马尾松花粉酯化多糖对MIN6细胞胰岛素分泌和[Ca^(2+)]_i的影响

Effects of Sulfated Polysaccharide from Masson Pine Pollen on Insulin Secretion and [Ca^(2+)]_i in MIN6 Cell

探讨马尾松花粉硫酸酯化多糖对MIN6细胞胰岛素分泌和[Ca2+]i的作用。选取60%乙醇沉淀的马尾松花粉多糖(PPM60),氯磺酸-吡啶法得到硫酸酯化物(SPPM60);检测PPM60及SPPM60刺激后MIN6细胞胰岛素分泌量和[Ca2+]i的变化。观察钙离子抑制剂维拉帕米和低分子肝素的影响。PPM60对MIN6细胞胰岛素分泌几乎没有影响,SPPM60能显著促进胰岛素的分泌和[Ca2+]i升高(P<0.05)。维拉帕米和低分子肝素钠均可以降低SPPM60引起的胰岛素分泌增加,但差异不显著。同样两者均可显著性降低SPPM60引起的[Ca2+]i的升高(P<0.05)。维拉帕米可以明显抑制3.6 mg/mL葡萄糖的促胰岛素分泌(P<0.05)和升高[Ca2+]i的作用(P<0.01)。SPPM60可以单独促进胰岛素的分泌,其作用机制与葡萄糖刺激胰岛素分泌(GSIS)有一定相似性,但同时存在其他的信号通路。

The former research of our laboratory has found that the Pinus massoniana pollen polysaccharides precipitated by 60% alcohol （PPM60） and its sulfated derivative （SPPM60） could increase intracellular free calcium concentration in rat myocardial ceils, splenocytes of mice and human chronic myelogenous leukemia ceils. Intracellular free Ca2. concentration can be one of the important factors affecting insulin secretion. However it is still unclear how about the PPM60 and SPPM60＇s role in secretion of insu- lin of pancreatic β-cells ,through changing the intracellular calcium concentration. This research aims at investigating the effects of masson pine pollen polysaccharide and its ester on insulin secretion and [ Ca2＋ ]i in MIN6 cell. Sulfated polysaccharide （SPPM60） was derivated from 60% ethanol precipitation of masson pine pollen polysaccharide （PPM60） modified by chlorosulfonic acid-pyri- dine method; enzyme-linked immunosorbent assay method was used to detect the insulin secretion in the MIN6 cells supema- tant. MIN6 ceils were treated with the calcium fluorescence probe Fura 2-AM, using a fluorescence spectrophotometer to detect [ Ca2 ＋ ]i changes affected by SPPM60. The concentration of insulin in MIN6 cell supernatant and [ Ca2＋ ]i in MIN6 cell was meas- ured. Verapamil and Low Molecular Weight Heparin （LMWH） were used to testify whether insulin secretion and [Ca2＋ ]i were affected. PPM60 had little impact on insulin secretion. However SPPM60 had significant role in promoting insulin secretion in MIN6 cell. SPPM60 increased [ Ca2＋ ]i significantly compared with the control group （ P 〈 0.05 ）. Verapamil and LMWH showed inhibition of insulin secretion caused by the SPPM60 in certain degree, but no significant difference. Similarly, they significantly inhibited the rise of [ Ca2＋ ]i caused by SPPM60 （P 〈 0. 05 ）. Verapamil significantly inhibited the rise of insulin secretion （ P 〈 0.05 ） and [ Ca2＋ ]i （P 〈0.01 ） caused by glucose （3.6 mg/mL）. SPPM60 could stimulate lonely the insulin secretion in MIN6 cell. It had similar mechanism to that of glucose-stimulated insulin secretion （GSIS） though there occured other signaling pathways.