Fas配体表达对同种胰岛移植的影响

目的探究睾丸细胞FasL表达能否对共移植的胰岛移植物提供免疫豁免作用以及胰岛细胞FasL基因转染对同种胰岛移植的影响。方法　将同种大鼠胰岛及睾丸细胞同时移植于糖尿病受体，重组腺病毒AdV-FasL感染胰岛细胞后移植，观察移植物存活情况、胰岛功能，并检测移植物内浸润淋巴细胞以及基因转染胰岛细胞凋亡情况。结果　单纯移植胰岛组平均存活期为(6.3±0.6)d。与胰岛细胞同时移植的睾丸细胞数增加至1×107时，存活期大于50d（P<0.05）。表达FasL的睾丸细胞在移植物内诱导浸润淋巴细胞凋亡。FasL基因转染组出现排斥加速，存活期缩短至(3.4±0.2)d。FasL转染的胰岛细胞在移植后24h见FasL表达，48h表达增强，移植后FasL转染胰岛细胞凋亡。结论　表达FasL的睾丸细胞与胰岛同时移植可诱导活化的淋巴细胞凋亡，使胰岛移植物获得免疫豁免、存活期延长，但通过FasL基因转染使胰岛细胞直接表达FasL引起胰岛细胞凋亡和粒细胞浸润，导致排斥加速。     

Immune privilege induced by cotransplantation of islet and allogeneic testicular cells

Objective To induce islet allograft long- term survival through cotransplantation of islet cells with sertoli cells. Methods Testicular sertoli cells were prepared by digestion with collagenase, trypsin an d DNase, and were cultured for 48 hours. Collagenase digested and Ficoll purifi ed donor (Wistar rat) islets were cotransplanted with allogeneic sertoli cells i n the absence of systemic immunosuppression. Terminal deoxynucleotidyl transfer ase- mediated X- dUTP nick- end labeling (TUNEL) was used to label apoptosis of lymphocytes surrounding the islet graft. Results Cotransplantation of islets and 1×10(7) sertoli cells reversed the diabetic sta te for more than 60 days in 100% (6/6) of the chemically diabetic Sprague Dawley rats. Grafts consisting of islets alone or islets plus 1×10(5) sertoli cells survived only for 5-6 days. Apoptosis of lymphocytes surrounding the islets was quite clear. Conclusion Cotransplantation of islets with FasL［+］ sertoli cells induces local immune priv ilege and allows long- term graft survival without systemic immunosuppression.     

The bone marrow as a potential receptor site for pancreatic islet grafts

BACKGROUND: Pancreatic islet transplantation in the human liver is being performed with increasing success to treat diabetes. However, the liver as a receptor site has many drawbacks due to immunological and non-immunological factors as well as important technical limitations. Bone marrow offers an easily accessible extrahepatic receptor site. Therefore, we attempted to explore the survival of pancreatic islets transplanted into the bone marrow of rats. METHODS: Pancreatic islets islografts and allografts were implanted into the bone marrow of rats. No immunosuppression was used. Morphology, presence of insulin, and glucagon and signs of apoptosis and rejection were explored. RESULTS: Pancreatic islets can be successfully engrafted into the bone marrow of rats, maintaining a normal histological appearance in insulin and glucagon content and no signs of apoptosis or rejection. CONCLUSIONS: These preliminary findings suggest that the bone marrow is capable of maintaining pancreatic islets in the absence of immunosuppression and, thus, can constitute an immunoprivileged environment for engraftment.     

