乙型肝炎病毒x基因转染肾小管上皮细胞导致细胞凋亡的机制探讨

目的:探讨乙型肝炎病毒x基因(HBx)转染人近端肾小管上皮细胞系(HK-2细胞)后对其凋亡及细胞因子分泌的影响及可能机制. 方法:体外培养HK-2细胞,用分子克隆法构建pcDNA3.1/myc-HBX质粒,采用脂质体转染法瞬时转染HK-2细胞,实时荧光定量PCR (RT-PCR)及蛋白印迹法验证HBx在HK-2细胞中的表达.以未转染质粒组和转染空载质粒pcDNA3.1/myc组作为对照,显微镜观察转染HBx基因后HK-2细胞形态,RT-PCR检测各组细胞Toll样受体4(TLR4) mRNA水平,蛋白印迹法检测各组细胞TLR4的表达,流式细胞术检测各组细胞凋亡情况,ELISA检测细胞培养上清中白细胞介素4(IL-4)、干扰素γ(IFN-γ)水平. 结果:转染pcDNA3.1/myc-HBx质粒后的HK-2细胞中HBx mRNA和蛋白表达水平明显增加,证实转染成功;转染HBx基因组细胞形态不规则,细胞状态受损;与对照组相比,转染HBx基因组TLR4的表达水平明显上调(P＜0.05),凋亡细胞数量明显增多(P＜0.05),细胞上清液中IFN-γ水平增加,但IL-4水平降低. 结论:经构建pcDNA/myc-HBx质粒并转染肾小管上皮细胞可成功制备乙肝病毒细胞感染模型,由此导致的肾小管上皮细胞凋亡可能与HBx引起肾小管上皮细胞TLR4的表达上调有关,HBx亦能导致细胞因子分泌紊乱.     

siRNA介导的E2F-1基因沉默对胃癌细胞MGC803中Rb蛋白表达水平的影响

目的应用RNA干扰技术研究E2F-1基因沉默在人胃癌MGC803细胞中对Rb蛋白表达的影响。方法将重组质粒E2F-1-siRNA转染MGC803细胞,筛选稳定株,利用RT-PCR检测转染前后细胞E2F-1mRNA表达水平,并通过蛋白免疫印记法(Western blot)检测各组细胞E2F-1蛋白的表达水平来验证质粒转入胃癌细胞的情况。采用Western blot检测三组细胞中总Rb蛋白和磷酸化Rb蛋白的表达情况,计算出非磷酸化Rb蛋白表达情况,统计各组间的差异。结果重组质粒E2F-1-siRNA成功转入胃癌细胞中;有效抑制了E2F-1 mRNA的表达,E2F-1蛋白水平显著下降,与阴性对照组及未转染组相比分别降低了83.2%和84.6%,去磷酸化Rb蛋白分别增加了61.22%(P0.05)和66.60%(P0.05),差异有统计学意义。结论沉默表达E2F-1基因后,可以有效降低Rb蛋白的水平,促进其活化形式去磷酸化Rb蛋白的产生。     

GLUT4 mRNA在糖尿病大鼠骨骼肌及脂肪组织中的表达及意义

目的探讨葡萄糖转运蛋白4（GLUT4）mRNA表达在2型糖尿病发病中的作用。方法将46只SD大鼠随机分为对照组及高脂组各10只、高血糖组及糖尿病组各13只,分别采用高脂饮食及腹腔内注射链脲佐菌素方法制备高脂、高血糖及2型糖尿病模型。10周末实验结束后,分别取血测空腹血糖（FBG）、空腹胰岛素（FINS）,计算胰岛素指数（ISI）;采用RT-PCR检测骨骼肌、脂肪组织中GLUT4 mRNA表达。结果与对照组比较,糖尿病组FBG、FINS及ISI显著升高、骨骼肌及脂肪组织中GLUT4 mRNA表达均明显下调,高血糖组、高脂组骨骼肌和脂肪组织中GLUT4 mRNA表达亦显著低于对照组,四组GLUT4 mRNA在骨骼肌中的表达均显著高于脂肪组织（P〈0.01、0.05）。结论 GLUT4 mRNA在骨骼肌中的表达高于脂肪组织;高脂饮食、高血糖可通过下调GLUT4 mRNA表达加重胰岛素抵抗,进而诱发2型糖尿病。     

