【作者】 任莉莉；

【导师】 龚非力； 李富荣；

【作者基本信息】 华中科技大学 ， 免疫学， 2010， 博士

【摘要】 第一部分人脂肪来源干细胞的分离、培养和鉴定目的分离、纯化人脂肪来源的干细胞(human adipose tissue-derived stem cell, hASC)并鉴定其生物学特性。方法采用zuk等建立的人脂肪间充质干细胞分离培养方法：借助普通外科手术获得脂肪组织；分离、纯化脂肪来源干细胞；流式细胞术分析第四代细胞表面标志和胞浆抗原表达；绘制细胞生长曲线；借助端粒酶活性检测,评价其体外增殖能力；借助吉姆萨染色,分析第4、8、12、16代细胞染色体核型,评价干细胞遗传稳定性；双层琼脂培养检测干细胞成瘤性。结果在培养第四代始,细胞出现均一的旋涡状生长,增殖速度较快。流式细胞术分析显示,第四代细胞表达CD73(99.1%)、CD90(97.9%)、CD105(97.2%),符合间充质干细胞特征。细胞生长曲线显示,细胞倍增时间约为36小时。染色体核型分析显示,hASCs为正常二倍体核型,G显带检测未见异常。体外双层琼脂检测未见成瘤性。结论hASC经分离、纯化,可在体外有效扩增,符合脂肪间充质干细胞表面标志物鉴定的国际标准和公认的干细胞特征。体外培养过程,hASC不表达胰腺相关标志物,染色体核型稳定且无变异,未见成瘤性。第二部分胎胰蛋白/损伤胰腺蛋白复合物诱导hASC向胰腺方向的分化目的评价大鼠胚胎胰腺/损伤胰腺组织的可溶性蛋白提取物诱导hASC向胰岛素分泌细胞分化的能力。方法制备大鼠胚胎胰腺/损伤胰腺的蛋白匀浆,提取可溶性细胞因子复合物,与hASC共培养20天,在不同时间点检测胰腺发育相关基因表达水平和胰岛素/C肽分泌水平。结果胚胎胰腺/损伤胰腺的蛋白提取物,可诱导hASC表达与胰腺发育密切相关的基因产物(如PDX-1、Pax-4、Pax-6等),部分分化细胞可表达胰岛素和C肽。结论提示损伤胰腺和胚胎胰腺内富含胰腺发育相关蛋白,可诱导hASC向胰腺方向分化,并最终诱生胰岛素、C-肽阳性细胞。第三部分胰岛新生相关蛋白肽方案促进hASC向胰腺方向的深度分化目的评价胰岛新生相关蛋白肽4步方案诱导hASC分化为胰岛样细胞团的效果。方法应用胰岛新生蛋白肽联合胰腺发育相关细胞因子及化学因子进行四步诱导,RT-PCR、实时定量PCR及细胞免疫荧光检测诱导后细胞胰腺发育及功能基因表达的改变,在不同浓度(5.6mM、25mM)葡萄糖刺激下,检测分化细胞胰岛素及C肽分泌量,评价细胞的葡萄糖应激能力。结果诱导过程中,hASC的干细胞相关基因Oct3/4表达降低至原始水平的1/20,而与胰腺系分化发育相关的基因(如ck19、nestin PDX-1、nkx2.2等)表达水平均显著升高。诱导分化后期,INGAP-pp诱导组出现直径约150-200μm的胰岛样细胞团,Scramble-pp诱导组出现直径约50-100μm的胰岛样细胞团,前者更接近天然胰岛的直径。免疫荧光检测显示,INGAP-pp所诱生的胰岛样细胞团内存在胰岛素和C肽双阳性细胞群,可对不同浓度葡萄糖刺激产生反应,分泌量约占天然胰岛细胞1/10-1/15。结论INGAP-pp诱导方案可经间质细胞向上皮细胞转化的途径,诱导hASC向胰腺系细胞分化,在体外形成具有胰岛素分泌功能的胰岛样细胞团。虽然与天然胰岛相比尚不完善,但提示在适当条件诱导下,hASC确可分化为具有葡萄糖应激能力的胰岛样细胞。第四部分胰岛样细胞团移植治疗糖尿病大鼠的效果评价目的评价纯化INGAP-pp蛋白及INGAP-pp诱导分化产生的ILC移植治疗糖尿病大鼠的效果。方法STZ诱导大鼠Ⅰ型糖尿病模型,在体视镜下新鲜分离人胰岛及INGAP-pp诱导组所产生ILC各200个,分别移植于糖尿病大鼠模型左肾包膜下,比较正常胰岛移植组、ILC移植组、INGAP-pp蛋白注射组、糖尿病组及正常大鼠组在实验周期的血糖水平,每3天检测1次。对各组进行腹腔内葡萄糖耐受实验检测,统计各组存活率。结果移植正常人胰岛的糖尿病大鼠血糖水平趋于正常,与正常大鼠血糖接近；移植ILC的糖尿病大鼠组血糖水平在术后短暂上升,遂即下降,并在稍高于正常大鼠血糖水平保持稳定；注射INGAP-pp蛋白的糖尿病大鼠在注射过程中血糖下降,停止注射蛋白后缓慢回升；糖尿病大鼠组血糖持续异常,至实验结束。腹腔内葡萄糖应激实验中,移植第20天,移植正常胰岛和ILC的糖尿病大鼠均出现接近于正常大鼠的反应曲线,但ILC移植组血糖水平在各时间点均高于胰岛移植组。由于血糖持续波动,INGAP-pp蛋白注射组未进行葡萄糖应激实验。另外,整个实验过程中,糖尿病组大鼠逐渐死亡,至51天全部死亡；整个实验过程中胰岛移植组和ILC移植组大鼠无一死亡。结论ILC移植可降低糖尿病大鼠血糖水平,并延长糖尿病大鼠生存时间。虽然与天然胰岛组相比,降低血糖的程度尚不足,但确已具有葡萄糖应激能力。提示：INGAP-pp诱生的ILC具有治疗糖尿病的临床应用前景。更多还原

【Abstract】 Part I:Objective:To isolate, purify and characterize human adipose tissue-derived stem cells.Methods:With slightly modified protocol demonstrated by zuk et al, human adipose tissue-derived stem cells (hASCs) were isolated and expanded in vitro. FACS analysis was performed to identify the cell surface and cytoplasm antigen.Tolemerase repeated amplification protocol (TRAP) and G-banding Karyotype analysis were performed to evaluate the safety of hASCs cultured in vitro. Results:Since passage 4, hASCs developed a uniform morphology resembling that of fibroblasts, FACS analysis of the fourth passage cells showed purified hASCs expressed high levels of cell surface markers, such as CD73(99.1%),CD90(97.9%), CD105(97.2%). These results are consistent with the definition that mesenchymal stem cells (MSCs) must express CD73, CD90, and CD105. Also, extremely low expression levels of CK-19 and Pdx-1, which supposed to