高葡萄糖条件对胰岛β细胞株NIT-1拉曼光谱的变化和马铃薯球蛋白tuberin的影响
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摘要
目的：探讨及研究不同糖浓度下对NOD鼠胰岛β细胞株NIT-1的损伤作用及罗格列酮对该细胞的保护作用。研究在不同浓度葡萄糖条件下,对β细胞内马铃薯球蛋白(tuberin)表达的影响,探讨马铃薯球蛋白tuberin对胰岛β细胞生长、功能的影响。
方法：将NIT-1细胞按5×104个细胞／孔置于24孔培养板,培养48小时后,随机分入各处理组5.6组(含葡萄糖5.6mmol／L)、7.8组(7.8mmol／L)、11.1组(11.1 mmol／L)、16.7组(16.7mmol／L)、22.2组(22.2mmol／L)及27.6组(27.6mmol／L)再培养24小时,分别施与罗格列酮干预48h,应用拉曼光谱技术检测不同糖浓度和各组经罗格列酮干预后48小时的单个细胞变化,应用蛋白免疫印迹方法(Western Blot)检测不同糖浓度组和各组药物干预后48小时的马铃薯球蛋白tuberin的表达。
结果：
(1)高糖组16.7mmol／L组和22.5mmol／LNIT-1细胞在培养至120小时后与11.1mmol／L组的拉曼光谱比较与核酸和蛋白的相关的峰值有降低其中788 cm-1,1002 cm-1,1085 cm-1,1450cm-1和1650 cm-1峰值下降明显,且随糖浓度的增加峰值下降幅度越大。
(2)当加入10-5mol／l罗格列酮干预后与对照组拉曼光谱比较,16.7组和22.5组与11.1mmol／L对照组比较788 cm-1,1002 cm-1,1085cm-1,1450cm-1和1650 cm-1峰值亦有下降变化,且22.5加药组下降幅度大于16.7加药组。在同一糖浓度下,加药组上述峰值均大于未加药组,差别有统计学意义。
(3)当葡萄糖浓度在5.6mmol／L时,tuberin蛋白表达含量最低,而随着葡萄糖浓度的不断升高,TSC2转录水平也呈不断增加状态,tuberin蛋白表达5.6mmol／L组显著低于其他各组(P<0.05)；27.6mmol／L组与22.5mmol／L显著高于5.6mmol／L组、11.1mmol／L及16.7mmol／L组(P<0.05)。
(4)在10-5mol／l的罗格列酮干预后,各葡萄糖组所表达tuberin较未干预组相比趋势不变,仍是5.6组<11.1组<16.7组<22.5组<27.6组,27.6mmol／L组显著高于其他各组(P<0.05),同一糖浓度下,未予罗格列酮干预组tuberin蛋白的表达均高于罗格列酮干预组(组间比较P均<0.05)。
结论：高浓度葡萄糖通过上调马铃薯球蛋白(tuberin)表达阻碍胰岛β细胞增殖、诱导胰岛细胞凋亡。罗格列酮可能通过作用于胰岛β细胞直接或间接的调节tuberin蛋白的表达来调控胰岛β细胞的增殖及功能、抑制凋亡；提示通过调控马铃薯球蛋白(tuberin)的表达的措施,可能有效影响胰岛β细胞的分泌功能和细胞的增殖、凋亡。
Objective:To investigate the role of rosiglitazone in different concentrations of glucose-induced protein tuberin ofβcells, and explore the effects of protein tuberin on the function and proliferation、apoptosis of pancreaticβ-cell.
Methods:The NIT-1 cells were puted by 5×104 cells/hole in 24-holes culture plate, and cultured for 48 hours later were random accessed to the treatment group 5.6 group (containing glucose 5.6mmol/L),7.8 group (7.8mmol/L),11.1 group (11.1 mmol/L),16.7 Group (16.7mmol/L),22.2 Group (22.2mmol/L) and 27.6 group (27.6mmol/L), and then 24 hours later were respectively intervented by 10"5 mmol/L rosiglitazone for 48h. Single NIT-1 cell was measured by Raman spectroscopy, the expression in protein lever of p-tuberin by Western blot in each group at the time of 48h intervention.
Results:(1) At the high concentration of glucose, NIT-1 cells were tested by Raman spectroscopy,The most noticeable changes were the decrease of spectral intensity at the788cm-1,1002 cm-1,1085 cm-1,1445 cm-1 and 1655 cm-1 peaks, mainly corresponding to protein and lipid, and the peaks were gradually decreased along with increasing glucose concentration(every group compared with 11.1 group P<0.05),11.1 group>16.7 group>22.5 group.
