睾丸支持细胞与肝细胞混合共微囊化移植治疗大鼠急性肝功能衰竭

目的 观察大鼠睾丸支持细胞(Sertoli细胞)对微囊化肝细胞腹腔移植治疗大鼠急性肝功能衰竭疗效的影响.方法 气流法制备含单独肝细胞或Sertoli细胞与肝细胞混合的微囊.D-氨基半乳糖诱导大鼠急性肝功能衰竭模型,设模型组、裸肝细胞移植组、微囊化肝细胞移植组和Sertoli细胞与肝细胞混合微囊化移植组.每组24只,检测ALT、AST、TBil、Alb水平,RT-PCR法测定肝脏凋亡指标Smac/Diabln、caspase-3表达情况,同时各组另取15只大鼠进行生存率分析.观察腹腔内微囊的变化以及腹水中淋巴细胞数的改变.样本比较采用重复测量的多元方差分析或单因素方差分析.组间比较采用t检验.结果 Sertoli细胞与肝细胞混合微囊化治疗组ALT、AST在48 h时即分别降至(533.7±76.5)U/L、(381.2±46.7)U/L,TBil在72 h降至(7.364±2.18)μmol/L.血清Alb水平在48 h已升至(28.4±2.5)g/L,与其他各组相比,差异有统计学意义(F值分别为10.7、6.5、12.2和8.4,均P＜0.05);且Smac/Diablo、caspase-3 mRNA在48、72 h的表达水平也较其他组低(F值分别为3.7、4.8和3.6、4.2,均P＜0.05).微囊化肝细胞移植组和Sertoli细胞与肝细胞混合微囊化移植组大鼠生存率无明显差别,但明显高于其他两组.不同组的微囊均未与腹腔粘连.混合微囊组腹水中的淋巴细胞数也较肝细胞微囊组少(t=4.21,P＜0.05).结论 Sertoli细胞与肝细胞混合微囊化移植治疗肝功能衰竭的效果优于单纯微囊化肝细胞移植.纯肝细胞微囊在腹腔内仍可引起一定量淋巴细胞聚集,而Sertoli细胞的存在有助于减少这一现象.     

胰岛与Sertoli细胞体外共同培养

目的 研究胰岛体外长期培养的新方法。方法 采用绿色荧光蛋白（GFP）标记成年SD大鼠胰岛，与Sertoli细胞共同培养20周，应用形态学方法和透射电镜观察胰岛细胞单独培养和共同培养的生长特性；放射免疫法测定胰岛素分泌量。结果 胰岛单独培养3周。细胞存活率显著下降，胰岛阳性细胞数目减少，胰岛素24h累积分泌量和对葡萄糖刺激的反应性下降，大部分胰岛β细胞超微结构破坏，分泌颗粒减少；与Sertoli细胞共同培养的胰岛存活时间显著延长，细胞存活率和胰岛素阳性细胞数目较单独培养显著增加，胰岛素分泌量始终处于高水平状态，培养20周胰岛β细胞超微结构基本正常，结论 大鼠胰岛与Sertoli细胞共同培养可以促进胰岛生长，显著延长存活时间，是一种新的体外长期培养胰岛的方法。     

猪胰岛与大鼠睾丸支持细胞共同移植对糖尿病大鼠胰岛生长的影响

目的　研究胰岛异种移植的方法。　方法　用SD大鼠睾丸支持细胞 (Sertoli细胞 )与新生猪胰岛样细胞团 (ICCs)共同培养后进行异种移植。　结果　对照组胰岛细胞存活率及存活时间分别为 (63 .6%± 4.2 %和 3 .4d± 1.2d) ,较共同培养组 (87.2 %± 2 .7%和 13 .5d± 4.6d)显著为低(P <0 .0 1) ;对照组大部分胰岛细胞超微结构破坏 ,而共同培养组胰岛细胞超微结构基本正常 ;对照组胰岛素 2 4h累积分泌量和对葡萄糖刺激的反应性下降 ,共同培养组胰岛素分泌量始终处于高水平状态 (P <0 .0 1)。　结论　猪胰岛细胞与Sertoli细胞共同培养移植可以促进胰岛生长 ,明显延长胰岛移植物的存活时间     

