微囊化胰岛细胞异种移植治疗小鼠糖尿病

我们进行空囊移植及胰岛、微囊化胰岛的异种移植 ,以期寻找影响胰岛存活的原因 ,现将结果报道如下。一、材料和方法1 .糖尿病小鼠的制备 :链脲菌素溶于枸橼酸钠溶液中 ( ｐＨ4.5)制成浓度1 0 ｇ/Ｌ溶液 ,2 2 0ｍｇ/ｋｇ体重小鼠腹腔注入。非空腹血糖连续 2次超过 3.5ｇ/Ｌ则定为已患糖尿病。剪尾以便携式血糖仪测定尾静脉血糖。2 .胰岛细胞的分离和纯化 :胰岛细胞的分离见文献 [1 ]。胰岛细胞纯化 :Ｆｉｃｏｌｌ离心。消化沉淀物和 2 5%Ｆｉｃｏｌｌ溶液混匀 ,其上依次加入 2 3.0 %、2 0 .5%、1 1 .0 %Ｆｉｃｏｌｌ各 2ｍｌ,80 0 ｇ离心 1 5…     

海藻酸-壳聚糖-聚乙烯乙二醇微囊生物相容性的研究

目的比较海藻酸-壳聚糖-聚乙烯乙二醇微囊(ACP微囊)和海藻酸-聚赖氨酸-海藻酸微囊(APA微囊)的生物相容性。方法(1)两种微囊(50、100和200个)与健康人血清共浴,检测微囊对补体的激活程度。(2)1000个APA和ACP微囊分别植入Wistar大鼠的腹腔,4d和3周时统计取出的微囊数和微囊的纤维化率。(3)Wistar大鼠胰岛用ACP微囊和APA微囊包裹,分别贯续置于含3.3mmol/L和16.7mmol/L葡萄糖的Hank's溶液中培养,测定培养液中胰岛素的浓度。结果(1)ACP微囊组残余补体活性高于APA微囊组。(2)4d时,ACP和APA微囊的取出数分别是845.0±40.4和807.6±45.7(P>0.05),囊周纤维化率分别是16.40%和65.68%(P<0.05);3周时两种微囊的取出数分别为715.0±133.0和367.5±105.6(P<0.05),囊周纤维化率为27.8%和83.9%(P<0.05)。(3)在含3.3mmol/L葡萄糖的Hank's液中,未微囊胰岛组、APA和ACP微囊化胰岛组的胰岛素浓度分别是(123.48±4.70)mIU/L、(110.11±12.18)mIU/L和(110.90±11.95)mIU/L,当葡萄糖浓度为16.7mmol/L时,胰岛素浓度分别是(754.75±13.81)mIU/L、(689.30±27.71)mIU/L和(684.28±70.10)mIU/L。结论海藻酸-壳聚糖-聚乙烯乙二醇微囊的生物相容性要优于海藻酸-聚赖氨酸-海藻酸微囊,前者更适合应用于微囊化胰岛移植。     

微囊化新生猪胰岛样细胞团异种移植

本实验研究新生猪与大鼠产的异种移植。结果显示，新生猪胰岛样细胞团可纠正糖尿病大鼠的高血糖状态；海灌酸钠－多聚赖氨酸微囊能有效保护移植物的存活，平均存活时间为４个月。而微囊作为一种新型防排斥手段，将应用于广泛的领域。     

大鼠胰岛微囊化延长异种移植物在糖尿病小鼠体内生存期

本实验用胶原酶消化法分离新生大鼠胰岛，培养和Ｆｉｃｏｌｌ密度梯度离心纯化胰岛。用海藻酸－聚赖氨酸－海藻酸生物膜包裹胰岛并进行体外胰岛素分泌的能测定和体内异种移植实验。１０只链脲霉素诱导的糖尿病小鼠腹腔内移植微囊化胰岛３天后，小鼠血糖降至正常，体重逐渐增加，糖尿病得到纠正。维持最长者达１３０天，平均８５．２天。明显优于单纯胰岛植组，其功能维持不足９天。肾脏病理组织染色和透射电镜显示早期微囊化胰岛移植     

微囊化新生猪胰岛异种移植治疗糖尿病大鼠的实验研究

目的 观察微囊化新生猪胰岛异种移植对糖尿病大鼠的治疗效果及其对糖尿病并发症的预防作用。方法 糖尿病大鼠随机分为3组：①海藻酸钠-聚赖氨酸-海藻酸钠(APA)微囊包膜胰岛移植组；②未微囊包膜胰岛移植组，两组均接受胰岛腹腔移植；③未移植组。定期监测移植后血糖，白内障发生情况。并进行肾脏组织病理学检查，比较各组结果。结果 微囊化胰岛移植逆转了糖尿病大鼠的高血糖状态，最长达235天，明显长于未微囊胰岛移植组。有效的早期微囊胰岛移植的大鼠未出现糖尿病性白内障及肾小球基底膜改变，而未移植组及未微囊化胰岛移植组大鼠2～3个月后均出现白内障及肾小球基底膜改变。结论 APA微囊能有效保护移植物的体内存活，微囊化新生猪胰岛移植对大鼠糖尿病及其并发症有较好的治疗及顶防作用。     

