肺炎衣原体通过c-jun氨基末端激酶信号通路上调胆固醇酰基转移酶1的表达

目的 观察c-jan氨基末端激酶(JNK)信号通路在肺炎衣原体(C.pn)调控人单核细胞株(THP-1)源性巨噬细胞酰基辅酶A:胆固醇酰基转移酶1(ACAT1)表达中的作用,初步探讨C.pn诱导泡沫细胞形成的机制和途径. 方法 THP-1单核细胞诱导分化为巨噬细胞后随机分为4组:对照组、C.pn感染组、SP600125(JNK特异性抑制剂)+C.pn组、SP600125组.分别用反转录聚合酶链反应(RT-PCR)法和Western blot法检测各组细胞ACAT1 mRNA和蛋白表达,油红O染色和酶荧光化学法检测泡沫细胞的形成. 结果 C.pn组ACAT1 mRNA(4.16±0.26)及蛋白(1.20±0.10)表达明显高于对照组(分别为2.17±0.18和0.61±0.03,均P<0.05),且可观察到C.pn诱导的泡沫细胞形成.而SP600125能呈浓度依赖性地抑制C.pn诱导的ACAT1 mRNA及蛋白表达上调(r值分别为-0.92和-0.96,均P<0.05)及泡沫细胞形成. 结论 C.pn通过JNK信号通路上调巨噬细胞ACAT1的表达,导致细胞内胆固醇酯蓄积,从而诱导巨噬细胞源性泡沫细胞形成.     

肺炎衣原体通过上调酰基辅酶A:胆固醇酰基转移酶1诱导人单核细胞株THP-1源性泡沫细胞形成的实验研究

目的 观察酰基辅酶A:胆固醇酰基转移酶1(ACAT1)在肺炎衣原体(C.pn)诱导泡沫细胞形成中的作用,初步探讨C.pn诱导泡沫细胞形成的机制.方法 人单核细胞株(THP-1)给予160 mmol/L佛波酯(PMA)孵育48 h,诱导分化为巨噬细胞后随机分为4组:阴性对照组、阳性对照组、C.pn感染组和ACAT抑制剂58-035+C.pn感染组.分别运用RT-PCR和Western blot检测各组ACAT1 mRNA和蛋白表达.运用油红O染色观察细胞浆内脂滴的变化,用酶荧光学法检测细胞内胆固醇酯含量的变化.结果 C.pn感染呈浓度和时间依赖性地上调ACAT1 mRNA和蛋白表达.高浓度的C.pn(5×105和1×106IFU)感染THP-1源性巨噬细胞48 h后,细胞浆内脂滴明显增多,胆固醇酯与总胆固醇的比值明显增加(＞50%).ACAT抑制剂58-035可以抑制C.pn诱导的细胞浆内脂滴的增多.随着58-035浓度的增加,逐渐抑制C.pn诱导的细胞内胆固醇酯含量的增加.结论 ACAT1表达上调是C.pn诱导泡沫细胞形成的机制之一,这可能为进一步阐明C.pn感染促进动脉粥样硬化发生发展提供一个新的理论依据.     

IL-10对TNF-α诱导的泡沫细胞三磷酸腺苷结合盒转运体A1表达的抑制作用

目的:观察白介素-10(IL-10)对肿瘤坏死因子-α(TNF-α)诱导的THP-1巨噬细胞源性泡沫细胞三磷酸腺苷结合盒转运体A1(ABCA1)表达的干预作用.方法:THP-1单核细胞经诱导转化为巨噬细胞源性泡沫细胞后随机分为4组:对照组,培养液中不加任何其他物质;TNF-α组,培养液中加10μg/L TNF-α;IL-10组,培养液中加10μg/L IL-10;IL-10 TNF-α组,培养液中加10μg/L IL-10预先孵育2 h后,再加10μg/L TNF-α.采用RT-PCR和Western-Blot检测各组0 h、6 h、12 h、24 h、48 h ABCA1 mRNA和蛋白表达.结果:TNF-α组ABCA1 mRNA和蛋白表达呈时间依赖性降低(F=782.94,F=768.12,P均＜0.05).IL-10组ABCA1 mRNA表达在24 h和48 h有轻微增加(P＜0.05),但ABCA1蛋白表达在各时间点无明显变化.IL-10 TNF-α组ABCA1 mRNA和蛋白表达也呈时间依赖性降低(F=697.23,F=749.64,P均＜0.05),但同TNF-α组相比,其下降程度有所减轻(P＜0.05).结论:IL-10可部分抑制TNF-α所引起THP-1巨噬细胞源性泡沫细胞ABCA1表达的下调.     

