Aim: To compare serum level of matrix metalloproteinase 3 (MMP3) and tissue inhibitor metallo-proteinase 1 (TIMP1) in vascular dementia patients and healthy control subjects. Methods: A case control study was carried ...Aim: To compare serum level of matrix metalloproteinase 3 (MMP3) and tissue inhibitor metallo-proteinase 1 (TIMP1) in vascular dementia patients and healthy control subjects. Methods: A case control study was carried out in Ain Shams University hospital, Cairo, Egypt. 32 cases with vascular dementia were collected and classified into 2 subgroups;vascular dementia of multiinfarct type (VDMI) 14 patients, and vascular dementia of subcortical type (VDSC) 18 subjects. 23 cases with normal cognitive functions were collected as control group. Cases were subjected to comprehensive geriatric assessment, neurological examination, neuropsychological testing and brain CT scan. Blood sample was collected to analyze serum level of matrix metalloproteinase 3 (MMP3) and tissue inhibitor metalloproteinase 1 (TIMP1). Results: Mean serum level of TIMP1 (20.85 × 103 picogram/ml) was significantly lower than mean serum level of TIMP1 in control group (27.69 × 103 picogram/ml) (p = 0.018). The same finding was also evident when comparing VDMI subgroup mean serum TIMP1 (18.71 × 103 pc/ml) to control group (p = 0.025). There was no significant difference between mean serum MMP3 levels in cases group (mean = 67.39 × 103) as compared to control group (mean = 61.65 × 103 pc/ml) (p = 0.519). Conclusion: Patients with VD particularly VDMI has lower serum level of TIMP1 as compared to control group.展开更多
目的构建大鼠TIMP1 miRNA慢病毒RNAi载体,并观察其转染HSC-T6细胞后对TIMP1基因表达的影响。方法根据BLOCK-iT Poll miR RNAi Expression System with EmGFP要求,应用在线miRNA设计工具http://rnaidesigner.invitrogen.com/rnaiexpress/...目的构建大鼠TIMP1 miRNA慢病毒RNAi载体,并观察其转染HSC-T6细胞后对TIMP1基因表达的影响。方法根据BLOCK-iT Poll miR RNAi Expression System with EmGFP要求,应用在线miRNA设计工具http://rnaidesigner.invitrogen.com/rnaiexpress/,针对大鼠TIMP1基因(GenBank:U06179)的598位点设计、合成Oligo DNA,退火形成双链DNA后,与载体pcDNATM6.2-GW/EmGFP-miR连接,转化Top10感受态细胞,构建TIMP1 miRNA慢病毒RNAi载体。经PCR扩增及测序鉴定正确后,以脂质体LipofectimineTM2000介导转染经TGF-β1刺激的大鼠肝星状细胞株HSC-T6,24、48、72h后收集细胞,应用RT-PCR鉴定TIMP1的表达情况。结果经PCR及测序鉴定构建的TIMP1 miRNA慢病毒RNAi载体正确;将其转染经TGF-β1刺激的大鼠肝星状细胞株HSC-T6,48 h即可见TIMP1 mRNA表达量下降,72 h检测不到TIMP1 mRNA的表达,而未转染组及转染pLacZ-miR/GFP(阴性对照)组未见此改变。结论成功构建大鼠TIMP1 miR-NA慢病毒RNAi载体pTIMP1-miR/GFP;pTIMP1-miR/GFP使大鼠肝星状细胞株HSC-T6 TIMP1基因沉默。提示了RNA干扰可使肝纤维化形成过程中的关键因子TIMP1基因沉默,为RNA干扰技术进一步应用治疗大鼠肝纤维化模型奠定了基础。展开更多