胰岛素通过激活P13K/mTOR通路诱导人肾小系膜细胞血管内皮细胞生长因子转录

目的 探讨胰岛素诱导人肾小球系膜细胞(HMC)血管内皮细胞生长因子(VEGF)基因转录机制. 方法 (1)用VEGF报告质粒或低氧诱导因子1(HIF-1)结合位点畸变的VEGF mut报告质粒转染HMC,荧光素酶分析法检测其转录活性;(2)应用Real-time PCR、Western blot法分别检测HIF-1α mRNA和蛋白表达. 结果 (1)胰岛素呈剂量依赖性增加VEGF基因转录,在100nmol/L时达高峰,为未刺激组的2.14±0.17倍(P<0.01),但对转染VEGF mut报告质粒的HMC VEGF基因转录无影响;(2)Ly294002和雷帕霉素均可抑制胰岛素诱导的VEGF基因转录(P<0.01);(3)胰岛素呈时间依赖性上调HIF-1α蛋白表达,4h达高峰,为对照组的2.35±0.35倍(P<0.01),但对其mRNA表达无影响. 结论 胰岛素通过激活P13K/mTOR通路和上调HIF-1α蛋白表达诱导HMC VEGF基因转录.     

HIF-1α对乳腺癌MCF-7细胞增殖的影响及其机制探讨

目的 观察缺氧诱导因子1α（HIF-1α）基因对乳腺癌MCF-7细胞增殖的影响,并探讨其可能的机制.方法 构建并筛选HIF-1 α基因的RNAi表达质粒,以脂质体LipofectamineTM 2000介导转染MCF-7细胞.转染后48h,采用实时定量RT-PCR技术检测转染细胞中HIF-1α mRNA的转录水平,筛选有效的HIF-1 αRNAi质粒;CCK-8法比较转染前后乳腺癌细胞增殖变化,实时定量RT-PCR检测顺滑蛋白（SMO）mRNA表达.结果 成功构建含HIF-1α短发夹状RNA（shRNA）1～4的RNAi表达质粒,其中HIF-1 αshRNA-4干扰抑制效率为74％,干扰效果最强（P均＜0.05）.HIF-1 αshRNA-4干扰48、72、96 h后,MCF-7细胞的生长抑制率明显升高（P均＜0.05）.HIF-1αshRNA-4转染后MCF-7细胞SMO mRNA的相对表达量为0.56 ±0.06,低于转染前的1.07 ±0.16（P ＜0.05）.结论 HIF-1α表达可促进乳腺癌细胞增殖,可能与其参与调控SMO的表达有关.     

亚油酸对纤溶酶原激活物抑制剂-1表达的影响及其机制

目的探讨过氧化体增殖物激活型受体α（PPARα）的特异性激活物亚油酸对HepG2细胞1型纤溶酶原激活物抑制剂（PAI-1）mRNA表达及其活性的影响和在该基因转录调控中的作用机制.方法用不同浓度亚油酸为诱导因素刺激HepG2细胞,采用半定量RT-PCR法检测PAI-1mRNA水平,发色底物法检测PAI-1的活性变化.构建四个含PAI-1启动子序列从-804～＋17间不同长度片段驱动的荧光素酶报告基因质粒,体外瞬时转染HepG2细胞,检测荧光素酶的活性.结果与对照组相比,亚油酸组能使HepG2细胞PAI-1mRNA表达及蛋白活性显著升高（P＜0.05,P＜0.01）,且呈一定剂量依赖性;亚油酸诱导可使PAI-1转录活性显著升高（P＜0.01）;与转染质粒PAI-pGL3-A（-804/＋17）相比较,当转染质粒含有PAI-pGL3-B（-636/＋17）、PAI-pGL3-C（-449/＋17）时,荧光素酶活性显著降低（P＜0.01）;共转染PPARα表达质粒（PPARα-pSG5）的细胞在亚油酸诱导下PAI-1转录活性显著升高（P＜0.01）.结论亚油酸可以增加HepG2细胞PAI-1mRNA表达及其蛋白活性,调节PAI-1的基因转录,PPARα参与亚油酸对PAI-1基因的表达调控;在PAI-1启动子-804～-636、-449～-276区域内存在亚油酸作用的调控PAI-1基因表达的序列.     

