胎鼠胰腺干细胞与胰岛联合移植保护胰岛

背景：胰腺干细胞可在体外维持胰岛的结构,减少胰岛细胞坏死及凋亡,延长胰岛的体外存活时间,保护胰岛的活性。 目的：探索胎鼠胰腺干细胞与胰岛共移植体内保护移植胰岛,提高胰岛移植疗效的可行性。 方法：将成年大鼠35只随机等分为联合移植组、单独胰岛移植组、单纯胰腺干细胞移植组、模型组及对照组,前4组均腹腔注射链脲佐菌素-柠檬酸盐缓冲液建立糖尿病模型。联合移植组、单独胰岛移植组、单纯胰腺干细胞大鼠分别在左侧肾包膜下移植分离纯化孕16 d SD大鼠胎鼠胰腺干细胞和/或成年SD大鼠胰岛。 结果与结论：联合移植组大鼠移植后5 d内血糖可降至正常,血浆胰岛素达到正常水平,胰岛存活时间(18.2±2.4) d;单独移植组大鼠血糖可于移植后1周内降至正常,胰岛存活时间(14.4±2.1) d;两组胰岛存活时间比较差异有显著性意义(P < 0.05)。而其他组糖尿病大鼠血糖均未能降至正常范围。说明胎鼠胰腺干细胞与胰岛共移植可延长胰岛体内存活时间,保护胰岛功能,提高移植疗效。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

胰腺干细胞的研究进展

供体的缺乏和移植排斥反应制约了胰岛移植的广泛应用 ,胰腺干细胞分离及诱导分化技术的进步将有助于解决这一矛盾     

胰腺干细胞的研究进展

供体的缺乏和移植排斥反应制约了胰岛移植的广泛应用,胰腺干细胞分离及诱导分化技术的进步将有助于解决这一矛盾.     

胰腺干细胞体外培养扩增的方法

背景：胰腺干细胞在体外培养时极易分化,很难获得足够数量的胰腺干细胞。 目的：探讨一种简便易行的胰腺干细胞体外扩增培养方法。 方法：分离新生昆明小鼠胰腺,原代培养三四天,成纤维细胞中出现呈集落生长的胰腺干细胞后,以0.02%EDTA消减成纤维细胞的数量,降低其比例后继续培养,待细胞铺满培养瓶底的80%时,重复三四次,逐渐提高胰腺干细胞的数量。检测胰腺干细胞特异性标志Nestin。尼克酰胺诱导胰腺干细胞分化;双硫腙染色对胰岛样细胞团进行检测。 结果与结论：消减胰腺成纤维细胞的数量及比例后,胰腺干细胞可持续表现出活跃的分裂增殖能力,胰腺干细胞的数量显著增加;细胞保持球形、单个大核、细胞质折光性强、表达(Nestin)。扩增的胰腺干细胞,以尼克酰胺诱导后分化为胰岛样细胞团,双硫腙染色阳性。提示,减少共培养中成纤维细胞的比例和数量,有利于胰腺干细胞的增殖,获得较多数量的胰腺干细胞。扩增后的胰腺干细胞仍保持未分化状态,在适当的体外诱导条件下能分化为胰岛样结构,具有一定的分化潜能。     

人胎胰腺GnRH免疫反应细胞

目的：探讨促性腺激素释的激素(GnRH)免疫反应细胞在人胎胰腺的存在部位和数量变化。方法：用免疫组织化学SABC法,对37例第10-32w人胎胰腺内的GnRH-IR细胞进行观察,并用体视方法分析其数量变化。结果：人胎胰腺GnRH-IR细胞出现于第13w,其数密度随胎龄增加而增大;分布于胰岛及外分泌部的腺泡上皮、导管上皮细胞间。位于胰岛的GnRH-IR细胞呈圆形、卵圆形或多边形。位于腺泡上皮细胞间的GnRH-IR细胞多为锥体形,外分泌部的GnRH-IR细胞均为开放型细胞。结论：胰腺GnRH-IR细胞于胚胎第13w出现,广泛存在于内、外分泌部,其数量随胎龄增加而增加。     