微囊化大鼠胰岛异种移植的实验研究

目的　通过动物实验明确微囊化胰岛是否具有免疫隔离作用。方法　SD大鼠胰腺原位消化 ,Ficoll间断密度梯度离心法纯化、分离胰岛 ,气流吹喷制作海藻酸钠 /聚赖氨酸 /海藻酸钠(APA)微囊化大鼠胰岛 ,比较微囊化与未微囊化胰岛的胰岛素释放试验 ;将微囊化 (实验组 )与未微囊化 (对照组 )大鼠胰岛植入链脲佐菌素 (STZ)诱导的I型糖尿病小鼠中 ,作两组间血糖正常持续时间比较。结果　实验组与对照组的胰岛素释放试验差异无显著性 (P >0 .0 5 ) ;实验组血糖正常持续时间为 2 3～ 6 5d(平均 48d) ,对照组为 3～ 6d(平均 5d) ,两组差异有极显著性 (P <0 .0 1)。已排斥的实验组小鼠腹腔灌洗发现部分微囊化胰岛存活 ,部分已坏死 ,但微囊膜皆完整 ,囊壁无纤维化。结论　微囊具有良好的免疫隔离作用 ,可使胰岛移植物存活时间明显延长。同时推测微囊内移植物死亡与细胞因子、自由基作用或营养不足等有关。     

微囊化人胎胰岛移植治疗小鼠实验性糖尿病的观察

目的 :探讨微包囊技术在解决胰岛移植免疫排斥问题中的作用。方法 :将用链脲霉素 (STZ)制备的合格糖尿病模型鼠 2 1只随机分为 3组 ,每组 7只。空囊组腹腔内植入 5 0 0～ 6 0 0个空囊 ,游离胰岛组植入经胶原酶消化制备的人胎胰岛细胞 10 0 0± 10 0个 ,微囊组植入 10 0 0± 10 0个微囊包裹的胰岛细胞。结果 :游离胰岛组和微囊组小鼠在完全停用胰岛素的情况下 ,术后血糖分别降至 7.94± 2 .36mmol.L-1和 7.0 7± 1.15mmol.L-1,与空囊组比较差异有统计学意义 (t=13.170　P <0 .0 0 1,t=2 4 .999　P <0 .0 0 1) ,分别持续 7.4 3± 3.4 2天和 78.4± 2 1.2 7天 (t =8.6 5P <0 .0 0 1)。结论 :该微囊化人胎胰岛移植具有良好的组织相容性和免疫隔离作用 ,明显延长移植胰岛的存活时间     

微囊化大鼠胰岛异种移植治疗小鼠实验性糖尿病的研究

目的 研究海藻酸钠－聚赖氨酸－海藻酸钠包裹胰岛进行移植的效果。方法 将Ｗｉｓｔａｒ大鼠的胰腺先行胶原酶胰管内注射消化,然后分离,纯化,所得胰岛经培养后制成微囊包膜的胰岛,微囊直径为０．４￣０．５ｍｍ,每个微囊内包１个胰岛。     

改良的微囊化新生猪胰岛细胞移植治疗Ⅰ型糖尿病八例报告

在动物实验的基础上，对８例Ⅰ型糖尿病患者进行了改良的微囊化新生猪胰岛细胞移植治疗。８例患者中，３例移植于网膜囊，５例移植于三角肌，于移植前及移植后１、６、１２个月观察空腹血糖（ＦＢＧ）、糖基化血红蛋白Ａ１ｃ（ＨｂＡ１ｃ）、Ｃ－酞、ＣＤ４阳性细胞、ＣＤ８阳性细胞、可溶性白细胞介素２受体和胰岛素用量（ＩＮＳ）的变化。结果显示，移植前后Ｔ细胞亚群无明显变化，提示无排斥反应发生；结果还显示移植的猪胰岛数量决定了疗效：移植量少于１０个猪胰的胰岛（约３２６１８０±１２１３０个胰岛），移植后ＩＮＳ减量少于４０％，Ｃ－酞升高不明显，移植量达到１０个猪胰的胰岛，术后ＩＮＳ减量＞７５％，Ｃ－酞峰值是术前的２．５６倍。提示此种方法移植的必需量为１０个新生猪胰的胰岛。     

