槟榔碱对STZ致INS-1细胞损伤的保护和修复作用

目的 体外建立链脲佐菌素( streptozotocin,STZ)致INS-1胰岛细胞损伤的模型,观察槟榔碱对STZ损伤INS-1 细胞的保护和修复作用,并初步探讨其作用机制.方法 将INS-1细胞悬液接种至培养板,C02孵育箱孵育,药物干预后,MTT法测定细胞增殖活力,放免法测定细胞上清胰岛素含量,比色法检测丙二醛(MDA)含量和总抗氧化能力(T-AOC).结果 与STZ组比较,槟榔碱保护组和修复组OD值均明显提高(P＜0.01),说明槟榔碱具有促进INS-1细胞增殖的作用;两组槟榔碱均明显促进了胰岛素的释放(P＜0.01);两组槟榔碱均显著降低了脂质过氧化产物MDA生成(P＜0.01),显著提高了T-AOC的含量(P＜0.01),具有抗氧化作用.结论 槟榔碱对STZ损伤的INS-1 细胞有一定保护和修复作用,其机制可能是通过降低MDA生成、提高T-AOC能力实现的.     

芍药苷对STZ损伤的INS-1细胞的保护和修复作用

目的 研究中药单体芍药苷对INS-1细胞的影响,及对链脲佐菌素(STZ)损伤的胰岛细胞的保护、修复作用,并初步探讨其保护机制.方法 INS-1细胞传代培养后,用四氮唑蓝(MTT) 比色法测定细胞增殖活力,并测定细胞培养液中胰岛素浓度、丙二醛( MDA) 浓度、总抗氧化能力(T-AOC ).结果 芍药苷可促进INS-1细胞释放胰岛素.STZ损伤后,INS-1细胞活力降低;胰岛素分泌量减少;MDA含量明显升高;T-AOC下降.芍药苷保护组能不同程度改善STZ引起的上述指标的改变.结论 芍药苷可促进INS-1细胞释放胰岛素,对STZ损伤的INS-1细胞有显著的保护作用,这种作用可能是通过提高INS-1细胞的抗氧化能力实现的.     

内源性雌激素对STZ诱导雌性小鼠糖尿病的保护作用研究

目的 研究内源性雌激素对糖尿病小鼠血糖、血清胰岛素水平和抗氧化能力的影响,探讨内源性雌激素对胰岛细胞和胰岛素敏感性可能的保护作用.方法 小鼠按体重随机分为4组,假手术组(Sham)、去卵巢手术组(Ovx)、假手术注射链脲佐菌素(streptozotocin,STZ)组(Sham+ STZ)、去卵巢手术注射STZ组(Ovx+STZ).切除小鼠两侧卵巢,腹腔注射STZ制糖尿病小鼠模型.跟踪测定体重、血糖,进行糖耐量试验.取血制血清,取出胰腺于4％多聚甲醛中固定.测定血清中胰岛素浓度,丙二醛(MDA)、总抗氧化力( T-AOC),所取胰腺做病理切片,观察胰岛细胞的变化.结果 Ovx组血糖和MDA值明显高于Sham组,总抗氧化力低于Sham组;Ovx+STZ血糖和MDA值高于Sham+STZ组,胰岛素水平和T-AOC要低于Sham+STZ组.结论 内源性雌激素对糖尿病小鼠具有一定保护作用.     

