GLUT4研究进展

葡萄糖转运蛋白 4 (GLUT4 )是脂肪细胞和骨骼肌细胞协助葡萄糖转运的主要蛋白质 ,基础状态时分布于细胞内 ,在胰岛素刺激或运动等刺激下转位至细胞膜上。对GLUT4表达的调节在转录水平和转录后水平都存在。GLUT4转位涉及胰岛素信号传导途径和一磷酸腺苷激活的蛋白激酶 (AMPK)途径。GLUT4分子内部结构变化也可影响葡萄糖的转运。     

高脂饮食喂养对大鼠骨骼肌细胞膜GLUT4含量的影响

目的:检测骨骼肌细胞膜GLUT4含量的改变,探讨高脂饮食喂养诱导胰岛素抵抗的受体后机制。方法:将动物分为3组:①正常对照组;②高脂饮食组;③高脂饮食+饮食控制组。通过8周高脂饮食喂养建立胰岛素抵抗大鼠模型,随后代以普通饮食继续喂养4周。用Westernblot方法检测骨骼肌细胞膜表面GLUT4蛋白表达。结果:在胰岛素刺激下,高脂饮食组大鼠骨骼肌细胞膜GLUT4蛋白表达显著少于正常对照组(减少约31%);饮食控制组骨骼肌细胞膜GLUT4蛋白表达明显高于高脂饮食组(约1.14倍)。结论:高脂喂养的方法可成功复制出胰岛素抵抗大鼠模型;高脂饮食可能通过影响胰岛素信号转导系统,使胰岛素刺激的GLUT4转位至细胞膜受阻,其在膜上的含量也降低,从而促进胰岛素抵抗的形成和发展。     

罗格列酮对实验性2型糖尿病合并高脂血症大鼠骨骼肌组织IRS-1及GLUT4表达的调节

目的： 观察罗格列酮对实验性2型糖尿病合并高脂血症大鼠骨骼肌组织胰岛素受体底物-1(IRS-1)表达的调节及大鼠骨骼肌细胞葡萄糖转运蛋白4(GLUT4)表达的影响，进一步探讨其可能的作用机制。方法: 采用低剂量链脲佐菌素尾静脉注射联合高脂饲料喂养建立2型糖尿病合并高脂血症大鼠模型。未予上述处理的大鼠作为正常对照组，成模大鼠随机分为糖尿病对照组和罗格列酮干预组。药物干预4周后，称重，检测血清葡萄糖、胰岛素、甘油三酯和胆固醇，应用Western印迹法检测大鼠肌肉组织中IRS-1蛋白的表达和细胞膜GLUT4的表达量。结果: (1)罗格列酮干预组大鼠空腹血糖、胰岛素、甘油三酯水平均低于糖尿病对照组(P<0.05)，高于正常对照组(P<0.05)；罗格列酮干预组血清胆固醇高于正常对照组(P<0.05)，与糖尿病对照组差异无显著(P>0.05)。(2)大鼠肌肉细胞膜GLUT4蛋白表达比较：糖尿病对照组明显少于正常对照组，罗格列酮干预组明显多于糖尿病对照组(P<0.05)。(3)大鼠肌肉组织IRS-1蛋白表达量及其酪氨酸磷酸化程度比较：糖尿病对照组明显少于正常对照组，罗格列酮干预组明显多于糖尿病对照组(P<0.05)。结论: 罗格列酮可促进大鼠肌肉细胞膜GLUT4的合成增加，可能是通过上调大鼠肌肉组织IRS-1蛋白的表达量及其酪氨酸磷酸化的程度实现的。     