血清半乳糖凝集素-3用于2型糖尿病相关肾病的诊断价值研究

目的 探讨血清半乳糖凝集素-3（galectin-3）用于2型糖尿病（type 2 diabetes mellitus,T2DM）患者发生糖尿病相关肾病（diabetic kidney disease,DKD）的诊断价值。方法 回顾性纳入2009年4月～2015年5月在笔者医院内分泌科及肾内科住院的T2DM患者130 例,根据尿白蛋白及接受透析情况分为尿白蛋白正常至中度增加（A₁期）组（n=49）、中度增加（A₂期）组（n=36）、重度增加（A₃期）组（n=28）及透析组（n=17）。就诊时纪录患者一般情况及常规实验室检测指标。检测患者血清galectin-3及胱抑素C（Csy-C）水平,分析galectin-3与临床指标的相关性。使用ROC曲线分析galectin-3单独及联合Csy-C用于诊断DKD肾功能损害的价值。结果 透析组血清galectin-3水平（87.47±26.65ng/ml）最高,随后依次为A₃期、A₂期、A₁期及健康对照组;eGFR≤15ml/（min·1.73m²）或透析患者galectin-3水平也显著高于其他eGFR情况的患者。所有患者中,galectin-3与尿白蛋白肌酐比（ACR）（r=0.760,P=0.000）及Csy-C（r=0.723,P=0.000）呈正相关,与eGFR（r=-0.768,P=0.000）呈负相关。Galectin-3单独诊断肾功能损害的AUC为0.908（95% CI：0.859～0.958）,联合Csy-C的AUC为0.920（95% CI：0.873～0.967）。结论 T2DM患者血清galectin-3水平升高提示更严重的白蛋白尿及肾功损害,galectin-3单用或联合Csy-C具有良好的对肾功能损害的诊断价值。     

肺炎衣原体通过上调酰基辅酶A:胆固醇酰基转移酶1诱导人单核细胞株THP-1源性泡沫细胞形成的实验研究

Objective To investigate the expression changes of acyl-coenzyme A: cholesterol acyhransferase 1 (ACAT1) on Chlamydia pneumoniae (C. pn) induced foam cell formation. Methods Human monocytic cell line (THP-1) was induced into macrophages by 160 nmol/L phorbol myristate acetate (PMA)for 48 h, and were randomly allocated into four groups: negative control group (50 μg/ml LDL for 48 h) ; positive control group (50 μg/ml ox-LDL for48 h) ; C. pn infection group (50 μg/ml LDL plus 1× 105,4×105,5×105and 1×106 IFU C.pn for 48 h or 1×106 IFU C.pn for 0,24,48 and 72h); ACAT inhibitor 58-035 plus C. pn infection group (1, 5, 10 μg/ml ACAT inhibitor 58-035 pretreatment for 1 h,50 μg/ml LDL and 1 × 106 IFU C. pn for 48 h). The mRNA and protein expressions of ACATI were determined by RT-PCR and Western blot, respectively. Lipid droplets in cytoplasm were observed by oil red 0 staining. The contents of intracellular cholesteryl esters were detected by enzyme-fluorescence. Results The mRNA and protein expressions of ACATI were significantly up-regulated in positive control cells compared those in negative control cells and further upregulated by C. pn infection in a time-dependent and concentration-dependent manner (all P ＜ 0.05). There were significantly increases in the accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol in positive control cells as compared with negative control cells and these were further aggravated by C. pn (at the concentrations of 5× 105 and 1×106IFU for 48 h) and C. pn infection induced increases in the accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol could he significantly attenuated by ACAT inhibitor 58-035 (all P ＜ 0.05). Conclusion Chlamydia pneumoniae induces THP-1-derived foam cell formation by up-regulating the expression of ACATI.     

