东莨菪碱对大鼠强迫游泳行为的影响及其作用靶点

目的 阐明东莨菪碱发挥抗抑郁作用的可能脑区和靶点。方法 明确东莨菪碱的抗抑郁样作用：将正常Sprague Dawley （SD）大鼠采用数字表法随机分为东莨菪碱组和0.9%（质量分数）氯化钠注射液对照组,腹腔注射25 μg/kg东莨菪碱或等体积0.9%（质量分数）氯化钠注射液,24 h后进行旷场实验和强迫游泳实验以评价东莨菪碱对大鼠抑郁样行为的影响;明确东莨菪碱发挥抗抑郁效应的脑区：在腹腔注射25 μg/kg东莨菪碱前1 h向大鼠内侧前额叶皮质（medial prefrontal cortex,mPFC）立体定位注射γ-氨基丁酸-A （γ-aminobutyric acid-A,GABA-A）受体激动剂蝇蕈醇（Muscimol）1.25 μg,24 h后观察东莨菪碱的行为学效应是否被阻断;探索东莨菪碱抗抑郁的作用靶点：通过微量给药系统分批次分别向大鼠侧脑室注射毒蕈碱型乙酰胆碱受体2（muscarinic acetylcholine receptor 2,M₂）选择性拮抗剂（methoctramine,MCT）0.5、1和2 μg,对照组均给予2 μL 0.9%（质量分数）氯化钠注射液,24 h后利用旷场实验和强迫游泳测试评价大鼠行为学变化。结果 腹腔注射东莨菪碱24 h后,大鼠强迫游泳不动时间低于对照组（P<0.05）,旷场总路程差异无统计学意义（P>0.05）;向mPFC注射Muscimol可明显增加东莨菪碱组大鼠的不动时间（P<0.01）,旷场总路程差异无统计学意义（P>0.05）;与侧脑室注射0.9%（质量分数）氯化钠注射液相比,0.5 μg MCT不影响大鼠在强迫游泳实验中不动时间（P>0.05）,1 μg MCT可明显降低影响大鼠不动时间（P<0.01）,而2 μg MCT则可明显增加影响大鼠不动时间（P<0.05）。结论 东莨菪碱可能通过阻断mPFC的M₂受体发挥抗抑郁作用。     

胃转流术对非肥胖型2型糖尿病大鼠骨骼肌组织中MG53蛋白表达的影响

目的研究胃转流术对非肥胖型2型糖尿病(T2DM)大鼠骨骼肌组织中E3泛素连接酶—mitsugumin53(MG53)蛋白及其mRNA,以及胰岛素受体底物1(IRS-1)mRNA表达的影响,探讨胃转流术改善骨骼肌胰岛素抵抗的可能机制。方法将24只雄性GK大鼠随机分为糖尿病手术组、糖尿病假手术组及糖尿病对照组,每组8只,若大鼠死亡,则重新制备模型补充;另外以8只Wistar大鼠作为正常对照组。应用Westem blot技术检测4组大鼠术后第8周时骨骼肌组织中MG53蛋白的表达水平,应用RT-PCR技术检测4组大鼠术后第8周时骨骼肌组织中IRS-1 mRNA和MG53 mRNA的表达水平。结果①糖尿病假手术组及糖尿病对照组大鼠的MG53 mRNA及其蛋白的表达水平均高于正常对照组和糖尿病手术组(P<0.05),但糖尿病手术组和正常对照组间比较、糖尿病假手术组和糖尿病对照组间比较差异均无统计学意义(P>0.05)。②与正常对照组比较,糖尿病手术组、糖尿病假手术组及糖尿病对照组大鼠的IRS-1 mRNA的表达水平均较低(P<0.05),但糖尿病手术组、糖尿病假手术组及糖尿病对照组大鼠的IRS-1 mRNA的表达水平比较差异无统计学意义(P>0.05)。结论胃转流术降低了非肥胖型T2DM大鼠骨骼肌组织中IRS-1蛋白的E3泛素连接酶—MG53蛋白的表达水平,减少了IRS-1蛋白的泛素化降解,这可能是骨骼肌胰岛素信号通路中IRS-1蛋白表达上调的主要机制之一,从而改善了骨骼肌的胰岛素抵抗,降低了血糖。     