不同浓度红景天苷对Ⅱ型糖尿病大鼠骨骼肌磷脂酰肌醇-3-激酶及葡萄糖转运蛋白-4表达的影响

目的:观察不同浓度红景天苷对Ⅱ型糖尿病大鼠血糖、血脂及骨骼肌磷脂酰肌醇-3-激酶(PI3K)和葡萄糖转运蛋白-4(GLUT4)表达含量的影响,探讨红景天苷降糖改善胰岛素抵抗的可能机制。方法:采用小剂量链脲佐菌素加高脂高热量饲料喂养方法建立Ⅱ型糖尿病大鼠模型,将建模成功后大鼠随机分为糖尿病模型组(DM),二甲双胍治疗组,红景天苷高、中、低剂量组。按规定药物剂量灌胃12周后测血糖、血脂及胰岛素水平,处死大鼠,取后肢骨骼肌,免疫印迹检测组织中PI3K和GLUT4的表达水平。结果:与对照组比较,DM组大鼠骨骼肌细胞中PI3K和GLUT4表达明显降低;与DM组比较,红景天苷各治疗组骨骼肌细胞中PI3K和GLUT4的表达明显增强。结论:红景天苷可能通过增强大鼠骨骼肌组织细胞中PI3K和GLUT4的表达以改善Ⅱ型糖尿病胰岛素抵抗。     

饮食因素对大鼠脂肪细胞葡萄糖转运蛋白4的影响

目的:研究饮食因素对大鼠脂肪细胞葡萄糖转运蛋白4(GLUT4)的影响。方法:实验大鼠随机分为正常组(n=10)、模型组(n=10)和饮食干预组(n=10)。正常组以基础饲料喂养10周;模型组以高糖高脂饲料喂养10周;饮食干预组以高糖高脂饲料喂养4周后,改喂基础饲料6周。Westernblot法检测各组大鼠脂肪细胞内、外膜GLUT4含量。结果:模型组大鼠脂肪细胞内、外膜GLUT4含量均低于正常组(P<0.05)。与模型组大鼠相比,饮食干预组大鼠脂肪细胞内膜GLUT4含量无显著差异,但细胞外膜GLUT4含量增加P<0.05)。结论:高糖高脂饮食可能通过降低脂肪细胞GLUT4含量及其转位使大鼠产生胰岛素抵抗。饮食干预可提高脂肪细胞GLUT4含量并改善其转位,增加葡萄糖的摄取,提高胰岛素敏感性。     

小鼠骨骼肌细胞离体胰岛素抵抗模型的建立和验证

目的:建立稳定可重复的骨骼肌细胞离体胰岛素抵抗模型,促进胰岛素抵抗病理机制的探索及药物的研发与筛选。方法:采用小鼠骨骼肌成肌细胞C2C12为研究对象,分别以正常分化液及含40和60 mmol/L葡萄糖的分化液诱导分化,每天用相差显微镜观察不同浓度葡萄糖处理对细胞汇聚、融合和形成多核肌管的影响;分别在分化1、3、5和7 d后应用2-NBDG法检测不同处理对细胞基础糖摄取和胰岛素刺激糖摄取的影响;在分化5 d和7 d后应用Western blot法检测不同干预对葡萄糖转运子4(glucose transporter 4,GLUT4)蛋白表达的影响;分化5 d后应用免疫荧光组化法检测不同干预对GLUT4蛋白分布的影响。结果:形态学结果显示,经60 mmol/L葡萄糖(高糖)处理3 d后明显抑制C2C12细胞的生长分化;葡萄糖摄取结果表明高糖处理5 d和7 d后均明显抑制C2C12的基础糖摄取和胰岛素刺激的糖摄取(P0.01),且处理5 d后胰岛素刺激的糖摄取与基础糖摄取的差异无统计学显著性(P0.05); Western blot检测结果表明高糖处理5 d和7 d后,胰岛素刺激的GLUT4表达与基础GLUT4表达的差异无统计学显著性(P0.05),而对照组差异显著(P0.05);免疫荧光组化检测结果表明高糖处理5 d后明显减少C2C12细胞胞膜上GLUT4蛋白分布水平(P0.01)。40 mmol/L葡萄糖处理也在一定程度上发挥作用,但效果不如60 mmol/L葡萄糖明显和稳定。结论:通过高糖刺激可成功构建较为稳定的小鼠骨骼肌细胞离体胰岛素抵抗模型,以60 mmol/L葡萄糖刺激5 d效果最好,通过形态学观察并检测基础和胰岛素刺激的葡萄糖摄取能力和GLUT4蛋白表达与分布能较好地评价骨骼肌细胞离体胰岛素抵抗水平。     