人参糖肽对大鼠胰岛β细胞损伤的改善作用

目的探讨人参糖肽对链脲佐菌素(STZ)引起大鼠胰岛β细胞损伤的改善和防护作用。方法腹腔注射STZ损伤雄性Wistar大鼠胰岛为胰岛损伤组;注射STZ前连续7 d腹腔注射人参糖肽保护胰岛为人参糖肽组;以不经任何处理的大鼠为对照组。实验结束时采用放射免疫分析法检测大鼠血清胰岛素和C肽含量,苏木素-伊红(HE)染色观察胰岛病理改变,免疫组织化学染色检测胰岛素蛋白表达水平,电镜观察胰岛细胞超微结构变化。结果与对照组相比,胰岛损伤组大鼠血清胰岛素和C肽含量显著降低(P0.01),而人参糖肽组的血清胰岛素和C肽含量较胰岛损伤组明显升高(P0.01)。HE染色观察显示胰岛损伤组胰岛细胞数量显著减少,细胞排列不整,细胞肿胀,结构破坏,人参糖肽组的胰岛细胞损伤明显改善。免疫组织化学研究显示,胰岛损伤组胰岛素阳性染色面积明显缩小,而人参糖肽组的胰岛素阳性染色面积明显扩大(P0.01)。胰岛损伤组胰岛β细胞超微结构为变性-坏死性改变且分泌颗粒极少,而人参糖肽组胰岛β细胞变性-坏死性改变明显改善,分泌颗粒显著增多。结论人参糖肽能明显改善STZ引起的大鼠胰岛β细胞损伤。     

熊脱氧胆酸减轻内质网应激介导的胰岛β细胞凋亡的机制

目的 探讨熊脱氧胆酸( Ursodeoxycholic acid,UDCA)通过减轻内质网应激保护链脲佐菌素诱导的糖尿病大鼠胰岛β细胞凋亡的作用.方法 链脲佐菌素(50 mg/kg)一次性腹腔注射建立糖尿病大鼠模型(n=40),并将14只造模成功大鼠按随机数字表法随机分为糖尿病组7只,UDCA组7只,另取正常对照组10只.自造模成功第10天开始以UDCA(40 mg·kg-1·d-1)给大鼠灌胃30 d.饲养期间观察大鼠血搪.处死后留取大鼠血清和组织标本,测定空腹胰岛素,TUNEL检测胰岛β细胞凋亡的形态学改变.胰腺组织总RNA采用定制基因芯片对89条凋亡相关基因的表达进行检测,以RT-PCR和Western印迹法验证相关基因的表达.结果 糖尿病大鼠血糖在给予UDCA治疗后逐渐下降,但仍然未降到正常水平.糖尿病大鼠空腹胰岛素降低,给予UDCA治疗后空腹胰岛素水平有所增加.TUNEL显示糖尿病组大鼠予UDCA治疗后减少了由链脲佐菌素引起的胰岛β细胞凋亡(P＜0.01).在89条基因中,糖尿病组上调基因42条,下调基因46条.UDCA可以逆转部分基因的影响.与对照组相比,糖尿病组大鼠Bax、Caspase-3、Bip、CHOPmRNA和CHOP蛋白显著上调(P＜0.05),而抗凋亡蛋白Bcl-2 mRNA显著下调(P＜0.05),给予UDCA治疗后这些参数均有明显改善(P＜0.05).结论 熊脱氧胆酸可能通过减轻内质网应激达到保护链脲佐菌素诱导的糖尿病大鼠胰岛β细胞凋亡的作用.     

人参糖肽对大鼠体外胰岛β细胞的保护作用

目的探讨人参糖肽对体外培养大鼠胰岛β细胞的保护效果。方法分离和培养大鼠胰岛β细胞。人参糖肽预处理β细胞之后用链脲佐菌素(STZ)引起细胞损伤(人参糖肽+STZ组),STZ引起β细胞损伤后再进行人参糖肽处理(STZ+人参糖肽组),以正常胰岛β细胞和单用人参糖肽处理的β细胞作为对照。检测培养上清中胰岛素和C肽分泌水平,噻唑蓝(MTT)法检测细胞存活率,免疫组织化学染色测量β细胞内胰岛素蛋白表达水平,电镜观察细胞超微结构变化。结果人参糖肽+STZ组胰岛β细胞的胰岛素和C肽分泌量与正常胰岛细胞比较差异无统计学意义(P>0.05),而在STZ+人参糖肽组二者分泌量均明显减少(P<0.05)。人参糖肽+STZ组胰岛β细胞的存活率有所下降,但与单独人参糖肽组比较差异无统计学意义(P>0.05),而STZ+人参糖肽组细胞存活率显著降低(P<0.05)。免疫组织化学定量研究显示,STZ+人参糖肽组胰岛β细胞内胰岛素阳性染色面积与细胞面积的百分比明显小于其他3组。STZ+人参糖肽组胰岛β细胞超微结构为变性及坏死性改变,且分泌颗粒极少,而人参糖肽+STZ组胰岛β细胞这些改变明显减轻,可见大量分泌颗粒。结论人参糖肽对STZ引起的大鼠体外胰岛β细胞损伤有防护作用。     