be markers of pancreatic stem cells, were detected. Negative expression of CD45 means no contamination of hemopoiesis stem cells. TRAP test revealed P4, P8, P12 had nomal tolemerase activity and G-banding analysis showed P4, P8, P12 and P16 had normal katyotypes. Conclusions:hASCs could expand in vitro efficiently after being isolated and purified. The hASCs we cultured in the study were consistent to those reported previously. When cultured in vitro, hASCs have stable chromosome structure as devided in vitro and being safe for cytothrapy.Part II:In Vitro differentiation of human adipose tissue-derived stem cells into Insulin/c-peptide positive cells by embryonic pancreas extractObjective:To evaluate the effect of embryo pancreas extract (EPE) from pre-born Sprague-Dawley rats on induction of pancreatic differentiation of human adipose tissue-derived mesenchymal stem cells(hASCs).Methods:By using real-time PCR and immunofluorescence technology, expression levels of genes related with pancreas development of hASCs cultured with EPE for 20 days were detected.Results:undifferentiated hASCs expressed key markers of embryo stem cells. Genes involved in early pancreas development showed increased expression in EPE-treated culture. Immunocytochemical analysis showed C-peptide/insulin positive cells in EPE-treated cells but not in undifferentiated hASCs.Conclusion:hASCs have the potential to differentiate into pancreas cell lineages with EPE induction, so could be substitution of embryo and bone-marrow derived stem cells for future cell therapy applications.Part III:IN Vitro Differentiation of human adipose-derived stem cells intoIslet-like cell clusters by INGAP-pp protocolObjective:To establish a novel method for the differentiation of islet-like clusters from human adipose-derived stem cells with INGAP-related pentadecapeptide (INGAP-PP) combined with chemical molecules and cytokines.Methods:the human adipose-derived stem cells were isolated, purified and expanded in vitro and differentiated using a four-step protocol that included TSA, INGAP-PP/Scrambled peptide (Scrambled-P), nicotinamide and exendin-4. By using real-time PCR and immunofluorescence technology, expression levels of genes related with pancreas development. Insulin and C-peptide released by differentiated cells in response to high/low glucose level were detected by ELISA.Results Undifferentiated hASCs expressed oct3/4, key markers of embryo stem cells and at the end of induction, Oct3/4 expression level decreased to 1/20 of original. The expression level of genes involved in early pancreas development increased in both INGAP-PP/Scrambled peptide culture significantly. Islet-like clusters were obtained in both INGAP-PP/Scrambled peptide group, but in INGAP-pp group, the diameters of clusters were much closer to native islets, which were about 150-200μm. Immunocytochemical analysis showed C-peptide/insulin positive cells in INGAP-pp induced group but not in undifferentiated hMSCs.Conclusions:By INGAP-pp