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     

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。     

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     

微囊化大鼠胰岛细胞移植治疗糖尿病小鼠的实验研究

观察微囊化和未囊化大鼠胰岛细胞移植治疗糖尿病小鼠的疗效。选用SD大鼠经胆总管原位灌注胶原酶液,分离消化胰腺组织,用淋巴细胞分离液(Histopaque-1077)纯化胰岛细胞,将微囊化和未囊化大鼠胰岛细胞移植于糖尿病小鼠腹腔。分离的胰岛细胞对刺激反应良好,微囊化和未囊化胰岛细胞移植后均可纠正糖尿病小鼠的高血糖状态,但未囊化胰岛细胞移植组只能维持血糖正常2~3d,而微囊化胰岛细胞移植组可持续降低血糖30d以上。微囊化胰岛细胞移植治疗糖尿病小鼠具有良好的效果,微囊具有较好的免疫隔离作用。     

人胎盘间充质干细胞诱导分化为胰岛素分泌细胞的研究

目的：探索人胎盘间充质干细胞（MSCs）分化为胰岛素分泌细胞的诱导条件。方法从胎盘组织中获取MSCs,采用激活素A、表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）等多种细胞因子诱导胎盘MSCs向胰岛素分泌细胞分化。利用免疫细胞化学染色、Western Blot及动物实验,对诱导后的细胞进行鉴定。结果经诱导后的细胞具备胰岛β细胞的部分特征,能表达胰十二指肠同源盒-1（PDX-1）、胰岛素和C肽,对糖尿病小鼠具有降糖作用。结论人胎盘MSCs经多种细胞因子诱导后可分化为胰岛素分泌细胞。     

Islet amyloid polypeptide in insulinoma and in the islets of the pancreas of non-diabetic and diabetic subjects

Summary Amyloid deposition is a common pathological feature in insulinoma and in the islets of the pancreas in type-2 diabetic patients. The present immunohisto-chemical study revealed that normal B-cells, insulinoma, and amyloid deposits in insulinoma and diabetic pancreatic islets were commonly immunoreactive with antiserum to C-terminal synthetic tetradecapeptide of human islet amyloid polypeptide (IAPP) (24–37). Amyloid fibrils in insulinoma were also positive to IAPP by immunoelectron microscopy. A high level of IAPP was detected in the plasma and tissue of a insulinoma patient by radioimmunoassay suggesting that amyloid deposition in insulinoma is due to overproduction of IAPP. Amyloid deposits immunoreactive to IAPP were also seen in all diabetic pancreatic islets, but in no non-diabetic islets. There was much amyloid deposition in the islets of severe diabetics, whose B-cells demonstrated decreased immunoreactivities for IAPP and insulin. The IAPP content of the pancreas was 649.0 and 847.7 pg/mg wet weight in each of two diabetic patients, and 1034.6 and 1447.7 pg/mg wet weight in two non-diabetic patients. The present study revealed that IAPP is a bioactive peptide secreted from islet B-cells and are amyloidogenic peptide concerned in diabetogenensis and/or the progression of type-2 diabetes mellitus.     

Immunohistology of islet amyloid polypeptide in diabetes mellitus: Semi-qantitative studies in a post-mortem series

Summary Immunoreactivity for islet amyloid polypeptide (IAPP) in the islets of Langerhans of non-insulin-dependent diabetic patients and non-diabetic patients of a non-selected post-mortem series was studied with a new polyclonal IAPP antibody. Out of 133 patients examined, 124 exhibited immunoreactivity for IAPP. Immunoreactivity was localized intra- and extracellularly and was limited to the islets of Langerhans. No extracellular immunoreactivity was observed in amyloid-negative cases. Co-localization of insulin and IAPP in the same islet-cells was verified by double staining with monoclonal insulin and polyclonal IAPP antibodies. Of 100 patients with non-insulin-dependent diabetes mellitus (NIDDM) and islet amyloid, 98 exhibited IAPP-positive deposits and 71 exhibited intracellular immunoreactivity. Evaluation of intracellular immunoreactivity and degree of islet amyloid deposition in cases of overt NIDDM revealed an inverse relationship, in that intracellular IAPP immunoreactivity were reduced in patients with developing islet amyloid deposition. Our data are consistent with the hypothesis of primary-cell dysfunction leading to amyloid formation, with subsequent disturbance of-cell homeostasis.Supported by the Johanna and Fritz Buch-Gedächtnisstiftung (Hamburg, FRG)     

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。     

Isokinetic gradient centrifugation prolongs survival of pig islets xenografted into mice

Highly purified porcine islets were prepared by isokinetic gradients performed subsequently to isopycnic gradients. This additional purification step separates ductal, vascular, and lymphoid tissue effectively from endocrine tissue. Although ductal, vascular, and lymphoid tissue comprises only a minor contamination of the islet suspensions, a significant prolongation of the survival of porcine islets xenografted into streptozotocin diabetic C57BL/6 mice can be achieved by the elimination of the non-endocrine tissue. Rejection after islet transplantation is delayed from 2.2±0.4 days (n=27) to 13.1±2.1 days (n=36), respectiveley, when conventionally purified and highly purified islets are compared. Irrespective of the purification state, pretreatment of islets by low temperature culture had no effect on xenograft survival.     

The current situation in human pancreatic islet transplantation: problems and prospects

In 2000, seven cases of pancreatic islet transplantation were documented by the researchers at the University of Alberta (Edmonton, Canada). The transplantation was performed using a protocol with a novel immunosuppressive regimen later named the Edmonton Protocol. The results showed that all seven patients became free of the need for insulin therapy. The success of the Edmonton Protocol has enhanced expectations for and interest in islet transplantation all over the world. Islet transplantation seems to be an almost ideal therapy for insulin-dependent patients. In our recent participation in transplant-related conferences in Europe or the USA, we have felt great enthusiasm for islet transplantation among clinicians, researchers, as well as medical staff. In Japan, the Organ Transplant Law that allows transplants from brain-dead patients was enacted in 1997. Since the passing of the law, peoples interest in tissue and organ transplantation, particularly islet transplantation, has been growing. Here we present general information on islet transplantation and discuss its problems and prospects.This is an updated version of an article that appeared in Organ Biology, Vol. 9 No. 3 (2002).