APP5肽类似物165对链脲佐菌素损伤SH-SY5Y细胞的保护作用

目的探讨体外APP5肽类似物165（下称P165）对链脲佐菌素（STZ）损伤SH-SY5Y细胞的保护作用。方法STZ0.8mmol/L作用于SH-SY5Y细胞的损伤模型。通过测定噻唑蓝（MTT）代谢率、乳酸脱氢酸（LDH）漏出率,瑞氏染色观察细胞形态,Western blot检测相关蛋白,观察P165的保护作用。结果与正常对照组细胞的MTT代谢率（1.00±0.01）和LDH漏出率（201.56±47.54）比较,STZ损伤组细胞MTT代谢率降低（0.84±0.01）,LDH漏出率升高（317.83±76.95）,差异有统计学意义（P〈0.05）,细胞胞体面积缩小,胰岛素信号通路相关蛋白IRS-1、PI3K表达减少;P16530μmol/L组细胞MTT代谢率升高（0.91±0.01）,LDH漏出率降低（228.53±35.65）,细胞形态恢复正常,胰岛素信号通路相关蛋白IRS-1、PI3K表达恢复正常。结论体外STZ与SH-SY5Y细胞共孵育,可能影响胰岛素/胰岛素受体信号转导系统对细胞的促生长作用,而P165可能通过激活该信号通路逆转这种损伤,可作为一种神经营养因子起到保护作用。     

山药多糖对链脲菌素糖尿病大鼠糖脂代谢及氧化应激的影响

目的探讨山药多糖对糖尿病大鼠糖脂代谢及氧化应激的影响。方法链脲佐菌素(STZ)制造糖尿病大鼠模型。实验设正常对照组、STZ模型组、高剂量山药多糖组、中剂量山药多糖组、低剂量山药多糖组及二甲双胍组。造模后给予山药多糖或二甲双胍治疗4W。实验结束处死大鼠,比较各组大鼠体重、12 h尿量、饮水量及摄食量。测定空腹血糖、糖化血清蛋白、血清胰岛素水平;检测甘油三酯(TG)、胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)及胰腺丙二醛(MDA)、谷胱甘肽(GSH)及总抗氧化能力(T-AOC)指标。结果与模型组比较,山药多糖有效缓解了糖尿病大鼠症状;与模型组比较,山药多糖明显降低血糖、血脂及MDA水平(P<0.05,P<0.01),增强GSH、T-AOC活性,升高HDL-C水平(P<0.05,P<0.01)。结论山药多糖具有较好调节糖脂作用,其作用机制可能与改善胰岛素敏感性及抗氧化作用相关     

盐酸小檗碱对糖尿病大鼠肠道菌群结构的影响

目的观察盐酸小檗碱对高糖高脂膳食联合链脲佐菌素(STZ)诱导的糖尿病(DM)大鼠肠道菌群结构的影响,探讨其治疗糖尿病的可能机制。方法将雄性SD大鼠随机分为3组:正常对照组(对照组,n=6)、高糖高脂膳食联合链脲佐菌素(STZ)诱导DM组(DM组,n=6)、高糖高脂膳食联合链脲佐菌素(STZ)诱导DM加小檗碱干预组(小檗碱组,n=6)。小檗碱干预12周后,微生物平板菌落计数法测定各组大鼠结肠内粪便中大肠杆菌和双歧杆菌的数量,并测定大鼠体重、空腹血糖(FBG)、空腹胰岛素(FINS)及血浆内毒素(LPS)水平,计算胰岛素抵抗指数(HOMA-IR)。结果与对照组大鼠相比,DM组大鼠粪便中双歧杆菌数量降低,大肠杆菌数量升高,差异有统计学意义(P0.05);DM组大鼠体重、LPS、FBG、FINS、HOMA IR升高(P0.05)。与DM组大鼠相比,小檗碱组大鼠粪便中双歧杆菌数量升高,大肠杆菌数量降低,差异有统计学意义(P0.05);小檗碱组大鼠体重、LPS、FBG、FINS、HOMA IR降低(P0.05)。结论盐酸小檗碱可能通过改善肠道菌群结构从而降低高糖高脂膳食联合链脲佐菌素诱导的糖尿病大鼠的血浆内毒素水平,改善其胰岛素抵抗程度,发挥降糖作用。     