输入供体血对大鼠移植胰腺白介素-2和TNF-α的影响

目的：了解输入供体血延长大鼠移植胰腺功能存活的机制。方法：取封闭群健康wistar大鼠作为供受体，制成糖尿病模型的受体大鼠分为对照和输入供体血二组。采用胰管开放诠胰腺腹腔内移植。术后7天处死动物。取移植胰腺作HE染色和胰岛gomori改变醛复红染色。碱性磷酸酶桥联酶标法检测淋巴细胞和细胞因子。结果：输入供体血组的移植胰腺腺泡和胰岛结构正常。CD^ 4T细胞显著增高。对照组有互腺腺泡和胰岛结构破坏。CD^ 8T细胞。IL-2，TNF-α显著增高，对照组CD^ 3T细胞、NK细胞均比输入供体血组高，但无显著差异。结论：输入供体血延长大鼠移植胰腺功能存活与IL-2，TNF-α降低有关。     

CD40LIg基因修饰骨髓间充质干细胞对异种胰岛移植排斥反应的抑制作用

 目的 探讨T细胞分化群40补体免疫球蛋白（CD40LIg）基因修饰骨髓间充质干细胞（MSCs）对异种胰岛移植排斥反应的抑制作用。方法 建立Wistar-SD大鼠异种胰岛移植模型,用携带CD40LIg基因的重组腺病毒感染的MSCs进行干预治疗,观察糖尿病大鼠胰岛移植后生存情况、血糖变化和移植物病理形态学改变,检测移植物CD40LIg和胰岛素的表达以及移植大鼠白细胞介素2（IL-2）和肿瘤坏死因子（TNF-α）的水平变化。结果 (1)糖尿病大鼠血糖平均在移植后2天恢复正常,对照组血糖平均在移植后7天升高,单纯MSCs组和转染MSCs组血糖分别在20天和47天升高。(2)对照组、单纯MSCs组和转染MSCs组,移植物存活时间分别为（9±3.2）d、(25±5.3)d和(53±7.5) d,各组间比较具有显著差异(F =5.362, P < 0.05);移植糖尿病大鼠生存时间分别为（24±6.8）d、(51±7.9)d、(95±12.7)d,各组间比较差异具有显著性(F =6.821, P < 0.05)。(3)对照组在胰岛移植后7d内,IL-2和TNF-α的水平均急剧上升,显著高于移植前水平(P < 0.01)。(4) 两个治疗组移植物内均可见成片的胰岛细胞团,未见淋巴细胞浸润,转染MSCs组移植物内可见CD40LIg和胰岛素的表达。结论 CD40LIg基因修饰骨髓间充质干细胞可以延长胰岛移植物的存活时间,抑制大鼠异种胰岛移植的排斥反应。     

生理盐水负荷对大鼠胰岛移植物原发性无功能的预防作用

目的 :观察生理盐水负荷对胰岛移植物原发性无功能的影响 ,以期寻找新的治疗原发性无功能的方法。方法 :建立链脲霉素诱导的大鼠糖尿病模型和左肾被膜下同种胰岛移植模型。术后实验组大鼠给予生理盐水负荷 ,对照组不给任何处理 ,通过测定血糖值判定移植物的功能。结果 :①注射链脲霉素后 4d～ 7d进行胰岛移植时 ,未见原发性无功能 (PNF)。注射链脲霉素后 9d以上时进行胰岛移植 ,在无生理盐水负荷的条件下 ,全部出现PNF ,两者差异显著 (P <0 0 1)。②对照组PNF的发生率为 10 0 % ,而附加生理盐水负荷后PNF发生率减少到 5 0 % ,两组差异非常显著 (P <0 0 1)。③把培养时间从 4h增加到 12h时 ,PNF的发生率从 5 0 %增加到 70 %和对照组无差异 (P >0 0 5 )。结论 :同种胰岛移植术后给予生理盐水负荷 ,可以预防胰岛移植物原发性无功能的发生。延长胰岛培养时间 ,可诱发胰岛移植物原发性无功能的发生。     