AMPK调节骨骼肌细胞GLUT4基因表达的机制研究

腺苷酸活化蛋白激酶(AMPK)能调节运动/肌肉收缩所引起的骨骼肌细胞葡萄糖转运蛋白4(GLUT4)基因的表达,但至今它的调节机制不清.研究显示在非运动刺激引起的细胞信号事件中由组蛋白去乙酰化酶(HDACs)以及组蛋白乙酰化酶(HATs)控制的组蛋白乙酰化状态是调节基因表达的重要机制,所以我们假设AMPK信号途径是通过征用HDACs中的HDAC5(在骨骼肌细胞内高表达)来实现对运动/肌肉收缩引起的GLUT4基因表达控制.细胞分为正常浓度葡萄糖对照组(NGLU组)、正常浓度AICAR组(NGLU AICAR组)、高浓度对照组(HGLU组)、高浓度AICAR组(HGLU AICAR组).用5 mmol/L和20 mmol/L葡萄糖浓度培养骨骼肌细胞后,NGLU AICAR组和HGLU AICAR组与肌肉收缩模拟信号刺激5-氨基-4-甲酰胺咪唑核糖核苷酸(AICAR)孵育.AICAR能激活NGLU组骨骼肌细胞AMPKα2、减少骨骼肌细胞核HDAC5蛋白、促使HDAC5与骨骼肌细胞加强因子(MEF2)蛋白分离和上调GLUT4基因的表达;相反,高浓度葡萄糖延迟由AICAR引起的AMPKα2磷酸化、AMPKα2向细胞核转入、HDAC5向细胞核转出和GLUT4基因的表达.实验结果说明在不同葡萄糖浓度下的骨骼肌细胞GLUT4基因表达变化都对应着上游AMPK蛋白和下游HDAC5蛋白的变化,AMPK可能是征用转录抑制子HDAC5来调节MEF2的活性而达到控制肌肉收缩所引起的GLUT4基因表达.     

辛伐他汀对小鼠胰腺β细胞功能的影响及机制研究

 目的：观察辛伐他汀对小鼠胰腺β细胞株MIN6胰岛素分泌功能的影响并探讨其可能机制。方法：将MIN6细胞随机分为正常对照组和低、中、高浓度辛伐他汀组,分别用含0、2、5、10 μmol/L辛伐他汀和15%胎牛血清的高糖DMEM培养基培养48 h。采用放射免疫分析法检测辛伐他汀对MIN6细胞胰岛素分泌功能的影响;生物化学发光法测定细胞内ATP含量;用实时荧光定量PCR检测内向整流钾离子通道62（Kir62）、电压依赖性钙离子通道12（CaV12）及葡萄糖转运体2（GLUT2）mRNA表达水平;用Western印迹检测Kir62、CaV12及GLUT2蛋白表达水平。结果：5和10 μmol/L的辛伐他汀能够明显减少MIN6细胞胰岛素的合成及分泌（P<005）;辛伐他汀处理组MIN6细胞内ATP水平较正常对照组明显降低（P<005）;辛伐他汀各处理组MIN6细胞Kir62 mRNA表达水平较正常对照组明显上调（均P<001）,5和10 μmol/L辛伐他汀组CaV12 mRNA水平明显下调（均P<001）,GLUT2 mRNA表达水平明显下调（P<005）;5和10 μmol/L辛伐他汀处理组Kir62蛋白表达较正常对照组明显升高（均P<001）,10 μmol/L辛伐他汀处理组CaV12及GLUT2蛋白表达水平明显下降（均P<001）,5 μmol/L辛伐他汀组CaV12蛋白表达水平较正常对照组亦有明显下降（P<001）。结论：辛伐他汀对小鼠胰腺β细胞株MIN6胰岛素的合成和分泌具有一定的抑制作用。辛伐他汀可能通过抑制MIN6细胞内ATP的生成以及上调MIN6细胞Kir62、下调CaV12和GLUT2的表达从而影响其胰岛素的合成和分泌。     