人胎胰腺内胰岛淀粉样多肽和胰岛素免疫反应细胞的个体发生

为了探讨人胎胰腺ＩＡＰＰ－与Ｉｎｓ－ＩＲ细胞的个体发生及其分布与共存，应用免疫组织化学ＰＡＰ法，对２２例１１￣３０周人胎胰内ＩＡＰＰ－和Ｉｎｓ－ＩＲ细胞进行定位研究。结果显示，１１周人胎儿胰内已可见到ＩＡＰＰ－ＩＲ细胞单个散在，Ｉｎｓ－ＩＲ细胞较多，未见ＩＡＰＰ与Ｉｎｓ共存的细胞。１６周以后，ＩＡＰＰ－ＩＲ细胞逐渐增多，或散在分布，或三五成群。与邻片比较观察，可见到ＩＡＰＰ与Ｉｎｓ定位的细胞相应，     

胎鼠胰腺干细胞原位移植治疗糖尿病的实验研究

目的：原位移植胎鼠胰腺干细胞，探讨其在糖尿病鼠胰腺微环境中转分化为胰岛样细胞团的可行性。方法：分离纯化SD大鼠胎鼠胰腺干细胞；荧光原位杂交（fluorescent in situ hybridization, FISH）检测SRY上的性别决定区（sex determining region on Y, SRY）以鉴别雄雌；免疫细胞化学检测巢蛋白（Nestin）、胰十二指肠同源异型盒基因（PDX-1）的表达及流式细胞术测定Nestin细胞含量以鉴定胰腺干细胞；分胰腺实质内移植组、实验对照组及空白对照组，10只/组；监测各组大鼠血糖及血浆胰岛素含量，8周后取大鼠胰腺组织切片观察，FISH检测SRY，逆转录-聚合酶链反应（RT-PCR）观察各组大鼠胰腺内Nestin、PDX-1及胰岛素等mRNA的表达情况，免疫印迹（Western blotting）检测PDX-1及胰岛素的蛋白表达水平。结果：有5只胎鼠经FISH检测为雄性。免疫组化示存在Nestin 和PDX-1阳性细胞，流式细胞术测定Nestin阳性细胞含量占74.1%。胰腺实质内移植组大鼠于移植后第3周血糖开始下降，血浆胰岛素水平逐渐升高；第5周血糖及血浆胰岛素均达到正常水平并维持。取第8周大鼠胰腺组织切片HE染色可见外源性细胞团，FISH检测SRY阳性。RT-PCR示胰腺实质内移植组胰腺组织内胰岛素的mRNA表达明显高于实验对照组（P<0.05），而Nestin及PDX-1的mRNA表达量高于实验对照组及正常组（P<0.05）。Western blotting示干细胞移植组胰腺组织内胰岛素含量接近正常大鼠（P>0.05），而PDX-1的含量高于正常大鼠(P<0.05)。结论：胎鼠胰腺干细胞原位移植后可在体内转分化为胰岛样细胞团且具有良好的功能，移植后可使血糖降至正常，胰腺干细胞原位移植可能为1型糖尿病的治疗提供了一个新的策略。     

胎肝滤液诱导骨髓间充质干细胞向肝细胞分化

目的 探讨胚胎肝组织滤液定向诱导大鼠骨髓间充质干细胞(BMSCs)分化为肝细胞的诱导条件,为肝组织工程提供新的种子细胞来源。 方法 在体外培养体系中加入胎肝滤液,模拟体内肝脏微环境,诱导BMSCs向肝细胞定向分化,以免疫细胞化学检测肝细胞标志物;PAS检测糖原表达;靛青绿染色检测转化细胞的分化程度;测定细胞培养上清液中丙氨酸氨基转移酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)的含量以检测其功能状态。 结果 BMSCs经胎肝滤液诱导14d时细胞呈现多角形、卵圆形或圆形细胞的特征性改变;甲胎蛋白(AFP)和白蛋白(ALB)免疫反应、PAS反应和吲哚靛青绿(ICG)摄入实验及上清液中ALT、AST、ALP等酶均在诱导的第7天开始出现,AFP和ALB免疫反应在21d时达高峰;PAS反应和ICG摄入实验随着时间的延长而增强;上清液中的各个酶在14d达高峰,之后呈下降趋势。 结论 胎肝滤液可诱导BMSCs形成具有肝细胞形态和功能特点的细胞。     