栀子苷对2型糖尿病大鼠胰岛细胞的保护作用

目的研究栀子苷对2型糖尿病大鼠胰岛细胞的保护作用。方法采用高脂高糖饲料喂养大鼠4周后,腹腔注射链脲佐菌素(STZ)复制2型糖尿病大鼠模型。药物干预4周后,微量血糖仪检测空腹血糖(FBG)、ELISA法测定空腹胰岛素(FINS);测定胰腺组织中超氧化物歧化酶(SOD)活性、谷胱甘肽过氧化物酶(GSH-PX)活性、丙二醛(MDA)含量。结果与糖尿病模型组比较,栀子苷治疗组大鼠血糖水平降低,而血清胰岛素水平升高。胰腺组织中SOD、GSH-PX活性升高,而MDA含量降低,表明栀子苷具有提高胰岛细胞抗氧化水平和抗炎症损伤的能力。结论栀子苷能有效提高胰岛素分泌并抑制胰岛细胞凋亡,提示栀子苷对2型糖尿病大鼠胰岛细胞具保护作用。     

Effects of cytokines on rat insulinoma INS-1 cells.

To investigate further the role of cytokines in the pathogenesis of type I insulin-dependent diabetes mellitus, the effects of interleukin-1 beta (IL-1), tumour necrosis factor-alpha (TNF) and gamma-interferon (IFN) were tested on rat insulinoma INS-1 cells. Whereas TNF and IFN had, respectively, a minor or no effect on insulin production, IL-1 caused a time- and dose-dependent decrease in insulin release and lowered the insulin content as well as the preproinsulin mRNA content of INS-1 cells. Both IL-1 and TNF exerted a cytostatic effect, estimated by a decrease in [3H]thymidine incorporation, while only IL-1 decreased cell viability as measured by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The glutathione content of INS-1 cells was shown to be modulated by the presence of 2-mercaptoethanol in the culture medium, but was not affected by IL-1 or TNF. In conclusion, INS-1 cell culture is considered to be a useful model for studying the effect of cytokines on insulin-producing cells. The differentiated features of these cells will permit several questions to be addressed regarding the mechanism of action of IL-1 and eventually other cytokines, both at the level of gene expression and of intracellular signalling.     

Long-term exposure of INS-1 rat insulinoma cells to linoleic acid and glucose in vitro affects cell viability and function through mitochondrial-mediated pathways

Obesity with excessive levels of circulating free fatty acids (FFAs) is tightly linked to the incidence of type 2 diabetes. Insulin resistance of peripheral tissues and pancreatic β-cell dysfunction are two major pathological changes in diabetes and both are facilitated by excessive levels of FFAs and/or glucose. To gain insight into the mitochondrial-mediated mechanisms by which long-term exposure of INS-1 cells to excess FFAs causes β-cell dysfunction, the effects of the unsaturated FFA linoleic acid (C 18:2, n-6) on rat insulinoma INS-1 β cells was investigated. INS-1 cells were incubated with 0, 50, 250 or 500?μM linoleic acid/0.5% (w/v) BSA for 48?h under culture conditions of normal (11.1?mM) or high (25?mM) glucose in serum-free RPMI-1640 medium. Cell viability, apoptosis, glucose-stimulated insulin secretion, Bcl-2, and Bax gene expression levels, mitochondrial membrane potential and cytochrome c release were examined. Linoleic acid 500?μM significantly suppressed cell viability and induced apoptosis when administered in 11.1 and 25?mM glucose culture medium. Compared with control, linoleic acid 500?μM significantly increased Bax expression in 25?mM glucose culture medium but not in 11.1?mM glucose culture medium. Linoleic acid also dose-dependently reduced mitochondrial membrane potential (ΔΨm) and significantly promoted cytochrome c release from mitochondria in both 11.1?mM glucose and 25?mM glucose culture medium, further reducing glucose-stimulated insulin secretion, which is dependent on normal mitochondrial function. With the increase in glucose levels in culture medium, INS-1 β-cell insulin secretion function was deteriorated further. The results of this study indicate that chronic exposure to linoleic acid-induced β-cell dysfunction and apoptosis, which involved a mitochondrial-mediated signal pathway, and increased glucose levels enhanced linoleic acid-induced β-cell dysfunction.     

Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells

Rat insulinoma-derived INS-1 cells constitute a widely used beta-cell surrogate. However, due to their nonclonal nature, INS-1 cells are heterogeneous and are not stable over extended culture periods. We have isolated clonal INS-1E cells from parental INS-1 based on both their insulin content and their secretory responses to glucose. Here we describe the stable differentiated INS-1E beta-cell phenotype over 116 passages (no. 27-142) representing a 2.2-yr continuous follow-up. INS-1E cells can be safely cultured and used within passages 40-100 with average insulin contents of 2.30 +/- 0.11 microg/million cells. Glucose-induced insulin secretion was dose-related and similar to rat islet responses. Secretion saturated with a 6.2-fold increase at 15 mm glucose, showing a 50% effective concentration of 10.4 mm. Secretory responses to amino acids and sulfonylurea were similar to those of islets. Moreover, INS-1E cells retained the amplifying pathway, as judged by glucose-evoked augmentation of insulin release in a depolarized state. Regarding metabolic parameters, INS-1E cells exhibited glucose dose-dependent elevations of NAD(P)H, cytosolic Ca(2+), and mitochondrial Ca(2+) levels. In contrast, mitochondrial membrane potential, ATP levels, and cell membrane potential were all fully activated by 7.5 mm glucose. Using the perforated patch clamp technique, 7.5 and 15 mm glucose elicited electrical activity to a similar degree. A K(ATP) current was identified in whole cell voltage clamp using diazoxide and tolbutamide. As in native beta-cells, tolbutamide induced electrical activity, indicating that the K(ATP)conductance is important in setting the resting potential. Therefore, INS-1E cells represent a stable and valuable beta-cell model.     

Lisofylline,a novel antiinflammatory agent,protects pancreatic beta-cells from proinflammatory cytokine damage by promoting mitochondrial metabolism

Proinflammatory cytokine-mediated pancreatic beta-cell dysfunction is a key pathological event in type I diabetes mellitus. Lisofylline (LSF), an anti-inflammatory agent, has been shown to protect pancreatic islets from IL-1 beta-induced inhibitory effects on insulin release. However, the mechanism of LSF action is not known. Increasing evidence suggests that the mitochondria play an important role in regulating the beta-cell insulin release capacity and the control of cellular viability. To examine the direct effects of LSF on beta-cells, insulin-secreting INS-1 cells were exposed to a combination of recombinant IL-1 beta, TNF alpha, and IFN gamma with or without LSF for 18 h. Basal and glucose-stimulated static insulin release were measured using RIA. INS-1 cell viability was determined using in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and LIVE/DEAD dual fluorescence labeling. To evaluate INS-1 mitochondrial function, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolism, change in mitochondrial membrane potential, and intracellular ATP levels were assessed. Cytokine addition reduced basal (7.8 +/- 0.30 vs. 10.0 +/- 0.46 ng/ml.h; P < 0.005), glucose-stimulated insulin secretion (11.6 +/- 0.86 vs. 17.4 +/- 1.86 ng/ml.h; P < 0.005), and MTT metabolism in INS-1 cells. Over 40% of the cytokine-treated beta-cells exhibited nuclear DNA breakage, whereas the control cell death rate remained at 1-2%. Simultaneous application of LSF and cytokines to INS-1 cells restored insulin secretion, MTT metabolism, mitochondrial membrane potential, and cell viability to control levels. LSF increased beta-cell MTT metabolism as well as insulin release and glucose responsiveness. In summary, proinflammatory cytokines lead to a reduction of glucose-induced insulin secretion, mitochondrial activity, and viability in INS-1 cells. LSF at concentrations achievable in vivo protected beta-cells from the cytokine effects. The mechanism of LSF-induced protection may be by promoting mitochondrial metabolism.     

