In vitro pancreas duodenal homeobox-1 enhances the differentiation of pancreatic ductal epithelial cells into insulin-producing cells

AIM: To observe whether pancreatic and duodenal homeobox factor-1 enhances the differentiation of pancreatic ductal epithelial cells into insulin-producing cells in vitro. METHODS: Rat pancreatic tissue was submitted to digestion by collegenase, ductal epithelial cells were separated by density gradient centrifugation and then cultured in RPMI1640 medium with 10% fetal bovine serum. After 3-5 passages, the cells were incubated in a six-well plate for 24 h before transfection of recombination plasmid XlHbox8VP16. Lightcycler quantitative real-time RT-PCR was used to detect the expression of PDX-1 and insulin mRNA in pancreatic epithelial cells. The expression of PDX-1 and insulin protein was analyzed by Western blotting. Insulin secretion was detected by radioimmunoassay. Insulin- producing cells were detected by dithizone-staining. RESULTS: XlHbox8 mRNA was expressed in pancreatic ductal epithelial cells. PDX-1 and insulin mRNA as well as PDX-1 and insulin protein were signifi cantly increased in the transfected group. The production and insulin secretion of insulin-producing cells differentiated from pancreatic ductal epithelial cells were higher than those of the untransfected cells in vitro with a significant difference (1.32 ± 0.43 vs 3.48 ± 0.81, P < 0.01 at 5.6 mmol/L; 4.86 ± 1.15 vs 10.25 ± 1.32, P < 0.01 at 16.7 mmol/L). CONCLUSION: PDX-1 can differentiate rat pancreaticductal epithelial cells into insulin-producing cells in vitro. In vitro PDX-1 transfection is a valuable strategy for increasing the source of insulin-producing cells.     

线粒体分裂对胰岛β细胞功能的影响

目的 探讨线粒体分裂对胰岛β细胞功能的影响.方法 通过可诱导表达野生型动力相关蛋白1(Drp-1 WT)基因和突变型Drp-1(Drp-1K38A)基因的大鼠INS-1 β细胞,分析不同葡萄糖条件下,线粒体分裂对胰岛β细胞功能的影响.结果 在高糖条件下,强力霉素诱导后的Drp-1WT细胞线粒体分裂过程增强,网络状结构部分断裂,多呈点状结构,细胞胰岛素分泌功能降低(P<0.01),线粒体膜电位降低(P<0.05),细胞内ATP含量减少(P<0.05),细胞色素C表达增加,而在Drp-1K38A细胞中,上述变化明显减轻.结论 线粒体分裂增强可抑制胰岛β细胞功能.

Abstract：

Objective To investigate the effect of mitochondrial fission on the function of pancreatic β cells.Methods INS-1 stable cell lines allowing inducible expression of either wild-type dynamin-related protein 1 (Drp-1 WT)or its dominant-negative mutant(Drp-1 K38A)were used.The effect of mitochondrial fission on the function of pancreatic β cells were investigated under different concentrations of glucose.Results There were increased mitochondrial fission and disintegration of the mitochondrial reticulum into multiple punctiform organelles in Drp-1 WT cells induced with doxycycline under high glucose condition.Insulin secretion(P<0.01),mitochondrial membrane potential(P<0.05),and ATP content(P<0.05)were decreased and cytochrome C expression was increased after the expression of Drp-1 WT under high glucose condition while these changes were markedly mild in Drp-1 K38A expression cells.Conclusion The increased mitochondrial fission inhibits pancreatic β cell function.     

Effects of emodin on treating murine nonalcoholic fatty liver induced by high caloric laboratory chaw

AIM: To investigate the effects of emodin on the treatment of non-alcoholic fatty liver in rats induced by high caloric laboratory chaw. METHODS: Non-alcoholic fatty liver model was successfully established by feeding with high caloric laboratory chaw for 12 wk. Then the model rats were randomly divided into 3 groups, namely model control group, emodin group and dietary treatment group. The rats in emodin group were given emodin at dose of 40 mg/(kg·d) while animals in other groups were given distilled water of the same volume. The rats in model control group were fed with high caloric laboratory chaw while animals in other groups were fed with normal diet. Four weeks later, liver index (liver/body weight ratio), serum activities of liver-associated enzymes, blood lipid, fasting blood glucose, fasting plasma insulin, HOMA insulin resistance index (HOMA-IR), hepatic triglyceride content and histology features of all groups were assayed. The expression of hepatic peroxisomal proliferator activated receptor (PPAR) gamma was determined by RT-PCR. RESULTS: The body weight, liver index, serum activities of alanine aminotransferase (ALT), blood lipid, hepatic triglyceride content of model control group were significantly elevated, with moderate to severe hepatocyte steatosis. The expression of hepatic PPAR gamma mRNA was obviously reduced in model control group. Compared with model control group, the body weight, liver index, serum activities of ALT, blood lipids and hepatic triglyceride of emodin group significantly decreased and hepatic histology display was also greatly improved. Meanwhile, the expression of hepatic PPAR gamma mRNA was elevated. However, high serum activities of ALT and hyperlipidemia were persisted in dietary treatment group although liver index was decreased and liver histology was somewhat improved. CONCLUSION: It is suggested that emodin might be effective in the treatment of non-alcoholic fatty liver in rats. Its therapeutic mechanism could be associated with increasing the expression of hepatic PPAR gamma mRNA.     

