促胰素对大鼠胰岛细胞增殖及功能的影响

背景：有研究报道,促进胰岛β细胞增殖分化和再生是2型糖尿病患者一种潜在的治疗方案。 目的：观察促胰素对体外培养大鼠胰岛细胞增殖及功能的影响。 方法：采用胶原酶消化和组织培养法分离纯化大鼠胰岛细胞,检测其纯度和活性,将胰岛细胞分为空白对照组和实验组,空白对照组加入普通培养基,实验组培养基中加入不同质量浓度(100,200,300,400 mg/L)促胰素,培养1,3,5 d后CCK-8法检测细胞增殖活性;培养5 d后行低糖和高糖刺激胰岛素释放实验。 结果与结论：随着促胰素质量浓度的增高,胰岛细胞增殖活性呈剂量依赖性增加(P < 0.05),但无明显时间依赖性。除100,200 mg/L组外,300,400 mg/L两组胰岛素分泌量均显著高于空白对照组(P < 0.05)。表明300,400 mg/L促胰素不仅可促进胰岛细胞增殖,而且可显著增强胰岛细胞分泌胰岛素的功能。 关键词：促胰素;胰岛细胞;增殖;胰岛功能;胰岛分离;纯化 doi:10.3969/j.issn.1673-8225.2012.05.027     

肝前性门脉高压大鼠胰岛功能的变化

目的：探讨肝前性门脉高压大鼠胰岛α、β细胞功能及其与循环血液中胰岛素及胰高血糖素水平变化的关系。方法：采用部分结扎门静脉的方法复制肝前性门脉高压,假手术及正常鼠的胰岛进行分离和纯化,并对其体外生物学活性进行检测。结果：肝前性门脉高压组大鼠循环血液中胰岛素及腹高血糖素水平明显高于正常组大鼠（P＜0．01）。肝前性门脉高压级大鼠分离纯化的胰岛经体外培养48h或在含糖基质中孵育2h后释放胰岛素量明显低于正常,而陵高血糖素明显高于正常（P＜0．01）。结论：胰岛α细胞分泌功能增加可能有助于解释高胰高血糖素血症的发生;门脉高压时,循环血液中胰岛素水平增加,但哪胞功能低于正常。     

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：     

与肌源性干细胞共培养修复大鼠胰岛功能

目的采用体外共培养方式研究肌源性干细胞(MDSCs)修复大鼠胰岛功能的作用。方法提取新生大鼠MDSCs原代细胞,差速贴壁培养后行结蛋白免疫细胞化学鉴定,取第2代MDSCs接种在transwell底板。提取大鼠胰岛后,双硫腙染色鉴定,胰岛放入transwell板上层与MDSCs共培养作为实验组,用放射免疫法测胰岛功能,Western blot检测胰岛Bax、caspase3蛋白表达。另选胰岛单独培养作为对照组。结果实验组胰岛素释放指数(SI)值降低不明显,Bax、capase3蛋白定量升高不明显,与对照组相同时间比较差异有统计学意义(P0.05)。结论 MDSCs与大鼠胰岛共培养后,大鼠胰岛功能被修复。     

大鼠胰腺导管上皮细胞及转分化胰岛样细胞免疫学特性研究

目的:通过对大鼠胰腺导管上皮细胞、转分化的胰岛样细胞与天然胰岛细胞进行免疫原性比较,了解胰腺导管上皮细胞及转分化胰岛样细胞的免疫学特性.方法:大鼠胰腺导管上皮细胞,转分化胰岛样细胞和天然胰岛在体外与淋巴细胞共培养,检测淋巴细胞MHCⅠ、MHCⅡ抗原决定簇,IFN-γ和IL-2、IL-4水平.将三组细胞分别植入到大鼠糖尿病模型皮下,流式细胞技术检测外周血MHCⅠ、MHCⅡ表达、ELISA检测IL-2、IL-4水平和机体对其反应的病理学变化.结果:三组细胞与淋巴细胞共培养,MHCⅠ的表达未见明显差异(P＜0.05),MHCⅡ的表达胰岛样细胞(29.5%±3.1%)和天然胰岛细胞(32.6%±3.6%)较胰腺导管上皮细胞(10.8%±0.9%)明显升高(P＜0.05).淋巴细胞分泌IL-2水平和Elispot检测分泌IFN-γ的细胞数,胰腺导管上皮细胞较胰岛样细胞和天然胰岛细胞组显著性降低(P＜0.01).植入糖尿病大鼠模型的外周血IL-2水平和淋巴细胞MHCⅡ表达均随着植入时间逐渐升高,胰岛样细胞和天然胰岛细胞组较胰腺导管上皮细胞组升高明显,IL-4水平在三组细胞无明显差异.病理结果显示胰腺导管上皮细胞组淋巴细胞浸润较胰岛样细胞和天然胰岛细胞组为轻.结论:胰腺导管上皮细胞具有低免疫原性,转分化的胰岛样细胞免疫原性近似天然胰岛.     