PLA2R、 THSD7A与IgG亚型在特发性膜性肾病中的诊断价值

目的探讨特发性膜性肾病(idiopathic membranous nephropathy,IMN)患者血清抗磷脂酶A2受体(phospholipase A2 receptor,PLA2R)抗体水平、肾小球PLA2R、1型血小板反应蛋白7A域(thrombospondin type I domain containing 7A,THSD7A)、免疫球蛋白G(immunoglobulin G,IgG)亚型的表达及其在IMN中的诊断价值。方法选取2016年1月至2019年6月在雅安市人民医院肾内科经肾活检并确诊的IMN患者72例,以同期72例非IMN肾小球疾病患者为对照。采用酶联免疫吸附法检测血清抗PLA2R抗体滴度,免疫荧光法检测肾小球PLA2R、THSD7A及IgG亚型表达。采用受试者工作特征(ROC)曲线分析血清抗PLA2R抗体、肾小球PLA2R、THSD7A、IgG4诊断IMN的价值。结果血清抗PLA2R抗体、肾小球PLA2R、IgG4、THSD7A诊断IMN的灵敏度分别为61.11%、80.56%、97.22%、8.33%,特异度分别为97.22%、100.00%、97.22%、100.00%。血清抗PLA2R抗体和肾小球PLA2R任一指标阳性即诊断IMN的敏感性为83.33%;肾小球PLA2R、THSD7A和IgG4中任一指标阳性即诊断IMN的敏感性为97.22%。结论血清抗PLA2R抗体、肾小球PLA2R、THSD7A及IgG4亚型对于IMN诊断具有较高的临床价值。血清抗PLA2R抗体及肾小球PLA2R抗原的联合检测,肾小球PLA2R、THSD7A与IgG4的联合检测可以增加IMN诊断的敏感性。     

NIMA相关蛋白激酶7、Nod样受体蛋白3与多囊卵巢综合征炎症指标相关性研究

目的探讨卵巢颗粒细胞中NIMA相关蛋白激酶7(NEK7)和Nod样受体蛋白3(NLRP3) mRNA表达量与多囊卵巢综合征(PCOS)患者炎症指标的相关性及其预测价值。方法选取2018年8月至2019年3月来山西省儿童医院/山西省妇幼保健院生殖中心就诊的30例PCOS患者为研究组和同期由于男方因素或输卵管因素就诊的34例非PCOS不孕症患者为对照组。取卵日收集行IVF/ICSI-ET患者的卵泡液和颗粒细胞,检测卵泡液中炎症因子白介素(IL)-1β、IL-18的表达以及颗粒细胞中NEK7、NLRP3 mRNA的表达,并将NEK7、NLRP3的mRNA表达量与各指标进行相关性分析。最后应用受试者工作特征曲线(ROC)分析NEK7、NLRP3 mRNA对PCOS的预测价值。结果研究组体重指数(BMI)和获卵数显著高于对照组,且LH、LH/FSH、睾酮(T)水平亦显著高于对照组(P均<0.05)。研究组卵泡液中炎症因子IL-1β、IL-18的表达显著高于对照组,卵巢颗粒细胞中NEK7、NLRP3 mRNA的表达亦显著高于对照组(P均<0.05)。相关性分析结果显示,研究组患者卵巢颗...     

PDGFR-α在胃肠道间质瘤中的表达及其临床意义

目的:通过检测胃肠道间质瘤中血小板衍生生长因子受体-α(PDGFR-α)的表达情况,探讨PDGFR-α在胃肠道间质瘤中的诊断及预后意义。方法:应用免疫组织化学EnvisionTM二步法检测了119例胃肠道间质瘤中的PDGFR-α蛋白的表达情况,并与非胃肠道间质瘤进行对照研究。结果:在胃肠道间质瘤中PDGFR-α的阳性表达率为65.5%(78/119),其中,极低危险性、低度危险性、中度危险性和高度危险性的阳性率分别为52.9%(9/17)、71.0%(22/31)、67.9%(19/28)和65.1%(28/43),但各组之间PDGFR-α的表达水平差异无统计学意义(P>0.05)。胃肠道间质瘤和非胃肠道间质瘤PDGFR-α的表达水平差异有统计学意义(P<0.05)。结论:PDGFR-α联合CD117应用于胃肠道间质瘤的检测,特别是对一些CD117表达阴性的胃肠道间质瘤诊断及鉴别诊断具有重要的临床意义,但其不能作为胃肠道间质瘤分化程度的指标。     