基因转移CTLA4-Ig对异种胰岛移植排斥反应的影响

目的 探讨基因转移细胞毒性T淋巴细胞相关抗原4免疫球蛋白(CTLA4-Ig)对异种胰岛移植排斥反应的影响.方法 用携带基因CTLA4-Ig的重组腺病毒转染人胰岛细胞,植人糖尿病大鼠肾胞膜下,建立人-大鼠异种胰岛移植模型,观察糖尿病大鼠移植后血糖变化、生存情况及移植物病理形态学改变,检测移植物CTLA4-Ig、胰岛素的表达和移植大鼠IL-2、TNF-α的水平变化.结果 (1)糖尿病大鼠胰岛移植后2 d血糖降至正常,对照组和转染组血糖平均分别在移植后8 d和25 d升高.(2)转染组移植物存活时间(28±6)d,较对照组(10±2)d显著延长(t=10.52,P＜0.01).(3)移植大鼠生存时间:转染组(48±8)d显著长于对照组(21±6)d(t=12.23,P＜0,01).(4)对照组在移植后1周内,IL-2、TNF-α水平均急尉上升,较移植前显著升高(P＜0.01);转染组则较移植前下降.(5)转染组移植物可见CTLA4-Ig和胰岛索的表达.结论 基因转移CTLA4-Ig可抑制异种胰岛移植排斥反应,延长移植大鼠和移植物的存活时间.     

Somatostatin receptor expression and biological functions in endocrine pancreatic cells: review based on a doctoral thesis

Type 1 diabetes is resulting from the selective destruction of insulin-producing betacells within the pancreatic islets. Somatostatin acts as an inhibitor of hormone secretion through specific receptors (sst1-5). All ssts were expressed in normal rat and mouse pancreatic islets, although the expression intensity and the co-expression pattern varied between ssts as well as between species. This may reflect a difference in response to somatostatin in islet cells of the two species. The Non-Obese Diabetic (NOD) mouse model is an experimental model of type 1 diabetes, with insulitis accompanied by spontaneous hyperglycaemia. Pancreatic specimens from NOD mice at different age and stage of disease were stained for ssts. The islet cells of diabetic NOD mice showed increased islet expression of sst2-5 compared to normoglycemic NOD mice. The increase in sst2-5 expression in the islets cells may suggest either a contributing factor in the process leading to diabetes, or a defense response against ongoing beta-cell destruction. Somatostatin analogues were tested on a human endocrine pancreatic tumour cell line and cultured pancreatic islets. Somatostatin analogues had an effect on cAMP accumulation, chromogranin A secretion and MAP kinase activity in the cell line. Treatment of rat pancreatic islets with somatostatin analogues with selective receptor affinity was not sufficient to induce an inhibition of insulin and glucagon secretion. However, a combination of selective analogues or non-selective analogues via costimulation of receptors can cause inhibition of hormone production. For insulin and glucagon, combinations of sst2 + sst5 and sst1 + sst2, respectively, showed a biological effect. In summary, knowledge of islet cell ssts expression and the effect of somatostatin analogues with high affinity to ssts may be valuable in the future attempts to influence beta-cell function in type 1 diabetes mellitus, since down-regulation of beta-cell function may promote survival of these cells during the autoimmune attack.     

Application of anti-CD103 immunotoxin for saving islet allograft in context of transplantation

Background Previous studies using knockout mice document a key role for the integrin CD103 in promoting organ allograft rejection and graft-versus-host disease. However, a determination of whether blockade of the CD103 pathway represents a viable therapeutic strategy for intervention in these processes has proven problematic due to the lack of reagents that efficiently deplete CD103＋ cells from wild type hosts. To circumvent this problem, in the present study, we invented an anti-CD103 immunotoxin （M290-SAP）. We investigated whether M290-SAP has capacity to eliminate CD103-expressing cells in vivo and protect transplanted islets from destroying by host immune cells.Methods Flow cytometry was used to analyze the efficacy of M290-SAP in depleting CD103-expressing cells in vivo.Then using allogenic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, the therapeutic efficacy of CD103-expressing cell depletion was addressed.Results M290-SAP dramatically reduces the frequency and absolute numbers of CD103-expressing leukocytes in peripheral lymphatic tissues of treated mice. Balb/c islets transplanted into streptozotocin-induced diabetic C57BL/6 mice under single M290-SAP treatment showed an indefinite survival time compared with untreated mice, M290-treated mice and IgG-SAP treated mice （mean survival time, ＞100 days vs. ＜20 days）. C57BL/6 islets transplanted into hyperglycemic NOD mice under single M290-SAP treatment showed a pronounced delay in allograft rejection compared with untreated mice （mean survival time 12-13 days vs. ＜7 days）. Immunological analysis of mice with long-term islet allograft survival revealed an obvious atrophy thymus and severe downregulation of alloimmunity of CD8 subpopulation response to allogenic stimulation.Conclusion Regardless of the underlying mechanisms, these data document that depletion of CD103-expressing cells represents a viable strategy for therapeutic intervention in islet allograft rejection.     

