大鼠胰岛β细胞分离与培养的初步研究

发布时间：2018-01-12 09:11

本文关键词：大鼠胰岛β细胞分离与培养的初步研究　出处：《四川大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的探索胰岛β细胞分离纯化的方法，寻找大鼠胰岛β细胞体外原代培养的适宜条件。方法选用正常喂养的Wistar大鼠(雌雄不限，7-8周龄，体重250-300克)，采用经胆总管插管注入胶原酶的方法对大鼠胰腺进行消化，然后分别以20目和80目的不锈钢网筛过滤初步纯化胰岛，双硫腙染色及葡萄糖刺激试验鉴定胰岛及其活性。分离纯化后的胰岛在RPMI-1640培养基中过夜培养。培养后的胰岛经洗涤沉降等处理后，以胰蛋白酶和DNA酶再次消化，，形成胰岛的单细胞悬液。将单细胞悬液置于2．8mmol／L葡萄糖液，用流式细胞仪(BD FACSAria)以100mW，488nm的激光照射，分选510nm～550nm处的细胞。用免疫组织化学染色法及不同浓度葡萄糖刺激试验鉴定分选细胞及其活性，将所分选β细胞置于不同葡萄糖和IBMX浓度的Ham's F-10培养基中培养，观察IBMX对β细胞活性的影响。结果实验大鼠胰腺经过胶原酶消化和初步纯化后，平均每只大鼠可获得550±95个胰岛，DTZ染色及葡萄糖刺激试验证明胰岛活性良好。进行FACS后，平均每只大鼠可得到约5688个β细胞，回收率为93．69％，分离纯度为85．5％。将β细胞置于不同浓度葡萄糖和IBMX的Ham'sF-10培养基中，在较高浓度葡萄糖条件下(10．0mmol／L及16．0mmol／L)，β细胞活性较高，死亡和凋亡也较少。未观察到IBMX对β细胞活性的影响。结论1、从正常喂养的健康大鼠分离纯化所得的胰岛经过胰蛋白酶及DNA酶再次消化后，形成的单细胞悬液，在外界葡萄糖浓度为2．8mmol／L时可用FACS对β细胞进行分选纯化，但是所得细胞的纯度尚不太理想； 2、在β细胞进行原代培养时，初步实验证明在外界葡萄糖浓度较高时(大于10．0mmol／L)，β细胞可保持较高的活性，尚未发现IBMX对细胞活性有无影响。因此，有待于大规模的实验进一步寻找最适宜的体外原代β细胞生长环境以及提高其成活率。
[Abstract]:Objective to explore the method of isolation and purification of islet 尾 cells and to find suitable conditions for primary culture of rat islet 尾 cells in vitro. Methods normal fed Wistar rats (male and female, 7-8 weeks old, weight 250-300g) were used to digest the pancreas by injecting collagenase into the common bile duct. The islets were purified by 20 mesh and 80 mesh stainless steel screen respectively. Dithizone staining and glucose stimulation test were used to identify the islets and their activities. The isolated and purified islets were cultured overnight in RPMI-1640 medium. The cultured islets were treated with washing and sedimentation. Trypsin and DNA enzyme were digested again to form the islet single cell suspension. The single cell suspension was placed in 2.8 mmol / L glucose solution. BD FAC SARIA was irradiated with a laser of 100mWN 488nm by flow cytometry. The cells at 510nm and 550nm were selected. The cells and their activities were identified by immunohistochemical staining and glucose stimulation test of different concentrations. The 尾 cells were cultured in Ham's F-10 medium with different glucose and IBMX concentrations to observe the effect of IBMX on the activity of 尾 cells. Results the pancreas of experimental rats was digested and purified by collagenase, and the average islets of each rat were 550 卤95. DTZ staining and glucose stimulation test showed that islet activity was good. After FACS, about 5688 尾 cells were obtained per rat, and the recovery rate was 93.69%. The purity of 尾 cells was 85.5%. The 尾 cells were placed in Ham'sF-10 medium with different concentrations of glucose and IBMX. The activity of 尾 cells was higher under the condition of higher glucose concentration (10.0 mmol / L and 16.0 mmol / L). The effect of IBMX on 尾-cell activity was not observed. Conclusion 1. The islets isolated from normal feeding healthy rats were digested again by trypsin and DNA enzyme, and the single cell suspension was formed. 2. When the concentration of glucose was 2.8 mmol / L, 尾 -cells could be separated and purified by FACS, but the purity of the cells was not ideal. 2. When 尾 cells were cultured in primary culture, the primary experiment showed that 尾 cells could maintain high activity when the concentration of glucose was higher (> 10.0 mmol / L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1)). It has not been found that IBMX has any effect on cell activity. Therefore, the most suitable growth environment and survival rate of primary 尾 -cells in vitro need to be further explored in large-scale experiments.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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