SM22α调节GLUT4转位的机制及其在增殖性血管疾病中的意义

目的:葡萄糖转运体4(GLUT4)在球囊损伤血管新生内膜中高表达,而其转位过程依赖于肌动蛋白(actin)细胞骨架的调节。平滑肌蛋白22α(smooth muscle protein 22α,SM22α)是一种actin细胞骨架相关蛋白,其在增殖性血管疾病中表达下调。本研究观察了SM22α是否参与血管损伤或者PDGF刺激诱导的GLUT4表达和转位活性升高。方法:用PDGF-BB刺激血管平滑肌细胞(vascular smooth muscle cell,VSMC),观察GLUT4膜转位和细胞骨架的变化;用荧光葡萄糖2-NBDG检测葡萄糖摄取;用特异性si RNA敲低内源性SM22α表达;Brd U实验检测细胞增殖;高效液相色谱法检测组织葡萄糖含量。结果:PDGFBB诱导VSMCs GLUT4转位和葡萄糖摄取依赖于皮层F-actin聚合,而敲低SM22α促进这一过程。损伤新生内膜处GLUT4表达显著增加,PDGF-BB刺激促进细胞GLUT4表达和葡萄糖消耗,抑制GLUT4活性则显著降低细胞增殖活性。相对于WT组,SM22α-/-小鼠颈总动脉2-NBDG摄取显著增加,结扎后28 d新生内膜明显增厚,损伤动脉组织GLTU4转位和葡萄糖含量均明显升高。结论:PDGF-BB诱导的GLUT4转位和糖摄取参与VSMCs增殖。缺失SM22α可诱导皮层细胞骨架聚合,增强PDGF-BB诱导的GLUT4膜转位和糖摄取及代谢活性。SM22α是一种新的增殖相关糖代谢调节因子。     

多囊卵巢综合征大鼠骨骼肌细胞胰岛素受体底物1及其丝氨酸307位点磷酸化的表达

背景：胰岛素受体底物1的丝氨酸307位点磷酸化程度的增加参与了骨骼肌胰岛素抵抗的发生。 目的：观察多囊卵巢综合征大鼠骨骼肌细胞胰岛素受体底物1的含量及其丝氨酸307位点磷酸化程度的变化。 方法：将大鼠随机分为模型组和对照组,模型组给予人绒毛膜促性腺激素联合胰岛素皮下注射,并配以高脂饮食,构建大鼠多囊卵巢综合征模型;对照组皮下注射生理盐水,正常饲料喂养。 结果与结论：干预6周后,Western blot检测结果显示,与对照组相比,模型组大鼠骨骼肌细胞胰岛素受体底物1表达量显著下降(P < 0.05),而其丝氨酸307位点磷酸化蛋白表达明显升高(P < 0.05)。结果提示,多囊卵巢综合征大鼠骨骼肌细胞中胰岛素受体底物1蛋白表达下调及其丝氨酸307位点磷酸化蛋白表达上调与多囊卵巢综合征大鼠骨骼肌胰岛素抵抗的发生密切相关。     

Heparin-binding EGF-like growth factor (HB-EGF) overexpression in transgenic mice downregulates insulin-like growth factor binding protein (IGFBP)-3 and -4 mRNA