(2) After the intervention of 10-5mol/1 rosiglitazone, the peaks mationed had showed no significant alterations. the peaks of none rosiglitazone intervention groups were higher than that of rosiglitazone intervention groups.
(3) The expression in tuberin was lowest at 5.6mmol/L glucose concentration(every group compared with 11.1 groups P<0.05), and tuberin increased along with the glucose's level. The expression of tuberin of the 5.6mmol/L group was the lowest,less than any other group(P<0.05); The 27.6mmol/L group had showed no significant alterations with the 22.5 mmol/L group(P>0.05) while significantly higher than the 5.6mmol/L and 11.1mmol/ L、16.7mmol/L group (P<0.05).
(4) After the intervention of 10-5mol/1 rosiglitazone,the expression of tuberin had the same trend that was still 5.6 Group<11.1 group<16.7 group <22.5 group<27.6 group, the 27.6mmol/L group was significantly higher than the other group (P<0.05);The expression of overall tuberin was decreased significantly than non-intervention group. At the same glucose concentration, The expression of tuberin of none rosiglitazone intervention groups were higher than that of rosiglitazone intervention group s(inter-group difference was significant P<0.05).
Conclusion:High concentration of glucose could hinder beta cell proliferation, induce islet cell apoptosis, and reduce insulin secretion, raise protein tuberin. Above it showed that the islet cell apoptosis、insulin resistance caused by high concentration of glucose may be related with the change of tuberin.
Rosiglitazone could improve the function and proliferation, and inhibit the apoptosis of pancreaticβcells, through directly regulating the protein tuberin. This prompted that the measures to regulate the expression of TSC2 can effectively influence the secretion、proliferation、apoptosis of pancreatic (3 cells.
方法：将NIT-1细胞按5×104个细胞／孔置于24孔培养板,培养48小时后,随机分入各处理组5.6组(含葡萄糖5.6mmol／L)、7.8组(7.8mmol／L)、11.1组(11.1 mmol／L)、16.7组(16.7mmol／L)、22.2组(22.2mmol／L)及27.6组(27.6mmol／L)再培养24小时,分别施与罗格列酮干预48h,应用拉曼光谱技术检测不同糖浓度和各组经罗格列酮干预后48小时的单个细胞变化,应用蛋白免疫印迹方法(Western Blot)检测不同糖浓度组和各组药物干预后48小时的马铃薯球蛋白tuberin的表达。
结果：
(1)高糖组16.7mmol／L组和22.5mmol／LNIT-1细胞在培养至120小时后与11.1mmol／L组的拉曼光谱比较与核酸和蛋白的相关的峰值有降低其中788 cm-1,1002 cm-1,1085 cm-1,1450cm-1和1650 cm-1峰值下降明显,且随糖浓度的增加峰值下降幅度越大。
(2)当加入10-5mol／l罗格列酮干预后与对照组拉曼光谱比较,16.7组和22.5组与11.1mmol／L对照组比较788 cm-1,1002 cm-1,1085cm-1,1450cm-1和1650 cm-1峰值亦有下降变化,且22.5加药组下降幅度大于16.7加药组。在同一糖浓度下,加药组上述峰值均大于未加药组,差别有统计学意义。
(3)当葡萄糖浓度在5.6mmol／L时,tuberin蛋白表达含量最低,而随着葡萄糖浓度的不断升高,TSC2转录水平也呈不断增加状态,tuberin蛋白表达5.6mmol／L组显著低于其他各组(P<0.05)；27.6mmol／L组与22.5mmol／L显著高于5.6mmol／L组、11.1mmol／L及16.7mmol／L组(P<0.05)。
(4)在10-5mol／l的罗格列酮干预后,各葡萄糖组所表达tuberin较未干预组相比趋势不变,仍是5.6组<11.1组<16.7组<22.5组<27.6组,27.6mmol／L组显著高于其他各组(P<0.05),同一糖浓度下,未予罗格列酮干预组tuberin蛋白的表达均高于罗格列酮干预组(组间比较P均<0.05)。
结论：高浓度葡萄糖通过上调马铃薯球蛋白(tuberin)表达阻碍胰岛β细胞增殖、诱导胰岛细胞凋亡。罗格列酮可能通过作用于胰岛β细胞直接或间接的调节tuberin蛋白的表达来调控胰岛β细胞的增殖及功能、抑制凋亡；提示通过调控马铃薯球蛋白(tuberin)的表达的措施,可能有效影响胰岛β细胞的分泌功能和细胞的增殖、凋亡。
Objective:To investigate the role of rosiglitazone in different concentrations of glucose-induced protein tuberin ofβcells, and explore the effects of protein tuberin on the function and proliferation、apoptosis of pancreaticβ-cell.
