【作者】 李娜；

【导师】 马小军； 修志龙；

【作者基本信息】 大连理工大学 ， 生物化工， 2015， 博士

【摘要】 目前全球约有3.5亿人患有糖尿病,世界卫生组织(WHO)预测,在接下来的10年里,由糖尿病并发症引发的死亡将会超过50%。糖尿病患者中1型糖尿病约占10%,目前主要采用外源性胰岛素替代治疗的方法,然而相对于传统的胰岛素注射治疗,移植治疗是控制并发症的产生、提高患者生存质量的糖尿病最佳治疗手段。胰岛是生物活性物质,因此胰岛移植的成功取决于胰岛分离提取后的活性、运输中活性的保持等。因此,本文以胰岛为研究对象,并结合本实验室多年从事海藻酸钙凝胶支架研究所积累的成功经验,对胰岛的获得、纯化以及包埋胰岛进行研究。主要研究结果如下：(1)建立了高渗-低渗法优化胰岛纯化的方法。胰岛细胞表面存在葡萄糖转运蛋白-2(Glut-2),而外分泌细胞表面不表达该种蛋白,根据葡萄糖可以自由进出胰岛细胞这一特点,采用高渗-低渗处理可以有效裂解外分泌细胞,从而将其从胰岛中去除,获得纯度较高的胰岛。采用Plackett-Burman实验设计法对高渗-低渗处理过程中的关键因素进行了筛选。确定高渗液浓度及高渗处理时问为影响胰岛纯度的显著性因素。并且,在保持了胰岛功能和活性基础上,优化条件下获得的胰岛纯度显著性高于空白组(P<0.05),其纯度从87%提高到98%。(2)将海藻酸钠包埋技术应用于胰岛运输过程中的短期保存,发现海藻酸钠包埋胰岛的活性(97.8%)显著高于裸胰岛对照组(90.2%)。其次采用单因素实验确定了影响海藻酸钠包埋胰岛运输过程中的关键因素为预培养时间(0-24h)、血清浓度(2%-20%)、保存时间(12-24h),并进一步采用响应面Box-Behnken法优化得到胰岛活性随预培养时间、血清浓度、保存时间变化的方程,通过实验验证发现,该方程的预测值与实验值具有高吻合度(91.7-99.2%),表明建立的方程具有可信度,能够用于指导胰岛运输过程中保存工艺的建立。(3)本论文建立了工程化胰岛。由于胰岛单细胞可以聚集重组为胰岛类似物,因此本文采用海藻酸盐包封胰岛单细胞的方法制备胰岛细胞团。其形态与天然胰岛相似,直径在100μm内,并且重建后的工程化胰岛存活率几乎高达到100%,显著性高于分离获得的胰岛。此外,重建后的工程化胰岛均表达ISL-1, GCG, INS-1基因,并且保持了胰岛素释放能力。本文研究结果提示,采用高渗-低渗原理可以实现高纯度高活性分离提取胰岛,而海藻酸钙凝胶微球提供的微环境可以用于胰岛运输过程的保存,并且可以利用该体系重新构建工程化胰岛,实现对胰岛的高活性维持,为成功胰岛移植奠定基础。更多还原

【Abstract】 Diabetes mellitus currently affects about 350 million people worldwide, and the World Health Organization (WHO) predicts that death due to the complications of diabetes will likely increase by more than 50%over the next 10 years. Type I diabetes mellitus, which represents approximately 10%of all diabetes cases, requires daily insulin injections. This treatment, however, is demanding for the patient, can be painful, and does not eliminate the risk of diabetic complications. Islet transplantation is a promising treatment for type I diabetes mellitus that achieves an insulin-independent, constant normoglycemic state and avoids diabetic complications. While islets are biologically active substances, so a successful transplantation depends on optimizing islet viability in vitro, in culture after isolation from the pancreas, and during transportation. So the aim of this paper was to establish a new islet purification process to improve the purity of islets for implantation, and Alginate gels (ALGs) encapsulation technology was used to improve the islet purity. The main work of this dissertation is summarized as follows:Firstly, a new islet purification process comprising a hypertonic-hypotonic treatment step to improve the purity of islets was established. Glucose transporter-2 (Glut-2) locates on islet cell membrane and exocrine cells without Glut-2 transporter. This transporter plays a key role in the extracellular glucose-sensing mechanism. And the Plackett-Burman method was used to determine which factors had a significant influence on the purity of islets obtained after the hypertonic-hypotonic treatment step. The hypertonic solution concentration and the incubation time were both found to have a significant effect on islet purity. The purity of islets obtained using the modified purification process was significantly higher than that of islets obtained by density gradient alone. Importantly, good cell viability and normal insulin secretion ability of islets were maintained following the modified purification. The new purification process allows isolation of islets with improved purity and does not compromise the viability or function of the islets.Secondly, a method was developed that uses alginate encapsulation to protect islets from mechanical damage during shipment. We tested several variables for their impact on islet viability during transportation and used the significant variable to build a response surface methodology (RSM) model by Box-Behnken design method. This model can be used to predict islet survival rate and can serve as a guide for optimizing the transportation method of islets and increasing the success rate of the transplant procedure.Finally, in this paper, a new engineered islet was established. Some researchers found that islet single cells can reaggregate islet cell clusters, so ALGs was used to encapsulate islet cells to form artificial islets after dispersion islets into single cells. The shape of the islet cell clusters was similar to native islets, and the size of the islet cell clusters was limited to a maximum diameter of 100 μm. By limiting the diameter of this engineered islet cell clusters, cell viability was nearly able to 100%significantly improvement over native islets. Importantly, islet cell clusters were express the genes of islets, including Isl-1, GCG and INS-1, and insulin secretion-ability was maintained in vitro.In this study, the hypertonic-hypotonic treatment was effectively improved the purity of islets; ALG bead was an efficient microenvironment culture system, which might provide a useful platform for protecting islets from mechanical damage during shipment and establishing a new engineering islet to maintain high viability of islets, and promote the development of islets transplantation.更多还原