD166,but not antigens of hematopoietic CD14、CD34 CD45,and not antigens such as AFP and HLA-DR.The hFLMSC_s showed normal karyotype at passage 10 after cryopreservation for six months.Exposure of hFLMSC_s to routine inductive agents resulted in morphological changes towards adipocytes,osteoblasts.When hFLMSC_s were induced by TAT-PDX1 protein primarily in virto,they changed quickly into round and oval shape and gathered more and more islet-like clusters.RT-PCR showed the islet-like clusters expressed islet-related genes including Neuro D、Nkx2.2、Nkx6.1、Pax4、Pax6、Isl-1 and PDX1.The islet-like clusters were stained crimson red by dithizone indicating the high zinc content in the cells.The immumofluorescence demonstrated that the islet-like clusters were positive for Insulin and C-peptide by Laser scanning confocal microscope.Chemiluminescence immunoassay demonstrated that the insulin accumulation quantity of secretion was increased and maintained at a higher level with the time.The test of glucose-simulated insulin release showed the islet-like clusters could elevate the insulin secretion upon glucose challenge.The results of Western blot showed that insulin were present in the protein extraction of the islet-like clusters.NOD/SCID mice were easily into models of diabetes mellitus by intraperitoneal injection of streptozotocin.After the islet-like clusters were transpanted into the left renal subcapsular space of diabetic mice,the blood glucose levels decrease gradually, but control animals that did not receive transplants exhibited persistent hyperglycemia.There was a significant difference in the level of insulin and C-peptide before and after transplantation.The test of intraperitoneal glucose tolerance showed these cells had the response to glucose.When the left kidneys that contain the transplanted cells were removed,the hyperglycemia reappeared.Immunohistochemistry staining showed the transplanted cells under the kidny capsule were positive for insulin and C-peptide.RT-PCR showed the transplanted cells under the kidny capsule expressed human islet-related genes including Nkx2.2、Nkx6.1、Pax6、Isl-1 and PDX1.ConcusionsThe MSCs from the aborted fetal liver of 12-20 week’s gestation can be cultured and expanded in vitro,hFLMSCs strongly expressed CD90,CD105,CD166,but no CD14,CD45,CD34,HLA-DR and AFP was found.The hFLMSC_s have the ability to differentiate into the derivative cell types,moreover,they have a lower immunogenic activity and A long time to maintain a normal karyotype,so provide the ideal source for tissue engineering and cellular therapeutics.The hFLMSC_s can be induced to differentiate into isletβ-like cells with TAT-PDX1 protein primarily and have the ability of Stable secretion of insulin in vitro.When these cells were transplanted into the left renal subcapsular space of diabetic mice,the blood glucose levels decrease gradually and steadily.The grafts cells removed,the hyperglycemia reappeared.更多还原