JAZF1基因抑制对3T3-L1脂肪细胞糖、脂代谢的影响

目的 探讨JAZF1基因抑制对3T3-L1脂肪细胞糖、脂代谢相关基因的影响.方法 构建JAZF1小发夹RNA (shRNA)表达载体并转染3T3-L1细胞,实时荧光定量PCR(RT-QPCR)和蛋白印迹法检测JAZF1 mRNA和蛋白水平的表达;氢三放射示踪法检测3T3-L1细胞糖摄取率;蛋白印记法检测糖、脂代谢相关基因蛋白水平;油红O染色检测脂肪细胞甘油三酯(TG)含量变化.结果 成功构建JAZF1-shRNA;转染脂肪细胞48 h后,JAZF1 mRNA和蛋白水平明显低于对照组(P＜0.05);氢3放射性示踪法显示转染组葡萄糖摄取率明显降低(P＜0.05);PPAR-γ蛋白表达升高(P＜0.05),激素敏感脂肪酶(HSL)、内脏脂肪素(Visfatin)、胰岛素诱导基囚-2 (Insig-2)蛋白表达降低(均P＜0.05);油红O染色显示JAZF1转染组细胞内脂质积聚明显,比对照组升高约25％(P＜0.05).结论 JAZF1基因抑制可减少基础糖转运,增加脂质与胆固醇合成,减少脂质分解并减少相关脂肪细胞因子的表达.     

糖尿病大鼠VFN水平与GLUT1、GLUT4基因表达的相关性

目的探讨内脏脂肪素(VFN)与葡萄糖转运蛋白(GLUT)1、GLUT4基因表达的相关性。方法健康雄性Wistar大鼠33只随机分为正常对照组(NC组),糖尿病模型组(DM组),高脂饮食组(HF组),分别为10只、13只、10只。检测3组大鼠体重、空腹血糖(FBG)和空腹血清胰岛素(FINS),用稳态模型评估法(HOMA)评价胰岛素抵抗(IR)指数,采尾血以酶联免疫吸附试验(ELISA)测定VFN的含量。处死,分离附睾、肠系膜、折返腹膜脂肪及肾脏脂肪垫,称重,计为内脏脂肪重量,利用RT-PCR法分别检测3组大鼠内脏脂肪中VFN的表达。RT-PCR方法检测各组大鼠心肌组织GLUT1、GLUT4的基因表达水平。结果成功建立大鼠糖尿病模型。DM组VFN表达明显增加,GLUT1、GLUT4的相对表达水平明显降低(P0.05)。结论糖尿病大鼠体内VFN水平可影响GLUT1、GLUT4 mRNA的表达,且具有负相关性。     

脂肪因子网膜素1与妊娠期糖尿病胰岛素抵抗的相关性研究

目的通过检测GDM患者血清及大网膜脂肪组织中网膜素1(Omentin-1)及胰岛素受体底物1(IRS-1)、胰岛素受体底物2(IRS-2)的表达,探讨脂肪因子Omentin-1与GDM患者IR的关系。方法将研究对象分为GDM组26例、正常对照组(GNGT)24名。产前ELISA测定空腹血清Omentin-1水平;剖宫产术中收集大网膜脂肪组织,RT-PCR及Western blot检测其Omentin-1、IRS-1、IRS-2mRNA及蛋白表达。结果 GDM组血清中Omentin-1较GNGT组低[(217.88±72.15)vs(272.08±107.30)ng/ml,P=0.048]。GDM组大网膜脂肪组织中IRS-1mRNA及蛋白表达较GNGT组降低(P0.05),IRS-2在两组大网膜组织表达比较,差异无统计学意义;Omentin-1在GDM组大网膜脂肪组织中及蛋白表达降低,但差异无统计学意义。大网膜脂肪组织中Omentin-1 mRNA表达与IRS-1(r=0.755,P=0.000)及IRS-2mRNA(r=0.352,P=0.012)呈正相关。结论 Omentin-1表达降低与GDM患者IR有关。     