多途径联合诱导器官移植耐受和促进免疫功能重建

器官移植是治疗终末器官功能衰竭的主要方法,由于组织配型和高效免疫抑制剂的应用,急性排斥的发生率已大大降低,但目前慢性排斥仍无有效的控制方法,致使器官移植患者的长期(5～10年或更长)存活率明显偏低,而移植患者终身使用免疫抑制剂使感染、肿瘤的发病率增高,不但增加患者经济负担,而且严重影响患者的生活质量.通过诱导免疫耐受(移植耐受)的方法是解决移植排斥,避免上述问题的理想方法.     

Effects of mature Sertoli cells on allogeneic islets cocultured in vitro

INTRODUCTIONNowadays 180 million people suffer from dia-betes mellitus in world. An exogenous insulininjection has been the gold standard treatment fortype I diabetic patients, which is complicated andcould never completely correct the underlying loss ofislet cells and the resulting unregulated glucose lev-els. Islet transplantation seems to be an almost idealtherapy for insulin-dependent patients [1]. However,in isolate islet transplants the early clinical islet graftloss in 1 month is a…     

糖尿病小鼠左肾被膜下胰岛细胞与胰腺外分泌细胞共同移植动物模型的建立

目的：建立糖尿病小鼠左肾被膜下同种异体胰岛细胞与胰腺外分泌细胞共同移植动物模型及探讨胰腺外分泌细胞对胰岛移植物的损伤作用.方法：（1）体内实验：采用胆总管内逆行灌注胶原酶联合淋巴细胞分离液的方法来分离纯化胰岛,人工挑取胰岛细胞并收集胰腺外分泌细胞.链脲佐菌素腹腔注射诱导BALB/C小鼠成为糖尿病小鼠.单纯移植组（n=10）每只小鼠于左肾被膜上极移植胰岛细胞250个,共同移植组（n =10）每只小鼠于左肾被膜上下极同时移植胰岛细胞250个和等体积的胰腺外分泌细胞,持续观测血糖及生命体征变化,1个月后切除左肾并继续检测血糖.（2）体外实验：利用双硫腙对胰岛进行特异性染色来计算胰岛产量及纯度,利用台盼蓝染色鉴定胰岛细胞的活性,以及用葡萄糖刺激胰岛素释放实验来检测胰岛功能.结果：（1）胰岛移植后,单纯移植组及共同移植组血糖均逐步降至正常,共同移植组较单纯移植组血糖恢复正常时间延迟,移植术后第2,3,4,5天,单纯移植组受鼠血糖低于共同移植组,差异有统计学意义（P＜0.05）.切除两组受鼠左肾3d后,两组受鼠血糖均＞21 mmol/L.（2）每只小鼠可获得150～200个高质量胰岛,纯度及活性均高于90％,葡萄糖刺激后胰岛素释放量明显增加（SI=2.90）.结论：（1）成功建立糖尿病小鼠左肾被膜下同种异体胰岛细胞与胰腺外分泌细胞共同移植动物模型.（2）胰腺外分泌细胞与胰岛细胞同时移植会延迟植入胰岛功能恢复正常的时间.     

Blockade of γc signals in combination with donor-specific transfusion induces cardiac allograft acceptance in murine models

The γc cytokines play an important role in proliferation and survival of T cells. Blocking the γc signals can cause the activated donor-reactive T cells losing the ability to proliferate, and getting into apoptosis pathway, which contributes to induction of the peripheral tolerance. In this study, we induced the transplant tolerance through blocking the γc in combination with donor-specific transfusion (DST) in the cardiac transplantation. Following DST, on the day 2, 4 and 6, C57BL/6 recipients received an...     