大鼠胰岛分离技术的建立及胰岛活性的影响因素

目的建立稳定有效的大鼠胰岛分离方法,探讨影响胰岛活性的因素。方法通过胆管注射胶原酶方法提取大鼠胰岛,通过水浴法比较不同条件下葡萄糖刺激的胰岛素分泌,用放免法(R IA)测定胰岛素。结果牛血清白蛋白(BSA)能明显提高葡萄糖刺激的胰岛素分泌水平,长时间培养(>7 d)的胰岛,其葡萄糖刺激的胰岛素分泌下降约25%,而新鲜分离的胰岛和经1~5 d培养的胰岛未见明显差异。RPM I1640培养液中葡萄糖浓度11.1 mmol/L组,其胰岛素分泌明显高于5.5 mmol/L和25 mmol/L这2组。结论BSA、RPM I1640培养液中葡萄糖浓度及培养时间均与分离胰岛活性有关。     

诱导型一氧化氮合酶对大鼠胰岛细胞凋亡的影响

目的:探讨细胞因子和诱导型一氧化氮合酶(iN-OS)对体外培养的大鼠胰岛细胞凋亡和功能的影响及其机制。方法:大鼠胰岛细胞体外培养,随机分组,培养液中分别加入细胞因子IL-1β、TNF-α和/或iNOS抑制剂氨基胍,分为空白对照组、细胞因子组、氨基胍组、氨基胍+细胞因子组。检测指标包括:培养液NO水平和组织iNOS、结构型NOS活性,RT-PCR检测胰岛组织中iNOS基因和凋亡相关基因Bax、Bcl-2的表达情况,AO/EB染色检测胰岛存活,胰岛素释放实验检测胰岛功能。结果:与细胞因子IL-1β和TNF-α共同培养后,大鼠胰岛组织中iNOS的表达增强,活性显著提高(38.93±4.72)U/mL,NO水平上升(313.0±35.4)mol/L,而结构型NOS没有变化;同时胰岛促凋亡基因表达上调,抗凋亡基因表达下降,细胞的存活率下降,胰岛素分泌大大减少。加入氨基胍后,随着胰岛组织中iNOS的活性明显受到抑制,胰岛的凋亡程度减轻,存活和胰岛素分泌情况都明显改善。结论:iNOS在细胞因子诱导的胰岛细胞凋亡中起到十分关键的作用,而氨基胍通过抑制iNOS活性,减轻了细胞因子的损害,降低了胰岛凋亡水平,改善胰岛的存活与功能。     

3D自组装肽纳米纤维支架对胰岛细胞分泌功能的影响

背景：3D自组装肽纳米纤维支架能很好模拟体内微环境,给予细胞必要的结构模式,促进细胞外基质的正确组成及细胞的生长,改善细胞功能。 目的：体外观察3D自组装肽纳米纤维水凝胶支架对胰岛细胞分泌功能的影响。 方法：将3D自组装肽纳米纤维水凝胶支架与成年大鼠胰岛细胞共培养,AO-PI荧光染色法检测胰岛细胞的活性及生存率;放射免疫法测定胰岛细胞的分泌功能;扫描电镜观察胰岛细胞包裹在3D纳米支架中成三维立体的生长状态。 结果与结论：在3D纳米支架培养环境中胰岛细胞纯度≥80%;3D纳米支架组胰岛生存率及胰岛细胞分泌功能明显高于无支架组(2D培养组)(P < 0.05);扫描电镜显示自组装肽纳米纤维支架形成了具有几何形状的纳米级薄层,将胰岛细胞包裹在3D纳米支架中,胰岛细胞成三维立体生长。表明3D自组装肽纳米纤维支架可为胰岛细胞体外生存提供3D培养环境,改善胰岛细胞的活性、分泌功能及形态,延长胰岛细胞体外生存期。     