RNA干扰Twist基因对人膀胱癌T24细胞化疗敏感性的影响

目的 构建转录因子Twist基因的短发卡状RNA ( shRNA) 质粒,转染人膀胱癌T24细胞株后检测T24细胞对羟基喜树碱(HCPT)的敏感性.方法 体外构建Twist基因shRNA的表达质粒,脂质体法介导将其转染入T24细胞.实验分为正常对照组、非特异性转染组和特异性转染组.采用RT-PCR法和Western印迹法检测转染后Twist mRNA 及蛋白的表达.使用四甲基偶氮唑蓝法(MTT)及流式细胞术(FCM)测定Twist shRNA 转染T24细胞后细胞对HCPT敏感性的改变.结果 Twist shRNA 转染组细胞Twist mRNA和蛋白的表达与其他两组相比明显下调(P＜0.05);HCPT敏感性与正常对照组相比明显提高(P＜0.05).结论 靶向Twist 基因的序列特异性shRNA 可增强T24细胞对HCPT的敏感性.     

肥胖小鼠脂肪组织缺氧对脂联素表达的转录抑制作用

目的观察缺氧对脂肪细胞脂联素mRNA和蛋白表达的影响,探讨肥胖小鼠脂肪组织缺氧导致脂肪组织脂联素表达下降的机制。方法采用实时定量聚合酶链反应（qRT—PCR）和蛋白免疫印迹法（Western blotting）检测遗传型肥胖小鼠（ob／ob,12周）和高脂饮食肥胖小鼠（HFD,53周）的附睾旁脂肪中脂联素mRNA和蛋白的表达;用小鼠3T3-L1脂肪细胞系为模型,采用RT—PCR和荧光素酶报告基因方法检测缺氧处理后脂联素和过氧化物酶体增殖物激活受体（PPAR）-mRNA的表达和稳定性、脂联素启动子的活性;用Western blotting和荧光素酶报告基因检测缺氧对PPAR-γ在核蛋白中集聚以及PPAR-γ转录因子活性的影响。组间数据比较采用t检验。结果（1）缺氧时两种肥胖小鼠的脂肪组织中脂联素mRNA和蛋白的表达均显著下降（P〈0．01）;333-L1脂肪细胞系在缺氧8h和24h后,脂联素mRNA表达量分别下降至0．65±0．05和0．29±0．05,较对照组（1．00±0．04）明显降低,差异有统计学意义（t=11．548、24．893,均P〈0．01）,但缺氧对脂联素mRNA的稳定性并没有影响;荧光素酶报告基因方法表明,脂联素启动子的活性受到缺氧的抑制。（2）在两种肥胖小鼠的脂肪组织中,PPAR-γmRNA和蛋白的表达均明显下降（P〈0．01）;小鼠333-L1脂肪细胞系在缺氧8h和24h后,PPAR- γmRNA的表达量分别下降至0．72±0．09和0．54±0．07,与对照组（1．00±0．09）相比,差异有统计学意义（t：5．134、9．876,均P〈0．01）;PPAR一1蛋白的核转位以及PPAR一^y转录因子活性也受到缺氧的抑制。结论肥胖小鼠脂肪组织缺氧抑制了脂联素的表达,抑制作用可能发生在转录水平;其机制可能是通过抑制PPAR-γmRNA的表达和PPAR-γ转录因子的活性而实现的。     

Troglitazone improves GLUT4 expression in adipose tissue in an animal model of obese type 2 diabetes mellitus