Blockade of the OX40/OX40L pathway and induction of PD-L1 synergistically protects mouse islet allografts from rejection

Background OX40/OX40 ligand （OX40/OX40L） and programmed death-1/programmed death ligand-1 （PD-1/PD-L1） co- stimulator/signals play important roles in T cell-induced immune responses. The aim of this study was to investigate the roles of OX40/OX40L and PD-1/PD-L1 costimulatory pathways in mouse islet allograft rejection. Methods Lentiviral vectors containing OX40L siRNA sequences and an adenovirus vector containing the PD-L1 gene were constructed. The streptozotocin-induced model of diabetes was established in C57BL/6 （H-2b） mice. Diabetic C57BL/6 mice were randomly allocated into five groups： group 1, untreated control; group 2, Ad-EGFP treatment; group 3, Ad-PD-L1 treatment; group 4, OX40L-RNAi-LV treatment; group 5, OX40L-RNAi-LV combined with Ad-PD-L1 treatment. Lentiviral vector and the adenovirus vector were injected, singly or combined, into the caudal vein one day before islet transplantation. The islets of DBA/2 （H-2d） mice were transplanted into the renal subcapsular space of the diabetic recipients. Recipient blood glucose and the survival time of the allografts were monitored. Antigen-specific mixed lymphocyte reaction was also evaluated.     

PPARγ配体对1型糖尿病大鼠的治疗作用探讨

目的探讨PPARγ配体罗格列酮对STZ诱导的1型糖尿病大鼠胰腺β细胞的保护作用及其机制.方法实验用STZ诱导1型糖尿病大鼠动物模型,以罗格列酮5 mg*kg-1*d-1连续给药30 d.记录体质量和禁食血糖的变化.治疗第31天(停药后第1天)及治疗第61天取材观察胰腺形态学变化,应用免疫组织化学染色显示胰岛β细胞和nNOS的表达情况.结果与未治疗糖尿病动物相比,罗格列酮治疗组大鼠的糖尿病体征显著减轻,胰腺未见明显的病理改变,胰岛内胰岛素阳性细胞数量增多、接近正常水平,与此同时,胰腺内一氧化氮合酶(inducible nitric oxide synthase, iNOS)活性降低,而nNOS表达增强.结论罗格列酮对STZ诱导的1型糖尿病大鼠胰岛有一定的保护作用,其机制可能是通过抑制巨噬细胞内iNOS的活性,减轻胰岛的炎症反应,避免β细胞损害,促进胰岛功能的恢复.     

Inhibition of rejection in murine islet xenografts by CTLA4Ig and CD40LIg gene transfer

Background Costimulatory signals play a vital role in T cell activation. Blockade of costimulatory pathway by CTLA4Ig or CD40LIg have enhanced graft survival in experimental transplantation models yet mechanisms remain undetermined.We investigated the effects of CTLA4Ig and CD40LIg gene transfer on islet xenografts rejection in rats.Methods Human islets were infected with recombinant adenoviruses containing CTLA4Ig and CD40LIg genes and implanted beneath the kidney capsule of diabetic rats. Levels of blood sugar, morphological changes, and survival of grafts were recorded. Expressions of CTLA4Ig, CD40LIg and insulin were detected by immunohistochemical staining and cytokines levels were quantified by enzyme-linked immunosorbent assay （ELISA）.Results Blood glucose levels in transplant rats decreased to normal level on the 2nd day post transplantation. The mean blood glucose in the control group, CTLA4Ig transfected group, CD40LIg transfected group and CTLA4Ig ＋CD40LIg cotransfected group increased on days 8, 24, 21, 68, post transplantation respectively. The grafts in control group, CTLA4Ig transfected group, CD40LIg transfected group and CTLA4Ig ＋ CD40LIg cotransfected group survived for （8±1）, （29±4）, （27±3）, and （74±10） days, respectively. Survival in CTLA4Ig ＋ CD40LIg cotransfected group was significantly longer. Survivals of CTLA4Ig transfected group and CD40LIg transfected group were significantly longer than control group. In controJ animals, serum interleukin-2 and tumor necrosis factor a concentration significantly increased within seven days post transplantation. Haematoxylin eosin staining of grafts showed live islets in situ of transplant rats without inflammatory cell infiltration. Immunohistochemical staining confirmed the expression of insulin at islets in all experimental groups.Conclusions Transfer of CTLA4Ig and CD40Llg genes, especially the cotransfer of both, inhibits rejection of murine islet xenografts. Downregulated expressions of Th1 cells related cytokines might be related to the beneficial effects.     