Fas配体阳性睾丸Sertoli细胞对异种胰岛细胞移植的影响

目的探讨睾丸Sertoli细胞预防异种胰岛移植排斥反应的效果。方法分对照组、1×106细胞组、2×106细胞组、4×106细胞组,每组8只糖尿病大鼠,分离纯化猪胰岛细胞,植入糖尿病大鼠肾被膜下,观察胰岛的有功能存活及排斥反应情况。结果术后胰岛有功能存活时间分别为(6.0±0.6)d、(21.6±5.7)d、(35.7±8.9)d、(48.9±12.7)d,共移植组有功能存活时间较对照组明显延长(P<0.01),移植物存活时间与睾丸Sertoli细胞数量呈剂量依赖,术后第5天移植肾脏免疫组化检查显示4×106睾丸Sertoli细胞移植组有更多的大量结构完整猪胰岛细胞存活,浸润淋巴细胞被诱导凋亡。结论Fas配体阳性表达的SC细胞共移植能明显有效地抑制异种胰岛细胞移植的排斥反应,诱导局部淋巴细胞凋亡。     

大鼠肝细胞与睾丸支持细胞混合共微囊化的体外功能研究

目的 观察大鼠肝细胞与睾丸支持细胞混合共微囊化对肝细胞生物学活性的支持作用。方法利用微囊发生器制备含肝细胞、睾丸支持细胞及两者混合细胞的微囊，根据微囊内包裹细胞种类的不同，分为微囊化肝细胞组，微囊化睾丸支持细胞组，肝细胞、睾丸支持细胞分别微囊后共同培养组和肝细胞、睾丸支持细胞混合共微囊组。观察囊内细胞形态，体外培养测定培养液中Alb与尿素分泌，判断各组囊内肝细胞活性和功能。样本比较采用重复测量数据的多元方差分析。结果体外培养96h时，光学显微镜下见微囊呈球形，表面光滑，细胞均匀、散在分布于微囊内，囊内肝细胞、睾丸支持细胞呈圆形，未贴壁。微囊化睾丸支持细胞组培养上清液中无法测及Alb与尿素。与微囊化肝细胞组相比，肝细胞、睾丸支持细胞分别微囊后共同培养组与肝细胞、睾丸支持细胞混合共微囊组肝细胞的存活时间延长，培养上清液中Alb（F=217．56，P〈0．01）和尿素（F=232．72，P〈0．01）明显增加。混合细胞共微囊组于培养第7天Alb与尿素含量最高，分别为（2．80±0．11）g／L、（1．92±0．10）μmol／L，而其他两组均于培养第3天Alb与尿素含量达到最高，分别为（2．48±0．08）g／L、（1．47±0．08）μmol／L和（2．27±0．10）g／L、（1．37±0．05）μmol／L。相对于混合细胞共微囊组，肝细胞、睾丸支持细胞分别微囊后共同培养组在培养中后期Alb、尿素含量下降趋势明显。结论肝细胞与睾丸支持细胞混合共微囊化能明显延长囊内肝细胞的寿命，维持肝细胞形态和功能。     

大鼠胰岛与睾丸细胞共移植诱导同种胰岛移植免疫豁免

目的　探究大鼠胰岛与睾丸细胞共移植后睾丸细胞Fas配体表达能否对胰岛移植物提供免疫豁免作用。方法　将同种大鼠胰岛及睾丸细胞移植于糖尿病受体，观察移植物存活情况，并体外检测睾丸Sertoli细胞对活化淋巴细胞的抑制作用以及移植物内淋巴细胞凋亡情况。结果　单纯移植胰岛组移植胰岛平均存活期为(6±1)天。睾丸细胞和胰岛细胞共移植，当睾丸细胞数量为5×10     

冷冻保存后成人胰岛细胞与小肠黏膜下层的共培养

目的:探讨猪小肠黏膜下层(SIS)对冷冻保存后的胰岛细胞的支持和保护作用．方法:分离纯化后的成人胰岛细胞经冷冻保存1 mo后分为 SIS组和对照组,在RPMI-1640培养液培养1 wk后分别测定两组的胰岛细胞回收率,观察胰岛细胞形态并进行胰岛素刺激释放试验．结果:SIS组胰岛细胞回收率为90．5±1．8％,较游离组62．7± 3．6％显著提高(P<0．05),胰岛形态较对照组完整．在高糖刺激下,SIS组胰岛素分泌量较游离组增高(25．8±1．7 mU／L vs 14．6±1．3 mU／L,P<0．05)．在含有茶碱的高糖溶液中,微囊组的刺激指数为游离组的3倍．结论:SIS作为一种天然的细胞外基质材料,可显著提高胰岛细胞冷冻保存后的回收率,并改善胰岛细胞的功能．     