Methods:The NIT-1 cells were puted by 5×104 cells/hole in 24-holes culture plate, and cultured for 48 hours later were random accessed to the treatment group 5.6 group (containing glucose 5.6mmol/L),7.8 group (7.8mmol/L),11.1 group (11.1 mmol/L),16.7 Group (16.7mmol/L),22.2 Group (22.2mmol/L) and 27.6 group (27.6mmol/L), and then 24 hours later were respectively intervented by 10"5 mmol/L rosiglitazone for 48h. Single NIT-1 cell was measured by Raman spectroscopy, the expression in protein lever of p-tuberin by Western blot in each group at the time of 48h intervention.
Results:(1) At the high concentration of glucose, NIT-1 cells were tested by Raman spectroscopy,The most noticeable changes were the decrease of spectral intensity at the788cm-1,1002 cm-1,1085 cm-1,1445 cm-1 and 1655 cm-1 peaks, mainly corresponding to protein and lipid, and the peaks were gradually decreased along with increasing glucose concentration(every group compared with 11.1 group P<0.05),11.1 group>16.7 group>22.5 group.
(2) After the intervention of 10-5mol/1 rosiglitazone, the peaks mationed had showed no significant alterations. the peaks of none rosiglitazone intervention groups were higher than that of rosiglitazone intervention groups.
(3) The expression in tuberin was lowest at 5.6mmol/L glucose concentration(every group compared with 11.1 groups P<0.05), and tuberin increased along with the glucose's level. The expression of tuberin of the 5.6mmol/L group was the lowest,less than any other group(P<0.05); The 27.6mmol/L group had showed no significant alterations with the 22.5 mmol/L group(P>0.05) while significantly higher than the 5.6mmol/L and 11.1mmol/ L、16.7mmol/L group (P<0.05).
(4) After the intervention of 10-5mol/1 rosiglitazone,the expression of tuberin had the same trend that was still 5.6 Group<11.1 group<16.7 group <22.5 group<27.6 group, the 27.6mmol/L group was significantly higher than the other group (P<0.05);The expression of overall tuberin was decreased significantly than non-intervention group. At the same glucose concentration, The expression of tuberin of none rosiglitazone intervention groups were higher than that of rosiglitazone intervention group s(inter-group difference was significant P<0.05).
Conclusion:High concentration of glucose could hinder beta cell proliferation, induce islet cell apoptosis, and reduce insulin secretion, raise protein tuberin. Above it showed that the islet cell apoptosis、insulin resistance caused by high concentration of glucose may be related with the change of tuberin.
Rosiglitazone could improve the function and proliferation, and inhibit the apoptosis of pancreaticβcells, through directly regulating the protein tuberin. This prompted that the measures to regulate the expression of TSC2 can effectively influence the secretion、proliferation、apoptosis of pancreatic (3 cells.
引文
1.高浓度葡萄糖阻碍胰岛β细胞增殖、诱导胰岛细胞凋亡。
2.高浓度葡萄糖可能通过上调马铃薯球蛋白tuberin的表达,抑制了细胞增殖和促进细胞凋亡。
3.罗格列酮可以通过作用于胰岛β细胞直接或间接调节TSC2信号分子改善胰岛β细胞的增殖及功能、抑制凋亡；通过调控tuberin蛋白的表达的措施,可能能够有效影响胰岛β细胞的分泌功能和细胞的增殖、凋亡。
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22 Sent hil D, Choudhury G G, McLaurin C, et al. Vascular endothelial growth factor induces protein synthesis in renal epithelial cells:a potential role in diabetic nephropathy[J]. Kidney Int,2003,64(2):468-479.
23 Valle A, Jofra T, et al. Rapamycin prevents and breaks the anti-CD3-induced tolerance in NOD mice. [J]. Diabetes.2009,58(4):875-81.
24 Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Regulation of hypoxia-inducible factor(HIF)-1 activity and expression of HIF hydroxylases in response to insulin-like growth factor 1[J]. Mol endocrinol, 2005,19(5):1304-1317.
25刘红艳,林碧,余励明.胰岛素抵抗与糖尿病肾病的关系[J].广西医学,2005,27(12)：1920-1922.
26 Sarafidis PA, Bakris GL. Protection of the kidney by thiazolidinediones:an assessment from bench to bedside[J]. Kidneylnt,2006,70 (7):1223-1233.
2.高浓度葡萄糖可能通过上调马铃薯球蛋白tuberin的表达,抑制了细胞增殖和促进细胞凋亡。
3.罗格列酮可以通过作用于胰岛β细胞直接或间接调节TSC2信号分子改善胰岛β细胞的增殖及功能、抑制凋亡；通过调控tuberin蛋白的表达的措施,可能能够有效影响胰岛β细胞的分泌功能和细胞的增殖、凋亡。
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