Xenotransplantation of microencapsulated fetal rat islets

Fetal pancreatic islets were isolated from 21-day pregnant Wistar rats and enclosed in semipermeable alginate-polylysine-alginate capsules. Encapsulated islets that had been previously cultured for eight days in vitro were shown to secrete insulin in response to glucose challenge: low-glucose, high-glucose, and high-glucose + 3-isobutyl-1-methyl-xanthine (IBMX). Transplants of 800-1000 encapsulated cultured fetal islets into the peritoneal cavities of BALB/c mice with streptozotocin-induced diabetes restored normoglycemia for up to 171 days without immunosuppression. When the capsules were removed from 2 of the recipients they both quickly regressed to a diabetic state. Control groups of diabetic mice received unencapsulated, uncultured islets or empty capsules. The mortality rate among these animals was high and none experienced relief from hyperglycemia for longer than 6 days. These results demonstrate that cultured microencapsulated fetal rat islets of Langerhans can release insulin in response to an in vitro glucose challenge, and that transplants of these islets into diabetic mice can restore normoglycemia without the need for immunosuppressive therapy.     

Xenotransplantation of microencapsulated pancreatic islets contained in a vascular prosthesis: preliminary results

Porcine and human pancreatic islets were microencapsulated in an alginate-polylysine biomembrane and put in a chamber of a new vascular prosthesis composed of an inner tubing of Dacron mesh and an outer tubing of expanded polytetrafluorethylene material. The vascular prosthesis was anastomized between the iliac artery and the contralateral vein of diabetic dogs. The recipients did not receive any immunosuppressive therapy. Function of porcine and human islets was monitored by measuring serum glucose levels and human C-peptide concentrations. After transplantation, serum glucose levels were maintained at values lower than 200 mg/dl, and C-peptide concentrations were between 0.8 and 3.2 ng/ml. Injected insulin requirements decreased by 50%–60%. Four to 8 weeks after transplantation, histologic examination showed well-preserved and functioning islets in the majority of intact microcapsules. Fibrin and inflammatory cells were not observed in the chamber. These data suggest long-term survival and function of microencapsulated pancreatic islets in the vascular prosthesis.     

Xenotransplantation of microencapsulated pancreatic islets contained in a vascular prosthesis: preliminary results

Abstract. Porcine and human pancreatic islets were microencapsulated in an alginate-polylysine biomembrane and put in a chamber of a new vascular prosthesis composed of an inner tubing of Dacron mesh and an outer tubing of expanded polytetrafluorethylene material. The vascular prosthesis was anastomized between the iliac artery and the contralateral vein of diabetic dogs. The recipients did not receive any immunosuppressive therapy. Function of porcine and human islets was monitored by measuring serum glucose levels and human C-peptide concentrations. After transplantation, serum glucose levels were maintained at values lower than 200 mg/dl, and C-peptide concentrations were between 0.8 and 3.2 ng/ml. Injected insulin requirements decreased by 50%-60%. Four to 8 weeks after transplantation, histologic examination showed well-preserved and functioning islets in the majority of intact microcapsules. Fibrin and inflammatory cells were not observed in the chamber. These data suggest long-term survival and function of microencapsulated pancreatic islets in the vascular prosthesis.     

微囊化人胎胰岛功能的体外观察

目的 :观察微囊的通透性及对微囊化胰岛分泌功能的影响。方法 :将用胶原酶消化获得的胰岛细胞分为 2份 ,1份微囊包膜 ,1份未予包膜作为对照 ,培养 7天 ,比较两组培养液中胰岛素含量并分别用2 .7mmol·L-1、16 .6mmol·L-1浓度的葡萄糖及 16 .6mmol·L-1的葡萄糖加 10mmol·L-1茶碱刺激胰岛B细胞 ,观察其胰岛素分泌功能。结果 :两组在培养液中胰岛素含量及葡萄糖、茶碱刺激的胰岛B细胞胰岛素分泌方面无显著性差异 (P >0 .0 5 )。结论 :该微囊具有良好的通透性 ,微囊化胰岛的活性和分泌功能未受成囊过程的影响     

微囊化猪胰岛肝动脉内移植治疗犬Ⅰ型糖尿病

目的　观察放射介入方法行微囊化猪胰岛细胞肝动脉肝内移植的可行性及有效性。方法　采用放射介入技术经股动脉穿刺将微囊猪胰岛经肝动脉植入糖尿病模型犬肝内 ,术后监测空腹血糖、C肽、胰岛素用量变化 ,行肝功能、肝脏组织病理学检查。结果　移植后糖尿病犬血清C肽水平升高 2～ 6倍 ,胰岛素用量逐步减少 ,3只犬停用胰岛素并维持空腹血糖正常 ;移植后肝转氨酶一过性升高 ,2周后降至正常。结论　经肝动脉肝内微囊猪胰岛素异种移植治疗移植Ⅰ型糖尿病犬安全、可行。     