体外诱导胰腺干细胞向胰岛样细胞团的分化***☆

背景：目前由于胰岛来源匮乏,使得胰岛细胞移植治疗糖尿病无法满足临床需求,故体外将胰腺干细胞诱导分化为胰岛成为研究焦点。 目的：于体外将小鼠胰腺干细胞诱导成胰岛样细胞团并对其进行相关检测,探寻一种胰腺干细胞体外诱导分化成胰岛及鉴定的技术和方法。 方法：体外获得纯化的小鼠胰腺干细胞,采用联合诱导剂对其进行成胰岛方向的诱导分化,并对诱导形成的胰岛样细胞团进行形态学观察、双硫腙染色、RT-PCR和Western blot检测。 结果与结论：实验通过细胞形态学和细胞生长特性的观察以及免疫细胞化学染色证实体外成功获得了小鼠胰腺干细胞,采用联合诱导剂将其诱导成胰岛样结构,呈球形,以较细长的蒂部与瓶底连接,双硫腙染色将其染成铁红色。RT-PCR和Western blot 法可分别检测到胰岛样细胞团的胰岛素mRNA和胰岛素蛋白。结果证实小鼠胰腺干细胞可体外诱导分化成含β细胞的胰岛样细胞团。 关键词：胰腺干细胞;诱导;胰岛样细胞团;干细胞培养;小鼠 doi:10.3969/j.issn.1673-8225.2012.10.020     

成体干细胞诱导多能干细胞的研究进展

通过存在于卵母细胞中的一些未确定的因子,以成年细胞对动物进行克隆证实成年细胞能被重编程为胚胎干细胞(ESCs).近年来,某些转录因子被发现具有诱导体细胞且有多潜能性的特性,从而可以在不使用卵母细胞的情况下获得性能上与胚胎干细胞大体一致的诱导多能干细胞(iPS).iPS细胞为细胞多能性机制的研究、特定疾病模型的建立以及在...     

葡萄糖转运蛋白2在人脐带间充质干细胞向胰岛前体细胞分化过程中表达的变化

目的 探讨葡萄糖转运蛋白2（Glut2）在人脐带间充质干细胞(hUMSCs)向胰岛前体细胞分化过程中的表达变化。 方法 分离、培养、鉴定及诱导hUMSCs,分别收集诱导过程中第7天、14天和21天细胞和细胞上清液,采用免疫细胞化学、ELISA、免疫荧光、Western blotting及Real-time PCR检测诱导后细胞相关蛋白和基因的表达。结果 免疫组织化学检测诱导后细胞胰腺十二指肠同源盒基因-1（PDX-1呈阳性表达;免疫荧光检测诱导后细胞Ngn3和胰岛素呈阳性表达;Western blotting检测Glut2在诱导过程中逐渐升高,诱导14 d达到峰值,与正常组比较P<0.01;Real-time PCR显示,Glut2基因自第7天即增高（P<0.05）。 结论 hUMSCs经诱导后分化为胰岛前体细胞,表达Glut2后能引起胰岛素分泌,已初步具有胰岛B细胞功能。     

Critical islet mass for successful porcine islet autotrasplantation

A major reason for the failure of clinical islet transplantations may be a limited islet mass. The aim of this study was to determine the critical islet mass necessary for normalization of glucose metabolism in a porcine model. Diabetes was induced by total pancreatectomy. The splenic lobe of the pancreas was intraductally distended with UW-solution containing 2.67–3.33 mg/ml collagenase, and the distended pancreas was digested in a continuous digestion filtration device. The islets were purified on a isoosmotic Ficoll-sodium-diatrizoate gradient. The survival period of the diabetic recipients in group 2 and 3 receiving, respectively, a low (2.14±0.39 μL/kg body weight) and a high (4.99±0.83 μL/kg body weight) islet mass was significantly prolonged compared to that of diabetic recipients in group 1 receiving no islet transplantation. However, the survival period of the recipients in group 2 was not significantly different to that in group 3. Three recipients of an islet mass of >5 μl/kg body weight became normoglycemic (fasting blood glucose <100 mg/dl) for more than two months. Furthermore, the glucose and insulin release reactions to the glucose challenge were comparable to that before pancreatectomy. Contrarily, another five diabetic recipients of an islet mass of <4 μL/kg body weight became a fasting blood glucose level of <200 mg/dl. The glucose and insulin release reactions to the glucose challenge were improved only, but not normalized compared to that before pancreatectomy. The data presented in this study demonstrate that metabolic normalization in pancreatectomized diabetic minipigs can be established by autotransplantation of an islet mass of >5 μl/kg body weight.     

