摘要
目的探讨不同浓度葡萄糖及罗格列酮干预对胰岛β细胞株NIT-1的FoxO1、TSC2基因表达的影响及对细胞生长、分泌功能的影响。
方法将NIT-1细胞用RPMI-1640培养基进行培养,按培养液里葡萄糖浓度不同,分为五组:1组(含5.6mmol/L葡萄糖)、2组(11.1mmol/L)、3组(16.7mmol/L), 4组(22.5mmol/L)及5组(27.6mmol/L),每组均先用11.1 mmol/L培养液常规培养48小时,再分别换成不同糖浓度培养液进行处理培养,培养至第120小时。每组细胞培养72小时后,分别加入不同浓度的罗格列酮进行干预,加药后继续培养48小时,来检测相关指标。采用MTT比色法检测各组NIT-1细胞生长增殖活性;放射免疫分析法检测各组NIT-1细胞胰岛素分泌水平;免疫荧光细胞核染色法检测各组NIT-1细胞凋亡水平;免疫荧光和蛋白印迹方法检测NIT-1细胞中p-FoxO1蛋白的表达水平;RT-PCR方法检测各组NIT-1细胞中FoxO1、TSC2基因mRNA的表达水平。
结果随着葡萄糖浓度的增加,NIT-1细胞增殖水平呈先升后降改变,葡萄糖浓度为11.1 mmol/L时,细胞增殖率最高,5.6mmol/L组低于11.1mmol/L组(P<0.05),16.7mmol/L组、22.5mmol/L组、27.6mmol/L组细胞增殖率逐渐降低(P<0.05);胰岛素分泌量11.1mmol/L组最高,随着葡萄糖浓度的增加16.7mmol/L组、22.5mmol/L组、27.6mmol/L组逐渐减低,组间差异明显(P<0.05),但均多于5.6mmol/L组(P<0.05);同5.6mmol/L组、11.1mmol/L组进行比较,16.7mmol/L组、22.5mmol/L组、27.6mmol/L组细胞凋亡逐渐出现(P<0.05),其中11.1mmol/L组细胞凋亡率最低;通过免疫荧光和蛋白印迹检测方法进行定位定量分析,NIT-1细胞中p-FoxO1蛋白表达水平在葡萄糖浓度为11.1mmol/L时最高,而随着葡萄糖浓度的逐级升高,p-FoxO1蛋白表达逐渐减少(P<0.05);RT-PCR结果显示NIT-1细胞中FoxO1与TSC2基因mRNA在葡萄糖浓度为5.6mmol/L时表达最低,而随着葡萄糖浓度升高,FoxO1与TSC2mRNA的表达呈不断增加状态,各组间均有差异(P<0.05);予罗格列酮干预后各组NIT-1细胞增殖水平、胰岛素分泌量、及p-FoxO1蛋白表达水平均上升,各组细胞凋亡率及FoxO1与TSC2mRNA的表达均减少。
结论体外培养NIT-1细胞的最适宜葡萄糖浓度为11.1mmol/L,在此葡萄糖浓度下,胰岛β细胞生长状态良好,胰岛素分泌和细胞增殖可达最高状态;随着葡萄糖浓度的升高(>11.1mmol/L),胰岛β细胞凋亡亦增高,细胞生长受到抑制,并出现胰岛素分泌功能缺陷导致胰岛素分泌水平下降;加入罗格列酮以后细胞增殖与胰岛素分泌水平增加,细胞凋亡减少。高浓度葡萄糖(>11.1mmol/L)使p-FoxO1蛋白表达水平降低,同时使FoxO1基因mRNA表达水平升高,因此我们推测通过FoxO1核定位的增加,一方面可增强细胞核内相关凋亡基因的转录,导致胰岛β细胞凋亡,另一方面通过抑制过氧化物酶体增殖激活受体γ(PPARγ)的转录活性,影响细胞胰岛素的分泌,并增加细胞对胰岛素的抵抗作用;给予PPARγ的强效激动剂罗格列酮干预以后,NIT-1细胞FoxO1表达减少,同时TSC2表达也减少。这一结果提示我们FoxO1与TSC2在信号转导通路中存在着某些联系,罗格列酮可以通过调节FoxO1与TSC2,改善胰岛β细胞的增殖及分泌功能、减少细胞凋亡及解除胰岛素抵抗。
Objective To study the FoxO1 and TSC2 expression and cell proliferat- ion, secretion of NIT-1 cell line treated with rosiglitazone intervention in different concentrations of glucose.
