嗜黏蛋白阿克曼菌对大鼠胰岛细胞瘤细胞增殖、凋亡及胰岛素分泌功能的影响

刘曼妮; 张玲; 董新燕; 刘敏; 成果; 张翔凌; 何方; 王国庆

doi:10.12182/20200160202

嗜黏蛋白阿克曼菌对大鼠胰岛细胞瘤细胞增殖、凋亡及胰岛素分泌功能的影响

Effects of Akkermansia muciniphila on the Proliferation, Apoptosis and Insulin Secretion of Rat Islet Cell Tumor Cells

摘要

摘要:
目的探讨嗜黏蛋白阿克曼菌（Akkermansia muciniphila, A. muciniphila）对大鼠胰岛细胞瘤细胞（INS-1）增殖、凋亡及胰岛素分泌功能的影响。
方法将INS-1细胞分为正常组、修复组和保护组，每组均用3种A. muciniphila干预物（活菌、灭活菌和分泌物）处理48 h，并设不干预的空白对照。正常组INS-1细胞直接干预；修复组INS-1细胞先用链脲佐菌素（streptozotocin, STZ）造模再加干预物；保护组INS-1细胞先用干预物作用再加STZ造模。干预结束，用四甲基偶氮唑盐微量酶反应比色法（MTT法）检测细胞增殖；使用葡萄糖刺激，用酶联免疫吸附实验（ELISA）检测胰岛素分泌水平；实时荧光定量PCR技术检测胰岛素分泌相关基因和凋亡基因表达，Western blot检测Bax凋亡蛋白的表达。
结果 A. muciniphila干预物对INS-1细胞作用48 h后，其细胞形态无明显变化。A. muciniphila活菌干预的修复组INS-1细胞增殖活性较空白对照降低（P<0.005）。A. muciniphila干预物对正常组、修复组和保护组INS-1细胞的胰岛素分泌没有影响（P>0.05）。A. muciniphila分泌物可促进正常组、修复组和保护组INS-1细胞葡萄糖转运蛋白2基因(glucose transporter 2, Glut2)和修复组葡萄糖激酶基因 (glucokinase, GCK)的表达（P<0.05）。3种A. muciniphila干预物处理后的正常组INS-1细胞Bcl₂-associated X基因 (Bax)的表达量减少（P<0.001），A. muciniphila死菌干预的修复组INS-1细胞Bax基因表达量减少（P<0.05）。A. muciniphila干预物处理的细胞Bax蛋白表达减少。
结论 A. muciniphila可促进INS-1细胞胰岛素分泌相关基因的表达，抑制凋亡基因和凋亡蛋白Bax的表达，为研究如何用A. muciniphila改善2型糖尿病提供了新的方向。

Abstract:
Objective To investigate the effects of Akkermansia muciniphila (A. muciniphila) on the proliferation, apoptosis and insulin secretion of rat pancreatic islet cell tumor cells (INS-1).
Methods INS-1 cells were divided into three groups, normal, repair, and protect groups, and subsequently every group was subjected with A. muciniphila metabolites, live A. muciniphila orpasteurized A. muciniphila for 48 h. A group that did not treat with anything was set as blank control. After intervention, the cell viability was determined by MTT method, the insulin secretion level stimulated by glucose was determined by ELISA, the expressions of the genes involved in insulin secretion and apoptosis were tested by qRT-PCR, and the expression of apoptosis related protein Bax was evaluated by Western blot.
Results There was no significant change in INS-1 cell morphology after co-incubation with 3 types of A. Muciniphila interventions for 48 h. The proliferative activity of INS-1 cells was decreased in the repair group that treated with live A. muciniphila than that of control (P<0.005). A. muciniphila intervention had no effect on insulin secretion in INS-1 cells in normal, repair or protection group (P>0.05). A. muciniphila secretions promoted the expression of glucose transporter 2 (Glut2) in 3 groups and the expression of glucokinase (GCK) in repair group (P<0.05). The expression of Bax of the INS-1 cell in the normal group was decreased after intervented with 3 kinds of A. muciniphila intervention materials (P<0.001).The expression of Bax gene of the INS-1 cell in the repair group that treated with dead A. muciniphila was decreased (P<0.05). The expression of Bax protein of INS-1 cells that treated with A. muciniphila interventions was decreased.
Conclusion A. muciniphila can promote the expression of insulin secretion-related genes in INS-1 cells, inhibit the expression of apoptotic genes and apoptosis protein Bax.This research provides a new direction for applying A. muciniphila in improving type 2 diabetes.

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