高糖对肾小管上皮细胞ACE/ACE2表达的影响

体外培养的HK-2细胞分为正常对照组(NG组)、高糖组(HG组)、渗透压对照组(MG组),检测细胞ACE、ACE2 mRNA和蛋白表达及细胞上清液血管紧张素Ⅱ的浓度.结果 在NG组,正常培养的HK-2细胞即存在ACE、ACE2 mRNA及蛋白表达.在HG组HK-2细胞,ACE mRNA及蛋白表达较NG组升高,而ACE2mRNA及蛋白表达降低.在HG组血管紧张素Ⅱ水平较NG组显著升高.结果 表明高糖可促进体外培养的HK-2细胞ACE表达、抑制ACE2表达,进而促进血管紧张素Ⅱ合成.

Abstract：

HK-2 cells cultured in vitro were divided into three groups: normal glucose group ( NG ), high glucose group( HG), and mannitol group(MG). The expression of angiotensin-converting enzyme( ACE ) and ACE2 mRNA in HK-2 cells was detected. The concentration of angiotensin Ⅱ ( Ang Ⅱ ) in the culture medium was detected. The mRNA and protein expression of ACE and ACE2 existed in normal cultured HK-2 ( NG group ). In comparison with NG group, the mRNA and protein expressions of ACE in HG group increased significantly ( P＜0. 01 ), and the expression of ACE2 mRNA decreased significantly( P＜0. 01 ). The level of Ang Ⅱ in HG group was significantly higher than in NG group( P＜0. 05 ). The result show that high glucose may induce ACE expression and inhibit ACE2 expression, then promote synthesis of Ang Ⅱ in proximal tubular cells.     

甲状旁腺素受体1表达对高糖状态下INS-1细胞功能影响研究

目的 观察甲状旁腺受体1(PTH1R)的表达对胰岛β细胞胰岛素合成与分泌功能影响.方法 构建出PTH1R基因沉默的细胞模型后,分别应用放射免疫法观察25 mmol/L D-葡萄糖处理后对照组、阴性基因克隆组(siPTH1R-NC)、PTHrP组及阳性基因序列组(siPTH1R)细胞内胰岛素含量、葡萄糖刺激胰岛素分泌能力、应用Fluo-3/AM 检测INS-1细胞内钙离子浓度及应用INS-1细胞2-脱氧-[3H]-葡萄糖摄入率检测INS-1细胞葡萄糖转运能力.结果 PTHrP组胰岛素分泌能力高于其他3组,siPTH1R组则低于对照组及siPTH1R-NC组(均P＜0.01);PTHrP组中细胞内胰岛素含量显著高于对照组、siPTH1R-NC及siPTH1R组(均P＜0.01),其他3组间差异无统计学意义;PTHrP组中钙离子浓度水平高于其他3组,siPTH1R组低于对照组及siPTH1R-NC组.PTHrP组中细胞2-脱氧-[3H]-葡萄糖摄入率高于其他3组.结论 高糖状态下PTH1R表达水平与INS-1细胞胰岛素合成与分泌功能有关,可能为INS-1细胞自我保护的一种作用.

Abstract：

Objective To observe insulin synthesis and secretion in INS-1 under high glucose, and to clarify the effect of PTH1R. Methods After successful construction of recombinant PTH1R-siRNA vectors in INS-1 cell, insulin secretion and intracellular insulin content of control group, siPTH1R-Negative control group, PTHrP group, and siPTH1R group under 25 mmol/L glucose were measured by radioimmunoassay in INS-1 cell. Intracellular calcium were detected by Fluo-3/AM and the capability of glucose transport was calculated by assaying the uptake of [3H]-2-deoxy-D-glucose in cells.Results Compared with control group, and siPTH1R-NC group, PTHrP group showed increased capability of insulin secretion; PTHrP group had higher intracellular insulin levels than others; PTHrP group showed increased intracellular calcium; the uptake of [3H]-2-deoxy-D-glucose under high glucose after 48h of PTHrP group was increased(all P＜0.01). Conclusion There is a close relationship between PTH1R activation and insulin secretion and synthesis, PTH1R activation may be one of the protective mechanisms in maintaining function of β-cell under high glucose.     