Exendin-4对不同热缺血时间心死亡供体大鼠胰岛功能的影响

背景：心死亡供体大鼠供胰的利用不可避免地经过热缺血损伤,因此热缺血时间的长短对获得的胰岛数量及功能有重要影响。 目的：观察Exendin-4对不同热缺血时间心死亡供体大鼠胰岛功能的影响。 方法：大鼠胰岛细胞体外培养,根据实验条件随机分为3大组：热缺血0,30和45 min组,每组又分为2亚组,其中对照组为胰岛细胞培养24 h;实验组为10 nmol/L Exendin-4与胰岛细胞培养24 h。应用双硫踪染色来计算分离胰岛数目并判断所提胰岛的纯度;丫啶橙/溴乙啶染色检测胰岛存活,胰岛素释放实验检测胰岛功能。 结果与结论：随着热缺血时间的延长各组获得胰岛细胞的数量、纯度、存活率及功能逐渐降低。热缺血0 min组、30 min组、45 min组加入10 nmol/L Exendin-4培养24 h后分离、纯化的胰岛数量及纯度、活性均比未加入Exendin-4培养的对照组有所提高,热缺血30 min及热缺血45 min组中的实验组与对照组相比差异均有显著性意义(P < 0.05)。提示Exendin-4对不同热缺血时间心死亡供体大鼠胰岛细胞具有保护作用,能减少胰岛细胞凋亡,改善胰岛的存活与功能;Exendin-4的使用可能成为胰岛移植早期缺血的边缘供体预处理的有效方法。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

同系大鼠骨髓间充质干细胞对同种异体胰岛刺激的T淋巴细胞的免疫调节作用

目的:研究同系大鼠骨髓间充质干细胞(Mesenchymal stem cell,MSC)对同种异体胰岛刺激的T淋巴细胞的免疫调节作用,以及对共培养胰岛胰岛素分泌的影响。方法:以Wistar大鼠胰岛作为刺激原,Lewis大鼠MSC及外周血T淋巴细胞作为共培养细胞,分为三组:实验组(A)20IEQ胰岛细胞、1×108 L-1 T淋巴细胞和1×107 L-1 MSCs共培养;药物对照组(B)20IEQ胰岛细胞与1×108 L-1 T淋巴细胞共培养并加入0.25μg/ml CsA;阳性对照组(C)20IEQ胰岛细胞与1×108 L-1 T淋巴细胞共培养。CCK-8检测共培养3天时T淋巴细胞对同种异体胰岛刺激的反应性,ELISA检测共培养3天时上清液IFN-γ、IL-2、IL-4和IL-10的含量,以及共培养胰岛的胰岛素分泌功能。结果:MSC与CsA均可抑制同种异体胰岛刺激的T淋巴细胞增殖,A组T淋巴细胞增殖率(17.10±2.7)%与B组(14.65±1.8)%间差异无统计学意义(P>0.05);C组IFN-γ、IL-2含量显著高于A组及B组,IL-10含量显著低于A及B组,差异有统计学意义(P<0.05);IL-4含量在A、B、C三组间差异均无统计学意义(P>0.05)。A组胰岛的胰岛素分泌水平显著高于B组和C组,差异有统计学意义(P<0.05);胰岛素刺激指数(2.37±0.52)显著高于B组(1.80±0.36),差异有统计学意义(P<0.05)。结论:MSC与CsA均可通过减少Th1类细胞因子的表达使受者Th1/Th2平衡向Th2方向偏移,抑制同种异体胰岛刺激的T淋巴细胞增殖。相对于CsA,MSC可以保持胰岛素高分泌水平和良好的糖刺激反应性,避免了CsA可能带来的毒性和副作用,具有一定的临床应用优势及前景。     