完全弗氏佐剂抑制NOD鼠胰岛β细胞凋亡及其机制

目的:探讨完全弗氏佐剂( complete Freund’s adjuvant, CFA)对非肥胖糖尿病( nonobese dia-betic, NOD)鼠胰岛β细胞凋亡及凋亡相关基因Fas,FasL和Bcl-x表达的影响。方法:将4周龄NOD雌鼠随机分为CFA组(n=5)和生理盐水( NS)对照组(n=5 ) ,给CFA组鼠后脚板注射50μLCFA,对照者鼠后脚板注射等量NS。监测血糖,若血糖连续2 d≥11.1 mmol /L即诊断为糖尿病。当NOD鼠发生糖尿病或至30周龄时,处死动物,取胰腺组织制成薄切片,HE染色观察胰岛炎,采用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记( TUNEL)及ABC免疫组织化学双标记染色观察并计数凋亡的胰岛β细胞,ABC免疫组织化学法染色观察并记数Fas,FasL和Bcl-x表达阳性细胞。结果:至NOD鼠30周龄时,CFA处理组鼠无1只发生糖尿病,对照组鼠有3只发生糖尿病;CFA处理组的胰岛炎积分低于NS对照组(1.820±0.962 vs. 3.020±1.040,P<0.05 ) ;CFA处理组胰岛β细胞凋亡率、Fas阳性细胞率、FasL阳性细胞率均低于NS对照组[ (10.2±2.8) % vs. (15.9±6.5) %,(54.9±14.5)% vs.(75.7±12.9) %,(20.3±10.4) % vs. (27.9±12.0) %,P<0.05] ,Bcl-x阳性细胞率高于NS对照组[ (74.9±10.7) % vs. (66.0±18.3) %,P<0.05]。结论:CFA能够减轻NOD鼠胰岛β细胞凋亡,其机制与减少促凋亡基因Fas和FasL表达及增加抑制凋亡基因Bcl-x表达有关。     

基因修饰造血细胞移植后重建免疫功能的实验研究

目的 :探讨mFasL基因转移造血细胞移植后 ,受者免疫功能的重建情况。方法 :采用脂质体法将FasL cDNA转入BALB C鼠造血细胞 ,体外与BAC鼠骨髓移植物混合培养后输注给经致死量照射的BALB C鼠 ,通过观察死亡率、存活受者混合淋巴细胞反应 ,以了解移植后免疫功能的重建。结果 :未经骨髓移植组于 1周内全部死亡 ;未转基因骨髓移植组 6 0d存活率为 30 % ,转基因组 6 0d存活率为 80 % ;存活受者淋巴细胞对刺激原均表现有增殖反应。结论 :mFasL cDNA基因转移的造血细胞能成功地重建免疫功能     

大囊包被胰岛异种移植治疗糖尿病小鼠的实验研究

为防止胰岛异种移植后的排斥反应，根据免疫隔离原理，采用琼脂糖和胶原包被大鼠胰岛异种并将其植入糖尿病小鼠体内，观察受者的血糖变化。结果显示，接受大囊包被胰岛移植后，９２．３％的糖尿病小鼠血糖恢复正常，在不使用任何免疫抑制的飞速２下，受者正常血糖持续时间达１２５．８±５７．９天，而对照组正常血糖持续时间仅６．７５±２．９９天。受者耐糖曲线与正常对照组相似；