不同糖代谢状态下大鼠肝X受体的表达及其对肝糖代谢的影响

目的 观察肝X受体(LXR)在不同糖代谢状态下肝内表达的变化情况及其对肝糖代谢酶磷酸烯醇式丙酮酸羧激酶(PEPCK)mRNA和葡萄糖激酶(GCK)mRNA表达的调节,阐明LXR信号通路对糖代谢的影响机制.方法 SD雄鼠36只分为正常对照组、糖尿病组、肥胖组和肥胖伴糖尿病组.测定各组大鼠的体重、血糖、胆固醇和甘油三酯.采用实时PCR检测各组大鼠肝脏LXR mRNA、PEPCK mRNA和GCK mRNA的表达情况,应用Western印迹测定肝脏LXR蛋白质的表达.结果 各实验组大鼠肝LXRmRNA表达均显著高于对照组(P＜0.05).Western印迹结果示各组LXR蛋白表达量和转录水平一致.与对照组和肥胖组相比,肥胖伴糖尿病组和糖尿病组肝PEPCK mRNA表达均明显上调,GCK mRNA均明显下调;与对照组相比,肥胖组PEPCKmRNA表达明显下降,GCKmRNA明显上升(均P＜0.05).结论 在非糖尿病阶段,LXR可能作为糖代谢的保护性受体,通过调节肝糖代谢酶,使血糖保持稳态.进入糖尿病阶段后,LXR的保护作用并不能逆转因胰岛素不足所致的糖代谢相关酶的异常改变,血糖不能恢复正常.

Abstract：

Objective To investigate the mechanism of liver X receptor(LXR)signal pathway in regulating glucose metabolism by observing the variety of LXR expression and its impacts on regulating the mRNA expression of PEPCK and GCK, the key enzyme of hepatic glucose metabolism in various glucose metabolic status in rats. Methods SD rats were chosen and divided into four groups:the CON group, the induced DM group, the OB group, and the induced OB+DM group. For each group of rats, body weight, blood glucose, serum triglycerides, and cholesterol were measured. Then the rats were sacrificed and the livers were collected and studied. Real-time PGR was used to measure the expressions of LXR mRNA, PEPCK mRNA, and GCK mRNA in the livers. Finally, the Western Blot assay was used to measure the liver LXR protein expression. Results The expression of LXR mRNA was significantly higher in DM,OB, and OB+DM groups than in CON group(P＜0.05).The Western blot results showed that the levels of protein were in accordance with the mRNA expression. Comparing to the CON and the OB groups, the PEPCK mRNA expression of the OB+DM and the DM groups was significantly higher, while the GCK mRNA expression of these two groups was significantly lower(P＜0.05). Comparing to the CON group, the PEPCK mRNA expression of the OB group was significantly lower, while the GCK mRNA expression of OB group was significantly higher(P＜0.05).Conclusions During non-diabetic phase, LXR could act as a protective receptor for glucose metabolism and keep glucose homeostasis by regulating the key enzymes of the hepatic glucose metabolism. While in the diabetic phase, the protective receptor LXR failed to reverse the change of the related enzymes caused by insulin deficiency, and finally the plasma glucose level was raised.     