腺病毒介导的过氧化物酶增殖激活受体-δ小发夹状RNA对胰岛细胞瘤细胞脂代谢的影响

目的 构建小发夹状RNA（shRNA）腺病毒沉默过氧化物酶增殖激活受体-δ（PPAR-δ）表达,探讨PPAR-δ在大鼠胰岛细胞瘤细胞（INS-1）脂代谢中的作用和地位.方法 用限制性内切酶Sal Ⅰ和HindⅢ将shRNA质粒pGenesil-1载体中的PPAR-δ-shRNA片段连同U6启动子一起切下,连接至线性化的穿梭质粒pAdtrack-CMV上,将pAdtrack-CMV与骨架质粒pAdeasy在腺病毒载体电转感受态细菌（BJ5183）中重组得到PPAR-δ-shRNA腺病毒重组质粒.限制性内切酶Pac Ⅰ线性化重组质粒后用脂质体转染试剂Lipofectamine 2000转染人胚肾细胞（HEK293）,包装得到含PPAR-δ-shRNA的病毒重组子,病毒重组子在HEK293细胞中扩增后,将收集的病毒液感染INS-1细胞、RT-PCR和Western blot检测PPAR-δ蛋白的表达.RT-PCR检测该病毒对INS-1细胞脂代谢相关基因酰基辅酶A氧化酶（ACO）、肉毒碱棕榈酸转移酶1（CPT1）、脂肪酸转运蛋白1（FATP1）、长链酰基辅酶A脱氢酶（LCAD）表达的影响,并检测INS-1细胞内甘油三酯含量的变化.采用SPSS 12.0软件进行统计学分析,组间差异采用方差分析.结果 成功构建了含有大鼠PPAR-δ-shRNA基因的重组腺病毒载体,病毒滴度为1×1010PFU/ml.重组腺病毒感染后INS-1细胞PPAR-δ mRNA和蛋白表达明显下降.与空病毒组相比,PPAR-δ-shRNA腺病毒使ACO的表达下降40%（分别为0.72±0.05,0.44±0.07,P＜0.05）,CPT1表达下降27%（分别为0.66±0.08,0.48±0.02,P＜0.05）,使FATP1的表达下降55%（分别为0.65±0.07,0.30±0.02,P＜0.05）,使LCAD的表达下降32%（分别为0.66±0.12,0.45±0.10,P＜0.05）.与空病毒组相比,PPAR-δ-shRNA腺病毒使INS-1细胞内甘油三酯含量上升65%（分别为5.27±0.19,8.68±0.34,P＜0.05）.结论 成功构建了PPAR-δ-shRNA腺病毒,该腺病毒能够抑制INS-1细胞的脂肪酸氧化,促进细胞内脂质沉积.     

Culture at low glucose up-regulates mitochondrial function in pancreatic β cells with accompanying effects on viability

We tested whether exposure of β cells at reduced glucose leads to mitochondrial adaptions and whether such adaptions modulate effects of hypoxia. Rat islets, human islets and INS-1 832/13 cells were pre-cultured short term at half standard glucose concentrations (5.5 mM for rat islets and cells, 2.75 mM for human islets) without overtly negative effects on subsequently measured function (insulin secretion and cellular insulin contents) or on viability. Culture at half standard glucose upregulated complex I and tended to upregulate complex II in islets and INS-1 cells alike. An increased release of lactate dehydrogenase that followed exposure to hypoxia was attenuated in rat islets which had been pre-cultured at half standard glucose. In INS-1 cells exposure to half standard glucose attenuated hypoxia-induced effects on several viability parameters (MTT, cell number and incremental apoptotic DNA). Thus culture at reduced glucose of pancreatic islets and clonal β cells leads to mitochondrial adaptions which possibly lessen the negative impact of hypoxia on β cell viability. These findings appear relevant in the search for optimization of pre-transplant conditions in a clinical setting.     

