【作者】 张旭辉；

【导师】 范明； 王常勇；

【作者基本信息】 中国人民解放军军事医学科学院 ， 细胞生物学， 2004， 硕士

【摘要】 近年来糖尿病的患病率在世界范围内呈明显上升趋势，并给患者家庭和社会带来了沉重的经济负担。胰岛移植治疗糖尿病受到国内外研究人员的广泛关注，是目前国际上糖尿病治疗研究热点之一。胰岛移植能够对胰岛素的释放在生理水平上进行及时、精确的调控，并可以避免一系列慢性综合症的发生。尽管如此，目前胰岛移植仍存在亟待解决的两大难题即胰岛来源不足和移植物引发的免疫排斥反应。睾丸支持细胞(Sertoli cell，SC)是睾丸组织维持免疫豁免的关键细胞，并具有重要的营养、支持、吞噬、分泌等功能。为此，本研究首先摸索和掌握了胰岛和SC体外分离、培养的关键技术，其次针对SC对共移植的胰岛在体内发挥的免疫豁免作用进行了相关的实验研究。在此基础上，应用旋转式细胞培养系统(Rotary cell culture system，RCCS)体外共培养胰岛和SC，使二者形成了支持细胞-胰岛共聚物(Sertoli-islet cell aggreg ates，SICA)，通过对SICA体外胰岛素分泌特性的检测及糖尿病大鼠体内移植的研究，为SC和胰岛联合体内移植治疗糖尿病奠定了实验基础。 研究简述如下： 在第一部分，采用V型胶原酶和应用Ficoll不连续密度梯度离心法，从成年大鼠胰腺分离、纯化胰岛，并对其进行了鉴定。在此基础上，将胰岛在几种不同细胞外基质材料包被的培养板上培养，并对其生物学特性进行了检测。结果表明：从大鼠胰腺中分离、纯化的胰岛，通过双硫腙染色鉴定，其纯度为80％，台盼兰染色结果表明其活力为90％。在不同基质材料包被的培养板上培养胰岛，经生物学特性检测发现Matrigel包被组上培养的胰岛，其胰岛素分泌特性最好。 在第二部分，用V型胶原酶和胰酶系列消化SC，并从组织学、免疫组织化学、透射电镜检测等多方面对SC进行鉴定。结果证实，本部分分离、纯化所得的细胞为纯度较高的SC。 在第三部分，首先建立了链尿佐菌素诱导的糖尿病SD大鼠模型，然后将2000IEQ胰岛和1×10~5、1×10~6、1×10~7SC分别植入糖尿病大鼠肾被膜下，用便携式血糖仪检测血糖，并进行组织学染色。研究发现在不使用任何免疫抑制剂硕十学位论文中文摘要的情况下，2000工EQ胰岛和数量为1 xlJ个SC的共移植物，能够将糖尿病大鼠的血糖值维持在正常水平达41士2.5天，与其它组相比具有显著性差异(p<0.05)，且取移植后40天血糖仍维持正常的大鼠移植侧肾脏标本进行胰岛素抗体和Fas一L抗体免疫组织化学染色，结果显示移植SC和胰岛均正常存活。 在第四部分，将胰岛和SC在RCCS中共培养，使二者形成SICA，首先应用免疫组织化学和扫描电镜观察S工CA，其次应用放射免疫方法检测S工CA体外胰岛素分泌特性。在此基础上，将Z000IEQ胰岛和1火107个SC在RCCS中所形成的SICA植入糖尿病大鼠，相同细胞数量的胰岛和SC混合物作为对照组。体内移植后监测大鼠血糖变化情况。扫描电镜结果显示:在RCCS中培养形成的S1以结构紧凑、呈圆型或椭圆型，其中体积较小的SC贴附在胰岛细胞团的周围。免疫组织化学染色结果显示，SICA球体的中心区域为胰岛素抗体染色阳性反应的胰岛，外围区域则为Fas一L抗体染色呈阳性反应的SC。S工以体外胰岛素分泌实验表明:5 ICA具有良好的胰岛素分泌特性，与其它各组相比具有显著性差异(p<0.05)。SICA体内移植实验表明，SICA移植组大鼠血糖正常水平平均维持时间为56.0士1.7天，与胰岛和SC混合移植组相比具有显著性差异(p<0 .05)。 综合上述结果表明:SC和胰岛体内共移植后，适量的SC能够为共移植的胰岛提供免疫豁免。S工CA与单纯SC和胰岛混合物移植相比，能够在较长时间内将糖尿病大鼠的血糖水平维持在正常范围内，推测原因是RCCS能模拟细胞及组织体内生存环境，使胰岛更易保持其在体内原有的生物学功能。上述结果也表明，在RCCS中联合培养胰岛和SC，可以构建出类胰岛样结构，该结构的形成更易于SC为中心的胰岛团提供营养支持和免疫豁免。 本论文研究通过对SC和胰岛体外分离、培养、生物学功能检测以及体内移植治疗糖尿病模型鼠的系统研究，为今后应用SC和胰岛共移植治疗糖尿病，积累了关键实验数据，并奠定了相关研究的基础。更多还原

