胰石蛋白减轻胰腺星状细胞对胰岛β细胞的毒性效应

发布时间：2018-05-03 05:18

本文选题：胰腺星状细胞 + 胰石蛋白/再生蛋白　；参考：《东南大学》2015年硕士论文

【摘要】：背景及目的病理研究显示,2型糖尿病胰岛内存在纤维化,胰腺星状细胞是胰岛纤维化的主要始动和效应细胞,并且对胰岛p细胞具有直接损伤作用。胰石蛋白／再生蛋白(Pancreatic stone protein/regeneration protein, PSP/reg)是再生基因家族的重要成员之一,与胰腺细胞的增殖密切相关。有研究显示,该蛋白促进胰岛p细胞增殖及功能,并且可抑制胰腺星状细胞活化及其致纤维化能力。本实验探讨PSP/reg对胰腺星状细胞(Pancreatic Stellate Cell, PSC)胰岛p细胞毒性效应的影响。方法分离培养美国癌症研究所(Institute of Cancer Research, ICR)小鼠的PSC,在含或不含有PSP/reg的培养液中培养24小时,收集PSC上清(PSC su pernatant, PSC-SN),将MIN-6小鼠胰岛p细胞株分别培养在：①无血清培养基、②含0/30/100/200/300 ng/ml PSP/reg的无血清培养基、③PSC-SN、④PSP/reg干预的PSC-SN (PSP-PSC-SN)、⑤含有100 ng/ml PSP/reg 的 PSC-SN (PSP +PSC-SN)中24小时。CCK-8比色法观察不同处理组小鼠胰岛β细胞存活率；流式细胞术检测AnnexinV-FITCPI双染细胞比例：葡萄糖刺激的胰岛素释放(Glucose Stimulated Insulin Secretion, GSIS)试验；胰岛素ELISA试剂盒检测上清胰岛素浓度；Western blot法检测P62、LC3Ⅱ/Ⅰ蛋白的表达。结果1.CCK-8法检测P SP/reg组MIN-6小鼠胰岛β细胞活力百分比高于对照组,流式细胞术分析结果显示两组AnnexinV-FITCPI双染细胞比例无明显差异,GSIS试验结果显示PSP/reg组葡萄糖刺激的胰岛素释放浓度高于对照组,WB检测PSP/reg组P62蛋白含量低于对照组、LC3Ⅱ/Ⅰ比值高于对照组；2. CCK-8法检测PSC-SN组MIN-6小鼠胰岛p细胞活力百分比低于对照组,流式细胞术分析结果显示PSC-S N组AnnexinV-FITCPI双染细胞比例高于对照组,GSIS试验结果显示PSC-SN组葡萄糖刺激的胰岛素释放浓度低于对照组,WB检测PSC-SN组P62蛋白含量低于对照组、LC3IⅡ/Ⅰ比值高于对照组；3. CCK-8法检测PSP-PSC-S N组MIN-6小鼠胰岛p细胞活力百分比高于PSC-SN组,流式细胞术分析结果显示PSP-PSC-SN组AnnexinV-FITCPI双染细胞比例低于PSC-S N组,GSIS试验结果显示PSP-PSC-SN组葡萄糖刺激的胰岛素释放浓度高于PSC-SN组,WB检测PSP-PSC-SN组P62蛋白含量高于对照组、LC3Ⅱ/Ⅰ比值低于对照组；4. CCK-8法检测PSP+PSC-SN组MIN-6小鼠胰岛p细胞活力百分比高于PSC-SN组,流式细胞术分析结果显示PSP+PSC-SN组AnnexinV-FITCPI双染细胞比例低于PSC-SN组,GSIS试验结果显示PSP+PSC-SN组葡萄糖刺激的胰岛素释放浓度高于PSC-SN组,WB检测PSP+PSC-SN组P62蛋白含量高于对照组、LC3Ⅱ/Ⅰ比值低于对照组。结论在一定的浓度范围内,PSP/reg浓度依赖性地促进MIN-6细胞存活、胰岛素释放作用及MIN-6细胞的自噬水平,过高浓度的PSP/reg可导致MIN-6细胞的过度自噬,对MIN-6细胞的存活及胰岛素释放产生抑制作用；P SC-SN抑制MIN-6细胞增殖及胰岛素释放,促进MIN-6细胞凋亡,并诱导MIN-6细胞的过度自噬;PSP/reg促进PSC-SN干预的MIN-6细胞存活及胰岛素释放,减少PSC-SN诱导的MIN-6凋亡及过度自噬；此外,PSP/reg干预后的PSC-SN (PSP-PSC-SN)对MIN-6活力及胰岛素释放的的抑制、促进MIN-6凋亡及过度自噬等作用减弱。综上所述,PSP/reg本身具有增加胰岛p细胞活力及功能等作用,还可减轻PSC-SN对胰岛p细胞的损伤,此外,PSP/reg可作用于PSC,抑制其致胰岛p细胞损伤的能力,间接发挥胰岛p细胞的保护作用。
[Abstract]:Background and objective pathological studies have shown that the pancreatic islets of type 2 diabetes are fibrosis, and pancreatic stellate cells are the main initiation and effector cells of islet fibrosis and have direct damage to the islet P cells. Pancreatic stone protein/ regeneration protein (PSP/reg) is an important part of the regenerative gene family. One of the members is closely related to the proliferation of pancreatic cells. Studies have shown that the protein promotes the proliferation and function of pancreatic islet P cells and inhibits the activation and fibrosis of pancreatic stellate cells. The effect of PSP/reg on the toxic effects of pancreatic stellate cells (Pancreatic Stellate Cell, PSC) on pancreatic islet p cells. The PSC in the Institute of Cancer Research (ICR) mice was cultured for 24 hours in the culture medium containing or without PSP/reg, and collected the PSC supernatant (PSC Su pernatant, PSC-SN). (3) PSC-SN, 4 PSP/reg intervention PSC-SN (PSP-PSC-SN), and PSC-SN (PSP +PSC-SN) with 100 ng/ml PSP/reg in the 24 hour.CCK-8 colorimetric method to observe the survival rate of islet beta cells in different treatment groups, and flow cytometry to detect the proportion of AnnexinV-FITCPI double stained cells: glucose stimulated insulin release Cretion, GSIS) test, insulin ELISA kit to detect the concentration of insulin in the supernatant, and Western blot method to detect the expression of P62 and LC3 II / I protein. Results 1.CCK-8 method detected the percentage of islet beta cell activity in P SP/reg group MIN-6 mice higher than that of the