Troglitazone has been shown to improve peripheral insulin resistance in type 2 diabetic patients and animal models. We examined the effect of troglitazone on the expression of glucose transporter 4 (GLUT4) in muscle and adipose tissue from Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model of obese type 2 diabetes mellitus. In addition, the effects of troglitazone on GLUT4 translocation and on glucose transport activity in adipocytes were also evaluated. Muscle and adipose tissues were isolated from 35-week-old male troglitazone-treated and untreated OLETF rats at a dose of 150 mg/kg per day for 14 days. In skeletal muscle, the protein and mRNA levels of GLUT4 were not significantly different between OLETF and control rats and they were not affected by troglitazone. On the other hand, GLUT4 protein and mRNA levels in adipose tissue from OLETF rats were significantly decreased (P<0.01) compared with control rats and they were significantly increased (1.5-fold, P<0.01) by troglitazone. Troglitazone had no major effect on GLUT4 translocation in adipocytes, but it significantly increased (1.4-fold, P<0.05) the basal and insulin-induced amounts of GLUT4 in plasma membrane (PM) in adipocytes from OLETF rats. Consistent with these results, the basal and insulin-induced glucose uptakes in adipocytes from troglitazone-treated OLETF rats were significantly increased (1.5-fold, P<0.05) compared with untreated OLETF rats. Our results suggest that troglitazone may exert beneficial effects on insulin resistance by increasing the expression of GLUT4 in adipose tissue.     

Expression of the mRNA coding for 11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue from obese patients: an in situ hybridization study

Glucocorticoids play an important role in determining adipose tissue metabolism and distribution. Patients with Cushing's syndrome or receiving corticosteroid therapy develop a reversible visceral obesity. In obese patients, although circulating concentrations of cortisol are not consistently elevated, local conversion of inactive cortisone to active cortisol in adipose tissue, catalyzed by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1), could amplify glucocorticoid signaling. We have studied, using semiquantitative in situ hybridization, 11beta-HSD-1 mRNA expression in the adipocyte and stromal compartments of sc abdominal adipose tissue obtained from 12 lean patients and sc abdominal and visceral adipose tissue obtained from 18 obese patients. 11beta-HSD-1 mRNA was expressed in adipocytes, stroma, and walls of vessels. Localization of 11beta-HSD-1 mRNA did not differ between lean sc and obese sc or visceral adipose tissue. 11beta-HSD-1 mRNA levels were significantly (P = 0.0106) increased in the adipocyte compartment of sc adipose tissue obtained from obese patients as compared with nonobese ones, whereas no significant change (P = 0.446) was found in the stromal compartment. In obese patients, 11beta-HSD-1 mRNA expression was increased (P = 0.0157) in the stromal compartment of visceral compared with sc tissue, whereas no significant change (P = 0.8767) was found in the adipocyte compartment. In summary, our data show that 11beta-HSD-1 mRNA is increased in adipose tissue from obese patients, in the abdominal sc fat in adipocytes and in the visceral fat in both adipocytes and stroma. This observation suggests that an overexpression of 11beta-HSD-1 may explain part of the glucocorticoid-induced metabolic disorders linked to obesity and may promote visceral fat deposition.     

Regional variation in adipose tissue insulin action and GLUT4 glucose transporter expression in severely obese premenopausal women

Summary Insulin action and GLUT4 expression were examined in adipose tissue of severely obese premenopausal women undergoing gastrointestinal surgery. Fat samples were taken from three different anatomical regions: the subcutaneous abdominal site, the round ligament (deep abdominal properitoneal fat), and the greater omentum (deep abdominal intraperitoneal fat). The stimulatory effect of insulin on glucose transport and the ability of the hormone to inhibit lipolysis were determined in adipocytes isolated from these three adipose depots. Insulin stimulated glucose transport 2–3 times over basal rates in all adipocytes. However, round ligament adipose cells showed a significantly greater responsiveness to insulin when compared to subcutaneous and omental adipocytes. Round ligament fat cells also displayed the greatest sensitivity and maximal antilipolytic response to insulin. We also investigated whether regional differences in fat cell insulin-stimulated glucose transport were linked to a differential expression of the GLUT4 glucose transporter. GLUT4 protein content in total membranes was 5 and 2.2 times greater in round ligament adipose tissue than in subcutaneous and omental fat depots, respectively. Moreover, GLUT4 mRNA levels were 2.1 and 3 times higher in round ligament than in subcutaneous or omental adipose tissues, respectively. Adipose tissue GLUT4 protein content was strongly and negatively associated (r = –0.79 to –0.89, p < 0.01) with the waist-to-hip ratio but not with total adiposity. In conclusion, these results demonstrate the existence of site differences in adipose tissue insulin action in morbidly obese women. The greater insulin effect on glucose transport in round ligament adipocytes was associated with a higher expression of GLUT4 when compared to subcutaneous abdominal and omental fat cells. Moreover, despite the regional variation in GLUT4 expression, an increased proportion of abdominal fat was found to be associated with lower levels of GLUT4 in all adipose regions investigated. [Diabetologia (1997) 40: 590–598] Received: 8 October 1996 and in revised form: 28 January 1997     

Expression of an insulin-responsive glucose transporter (GLUT4) minigene in transgenic mice: effect of exercise and role in glucose homeostasis.