【Abstract】 Diabetes is a worldwide health problem characterized with high morbidity and mortality. Islet transplantation can restore the glucose homeostasis to the physiological level. However two major obstacles remain before islet transplantation hits the medical market.: the source of islets and the host immune attack on the islets. The purpose of this study is to investigate the Sertoii cells’ immune privilege protection effect on cotransplanted islets and the potential application in reverse of the diabetic state by Sertoli-islet cell aggreg ates (SICA).In the first part of the study, adult rat islets were isolated with type V collagenase and purified by Ficoll gradient centrifugation at densities of 11%,20%,23% and 25%. The islets were then cultured on extracelluar matrix-coated surfaces to investigate the supporting effect of different extracellular matrix, including type I and IV collagens, and Matrigel,on the viability and function of cultured islets. Islets were stained with diphenylthiocarbazone (DTZ) and trypan blue. The results showed that the purity and viability were 80% and 90%, respectively. Cell viability and insulin secretion level were the best in islets cultured in Matrigel-coated group.In the second part of the study, testicular Sertoii cells were prepared by serial digestion of rat testis using type V collagenase (2.5mg/ml), trypsin (25μ g/ml) and Dnase (4 μ g/ml). Then, Sertoii cells were identified by histological, immunohistochemical, and electron microscopic analysis. All the results firmly holded that a high purity sertoli cells were got through our established culture method.In the third part of the study, Sertoii cells and islets were cotransplanted under therenal subcapsular space of diabetic rats. Each diabetic recipient received 2,000 IEQ islets together with different dosage of Sertoli cells which were 1 X 105, 1X 106and 1 X 107. Blood glucose was monitored every other day. In the absence of the immunosuppression, the transplantation of a quantity of Sertoli cells (1 X 107) together with 2,000 purified islets, reversed the diabetic state for 41 ?.5 days. Statistical analysis showed significant difference among the five groups and the best efficiency was the Sertoli cells group at a density of 1 X 107 cells (p<0.05).The immunohistochemical study of the Sertoli cells (1 X 107) group demonstrated positive staining of Fas-L and insulin antibody.In the fourth part of the study, 2,000 IEQ islets and 1X107 Sertoli cells were cocultured in RCCS. Three dimensional Sertoli-islet cell aggregates were obtained. The basal insulin release level and glucose-stimulated insulin secretion level of SICA were assessed by radioimmunoassay. Immunohistochemical and electron microscopic analysis were also performed. SICA or a mixture of islets and Sertoli cells was transplanted under the renal subcapsular space of diabetic rats. In vitro insulin secretion test indicated that the best activity islets appeared in the SICA group. During the in vivo transplantation test, SICA group reversed the diabetic state for 56.0 + 1.7 days, which was of statistical significance in comparison with the Sertoli cells and islets cotransplantation group. The results of scanning electron microscopy of SICA showed intact islets surrounded by Sertoli cells. Fas-L and insulin immunostaining were positive.We concluded that cotransplantation of islets in accordance with certain ratio of Sertoli cells can induce local immune privilege. Secondly, SICA can help improve graft survival by inducing immunosuppression, since the structure of SICA makes it easier for the SC to provide immunosuppression and trophic support for the islets.更多还原