control group. The results of flow cytometry analysis showed that the proportion of two groups of AnnexinV-FITCPI double stained cells was not obvious. The results of GSIS test showed that the insulin release concentration of glucose stimulated by PSP/reg group was higher than that of the control group. The content of P62 protein in the PSP/reg group was lower than that of the control group, and the ratio of LC3 II / I was higher than that of the control group; the percentage of P cell viability in the MIN-6 mice of PSC-SN group MIN-6 was lower than that of the control group by 2. CCK-8 method, and the result of flow cytometry analysis showed PSC-S N. The ratio of AnnexinV-FITCPI double staining cells in group PSC-SN was higher than that in the control group. The results of GSIS test showed that the concentration of insulin release in PSC-SN group was lower than that of the control group. The content of P62 protein in PSC-SN group was lower than that of the control group, and the ratio of LC3I II / I was higher than that of the control group. The percentage of LC3I II / I ratio was higher than that of the control group; the percentage of the viability of the islet P cells in the MIN-6 mice of the PSP-PSC-S N group was higher than that of the control group. C-SN group, flow cytometry analysis showed that the proportion of AnnexinV-FITCPI double staining cells in group PSP-PSC-SN was lower than that of PSC-S N group. The results of GSIS test showed that the concentration of glucose stimulated insulin release in PSP-PSC-SN group was higher than that of PSC-SN group, WB detection PSP-PSC-SN group P62 protein content was higher than that of control group, LC3 II / I ratio was lower than that of control group, and the ratio of LC3 II / I was lower than that of the control group; 4. The percentage of P cell viability of MIN-6 mice in group P+PSC-SN was higher than that in group PSC-SN. The result of flow cytometry analysis showed that the proportion of AnnexinV-FITCPI double stained cells in group PSP+PSC-SN was lower than that of PSC-SN group. The result of GSIS test showed that the concentration of insulin release in PSP+PSC-SN group was higher than that of PSC-SN group, and WB detection of PSP+PSC-SN group protein content was higher than that of control group. The ratio of LC3 II / I was lower than that in the control group. Conclusion in a certain concentration range, PSP/reg concentration depended on the survival of MIN-6 cells, insulin release and the autophagy level of MIN-6 cells. Excessive concentration of PSP/reg could lead to excessive autophagy of MIN-6 cells, inhibit the survival of MIN-6 cells and release insulin, and P SC-SN. Inhibition of MIN-6 cell proliferation and insulin release, promoting apoptosis of MIN-6 cells, and inducing hyperautophagy of MIN-6 cells; PSP/reg promotes the survival and insulin release of PSC-SN interfered MIN-6 cells, reduces PSC-SN induced MIN-6 apoptosis and excessive autophagy; furthermore, PSC-SN (PSP-PSC-SN) prognosis of PSP/reg dry to MIN-6 activity and insulin release Inhibition, promote the effect of MIN-6 apoptosis and excessive autophagy. In summary, PSP/reg itself can increase the activity and function of islet P cells, and reduce the damage of PSC-SN to islet P cells. In addition, PSP/reg can act on PSC, inhibit the energy of islet P cell damage and indirectly protect the islet P cells.

【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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