Fas配体表达对甲状旁腺细胞移植的影响

目的探究睾丸细胞Fas配体(FasL)表达对联合移植的甲状旁腺细胞的影响。方法将不同数量的大鼠睾丸Sertoli细胞与同种的甲状旁腺细胞联合移植,观察移植物的存活情况;并对移植物进行细胞成分、淋巴细胞凋亡及甲状旁腺功能检查。结果单纯甲状旁腺细胞移植组,存活期为(17±4)天。睾丸Sertoli细胞与甲状旁腺细胞联合移植组存活时间延长,存活期可超过60天。当Sertoli细胞数增加到4×106时,在60天观察期内,8只鼠中的6只血清钙及PTH值维持在正常范围内(P<0.01)。移植物内可见甲状旁腺细胞、表达FasL的睾丸细胞及凋亡的淋巴细胞,甲状旁腺细胞仍具有旺盛的分泌能力。结论睾丸Sertoli细胞FasL的表达诱导了活化的淋巴细胞凋亡,对联合移植的甲状旁腺细胞起到了保护作用。     

胰岛素产生细胞微囊化后的活力观察

目的观察胰岛素产生细胞经微囊化后活力变化,为糖尿病的治疗寻找可行方案。方法采用贴壁法获得纯化的BM-MSCs。制备RPE进行诱导分化,同时制备微囊并将诱导后的细胞包裹。观察细胞的形态并进行胰岛素分泌能力检测。结果经微囊包裹后的细胞呈聚集状态,并且培养一段时间后未发现微囊破裂、变形、囊内细胞溢出的现象。功能学检测结果显示,BM-MSCs不分泌胰岛素,未微囊化的IPCs和微囊化的IPCs均具有分泌胰岛素功能,且微囊不影响胰岛素的释放。结论胰岛素产生细胞经微囊化后活性依然良好,且RPE可诱导BM-MSCs分化为IPCs,此为糖尿病的治疗提供了新思路。     

脂联素抑制高浓度葡萄糖诱导的SD大鼠原代胰岛β细胞凋亡的研究

目的探讨脂联素对高浓度葡萄糖诱导的原代大鼠胰岛β细胞凋亡的影响及其可能机制。方法分离和培养原代胰岛细胞,分组干预并检测胰岛细胞凋亡率及相关基因表达水平。结果高糖组胰岛β细胞凋亡率、Caspase-3和BaxmRNA表达量显著高于正常葡萄糖和高糖联合脂联素组（P〈0．01）。结论脂联素可以抑制高糖诱导的大鼠胰岛β细胞凋亡,其作用机制可能与凋亡相关基因Caspase-3与Bax基因表达下调有关。     

Microencapsulated hepatocytes and islets as in vivo bioartificial liver support system

AIM: To confirm the xenotransplantation of microencapsulated hepatocytes and islets as a temporary bioartificial liver support system for mice with acute liver failure (ALF).METHODS: Mice were rendered ALF by a single intra-peritoneal injection of D-galactosamine (D-gal) and their tail blood was sampled to examine differences in blood ALT,albumin (ALB), total bilirubin (TB) and glucose (GLU) between 4 experimental groups. Rat hepatocytes and islets were collected and microencapsulated referring to both Sun‘s and Fritschy‘s methods. Mice were grouped into control group (CG), free hepatocyte group (FHG), microencapsulated hepatocyte group (MHG) and microencapsulated hepatocyte plus islet group (HIG). Tissue samples were subjected to microscopic and electron microscopic (EM) examinations.RESULTS: The highest survival was observed in HIG,surprisingly at 100%(16/16), while the lowest was in CG at 12.5%(2/16), with inter-group statistical difference P＜0.05. ALT levels revealed no statistical difference between groups but the ALB level of HIG descended by the slightest margin{q=(0.54, 0.24, 1.33), P＜0.05} at the time when it reached the lowest point in all groups. TB of HIG returned to normal reference range (NRR) statistically sooner than that of others after a fierce elevation. No statistical inter-group difference was observed in GLU levels. Fusion between hepatocytes and beta cells was demonstrated giving rise to theoretical assumptions.CONCLUSION: Hepatocytes to be microencapsulated together with islets should be a preferred in vivo hepatic functional supporting system, which can dramatically prolong survival and improve living status.     