Glucose sensitivity of porcine and human islets in vitro

Preliminary experiments about the suitability of different commonly used culture media in our laboratory indicated, that prolonged exposure to high glucose concentrations during low temperature culture (LTC) impairs the viability of long term cultured human islets. As a consequence of the heterogenity of tested media the present study was aimed to evaluate the influence of different glucose concentrations on survival, viability and in-vitro function of cultured human islets in order to optimize islet survival until transplantation and to compare species dependent differences in glucose sensitivity. Quantified aliquots of freshly isolated (digestion-filtration, ficoll gradient purification) islets from consecutively processed human (n=6) and porcine (n=11) pancreata were subjected to different glucose concentrations (human islets: 500, 750, 1000 and 2000 mg/l; porcine islets: 1000 and 2000 mg/l) in CMRL (22°C) for 8–10 days. After LTC survival, viability and glucose-stimulated insulin release of incubated tissue was assessed. A reduction of glucose concentration promotes survival and viability of human islets but impairs in vitro function at the same time, presumably due to a reduced glucose oxidation as expressed by the significantly reduced stimulation index. In contrast to these findings in the human, elevated glucose concentration in porcine islet culture increases survival but reduces the glucose-stimulated insulin release and the viability of cultured islets. The contradiction of the results in regard to islet survival related to islet viability are still unclear in the pig and needs further evaluation.     

Functional abnormalities of islets of Langerhans of obese hyperglycemic mouse.

Glucose-induced insulin release was studied in vitro with isolated islets of Langerhans obtained from obese hyperglycemic C57Bl/6J-ob/ob (ob/ob) and lean C57Bl/6J-+/+ (control) mice. The threshold concentrations of glucose for insulin release were determined. In addition, the effect of total fast and of chronic food restriction on in vitro insulin release were studied. The following was observed: 1) with fasting, islet volume decreased. Islets obtained from ob/ob mice were larger than control islets, except for the chronic food restricted group. 2) Ob/ob islets were more sensitive to glucose than were controls in that the threshold for glucose-induced insulin release occured at lower glucose concentrations. 3) Fasting for 48 h completely abolished glucose-induced insulin release in control islets, whereas glucose-induced insulin release was maintained in 48-h and 7-day fasted ob/ob islets. 4) The increased glucose sensitivity of the ob/ob islets was maintained despite chronic food restriction.     

人胚胎胰岛分泌白细胞介素6的动力学研究

目的：阐述ＩＬ－６与胰岛功能或胰岛病变的可能关系。方法：采用常规消化方法分离人胚胎胰岛并进行原代培养,培养上清进行ＩＬ－６、胰岛素、胰高血糖素活性测定和ＩＬ－６ＭｃＡｂ中和实验,同时对胚胎胰岛进行体外葡萄糖刺激实现。结果：原代培养的胚胎胰岛在４０ｈ内ＩＬ－６分泌呈上升趋势（３７～１５３ｍＵ／胰岛）,每２４ｈ重新换取新培养液作为一个分泌周期,ＩＬ－６分泌以第１周期最高（１０６ｍＵ／胰岛）,体外连续培养ＩＬ－６的分泌以１ｄ最高（１４５ｍＵ／胰岛）,用ＩＬ－６ＭｃＡｂ可以阻断胰岛培养上清中ＩＬ－６的活性;胰岛素、胰高血糖素的分泌周期与ＩＬ－６不同;葡萄糖刺激明显促进ＩＬ－６的分泌,其分泌趋势与胰岛素分泌一致。结论：原代培养的人胚胎胰岛在无任何刺激情况下可自发分泌ＩＬ－６,而且ＩＬ－６的分泌有自己的分泌环路。     

Increased glucagon-stimulated insulin secretion of cryopreserved rat islets transplanted into nude mice

Cryopreservation is the only available technique for long-term storage of pancreatic islets. The freezing/thawing protocol may cause considerable loss of viable islet tissue and impair its function in vivo. The aim of this study was to investigate glucose and insulin levels after transplantation of fresh and cryo/thawed rat islets. Rat pancreatic islets were isolated following intraductal collagenase injection and Ficoll gradient purification. After isolation, islets were cultured for 24 h and then either transplanted or frozen after stepwise addition of DMSO according to Rajotte et al. and stored in liquid nitrogen. After rapid thawing islets were stepwise transferred into RPMI medium and cultured for another 24 h. The recipients were athymic mice with streptozotocine-induced diabetes. Two hundred fresh (n=13) or cryo/thawed (n=15) islets were transplanted beneath the renal capsule. Glucose levels were measured for 14 days and blood samples for insulin determination were obtained 15 min after i.p. glucagon (10 mg/kg) administration on day 14. Glucose levels were normalized (<9 mmol/l) in all recipients within 3 days since transplantation. On day 14, mean fasting values±SE in fresh and cryo/thawed islet groups were 4.0±0.6 and 4.4±0.4 mmol/l, respectively (P>0.05). Fasting insulin levels were higher in the cryo/thaw than in the fresh islet group (1.67±0.33 vs 0.57±0.13 ng/ml; P<0.01). Post-glucagon levels did not differ significantly (1.45±0.24 vs 0.86±0.24 ng/ml; P=0.06). While glucagon significantly increased insulin levels (P<0.01) in the fresh islet group, no change in insulin levels was observed (P>0.05) in the cryo/thaw group. Immunohistochemical staining demonstrated fragmentation of viable islet tissue which was more apparent in the cryo/thaw group. We conclude that in a short-term study cryo/thawed rat islets produce higher insulin levels than fresh islets transplanted into nude mice. This may be due to better islet survival or loss of feed-back regulation.     