An in vivo approach was taken to assess the biological significance of heparin-binding EGF-like growth factor (HB-EGF) using transgenic mice. Transgenic mice were generated using the pIRES-EGFP vector expressing a bicistronic mRNA containing both human HB-EGF (hHB-EGF) and enhanced green fluorescent protein (EGFP) coding sequences under the regulation of the cytomegalovirus immediate–early (CMV-IE) promoter. As a marker for transgene expression, EGFP fluorescence in 5?μm tissue sections was evaluated. To confirm HB-EGF expression in EGFP-containing tissues, HB-EGF mRNA was analyzed by RT-PCR and Northern blot analysis. Protein levels of HB-EGF and insulin-like growth factor binding protein-3 (IGFBP-3), a molecule that stabilizes IGFs, which in turn helps to promote growth, were analyzed by Western blot. Also, the weights of transgenic mice were compared with the weights of wild type non-transgenic littermates over a 10-week period. EGFP fluorescence, RT-PCR and Northern analysis of a variety of tissues from hHB-EGF transgenic mice indicate recombinant EGFP/hHB-EGF mRNA expression in kidney, liver, lung and stomach. Western blot analysis confirmed that HB-EGF protein levels were greater in these tissues from hHB-EGF transgenic mice compared to wild type non-transgenic littermates. IGFBP-3 protein was absent in serum of transgenic mice prior to the onset of puberty, but indistinguishable from wild type non-transgenic mice after puberty. Furthermore, IGFBP-3 and IGFBP-4 mRNA were downregulated in the kidney, but not liver or lung of the transgenic mice. In accordance with reduced IGFBP-3 and -4 levels, hHB-EGF transgenic mice exhibited a 20% decrease in weight prior to 6 weeks of age compared to wild type non-transgenic littermates. Our laboratory has generated a biologically functional transgenic mouse model exhibiting increased expression of hHB-EGF in kidney, liver, lung and stomach. Overexpression of hHB-EGF affected the growth rate of these transgenic mice possibly through a pathway involving IGFBP-3 and IGFBP-4.     

Modeling of the expiratory flow pattern of spontaneously breathing cats

A mathematical model was developed describing the entire expiratory flow pattern during spontaneous, tidal breathing in the absence of expiratory muscle activity. It provides estimates for the time constants of the respiratory system (tau RS(model)) and of the decay of continuing inspiratory muscle activity in early expiration (tau mus(model)). In ten anesthetized, tracheostomized cats flow, tracheal pressure and diaphragmatic EMG were measured during normal expirations and expirations with four different added resistances. No significant differences were found between tau RS(model) (0.21-0.49 sec) obtained by fitting the model to the flow data and tau RS obtained from the straight part of the expiratory flow-volume curve. tau mus(model) (0.050-0.052 sec) was comparable to similar time constants obtained from the integrated diaphragmatic EMG or from end-inspiratory, tracheal occlusion pressure. Fitted peak flow and time to peak tidal expiratory flow were not significantly different from those measured. In conclusion, for spontaneously breathing, anesthetized cats our model provides a close fit of the expiratory flow and parameter estimates were comparable with independently measured values.     

Immunohistochemical evidence of substance P-like immunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervous system.

With the indirect immunofluorescence technique of Coons and collaborators a possible coexistence of 5-hydroxytryptamine (5-HT) and substance P in neurons of the lower medulla oblongata was explored. Antisera to 5-HT and to dopadecarboxylase (aromaticl-aminoacid decarboxylase), an enzyme probably present in immunologically indistinguishable forms both in catecholamine and 5-HT neurons, were used as markers for 5-HT neurons and an antiserum raised to synthetic substance P conjugated with bovine serum albumin for substance P-containing neurons. Five or 10 μm thick, consecutive sections were stained with the three antisera. Numerous cell somata in nucleus raphe magnus, nucleus raphe obscurus, nucleus raphe pallidus, pars α of the nucleus reticularis gigantocellularis and nucleus interfascicularis hypoglossi contained both substance P-like immunoreactivity and 5-HT (and dopadecarboxylase) immunoreactive material. After intraventricular or intracisternal injections of 5,6- or 5,7-dihydroxytryptamine, two neurotoxins assumed to cause degeneration mainly of 5-HT neurons, enlarged substance P and 5-HT (and dopadecarboxylase) positive fibres were seen in, around and lateral to the olivary complex. Furthermore, in these rats both substance P and 5-HT positive nerve terminals in the ventral horns of the spinal cord disappeared.These findings indicate that substance P and 5-HT may coexist not only in some cell bodies but also in axons and nerve endings. The latter conclusion must, however, remain tentative since the neurotoxins may cause unspecific damage and thus not only damage 5-HT (and postulated ‘SP-5-HT’) neurons.In further experiments reserpine was used, a drug known to deplete monoamines by affecting their storage sites. With a high dose of reserpine a marked depletion of 5-HT was obtained both in nerve terminals and cell bodies whereas substance P immunoreactive material seemed unaffected. Possible interpretations of these findings is that substance P and 5-HT have different storage sites within the neuron, or that reserpine selectively causes loss of 5-HT and not substance P from the same storage site.     