The effects of a GLUT4 mini-transgene (containing 7 kb of 5' flanking and 1 kb of 3' flanking sequence and all exons and introns of the GLUT4 gene as well as a small foreign DNA tag) and of exercise training on expression of GLUT4 and glycemic control in mice were investigated. Transgenic mice harboring the minigene expressed < or = 2-fold the normal level of GLUT4 mRNA and protein in skeletal (gastrocnemius) muscle and adipose tissue. This modest tissue-specific increase in GLUT4 expression led to an unexpectedly rapid blood glucose clearance rate following oral glucose administration. In nontransgenic animals exercise caused a 1.5-fold increase in expression of GLUT4 mRNA and protein as well as a significant improvement of glycemic control. In transgenic animals harboring the minigene exercise increased expression of GLUT4 mRNA and protein derived from the minigene and endogenous gene and led to a further improvement of glycemic control. These findings indicate that the cis-regulatory element(s) controlling exercise-induced expression of the GLUT4 gene is located within the nucleotide sequence encompassed by the GLUT4 minigene. The fact that glycemic control is markedly improved by a relatively low level of expression of GLUT4 caused by the transfected minigene and is further enhanced by exercise in transgenic animals demonstrates that GLUT4 plays a pivotal role in glucose homeostasis in vivo. Of the effectors--i.e., cAMP, insulin, and arachidonic acid--known to down-regulate expression of GLUT4 by 3T3-L1 adipocytes in culture, only the decline in circulating arachidonate level in vivo correlated with up-regulation of GLUT4 caused by exercise.     

Glucose transporter 4 and insulin receptor substrate-1 messenger RNA expression in omental and subcutaneous adipose tissue in women

Insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) expression may provide an indirect reflection of the capacity of adipocytes to respond to insulin stimulation. We examined messenger RNA (mRNA) expression of these genes in omental and subcutaneous adipose tissue of women. Paired omental and subcutaneous adipose tissue samples were obtained from 36 women (age, 47 ± 5 years; body mass index, 28.0 ± 5.4 kg/m²) undergoing gynecologic surgeries. Total adiposity and visceral adiposity were assessed by dual-energy x-ray absorptiometry and computed tomography. The GLUT4 and IRS-1 mRNA expression levels were both significantly higher in subcutaneous compared with omental adipose tissue. A negative correlation was observed between body fat percentage and subcutaneous adipose tissue GLUT4 (r = −0.39, P < .05) and IRS-1 (r = −0.30, P < .08) mRNA abundance. However, in omental fat, only GLUT4 mRNA was inversely associated with body fat percentage (r = −0.53, P < .001). Moreover, the homeostasis model assessment of insulin resistance index was associated with mRNA expression of subcutaneous GLUT4 (r = −0.56, P < .001), subcutaneous IRS-1 (r = −0.51, P < .01), and omental GLUT4 (r = −0.54, P < .001), but not omental IRS-1. Interestingly, plasma adiponectin was only associated with subcutaneous GLUT4 (r = 0.48, P < .01) and IRS-1 (r = 0.48, P < .05) mRNA expression. The GLUT4 protein, unlike mRNA expression, was higher in omental than in subcutaneous adipose tissue. However, abdominal obesity-related differences in protein or mRNA expression were similar. Omental IRS-1 expression was low and unaffected by visceral obesity. In contrast, omental and subcutaneous GLUT4 as well as subcutaneous IRS-1 were reduced in visceral obesity. This divergent pattern of expression may reflect a lower capacity of omental adipose tissue to respond to insulin stimulation at all adiposity levels.