Protection of islet allografts transplanted together with Fas ligand expressing testicular allografts

Summary Fas ligand (FasL) is highly expressed in testicular tissues and thought to be responsible for protection from allograft rejection by inducing apoptosis of anti-graft activated T cells. FasL-expressing islets have been shown to induce a granulocyte-mediated inflammatory reaction. We investigated whether a graft can be protected from alloimmune responses by manipulating the Fas/FasL-system. We transplanted allogeneic islets under the kidney capsule of streptozotocin-induced diabetic mice together with testicular tissue. Significant prolongation of survival of C3H islet allograft was observed in C57BL/6 (B6) recipients transplanted with C3H testicular tissue, but not in those transplanted with C3H-gld testicular tissue expressing non-functional FasL. No significant prolongation was observed in B6-lpr recipients expressing non-functional Fas. Immunohistochemical staining of C3H testicular tissue in the composite graft showed a high expression of FasL, but not that of the C3H-gld testicular tissue. In situ terminal deoxynucleotidyl transferase-mediated dUDP-biotin catalysed DNA nick-end labelling (TUNEL) staining of a composite graft of C3H islet and testicular tissue in B6 recipients demonstrated extensive apoptosis of infiltrating mononuclear cells around the graft. The protective effect of C3H testicular tissue was abrogated when anti-FasL monoclonal antibody was administered i. p. postoperatively. Our results suggest that FasL-positive testicular allografts protect composite islet allografts and indicate that manipulation of Fas/FasL mediated apoptosis is a suitable strategy for controlling rejection of islet allografts. [Diabetologia (1998) 41: 315–321] Received: 12 September 1997 and in revised form: 14 November 1997     

表皮生长因子在食蟹猴睾丸不同发育阶段表达的研究

目的观察表皮生长因子（EGF）在出生后食蟹猴睾丸发育成熟过程中的表达特征,探讨其在睾丸发育成熟中的作用。方法用免疫组化技术检测食蟹猴从新生猴期至成年猴期不同发育阶段睾丸EGF的表达情况。结果 32只食蟹猴睾丸均表达EGF。EGF主要表达于食蟹猴曲细精管内的精原细胞及一些间质细胞的胞质中,偶见表达于支持细胞胞质。各发育阶段睾丸精原细胞EGF阳性率存在差异（P〈0.01）,以新生猴期最低,童猴后期最高。结论 EGF在食蟹猴睾丸不同发育阶段的表达差异,提示EGF对睾丸的形态发育及功能起调节作用。     

转NCF基因3T3细胞在大鼠坐骨神经缺损中的修复作用

目的 了解微囊化转神经生长因子（NGF）基因3T3细胞在修复周围神经缺损中的作用。方法 异体静脉桥接神经缺损，形成神经再生室，其内填充微囊化转NGF基因3T3细胞，修复大鼠的坐骨神经10mm失。用微囊化3T3细胞作为对照组。术后12w ，进行辣根过氧化物酶（HRP）示踪实验。结果 术后12w，实验动物均未出现明显炎症及排斥反应。实验组脊髓灰质前角被HRP标记的阳性神经元数明显多于对照组，其差异具有显著性意义（P〈O.05）。结论 聚赖氨酸／海藻酸钠（APA）微胶囊与周围神经组织有良好的生物相容性；辅加转NGF基因3T3细胞对修复缺损的神经具有良好的桥梁作用和促神经生长作用。     

Microencapsulated islet grafts in the BB/E rat: a possible role for cytokines in graft failure

Summary Alginate-polylysine microencapsulation has been proposed as a method of protecting transplanted pancreatic islets against immunological attack. Using this technique, prolonged graft survival has been reported in some diabetic animals. However, in the spontaneously diabetic insulindependent BB/E rat we found that intraperitoneal implantation of microencapsulated islets had only a short-lived effect on hyperglycaemia. Recovered microcapsules (both those implanted empty and containing islets) were surrounded by a foreign body type cellular overgrowth and, although many capsules remained intact, encapsulated islets were observed to be disintegrating. Loss of Beta cells was confirmed by immunohistology. Various polymer materials used in artificial membranes have been shown to activate macrophages involved in foreign body reactions and induce synthesis of interleukin-1, a known Beta-cell toxin. Reduced secretion of insulin and progressive islet damage (indicated by a significant reduction in residual islet insulin and DNA content) were demonstrated when microencapsulated islets were incubated with interleukin-1in vitro for 9 days. Similar effects were seen following exposure to a combination of gamma interferon and alpha tumour necrosis factor. Successful use of microencapsulation in islet transplantation depends upon the development of biocompatible membranes. The exclusion of smaller molecules, such as cytokines, which may be involved in foreign body mediated damage and microencapsulated islet graft rejection, could also be important.     