应用Dil标记的氧化高密度脂蛋白观察人单核细胞源性的树突状细胞成熟的研究

目的探讨氧化修饰高密度脂蛋白(OxHDL)和高密度脂蛋白(HDL)对人单核细胞源的树突状细胞(DCs)免疫功能的影响。方法从人外周血清中离心提取高密度脂蛋白,经氧化后,分别用Dil荧光标记。将人外周血分离的单核细胞加入包含rhGM-CSF(20ng/mL)和rhIL-4(20ng/mL)的培养基中培养,使其分化为DCs。以PBS作阴性对照,分别与50μg/mL的标记好的OxHDL和HDL混合培养48h后,动态观察DCs成熟过程中的形态学变化,流式细胞术检测DCs成熟表型(CD1#、CD80、CD86、HLA-DR)。结果使用Dil标记的方法良好地显示了DCs摄取OxHDL的形态学变化,OxHDL可促进DCs成熟表型HLA-DR、CD1$的表达(P<0.05),而HDL无此作用。结论Dil是适合DCs形态学研究的良好的荧光标记物,OxHDL可促进DCs成熟。     

小鼠脊髓发育候选基因Wnt7b的初步研究

目的:探讨Wnt7b基因及Wnt信号系统分子对小鼠脊髓发育的作用。方法:基因测序比较A/J和C57BL/6J两种小鼠Wnt7b基因开放阅读框架序列差异;以RT-PCR方法测定Wnt7b基因在两种小鼠组织中的表达差异;免疫组化法测定Frizzled在2种孕14天胎鼠脊髓上的表达和分布。结果:测序结果中发现两种小鼠Wnt7b开放阅读框架序列结构具有差异;在2种小鼠8种组织中,脊髓、肌肉、脾和肾中的Wnt7b基因表达具有明显差异;免疫组化的测定表明,发育14天的胎鼠脊髓上的Frizzled表达强度和分布范围具有显著差异,尤其是A/J小鼠在神经管周围表达明显强于C57BL/6J。结论:基因表达差异和基因结构差异提示,Wnt7b基因与脊髓发育具有一定的关联,应为一脊髓发育候选基因;Frizzled的强表达与脊髓发育的抑制相关,Wnt7b的信号转导途径介导了控制脊髓发育的过程。     

Effects of curcumin on peroxisome proliferator-activated receptor γ expression and nuclear translocation/redistribution in culture-activated rat hepatic stellate cells

Background The function of peroxisome proliferator-activated receptor γ （PPARγ） in hepatic fibrogenesis remains largely unknown. Curcumin is a natural substance extracted form Curcuma Longa Linn and has a variety of pharmacological effects. In this study, the effects of curcumin on the proliferation, activation and apoptosis of rat hepatic stellate cells （HSCs） through PPARγ signaling were investigated. Methods HSCs were isolated from the normal Sprague Dawley rats through in situ peffusion of the liver with Pronase E and density-gradient centrifugation with Nycodenz. Cells were treated with curcumin, troglitazone, salvianolic acid B or GW9662. The effect on HSCs proliferation was determined by MTT colorimetry. Total RNA was extracted by TRizol reagent and gene levels were determined by semi-quantitative RT-PCR. Total cellular and nuclear protein were isolated and separated by 10% sodium dodecy Isulfate polyacrylamide gel electrophoresis. Protein levels were determined by Western blot. Cell apoptosis was detected by Hoechst 33258 staining. PPARγ subcellular distribution was detected by immunofluorescent staining. The activities of MMP-2 and 9 were measured by Gelatin zymograph assay. Results Curcumin suppressed HSCs proliferation in a dose-dependent manner. As HSCs underwent gradual activation with culture prolongation the PPARγ nuclear expression level decreased. Curcumin up-regulated PPARγ expression and significantly inhibited the production of a-SMA and collagen Ⅰ. PPARγ is expressed in the cytoplasm and nucleus and is evenly distributed in HSCs, but accumulated in the nucleus of HSCs and disappeared from cytoplasm after curcumin treatment. Hoechst 33258 staining showed that curcumin induced the apoptosis of culture-activated HSCs and significantly increased pro-apoptotic Bax expression and reduced anti-apoptotic Bcl-2 expression. Cyclin D1 gene, activated NFKB p65 protein and TGFβR-Ⅰ protein expression were down-regulated significantly by curcumin. The activities of MMP-2 and MMP-9 were enhanced significantly by curcumin. Conclusions Curcumin can inhibit the proliferation and activation of HSCs, induce the apoptosis of activated HSCs and enhance the activities of MMP-2 and MMP-9. The effects of curcumin are mediated through activating the PPARγ sianal transduction pathway and associated with PPARγ nuclear translocation/redistribution.     