甲状腺素对甲状腺功能减低大鼠子代脑组织中同源盒基因Nkx6.1 mRNA表达的影响

目的 通过对妊娠期甲状腺功能减低(简称甲低)大鼠补充甲状腺素,探讨妊娠不同时间补充不同剂量甲状腺素对子代大鼠脑组织中同源盒基因Nkx6.1 mRNA表达的影响.方法 1月龄Wistar大鼠240只,雌雄各半,体质量100～120 g.按体质量将雌性大鼠随机分为8组:对照组,甲低组,甲低孕鼠妊娠早期(1～17 d)补充甲状腺素(妊早甲低)高、中、低剂量组,甲低孕鼠妊娠晚期(18～20 d)补充甲状腺素(妊晚甲低)高、中、低剂量组,每组15只.高、中、低剂量组每天补充的甲状腺索分别为3.5、2.0、0.5μg/100 g体质量.8组大鼠均饲以重度缺碘地区粮食配制的饲料,对照组饮用含碘200μg/L的碘酸钾溶液,7个甲低组饮用去离子水.雄性大鼠用正常食料饲养,3个月后按1:1交配.8组大鼠分别取孕17 d、新生、生后20 d子代大鼠脑组织,采用实时荧光定量PCR法检测脑组织中Nkx6.1 mRNA表达.结果 ①成功建立了甲低动物模型,8组大鼠血清TT_3、TT_4、FT_3、FT_4组间比较差异有统计学意义(F值分别为4.08、31.99、5.79、26.34,P均<0.01).②在孕17 d、新生、生后20 d,Nkx6.1 mRNA表达组间比较差异有统计学意义(F值分别为758.720、1121.589、144.716,P均<0.01);组内不同时间比较差异有统计学意义(F值分别为2898.863、325.605、716.285、56.329、236.727、196.678、7115.752、9152.306,P均<0.01).③时间因素和剂量因素对Nkx6.1 mRNA表达有影响(F值分别为1176.655、246.530,P均<0.01);时间与剂量因素间存在交互作用(F=1249.934,P<0.01).④在孕17 d、新生、生后20 d.甲低组Nkx6.1 mRNA表达与对照组比较,差异有统计学意义(P均<0.01).⑤在新生、生后20 d,6个甲低补充甲状腺素组Nkx6.1 mRNA表达与甲低组比较.差异有统计学意义(P均<0.01).⑥在孕17d、新生、生后20 d,除妊早甲低中剂量组外,其余5个甲低补充甲状腺素组Nkx6.1 mRNA表达与对照组比较,差异有统计学意义(P均<0.01).⑦在孕17 d、新生、生后20 d,妊早甲低高、低剂量组Nkx6.1 mRNA表达与中剂量组比较,差异有统计学意义(P均<0.01);在孕17 d、生后20 d,高剂量组与低剂量组比较,差异有统计学意义(肭<0.01).⑧在新生和生后20 d,妊晚甲低组组间Nkx6.1 mRNA表达比较,差异有统计学意义(P均<0.01).结论 甲低孕鼠子代大鼠脑组织同源盒基因Nkx6.1的表达受补充甲状腺素剂量及时间影响.     

球型脂联素对高糖环境NIT-1胰岛β细胞功能的影响

用β细胞株NIT-1来研究球型脂联素对高糖损伤β细胞分泌功能及相关基因表达的影响.结果 显爪球型脂联素可恢复部分β细胞功能相关基因的表达,抑制高糖诱导NAD(P)H氧化酶组分p47phox表达增加,但未能改善高糖诱导的胰岛素mRNA水平下降及胰岛素分泌缺陷.     

球型脂联素对高糖环境NIT-1胰岛β细胞功能的影响

用β细胞株NIT-1来研究球型脂联素对高糖损伤β细胞分泌功能及相关基因表达的影响.结果 显爪球型脂联素可恢复部分β细胞功能相关基因的表达,抑制高糖诱导NAD(P)H氧化酶组分p47phox表达增加,但未能改善高糖诱导的胰岛素mRNA水平下降及胰岛素分泌缺陷.     

球型脂联素对高糖环境NIT-1胰岛β细胞功能的影响

用β细胞株NIT-1来研究球型脂联素对高糖损伤β细胞分泌功能及相关基因表达的影响.结果 显爪球型脂联素可恢复部分β细胞功能相关基因的表达,抑制高糖诱导NAD(P)H氧化酶组分p47phox表达增加,但未能改善高糖诱导的胰岛素mRNA水平下降及胰岛素分泌缺陷.     

球型脂联素对高糖环境NIT-1胰岛β细胞功能的影响

用β细胞株NIT-1来研究球型脂联素对高糖损伤β细胞分泌功能及相关基因表达的影响.结果 显爪球型脂联素可恢复部分β细胞功能相关基因的表达,抑制高糖诱导NAD(P)H氧化酶组分p47phox表达增加,但未能改善高糖诱导的胰岛素mRNA水平下降及胰岛素分泌缺陷.     

球型脂联素对高糖环境NIT-1胰岛β细胞功能的影响

用β细胞株NIT-1来研究球型脂联素对高糖损伤β细胞分泌功能及相关基因表达的影响.结果 显爪球型脂联素可恢复部分β细胞功能相关基因的表达,抑制高糖诱导NAD(P)H氧化酶组分p47phox表达增加,但未能改善高糖诱导的胰岛素mRNA水平下降及胰岛素分泌缺陷.