胰岛淀粉样多肽、葡萄糖转运蛋白2在海洛因戒断、脱毒和复吸大鼠胰岛β细胞中的表达

Objective To investigate the expression changes of islet amyloid polypeptiole(IAPP) and glucose transporter 2(GLUT2) in islet β cells during heroin withdrawal induced by naloxone, methadone detoxification treatment, and heroin relapse. Methods Male SD rats (n=32) were randomly divided into a normal control group (NCG, n=8) and an experiment group (EG, n=24). The EG was further divided into heroin withdrawal group (HWG), methadone detoxication group (MDG), and heroin relapse group (HRG). Pancreas was drawn from each group. Immunohistochemistry and image analysis were used to detect the expression of IAPP and GLUT2. Results As compared with the normal control, the immunostainning of IAPP and GLUT2 was stronger in β cells of islets from heroin withdrawal and relapse rats than that in controls; the numerical density on area of IAPP also increased; the mean gray values decreased in these two groups. No difference was found in the immunostainning, the mean gray values and the numerical density on area during detoxification treatment compared to controls. Conclusion During heroin withdrawal and relapse, the synthesis of IAPP and GLUT2 increased, while they became decreased in methadone detoxification. The changes of IAPP and GLUT2 were consistent and had same rhythm in three stages. IAPP and GLUT2 took part in energy metabolic process, which may be related to food intake and body weight, or protective function toβcells.     

Isolation and characterisation of a human monoclonal autoantibody to the islet cell autoantigen IA-2

A hybridoma secreting a human monoclonal autoantibody to the islet cell autoantigen IA-2 was prepared from peripheral lymphocytes of a patient with type 1 diabetes and Graves' disease using EBV infection followed by fusion with a mouse/human hybrid cell line. The monoclonal antibody (M13) is an IgG1/kappa and in an immunofluorescence test M13 at 1 μg/mL showed islet cell antibody reactivity equivalent to 40 JDF units. M13 IgG bound ³⁵S-labelled IA-2 (26% at 100 μg/mL) and ¹²⁵I-labelled IA-2 (34% at 100 μg/mL) in an immunoprecipitation assay and reacted well with IA-2 in western blotting analysis. Amino acids 777–808 in the PTP domain of IA-2 were found to be important for M13 binding in an analysis using modified ³⁵S-labelled IA-2 proteins. M13 V region genes were from VH1-3, D3-22, JH4b, VKI DPK8/Vd+ and JK3 genes and showed a high replacement/silent mutation ratio for both the heavy (11.0) and the light (6.0) chain genes. Mouse monoclonal antibodies (mMAbs) reactive with at least three different epitopes within IA-2 aa 604–686 corresponding to the juxtamembrane domain were also obtained. F(ab′)₂ or Fab from the mMAbs inhibited serum IA-2 autoantibody binding to IA-2 in 20/22 diabetic sera whereas M13 F(ab′)₂ caused inhibition in only 6/22 sera. M13 is representative of some patient serum IA-2 autoantibodies and as such provides a useful tool to study autoimmune responses to IA-2.     

胰岛β细胞发育与再生的研究进展

Islet β cells excrete insulin and play a crucial role in glucose homeostasis. Decreased β cell mass or/and β cell dysfunction are one of the fundamental characteristics of diabetes. The advance in understanding β cell development and regeneration provides new approaches to diabetes treatment. This review focuses on the molecular mechanism about β cell development and the sources for β cell regeneration.     

CTLA4Ig基因修饰BMSCs对异种胰岛移植排斥反应的影响

目的 探讨细胞毒性T淋巴细胞相关抗原4免疫球蛋白( CTLA4Ig)基因修饰骨髓间充质干细胞(BMSCs)在异种胰岛移植排斥反应中的作用.方法 构建携带CTLA4Ig基因的重组腺病毒载体,用其感染BMSCs并和人胰岛细胞移植到糖尿病大鼠肾包膜下,建立人-大鼠异种胰岛移植模型.观察胰岛移植后糖尿病大鼠血糖变化、生存情况以...     

