本文關(guān)鍵詞：大鼠胰島β細(xì)胞分離與培養(yǎng)的初步研究　出處：《四川大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 胰島β細(xì)胞 細(xì)胞的分離純化 原代培養(yǎng)

【摘要】： 目的探索胰島β細(xì)胞分離純化的方法，尋找大鼠胰島β細(xì)胞體外原代培養(yǎng)的適宜條件。 方法選用正常喂養(yǎng)的Wistar大鼠(雌雄不限，7-8周齡，體重250-300克)，采用經(jīng)膽總管插管注入膠原酶的方法對大鼠胰腺進(jìn)行消化，然后分別以20目和80目的不銹鋼網(wǎng)篩過濾初步純化胰島，雙硫腙染色及葡萄糖刺激試驗(yàn)鑒定胰島及其活性。分離純化后的胰島在RPMI-1640培養(yǎng)基中過夜培養(yǎng)。培養(yǎng)后的胰島經(jīng)洗滌沉降等處理后，以胰蛋白酶和DNA酶再次消化，，形成胰島的單細(xì)胞懸液。將單細(xì)胞懸液置于2．8mmol／L葡萄糖液，用流式細(xì)胞儀(BD FACSAria)以100mW，488nm的激光照射，分選510nm～550nm處的細(xì)胞。用免疫組織化學(xué)染色法及不同濃度葡萄糖刺激試驗(yàn)鑒定分選細(xì)胞及其活性，將所分選β細(xì)胞置于不同葡萄糖和IBMX濃度的Ham's F-10培養(yǎng)基中培養(yǎng)，觀察IBMX對β細(xì)胞活性的影響。 結(jié)果實(shí)驗(yàn)大鼠胰腺經(jīng)過膠原酶消化和初步純化后，平均每只大鼠可獲得550±95個胰島，DTZ染色及葡萄糖刺激試驗(yàn)證明胰島活性良好。進(jìn)行FACS后，平均每只大鼠可得到約5688個β細(xì)胞，回收率為93．69％，分離純度為85．5％。將β細(xì)胞置于不同濃度葡萄糖和IBMX的Ham'sF-10培養(yǎng)基中，在較高濃度葡萄糖條件下(10．0mmol／L及16．0mmol／L)，β細(xì)胞活性較高，死亡和凋亡也較少。未觀察到IBMX對β細(xì)胞活性的影響。 結(jié)論1、從正常喂養(yǎng)的健康大鼠分離純化所得的胰島經(jīng)過胰蛋白酶及DNA酶再次消化后，形成的單細(xì)胞懸液，在外界葡萄糖濃度為2．8mmol／L時可用FACS對β細(xì)胞進(jìn)行分選純化，但是所得細(xì)胞的純度尚不太理想； 2、在β細(xì)胞進(jìn)行原代培養(yǎng)時，初步實(shí)驗(yàn)證明在外界葡萄糖濃度較高時(大于10．0mmol／L)，β細(xì)胞可保持較高的活性，尚未發(fā)現(xiàn)IBMX對細(xì)胞活性有無影響。因此，有待于大規(guī)模的實(shí)驗(yàn)進(jìn)一步尋找最適宜的體外原代β細(xì)胞生長環(huán)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 鹵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.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 王�；�;陳兵;張平;王富華;蔡紅衛(wèi);;人胎胰島細(xì)胞的分離培養(yǎng)及其意義[J];重慶醫(yī)學(xué);2006年09期

2 賈延R

本文編號：1413599