Glycine receptors contribute to cytoprotection of glycine in myocardial cells

Background The classic glycine receptor （GlyR） in the central nervous system is a ligand-gated membrane-spanning ion channel. Recent studies have provided evidence for the existence of GlyR in endothelial cells, renal proximal tubular cells and most leukocytes. In contrast, no evidence for GlyR in myocardial cells has been found so far. Our recent researches have showed that glycine could protect myocardial cells from the damage induced by lipopolysaccharide （LPS）. Further studies suggest that myocardial cells could contain GlyR or binding site of glycine. Methods In isolated rat heart damaged by LPS, the myocardial monophasic action potential （MAP）, the heart rate （HR） the myocardial tension and the activities of lactate dehydrogenase （LDH） from the coronary effluent were determined. The concentration of intracellular free calcium （[Ca^2＋]i） was measured in cardiomyocytes injured by LPS and by hypoxia/reoxygenation （H/R）, which excludes the possibility that reduced calcium influx because of LPS neutralized by glycine. Immunohistochemistry was used to detect the GlyR in myocardial tissue. GlyR and its subunit in the purified cultured cardiomyocytes were identified by Western blotting. Results Although significant improvement in the MAP/MAPD20, HR, and reduction in LDH release were observed in glycine ＋ LPS hearts, myocardial tension did not recover. Further studies demonstrated that glycine could prevent rat mycordial cells from LPS and hypoxia/reoxygenation injury （no endotoxin） by attenuating calcium influx. Immunohistochemistry exhibited a positive green-fluorescence signaling along the cardiac muscle fibers. Western blotting shows that the purified cultured cardiomyocytes express GlyR β subunit, but GlyR α1 subunit could not be detected. Conclusions The results suggest that glycine receptor is expressed in cardiomyocytes and participates in cytoprotection from LPS and hypoxia/reoxygenation injury. Glycine could directly activate GlyR on the cardiomyocytes and prevent calcium influx into the cardiomyocytes.     

肺癌外周血中EGFR mRNA、SP-D mRNA、LUNX mRNA表达的临床意义

目的:应用逆转录聚合酶链反应(RT-PCR)技术检测肺癌患者肿瘤组织和外周血中EGFRmRNA、SP-DmRNA、LUNXmRNA的表达,探讨其作为肺癌微转移检测分子标志物的可行性。方法:应用RT-PCR技术检测40例非小细胞肺癌组织和15例肺良性病变组织中EGFRmRNA、SP-DmRNA、LUNXmRNA的表达;检测40例肺癌患者外周血中靶基因的表达,并以15例良性肺疾病患者和10名健康人外周血作为对照。结果:40例肺癌患者病理组织中EGFRmRNA、SP-DmRNA、LUNXmRNA的表达率为77.5%(31/40)、100%(40/40)、100%(40/40),外周血中EGFRmRNA、SP-DmRNA、LUNXmRNA表达阳性率分别为55.0%(22/40)、52.5%(21/40)和57.5%(23/40);对照组中15例肺良性病变患者病理组织中EGFRmRNA表达率为13.3%(2/15),无SP-DmRNA和LUNXmRNA表达,15例肺良性病变患者和10名健康人的外周血中无EGFRmRNA、SP-DmRNA、LUNXmRNA表达。结论:EGFRmRNA、SP-DmRNA、LUNXmRNA作为检测肺癌组织及肺癌外周血微转移的分子标志物具有良好的敏感度和特异性;三者表达与肺癌TNM分期、组织学类型和癌细胞分化程度关系密切。