体外共培养诱导骨髓间充质干细胞向胰岛样细胞分化

目的 观察成熟胰岛细胞对小鼠骨髓间充质干细胞(bone marrow derived mesenchymalstem cells,mMSCs)的诱导分化作用,为胰岛细胞移植治疗糖尿病提供移植细胞来源.方法 采用贴壁法分离培养小鼠mMSCs,并传代扩增.应用共聚焦显微镜观察细胞形态,流式细胞仪分析其表面分子.经胆总管注入Ⅳ型胶原酶消化胰腺,行密度梯度离心获得胰岛细胞.运用Transwell共培养体系,取第3代mMSCs与胰岛细胞共培养,倒置显微镜观察细胞的生长及形态变化,细胞免疫化学法检测mMSCs胰岛素的表达,胰岛素释放试验检测胰岛素分泌.以单独培养mMSCs作为对照组.结果 从小鼠骨髓中获得的细胞培养48 h后呈长梭形,体积较大,1周后呈集落式生长,可传代培养.细胞表面分子Sca-1、CD29、CD44、CD105呈阳性,且表达水平较高,而CD34、CD45阴性,证实为mMSCs.与小鼠胰岛细胞共培养7 d后,部分mMSCs细胞胰岛素免疫组化染色呈阳性,胰岛素分泌量为(16.83±0.15)μIU/ml.结论 从小鼠骨髓中分离培养的mMSCs与胰岛细胞共培养后可以被诱导分化为胰岛样细胞,为在胰岛细胞移植治疗糖尿病中存在的供体缺乏和免疫排斥问题提供了新的解决方法 .     

茶多酚减轻代谢综合征大鼠胰岛β细胞炎性损伤的机制研究

通过高糖高脂饮食诱导建立SD大鼠代谢综合征(MS)模型.茶多酚干预10周后大鼠空腹血糖、甘油三酯、胆固醇、低密度脂蛋白胆固醇、游离脂肪酸水平均较MS组明显下降(均P＜0.05),茶多酚组胰腺组织肿瘤坏死因子α、干扰素、诱导型一氧化氮合酶的mRNA和蛋白表达较MS组明显下降(均P＜0.05),白细胞介素1β蛋白表达也明显降低(P＜0.05).电镜观察茶多酚组大鼠胰岛β细胞的分泌颗粒较MS组增多,细胞器结构破坏减轻.提示茶多酚可通过抑制炎性细胞因子的产生,保护胰岛β细胞免受损伤.     

Immunohistochemical studies on vascular endothelial growth factor and platelet endothelial cell adhesion molecule-1/CD-31 in islet transplantation

Neovascularization may be necessary for better and longer function of transplanted islets. Vascular endothelial growth factor (VEGF) is known to be one of the most important factors of angiogenesis. Recently, VEGF was reported to be expressed in islets of normal pancreas. We studied the expression of VEGF and neovascularization related peptides in transplanted islets. To determine the angiogenic microcapillary, immunochemical staining was performed for Factor VIII-related antigen (von Willebrand factor [vWF]) and platelet endothelial cell adhesion molecule-1/CD31 (PECAM-1), both of which are known as markers of the angiogenic microvessel. Transplantable islets were isolated from Lewis rats (8-10 weeks of age) by discontinuous dextran gradient after collagenase digestion. Seven to twelve hundred islets were injected into the portal vein (IPV group, n = 7) or transplanted into subnephrocapsular cavity (SNC, n = 12) of the same descent rats. In the IPV group, the liver was resected 1 hour, 1 week, or 4 weeks after transplantation (Tx). In the SNC group, the kidney was resected 1, 3, 7, or 28 days after Tx. Each tissue was fixed in formaldehyde and embedded in paraffin. Serial 4-microm slices were immunostained for insulin, VEGF, PECAM-1, or vWF using specific antibodies. In IPV group, insulin-positive cells were VEGF positive as were in the normal pancreas at all time points. Islets of 1 hour after Tx were barely PECAM-1 positive as were in normal pancreas, but islets became weakly stained at 7 and 28 days after Tx. In vWF staining, transplanted islets showed stronger staining than those in the normal pancreas. In SNC group, VEGF was also stained in insulin-positive cells at 1, 3, 7, and 28 days. In PECAM-1 staining, islets of 1 day after Tx were barely stained as were in normal pancreas. However, the staining was increasingly enhanced from 3 to 7 days and then appeared weakened at 28 days after Tx. In vWF staining, islets were always vWF positive, as was seen in IPV group. This study revealed that PECAM-1 appeared in islets after islet Tx, suggesting that neovascularization occurs within the islet grafts. On the other hand, VEGF of transplanted islet did not obviously vary with time. Enhancement of the neovascularization may lead to better results of islet Tx.