RNAi靶向沉默预处理星形胶质细胞胶质细胞源性神经营养因子对神经元凋亡的影响

研究表明外源性的胶质细胞源性神经营养因子可对脑缺血损伤时的神经元有保护作用,但关于内源性的胶质细胞源性神经营养因子的神经元保护作用目前机制不清。鉴于此,实验以正常培养的星形胶质细胞培养基,胶质细胞源性神经营养因子高表达星形胶质细胞培养基和采用RNAi技术沉默胶质细胞源性神经营养因子表达的星形胶质细胞培养基,作用于缺血神经元,观察不同条件培养基对神经元凋亡的影响。结果验证RNAi靶向沉默预处理星形胶质细胞胶质细胞源性神经营养因子的表达可促进神经元凋亡,氧糖剥夺预处理可上调星形胶质细胞的胶质细胞源性神经营养因子的表达,能明显降低神经元的凋亡。     

脑缺血损伤ActA与跨膜受体ActRⅡA在Smads转导通路中的表达

目的 探讨脑缺血损伤ActA/Smads通路主要位点基因表达在信号转导中的作用及其机制.方法 建立PC12细胞神经元氧糖剥夺(OGD)体外脑缺血模型,应用Real-time PCR技术,检测OGD3h、6h、9h、12h、16h、24h ActA及其受体ActRⅡA和下游Smad3 mRNA表达变化.结果 OGD3h PC12细胞ActβA、ActRⅡA及Smad3 mRNA表达均显著升高且达高峰;OGD6h ActβA mRNA表达有所降低并呈逐渐下降趋势;OGD6h ActRⅡA表达开始下降,但其下降趋势弱于ActβA,在OGD9h仍高于正常对照组,OGD24h达最低点;OGD6hSmad3 mRNA表达也开始下降,但其下降趋势弱于ActRⅡA,在OGD16h仍高于正常对照组,OGD24h达最低点(组间比较P＜0.05).结论 ActA/Smads通路的活性随OGD时间呈动态变化,短时程OGD可能通过诱导神经元跨膜受体ActRⅡA的上调激活该信号转导通路.     

西洛他唑对慢性脑缺血大鼠学习记忆能力的影响

目的观察慢性脑缺血致大鼠学习记忆功能及西洛他唑对其的影响。方法结扎大鼠双侧颈总动脉（2VO）建立慢性脑缺血模型。雄性Wistar大鼠随机分为假手术组、缺血组和西洛他唑组,每组又分为3、6、9周三个时间点;应用Morris水迷宫评定不同时期大鼠认知功能,通过HE染色观察神经细胞形态学改变。结果Morris水迷宫表明,各时间点西洛他唑组和假手术组大鼠游迷宫平均潜伏期均明显低于缺血组（P〈0.05）;HE染色显示假手术组和西洛他唑组皮层、海马CA1区神经元缺血性改变较缺血组改变轻,6、9周较3周给药组缺血改变明显。结论西洛他唑能改善慢性脑缺血大鼠的学习记忆能力,可能通过减轻神经元损伤作用改善慢性脑缺血大鼠学习记忆障碍。     

肺炎衣原体通过上调酰基辅酶A:胆固醇酰基转移酶1诱导人单核细胞株THP-1源性泡沫细胞形成的实验研究

Objective To investigate the expression changes of acyl-coenzyme A: cholesterol acyhransferase 1 (ACAT1) on Chlamydia pneumoniae (C. pn) induced foam cell formation. Methods Human monocytic cell line (THP-1) was induced into macrophages by 160 nmol/L phorbol myristate acetate (PMA)for 48 h, and were randomly allocated into four groups: negative control group (50 μg/ml LDL for 48 h) ; positive control group (50 μg/ml ox-LDL for48 h) ; C. pn infection group (50 μg/ml LDL plus 1× 105,4×105,5×105and 1×106 IFU C.pn for 48 h or 1×106 IFU C.pn for 0,24,48 and 72h); ACAT inhibitor 58-035 plus C. pn infection group (1, 5, 10 μg/ml ACAT inhibitor 58-035 pretreatment for 1 h,50 μg/ml LDL and 1 × 106 IFU C. pn for 48 h). The mRNA and protein expressions of ACATI were determined by RT-PCR and Western blot, respectively. Lipid droplets in cytoplasm were observed by oil red 0 staining. The contents of intracellular cholesteryl esters were detected by enzyme-fluorescence. Results The mRNA and protein expressions of ACATI were significantly up-regulated in positive control cells compared those in negative control cells and further upregulated by C. pn infection in a time-dependent and concentration-dependent manner (all P ＜ 0.05). There were significantly increases in the accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol in positive control cells as compared with negative control cells and these were further aggravated by C. pn (at the concentrations of 5× 105 and 1×106IFU for 48 h) and C. pn infection induced increases in the accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol could he significantly attenuated by ACAT inhibitor 58-035 (all P ＜ 0.05). Conclusion Chlamydia pneumoniae induces THP-1-derived foam cell formation by up-regulating the expression of ACATI.