Mapping of the binding sites for the OX1 orexin receptor antagonist, SB-334867, using orexin/hypocretin receptor chimaeras

The binding sites for agonists and antagonist of orexin receptors are not know, hampering progressive drug design approaches. In the current study, we utilized chimaeric orexin receptor approach to map the receptor areas contributing to the selectivity of the classical antagonist, SB-334867, for OX₁ receptors. Altogether ten chimaeras between OX₁ and OX₂ orexin receptors were utilized. The receptors were transiently expressed in HEK-293 cells. The ability (K_B) of SB-334867 to inhibit orexin-A-induced inositol phosphate release (phospholipase C activity) was measured. The results, in synthesis, suggest that there are several possible interactions contributing to the high affinity binding, all of which are not required simultaneously. This is indicated by the fact that most of the chimaeras display affinity (at least somewhat) higher than OX₂. As previously shown for the agonist distinction, the second quarter of the receptor, from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 seems to be most central also for SB-334867 binding, but also the third quarter, from the transmembrane helix 4 to the transmembrane helix 6 is able to contribute (and compensate for loss of other sites). A previous study has suggested that amino acids conserved between OX₁ and OX₂ receptors would somehow confer selectivity for subtype-selective antagonists. In contrast to previous findings, our results indicate that the amino acids distinct between the receptor subtypes are in key position.     

A rapid flow cytometry assay for the assessment of calcium mobilization in human neutrophils in a small volume of lysed whole-blood

Flow cytometry-based methods have been developed to measure most neutrophil responses. The assessment of the mobilization of calcium, however, is routinely performed on neutrophils isolated from whole blood. This report describes a flow cytometry-based assay to measure the mobilization of calcium in neutrophils directly in whole blood. This method requires minimal sample manipulation, small volumes of blood and is performed in a short period of time. Both clinical and research laboratories will be able to assess neutrophil function and the quality of granulocyte preparations using a more time and cost effective calcium mobilization test.     

Nitric oxide synthase inhibition speeds oxygen uptake kinetics in horses during moderate domain running

Within the moderate exercise intensity domain, the speed of oxygen uptake (V(O(2))) kinetics at the transition to a higher metabolic rate is thought to be limited by an inertia of the oxidative machinery. Nitric oxide (NO)-induced inhibition of O(2) consumption within the electron transport chain may contribute to this inertia. This investigation tested the hypothesis that a reduction or removal of any such NO effect via infusion of N(omega)-nitro-L-arginine methyl ester (L-NAME; a NOS inhibitor) would speed V(O(2)) kinetics at the onset of moderate exercise. Five Thoroughbred geldings underwent four transitions to running speeds of 7 m sec(-1) (two control, C, 2 L-NAME [20 mg kg(-1)]) on an equine treadmill during which pulmonary gas exchange was determined using a bias flow system. Consistent with exercise in the moderate intensity domain, in none of the transitions was a V(O(2)) slow component elicited. The L-NAME treatment significantly accelerated V(O(2)) kinetics via a reduction of the primary amplitude time constant (C, 17.3 +/- 1.7; L-NAME, 11.8 +/- 1.5 sec, P < 0.05) concomitant with faster overall dynamics (i.e. T(50) and T(75) both P < 0.05) and a trend toward a decreased O(2) deficit (C, 6.4 +/- 0.7; L-NAME, 4.7 +/- 1.2 L; P = 0.06). These data support the notion that NO contributes prominently to the oxidative enzyme inertia and thus the speed of V(O(2)) kinetics at the onset of moderate intensity exercise in the horse.     