Synergistic effect of microencapsulation and immunoalteration on islet allograft survival in bioartificial pancreas

Recently, we reported successful transplantation (Tx) of microencapsulated (mc) islets. However, graft failure observed in several cases was associated with an increased foreign body reaction compared to long-term functioning grafts. This study was performed to investigate the impact of an immunoalterating islet pretreatment (12–14 days culture at 22°C) on graft function. After microencapsulation in barium alginate beads the islets were cultured for another day. Diabetic LEWIS rats (blood glucose >19 mM) were transplanted with 3500 immunoaltered mc-Wistar islets intraperitoneally. Controls were transplanted with 3500 non-cultured syngeneic or allogeneic mc-islets. Additional syngeneic and allogeneic controls were transplanted with 6000 non-cultured, non-encapsulated islets intraperitoneally. Seventy percent of the recipients of microencapsulated, long-term low temperature cultured islets maintained normoglycemia at least for 15 weeks, while this was true in only 17% of those animals receiving microencapsulated non-pretreated allogeneic islets. Islets in non-encapsulated controls were rejected within several days. Graft function correlated with histologically proven viable islets within the capsules. Microencapsulation of islets markedly prolonged allograft survival compared to non-encapsulated islets; application of an immunoaltering low-temperature culture further improved graft function significantly. These data may support the hypothesis of induction of a reaction against microcapsules by the antigen release from the graft which may be avoided by immunoaltering islet pretreatment.     

Perfluorodecalin-enriched fibrin matrix for human islet culture

Disruption of microenvironment and decrease in oxygen supply during isolation and culture lead to pancreatic islet injury and their poor survival after transplantation. This study aimed to create a matrix for culturing islets, using fibrin as scaffold and perfluorodecalin as oxygen diffusion enhancing medium. Human pancreatic islets were divided in four groups: control, islets cultured in fibrin, islets in fibrin containing non-emulsified perfluorodecalin, and finally islets in fibrin supplemented with emulsified perfluorodecalin. After an overnight culture, cell damage (viability, proinsulin and insulin unregulated release, apoptosis (caspase-3 activation), secretory function, and presence of hypoxia markers (HIF-1a and VEGF expression) were assessed. Islets cultured in a matrix, had similar islet viability to controls (no matrix) but decreased levels of active caspase-3 and unregulated hormone release, but high level of hypoxia markers expression. Although the supplementation of fibrin with non-emulsified perfluorodecalin improves secretory response, there was no decrease in hypoxia markers expression. In contrast, emulsified perfluorodecalin added to the matrix improved islet function, islet viability and maintained level of hypoxia markers similar to control. Fibrin matrix supplemented with emulsified perfluorodecalin can provide a beneficial physical and chemical environment for improved pancreatic human islet function and viability in?vitro.     

Endotoxin impairs the engraftment of rat islets transplanted beneath the kidney capsule of C57BL/6-mice

The primary objective of this investigation was to determine the effect of endotoxin on islet xenograft survival within the first three days after transplantation. Pancreatic islets from Lewis rats were prepared under endotoxin-free conditions with Liberase (Boehringer) and purified by centrifugation on endotoxin-free Ficoll/Histopaque. After overnight incubation, with or without 10 μg/ml endotoxin, the islets were transplanted beneath the kidney capsule of normoglycemic C57Bl/6-mice. Three days later, kidneys were removed and their insulin content were measured. We could demonstrate significant differences (P<0.01) in insulin recovery between lipopolysaccharide-free and lipopolysaccharide-containing grafts. In case of endotoxin contaminated islets, we found only 13±2% (n=9) of the original insulin content, in contrast to 53±7% (n=9) when endotoxin-free islets where grafted. In experiments with islets isolated by use of conventional (lipopolysaccharide-containing) collagenase, and then cultured in endotoxin-free medium, insulin recovery three days after transplantation was 36±1% (n=13).     

