壳寡糖及其配合物对胰岛细胞增殖、胰岛素分泌的促进作用

Promotion effect of chitooligosaccharides and its derivatives on pancreatic islet cells proliferation and insulin secretion

背景:壳寡糖与有益于糖尿病治疗的微量元素形成配合物兼备了壳寡糖及微量元素的各种特性,很有可能在糖尿病及其并发症预防与治疗的研究方面取得新的进展。目的:观察壳寡糖及其铬、钒、硒配合物对胰岛β细胞系NIT-1的促增殖及促胰岛素分泌作用。设计、时间及地点:对比观察实验,于2008-07/09在中国海洋大学生物化学实验室完成。材料:壳寡糖及其铬、钒、硒配合物由中国海洋大学生物化学实验室制备;转基因小鼠胰岛β细胞系NIT-1细胞株由中国海洋大学药物所提供。方法:①胰岛β细胞系NIT-1细胞株接种于96孔板中,实验组加入100mg/L壳寡糖及其配合物的培养液培养,并设加入不含样品的培养液作为对照组,分别在加样后24,48,72,120,168,216h取出,MTT法测定细胞生长曲线。②胰岛β细胞系NIT-1细胞株接种于24孔培养板中,实验组加入100mg/L壳寡糖及其配合物的培养液,并设加入不含样品的培养液作为对照组,分别在加样后2,4,6,8,10,12,14d取出,用放射免疫法测定培养液中的胰岛素含量。同时在培养6d后进行胰岛素刺激释放试验,测定胰岛素释放量,计算刺激指数。主要观察指标:壳寡糖及其配合物对胰岛细胞促增殖作用及促胰岛素分泌作用。结果:壳寡糖及铬、硒配合物最适质量浓度为100mg/L,对胰岛β细胞有明显的增殖作用。壳寡糖铬、硒配合物在48h促胰岛β细胞增殖明显,且在120h细胞进入生长平台期,壳寡糖铬配合物组细胞密度达到最大,活力高于其他组。壳寡糖及其铬、钒、硒配合物组在第10天胰岛素释放量均高于对照组(P<0.05),壳寡糖组促胰岛素分泌作用最为显著。培养第6天壳寡糖及其铬、钒、硒配合物组胰岛素刺激指数均高于对照组(P<0.05)。结论:壳寡糖及其配合物对于胰岛β细胞体外增殖具有明显的促进作用,并可以显著促进胰岛β细胞的胰岛素分泌。

BACKGROUND： The coordination compound of chitooligosaccharides and microelement possess both biological activities of chitooligosaccharides and microelement, which have a considerable potential to be utilized in prevention and treatment of diabetes and its complications. OBJECTIVE： To detect the effect of chitooligosaccharides and its Cr, VO, Se derivatives on the pancreatic islet cells proliferation and insulin secretion. DESIGN, TIME AND SETTING： The comparative observation was performed at the Biochemistry Laboratory of Ocean University of China from July to September 2008. MATERIALS： Chitooligosaccharides and its derivatives were prepared at the Biochemistry Laboratory of Ocean University of China. SV40-transformed cell line （NIT-1） was provided by Medicine Institute of Ocean University of China. METHODS： （1）NIT-1 was inoculated into 96-well plate, 100 mg/L chitooligosaccharides and its derivatives were cultured, and the culture medium without chitooligosaccharides was served as the control group. MTT assay was used to detect the cell growth curve at hours 24, 48, 72, 120, 168, 216, respectively. （2）NIT-1 was inoculated into 24-well plate, 100 mg/L chitooligosaccharides and its derivatives were cultured, and the culture medium without chitooligosaccharides was served as the control group. The radioimmunoassay method was used to measure the insulin concentration at the days 2, 4, 6, 8, 10, 12, 14, respectively. Meanwhile, at the days 6, stimulated insulin release test was performed to determine insulin secretion and calculate stimulation index. MAIN OUTCOME MEASURES： Promotion effect of chitooligosaccharides and its derivatives on pancreatic islet cells proliferation and insulin secretion was observed. RESULTS： The results indicated that chitooligosaccharides and its Cr, Se derivatives could effectively accelerate the proliferation of the pancreatic β cells at the mass concentration of 100 mg/L at the 48 hours, and cells reach a stationary phase at the 120 hours. Chitooligosaccharides and its Cr derivatives group had the ultimate cell density and higher activity than other groups. The insulin secretion of chitooligosaccharides and its Cr, VO, Se derivatives groups was higher than the control group at the 10 days （P 〈 0.05）, especially significant in chitooligosaccharides group. At days 6, the stimulation index of chitooligosaccharides and its Cr, VO, Se derivatives groups was higher than the control group （P 〈 0.05）. CONCLUSION： Chitooligosaccharides and its derivatives possess obvious promotion effect on the pancreatic islet cells in vitro proliferation as well as insulin secretion of pancreatic β cells.