Biodritin microencapsulated human islets of Langerhans and their potential for type 1 diabetes mellitus therapy

BACKGROUND: Microencapsulation of pancreatic islets with polymeric compounds constitutes an attractive alternative therapy for type 1 diabetes mellitus. The major limiting factor is the availability of a biocompatible and mechanically stable polymer. We investigated the potential of Biodritin, a novel polymer constituted of alginate and chondroitin sulfate, for islet microencapsulation. METHODS: Biodritin microcapsules were obtained using an air jet droplet generator and gelated with barium or calcium chloride. Microencapsulated rat insulinoma RINm5F cells were tested for viability using the [3-(4,5-dimetyl-thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] [MTT] colorimetric assay. Microencapsulated rat pancreatic islets were coincubated with macrophages derived from mouse peritoneal liquid to assess the immunomodulatory potential of the microcapsules, using quantitative real time-PCR (qPCR). Biodritin biocompatibility was demonstrated by subcutaneous injection of empty microcapsules into immunocompetent Wistar rats. Insulin secretion by microencapsulated human pancreatic islets was evaluated using an electrochemoluminescent assay. Microencapsulated human islets transplanted into chemically induced diabetic mice were monitored for reversal of hyperglycemia. RESULTS: The metabolic activity of microencapsulated RINm5F cells persisted for at least 15 days. Interleukin-1beta expression by macrophages was observed during coculture with islets microencapsulated with Biodritin-CaCl2, but not with Biodritin-BaCl2. No statistical difference in glucose-stimulated insulin secretion was observed between nonencapsulated and microencapsulated islets. Upon microencapsulated islet transplantation, the blood glucose level of diabetic mice normalized; they remained euglycemic for at least 60 days, displaying normal oral glucose tolerance tests. CONCLUSION: This study demonstrated that Biodritin can be used for islet microencapsulation and reversal of diabetes; however, further investigations are required to assess its potential for long-term transplantation.     

Prolonged reversal of diabetic state in NOD mice by xenografts of microencapsulated rat islets

Transplantation of the islets of Langerhans could be the most promising approach to the clinical treatment of insulin-dependent (type I) diabetes mellitus. In this study, we report on a modified encapsulation technique that produces small alginate-polylysine capsules (0.25-0.35 mm diam). In an in vitro study, both encapsulated and unencapsulated islets showed comparable responses to glucose challenge in terms of insulin secretion. With the new capsules, 16 spontaneously diabetic NOD mice received transplants of 800 encapsulated rat islets/animal. Nonfasting blood glucose concentration decreased from 24.4 +/- 1.4 to 4.0 +/- 1.3 mM. At 4 and 5 mo posttransplantation, the capsules were removed from 2 recipients. Both animals regressed to a hyperglycemic state after capsule removal. However, after another islet transplantation, normoglycemia was again restored in these 2 animals. In control mice, which received unencapsulated islets, the xenografts remained functional for less than 10 days. A high mortality rate was observed among these animals within 2 mo of the recurrence of the hyperglycemic state. Our results clearly indicate that encapsulation of pancreatic islets in the improved capsules can effectively prolong xenograft survival without immunosuppression in an animal model that mimics human type I diabetes mellitus.     

Cytokine-induced functional suppression of microencapsulated rat pancreatic islets in vitro

BACKGROUND: It is likely that inflammatory cytokines are released near microencapsulated islets in vivo. METHODS: Rates of insulin release or glucose oxidation were measured after culture of microencapsulated rat islets with interleukin (IL)-1beta and tumor necrosis factor-(TNF-alpha). Their ability to recover from IL-1beta-induced suppression was also investigated. RESULTS: Microencapsulated islets were suppressed after exposure to IL-1beta, which was potentiated by TNF-alpha. After exposure to lower IL-1beta concentrations, microencapsulated islets had similar oxidation rates as corresponding controls. At higher concentrations, microencapsulated islets were more suppressed than nonencapsulated islets. Microencapsulated and control islets were able to recover from suppression after exposure to 2.5 U/ml of IL-1beta. CONCLUSIONS: Microencapsulation using the present alginate/poly-L-lysine/alginate capsules does not protect islets against the detrimental effects of IL-1beta and TNF-alpha. Indeed, microencapsulated islets seem to be more susceptible to suppression at higher concentrations of IL-1beta. However, after exposure to a lower concentration of IL-1beta, microencapsulated islets can recover.