Early islets and mesenchyme from an injured adult pancreas improve syngeneic engraftments and islet graft function in diabetic rats

A decrease in mass of isografts and a decline in islet function are major challenges in islet transplantations. Despite this, transplantation of 84?h harvested pancreatic duct ligation (PDL) tissues have been shown to have the same functional ability to foetal pancreata, but there was only 40% success in reverting hyperglycaemia. We tested the potential of early islets with mesenchymal stromal cells (MSCs) to promote isogeneic grafts survival and to restore normoglycemia in diabetic rats, in comparison with late islets. Islets were isolated from injured adult pancreata of donor rats at 24?h post ligation either with MSCs (24?h islet/MSC+) or without MSCs (24?h islet/MSC-), and at 84?h without MSCs (84?h islet/MSC-). These cells were transplanted under the renal capsule of syngeneic STZ-diabetic recipient rats. The islet grafts were monitored using the BGLs of recipients and the immunohistomorphology of the grafts were analysed using anti-insulin and anti-Ki67 antibodies. The mean BGL in 24?h islet/MSC+ recipients was reduced over time toward the control value. The curves of the mean BGLs in the control islet/MSC- and the 24?h islet/MSC- recipients dropped significantly below the control normal glucose group’s levels to reach their nadirs on weeks 4 and 6, respectively. Both curves had a peak overshoot on week 9, with no statistical significant difference between them. Engrafted islets were evident in these recipients, lasted for 5 and 6 weeks and correspondingly survived failure. However, insulin+ cells were present in the isografts of all recipients; but, only isografts in the 24?h islet/MSC+ presented with a homogenous subcapsular beta cell mass. In addition, the tendency of 24?h islet/MSC- to restore normoglycaemia with its survival capacity was statistically highly significant compared to the 84 islet/MSC- recipients (80%; 20%; p?=?0.001). Transplantation of early islets with MSCs from injured adult pancreata prolongs islet graft survival and improves isograft function in diabetic rats. This novel observation requires much further exploration for its clinical application, but this model already provides hope for new sources of donor islets for transplantation.     

Apoptosis in cultured rat islets of langerhans and occurrence of Bcl-2, Bak, Bax, Fas and Fas ligand

Isolated Langerhans islets are widely used for diabetic transplantation experiments and investigations of the mechanisms leading to the death or survival of insulin-producing cells in cultured islets. The present study was aimed at investigating programmed cell death and the role of apoptosis-associated peptides in insulin and glucagon cells of islets isolated from untreated rats and held in cultured suspension. Islets were removed from medium on days 0, 7, 14, 21 and 29, embedded in Epon, and semi-thin serial sections were prepared. At designated intervals, histologic sections were treated with the direct fluorescein-labelled TUNEL method and immunostained for pancreatic hormones (glucagon, insulin) and apoptotic peptides [Bak, Bax, Fas, Fas ligand (FasL)], as well as for the anti-apoptotic peptide Bcl-2. All tissue sections were investigated using confocal laser scanning microscopy under identical setting for semiquantitative estimation of staining intensity. The percentage of apoptotic cells was between 1.6 and 2.1% and most apoptotic cells were beta-cells. Corresponding cells often contained Bak and Bax. Fas and FasL were mostly detected in islet cells within the first week after preparing the cultured suspension. The insulin content was low (1.1 +/- 0.22 ng per islet) directly after isolation. It then increased progressively up to day 14, after which it began to decrease. Glucagon expression, on the other hand, remained high for the entire duration of the investigation. In conclusion, the islet beta-cells may recover after the isolation procedure, but after 4 weeks in culture, both the insulin content and Bcl-2 staining decrease. Moreover, apoptosis is mediated by different mechanisms after the isolation procedure and after culturing the islets for 1 month. The present data may be important for further studies on isolated, cultivated or transplanted islets.