protocol, hASCs could differentiate into islet-like clusters more efficiently through Mesenchymal/epithelium transition. It suggests hASCs have the potential to differentiate into pancreas cell lineages by INGAP-pp protocol; so could be substitution of embryo or bone-marrow derived stem cells for future cell therapy applications.Part IV:Objective:To evaluate the effects of transplantation of ILCs induced by INGAP-pp and INGAP-pp injection to cure diabetic rats.Methods:Diabetes was induced in 8-week-old male Sprague-Dawley rats by streptozocin (Sigma,55 mg/kg body weight in citrate buffer, pH 4.4, i.p).48 hours after injection, animals with blood glucose levels≥16.65 mmol/L for two consecutive days were transplanted with ILCs induced by INGAP-pp (200IEQ/l00μ1 High glucose DMEM) into the left subrenal capsule (n=10). As controls, fresh isolated human islets (200IEQ/100μ1 High glucose DMEM) and saline without cells were transplanted respectively. Two transplantation group were administered acrolimus (50ng/kg.d,p.o.) to avoid of immune rejection. To evaluate the effects of INGAP-pp protein on remission of diabetes, we also injected purified INGAP-PP (250μg/0.5mL, i.p.) to diabetic rats twice a day for twenty days. Blood glucose levels of all groups were measured every three days. Glucose tolerance tests were performed after injection of glucose (2g/kg bodywight, i.p.). Diabetic rats transplanted with normal islets and normal rats served as positive controls. Diabetic rats were used as negative control. Results:Blood glucose levels of rats transplanted with INGAP-induced ILCs fell to 7.8±0.8 mmol/L and kept this level for the whole experimental process while diabetic rats transplanted with normal islets, as a control, showed blood glucose levels similar to normal rats after 1-2 weeks. In addition, non-transplanted diabetic rats remained hyperglycemic and died consecutively. Interestingly, the group treated with INGAP-pp protein injection also showed a decrease of blood glucose level after 5-day injections, but never fell to normal level and re-raised 2 weeks later since the day INGAP-PP injection stopped. Diabetic rats transplanted with INGAP-induced ILCs or normal islets survived the whole 69-day process while diabetic rats without treatment suffered from hyperglycemic and died off within 51 days. Diabetic rats undergone INGAP-pp injections also all survived until the end of experiment. Intraperitoneal glucose tolerance tests showed SD rats transplanted with ILCs induced by INGAP-pp had the similar kinetics of the glucose clearance to the group transplanted with normal islets, although the blood glucose levels were higher at all time points. In contrast, untreated diabetic rats revealed no reaction to the high glucose concentrationConclusions:The results of transplantation of ILCs and normal islets to diabetic rats showed ILCs did decrease the blood glucose levels and extended the life-span of diabetic rats. ILCs transplantation reversed diabetes partially in animal models.更多还原