Aminoguanidine exerts a beta-cell function-preserving effect in high glucose-cultured beta-cells (INS-1)

We investigated the effects of aminoguanidine (AG) on beta-cell functions in an insulin secreting cell line (INS-1). Culture with 27mM glucose for one week markedly decreased both insulin release and insulin content compared to culture in 0.8 mM or 3.3 mM glucose. Relative to culture at 27 mM glucose alone, the co-exposure to 1 mM AG almost doubled basal as well as glucose or 25 mM KCl-stimulated insulin release and increased insulin content by 42%. AG failed to affect release and content in cells cultured at 0.8 or 3.3 mM glucose. Preproinsulin mRNA content in 27mM glucose-cultured cells was 52% suppressed compared to 0.8mM glucose-cultured cells, and AG treatment partially counteracted this decline. Advanced glycosylation end product (AGE)-associated fluorescence (370nm excitation and 440 nm emission) of cells' extracts did not differ between 27mM and 0.8mM glucose-cultured cells after 1 week of culture and fluorescence was unaffected by AG. Accumulation of nitrite into culture media was markedly increased from 27mM glucose-cultured cells, and this accumulation was 33% suppressed by AG. In conclusion, AG partially protects against glucotoxic effects in INS-1 cells. These beneficial effects may involve a decrease in early glycation products and/or nitric oxide synthase (NOS) activity. The effects which were obtained after one week of high glucose exposure may supplement AGE-associated effects seen after chronically elevated glucose.     

长春新碱对INS-1细胞hTERT、Sp1、c-myc基因表达影响的研究

目的观察抗肿瘤药物长春新碱（Vincristine,VCR）对大鼠胰岛B细胞瘤株INS-1细胞增殖和侵袭的抑制作用和hTERT、Sp1、c-myc表达的影响及其意义,探讨胰岛B细胞瘤的发病机制。方法 INS-1分为对照组（不加VCR）和实验组（加入浓度分别为0.008 mg/mL、0.04 mg/mL、0.2 mg/mL、1 mg/mL的VCR）。两组培养24 h后,用MTT比色法检测INS-1细胞的增殖抑制率;用Transwell侵袭试验检测INS-1细胞的侵袭力;RT-PCR检测各组细胞中hTERT、Sp1、c-myc基因的表达。结果实验组抑制率明显高于对照组（P〈0.05）;且随着VCR浓度的增加,INS-1细胞生长抑制率逐渐增加（P〈0.05）;Transwell小室培养细胞24 h后,实验组侵袭到下层膜的细胞数为（63.26±5.12）个,对照组为（150.65±4.02）个（P〈0.01）;与对照组比较,随着时间的延长,实验组的hTERT、Sp1、c-myc mRNA基因在INS-1细胞的表达也逐渐减弱（P均〈0.05）。结论 VCR能抑制INS-1细胞增殖和侵袭,可能通过抑制Sp1、c-myc和hTERT表达,从而抑制INS-1的端粒酶活性,进而抑制细胞增殖和降低侵袭能力。     

过氧化物酶体增生因子激活受体α和γ配体对游离脂肪酸介导的β细胞损害的干预作用

目的：研究过氧化物酶体增生因子激活受体α和γ(PPARα和PPARγ)配体对游离脂肪酸(FFA)介导的胰岛β细胞损害的干预作用。方法：应用PPARα配体氯贝丁酯及PPARγ配体曲格列酮(troglitazone,TGZ)和噻唑烷二酮(thiazolidinedione,TZD)处理大鼠胰岛素瘤细胞系β细胞(INS-1细胞),采用细胞活力和DNA片段梯度分析评价PPARα和PPARγ配体对FFA诱导的INS-1细胞损害的影响。结果：INS-1细胞与0．25～1mmol／L的FFA孵育24h后细胞活力下降,1mmol／L的FFA可诱导INS-1细胞发生凋亡。比较是否使用氯贝丁酯(100μmol／L)、TGZ(10μmol／L)和TZD(100μmol/L)处理β细胞的结果,发现这些配体可保护INS-1细胞免于FFA的细胞毒性(包括脂性凋亡)作用。结论：FFA可介导β细胞发生明显的脂毒性和脂性凋亡,应用PPARα和PPARγ配体可能具有保护β细胞免于FFA的细胞毒性的作用。