Venous thromboembolism in IBD: Increased risk for women in CD?

Although coagulation disturbances have been described in inflammatory bowel disease (IBD), it remains unclear how common venous thromboembolism (VTE) is in IBD, and what factors influence VTE frequency. We evaluated VTE in Crohn's disease (CD) and ulcerative colitis (UC) at LSUHSC-S, a southern US medical center with an approximately equal White: African-American (AA) (1.12:1) patient base. This retrospective study evaluated VTE as a co-morbidity in IBD as a function of age, gender and race based on ICD-10 coding (2011?2015.) Results. Of 276 IBD diagnostic records, 213 were for CD (77.17%) and 63 for UC (22.8%). 52% of the CD patients were white, 42% were AA, and 6% were other. 42% of CD patients were male, with 58% were female. 6.1% (13 patients) of the 213 CD patients had a VTE. Of these 13 CD patients, 9 had active disease and 4 were in remission. 9 of 13 were female and 4 were male, with 5 white patients and 4 A A patients. 63 patients were diagnosed with UC, 3.38-fold fewer cases than CD. 25 UC patients were white, 25 were AA and 13 were in other ethnic groups. Of 63 UC cases, 2 UC patients had a VTE, both with active disease. At our institution, VTE appears to be 3x more frequently associated with CD than UC and was more common in white female patients. The recognition of VTE risk in CD, particularly in women, may be an important observation which may guide therapy and limit potentially life-threatening consequences.     

RNA干扰技术抑制Polo-like激酶1表达对A549细胞的影响

目的： 观察RNA干扰技术能否有效抑制非小细胞肺癌细胞株A549细胞中Polo-like激酶1（Plk1）的表达水平，以及抑制后对A549细胞生长的影响。方法： 运用脂质体法，以Plk1为靶点，构建能产生siRNA的质粒载体psiRNA-hH1-Plk1并转入A549细胞。RT-PCR检测Plk1 mRNA表达的变化、Western blotting检测Plk1、cyclin B1、p53蛋白的表达变化、细胞计数分析细胞增殖、流式细胞术分析细胞周期变化和凋亡、免疫荧光染色检测α微管蛋白的表达。结果： psiRNA-hH1-Plk1质粒能特异地抑制Plk1基因的表达并使其活性下降，致使cyclin B1及p53蛋白的表达水平升高，微管聚集障碍或形成单极的纺锤体；A549细胞增殖减慢，出现G2/M期阻滞和凋亡。结论： 上述结果提示针对Plk1基因的RNA干扰有望用于肿瘤的基因治疗。     

苦参凝胶对银屑病大鼠皮肤损伤组织病理学及IL-6、PAR-2水平的影响

目的 研究苦参凝胶对银屑病大鼠皮肤损伤组织病理学及IL-6、蛋白酶活化受体-2(monoclonal antibody to protease activated receptor 2,PAR-2)水平的影响。方法 将60只Wistar大鼠分为对照组、模型组、维A酸组、苦参凝胶高、中、低剂量组，每组10只，观察各组大鼠一般情况、皮肤损伤病理改变、炎症细胞浸润评分、Baker评分及血清和皮肤组织中IL-6、PAR-2水平。结果 维A酸组和苦参凝胶各剂量组大鼠背部皮肤鳞屑、红斑较模型组有所减轻，苦参凝胶组大鼠症状减轻程度优于维A酸组。维A酸组和苦参凝胶各剂量组大鼠背部皮肤病理改变较模型组有不同程度的改善，苦参凝胶中、高剂量组改善程度优于维A酸组。炎症细胞浸润评分、Baker评分及IL-6、PAR-2水平测试，模型组较对照组明显升高（P<0.05），各治疗组较模型组明显降低（P<0.05），苦参凝胶中、高剂量组较维A酸组明显降低（P<0.05）。结论 苦参凝胶对银屑病大鼠皮肤损伤组织病理学表现及IL-6、PAR-2水平具有改善作用。