Methods The NIT-1 cells were cultured in RPMI-1640 medium, then divided into five groups according to different concentrations glucose culture medium, in which the first group(containing 5.6mmol/L glucose), the second group(11.1mmol/L), the third group (16.7mmol/L), the fourth group (22.5mmol/ L) and the fifth group(27.6mmol/L) differently.Firstly all groups were cultured with 11.1 mmol/L density of glucose which was called normal culture medium for 48 hours and then changed medium to different concentrateions glucose of culture medium for processing culture to 120 hours. The each groups of NIT-1 cells were added different concentrations of rosiglitazone intervention diferently after 72 hours and continue to culture for 48 hours and then cell proliferation detected with MTT assay,cell insulin secretion measured with radioimmunoas- say,cell apoptosis detected with immunofluorescence,p-FoxO1 expression were checked with western blot and immunofluorescence assay and FoxO1、TSC2 mRNA expression measured with RT-PCR assay.
Results The proliferation of NIT-1 cells were raised with glucose concen- tration increased. When the glucose concentration is up to 11.1mmol/L, the rate of cell proliferation reach the highest, when the glucose concentrations were over 11.1mmol/L, the cell proliferation were descended accompany with glucose concentrations growth (the results of cell proliferation rate as follow:5.6mmol/L group was lower than 11.1mmol/L group (P<0.05), 16.7mmol/L group, 22.5 mm ol/L group, 27.6mmol/L group compared with 5.6mmol/L group (P<0.05). Insul- in secretion of the cell is the highest in 11.1 mmol/L group. With glucose concentration increase arrived at 16.7mmol/L, 22.5mmol/L and 27.6mmol/L group the cell secreted insulin reduced gradually, there were significant different between every two groups (P<0.05), but insulin secretion in those of groups were much more than 5.6mmol/L group (P<0.05). The cell apoptosis progressi- vely increased in 16.7mmol/L group, 22.5mmol/L group, 27.6mmol/L(P<0.05) compared with 5.6mmol/L group and 11.1mmol/L group differently and rate of cell apoptosis was lowest in 11.1mmol/L group. p-FoxO1 protein expression was the highest in level of glucose concentration at 11.1 mmol/L, but the level of p-FoxO1 protein expression were descended followed by glucose concentra- tion increased progressively (P<0.05) compered with 11.1mmol/L glucose con- centration. FoxO1 and TSC2 results were showed that the two gene expressed were lowest in the glucose concentration at 5.6mmol/L and FoxO1 and TSC2 expression were increased with glucose concentration increased, it had much differece between each group (P<0.05). Cell proliferation,insulin secretion and p-FoxO1 expression increased after the intervention of rosiglitazone in each group, the rate of cell apoptosis and FoxO1、TSC2 mRNA expression were descended in each group.
Conclusions It is most suitful condition for cell culture,in that case, pan- creaticβ-cell growth better, cell proliferation and insulin secretion increased at glucose concentration of 11.1mmol/L. With the glucose concentration(>11.1 mm ol/L)enhenced the NIT-1 cells growth were inhibited but the rate of apoptotic cells were increased,meanwhile the level of insulin secretion, were reduced and caused the emergence of functional defects. After rosiglitazone added the cell proliferation increasing growth and insulin secretion levels was also increased but cell apoptosis significantly decreased. High concentrations of glucose (>11.1 mmol/L)can caused p-FoxO1 protein levels decrease,meanwhile, mRNA expres- sion levels of FoxO1 increased. In this results we speculated by the increased of nuclear localization of FoxO1, on the one hand,it can enhance the transcription of correlative apoptotic genes, which can lead to isletβ-cell apoptosis,and on the other hand by inhibiting the transcription activity of peroxisome proliferator- activated receptorγ(PPARγ), to affect cell insulin secretion and increase cells insulin resistance.After treated with PPARγagonist rosiglitazone FoxO1 expres- sion decreased in NIT-1cell line and TSC2 expressionis alsodecreased. The resu- lts clue to us that maybe have some corelation between TSC2 and FoxO1 in cell signaling pathway. It can be also inferred that rosiglitazone may regulate both FoxO1 and TSC2 in different way to improve the isletβ-cell proliferation,cell secretory function,and to release cell insulin resistance.
引文
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