间充质干细胞联合胰岛移植对受体鼠T淋巴细胞亚群的影响

目的研究骨髓间充质干细胞(MSCs)与胰岛共移植时,探讨MSCs如何通过T淋巴细胞亚群发挥免疫抑制作用。方法 Balb/c小鼠骨髓MSCs,与C57LB/6小鼠脾脏分离的T淋巴细胞和Balb/c小鼠脾淋巴细胞共培养,监测对T淋巴细胞增殖能力和细胞周期影响。与C57LB/6小鼠来源的T淋巴细胞共培养,流式细胞术分析T淋巴细胞亚群的变化。用Balb/c小鼠的骨髓MSCs和胰岛联合移植到C57BL/6糖尿病模型鼠,流式细胞术分析受体鼠T淋巴细胞亚群的变化。结果 MSCs可明显抑制T淋巴细胞增殖能力,使T细胞滞留在G0/G1期。使Th1/Th2和Tc1/Tc2比值向Th2和Tc2倾斜,对初始和记忆T细胞具有明显的抑制作用。MSCs联合胰岛移植可使免疫排斥反应减轻。结论 MSCs可抑制T淋巴细胞的增殖,Th1/Th2和Tc1/Tc2比值向Th2和Tc2倾斜,下调初始和记忆T细胞,以发挥免疫抑制作用。     

Prevalence of cytoplasmatic islet cell antibodies and insulin autoantibodies is increased in subjects with genetically defined high risk for insulin-dependent diabetes mellitus

Summary The presence of cytoplasmatic islet cell antibodies (ICA) and IgG insulin autoantibodies (IgG-IAA) has been observed in the prediabetic state of type 1 (insulin-dependent) diabetes (IDDM). We therefore analyzed the prevalence of these markers in sera from 1117 healthy HLA-typed first-degree relatives (1° Rel) of IDDM patients. ICA was determined by indirect immunofluorescence on cryostat sections of human pancreas. For IgG-IAA measurement a competitive solid-phase ELISA was used. ICA were present in 3.5% of 1° Rel vs 0.4% of controls (P<0.025). The highest frequencies of ICA were found in individuals of IDDM multiplex families (7.7%) and HLA-DR1,3 (5.4%), -DR1,4 (5.8%), and -DR3,4 (6.7%) positive subjects. We therefore conclude that the prevalence of ICA is increased in 1° Rel with high genetic risk for diabetes. IgG-IAA occurred in 9.9% of 1° Rel vs 1.4% of controls (P<0.01). Like ICA, IgG-IAA were significantly increased in a group of subjects being positive for either HLA-DR1,3-DR1,4, or -DR3,4 (16.5%,P<0.01). In multiplex families, however, prevalence of IgG-IAA was not increased. In contrast to ICA there was an additional influence of age and sex: IgG-IAA were found more often in siblings (mean age, 16.6 years; prevalence, 15.0%) than in parents (mean age, 44.1 years; prevalence, 8.3%) of IDDM patients (P<0.01). In brothers the prevalence of IgG-IAA is higher than in other 1° Rel. Only a weak association between ICA and IgG-IAA was observed in subjects (n=810) tested for both antibodies. IgG-IAA occurred in 6/35 (17%) ICA positive 1°Rel, while ICA were found in 6/79 (8%) IgG-IAA positive relatives.Abbreviations ICA islet cell antibodies - IgG-IAA immunoglobulin G-insulin autoantibodies - IDDM type 1 (insulin-dependent) diabetes mellitus - 1° Rel first-degree relatives - JDF Juvenile Diabetes Foundation - WHO World Health Organization - OD delta optical density     

通过基因转染诱导骨髓间充质干细胞为胰岛样细胞的进展

随着器官和组织移植技术的进步,胰腺和胰岛移植已开始用于治疗糖尿病并成为热点,但是关于胰岛细胞的来源问题还是存在着许多不足和改进之处。其中骨髓间充质干细胞(BMSCs)因为具有获取简单、易于培养和免疫抑制等优点成为被诱导为胰岛样细胞的热点。近几年来人工构建类胰岛细胞（即利用基因重组及基因转染再造胰岛样细胞）的技术提高了单...