目的:观察肝爽颗粒对HSC-T6细胞Ⅰ型胶原(CollagenⅠ,ColⅠ)、Ⅲ型胶原(CollagenⅢ,ColⅢ)、基质金属蛋白酶组织抑制因子1(Tissue Inhibitor of Metalloproteinase1,TIMP1)基因及蛋白表达的影响。方法:用浓度0.025、0.05、0.1、0.15、0....目的:观察肝爽颗粒对HSC-T6细胞Ⅰ型胶原(CollagenⅠ,ColⅠ)、Ⅲ型胶原(CollagenⅢ,ColⅢ)、基质金属蛋白酶组织抑制因子1(Tissue Inhibitor of Metalloproteinase1,TIMP1)基因及蛋白表达的影响。方法:用浓度0.025、0.05、0.1、0.15、0.2、0.35、0.5、1.0、1.5、2.0、4.0、8.0、16.0mg/ml肝爽颗粒作用于HSC-T6细胞48小时,采用MTT比色法观察其对HSC-T6细胞生长的影响;以0.05、0.1、0.2mg/ml肝爽颗粒作用于HSC-T6细胞48小时,采用逆转录PCR与ELISA方法分别测定其对HSC-T6细胞ColⅠ、ColⅢ、TIMP1基因及蛋白表达的影响。结果:空白对照组ColⅠ、ColⅢ、TIMP1基因表达水平分别为0.91±0.11、1.54±0.09、1.83±0.13辉度值,蛋白表达水平分别为(187.63±4.11)、(7.59±1.04)、(23.85±2.13)ng/ml,以0.05、0.1、0.2mg/ml肝爽颗粒作用于HSC-T6细胞48小时,浓度0.05mg/ml肝爽颗粒仅能降低ColⅠ蛋白的表达;当浓度升为0.10mg/ml时不仅可显著降低ColⅠ蛋白的表达,而且对ColⅢmRNA与蛋白的表达亦产生明显抑制作用;当浓度达到0.20mg/ml时作用更明显,可显著降低ColⅠ、ColⅢmRNA,ColⅠ、ColⅢ、TIMP1蛋白的表达。结论:肝爽颗粒能明显抑制HSC-T6细胞ColⅠ、ColⅢ基因表达,抑制ColⅠ、ColⅢ、TIMP1蛋白表达,从而可抑制Ⅰ、Ⅲ型胶原的合成,减弱TIMP1对基质金属蛋白酶的抑制作用,这可能是其抗纤维化的作用机制之一。展开更多
Small hairpin RNA （shRNA） was used to silence the HIF1α gene in human retinal pigment epithelial cells （RPE） under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloprote...Small hairpin RNA （shRNA） was used to silence the HIF1α gene in human retinal pigment epithelial cells （RPE） under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloproteinase tissue inhibitor 1 （TIMP1）. By using chemical hypoxic inducer CoCl2 to mimic RPE hypoxic environment, shRNA against the targeting region of HIF1α mRNA sequence was synthesized by a method of in vitro transcription, and the HIF1α was interfered in RPE cultured under hypoxia （induced by 150 μmol/L CoCl2 ）. RT-PCR was employed to detect the expression of HIF1α and TIMP1. The expression levels of HIF1α and TIMP1 were measured by using Western blotting. The results showed that after the RPE were transfected with specific shRNA against HIF1α mRNA, RT PCR revealed that under hypoxia, the efficacy of HIF1α gene silencing in RPE was 83.4 %. Western blotting revealed that the expression levels of HIF1α protein was dramatically dropped. In addition, RT-PCR results demonstrated that the expression of TIMP1 mRNA was decreased by 28.9 %, and the expression levels of TIMP1 protein were also significantly reduced by Western blotting. It was suggested that shRNA targeted against HIF1α mRNA could effectively silence the HIF1α gene, subsequently effectively inhibit the hypoxia-induced up-regulation of TIMP1.展开更多
文摘Aim: To compare serum level of matrix metalloproteinase 3 (MMP3) and tissue inhibitor metallo-proteinase 1 (TIMP1) in vascular dementia patients and healthy control subjects. Methods: A case control study was carried out in Ain Shams University hospital, Cairo, Egypt. 32 cases with vascular dementia were collected and classified into 2 subgroups;vascular dementia of multiinfarct type (VDMI) 14 patients, and vascular dementia of subcortical type (VDSC) 18 subjects. 23 cases with normal cognitive functions were collected as control group. Cases were subjected to comprehensive geriatric assessment, neurological examination, neuropsychological testing and brain CT scan. Blood sample was collected to analyze serum level of matrix metalloproteinase 3 (MMP3) and tissue inhibitor metalloproteinase 1 (TIMP1). Results: Mean serum level of TIMP1 (20.85 × 103 picogram/ml) was significantly lower than mean serum level of TIMP1 in control group (27.69 × 103 picogram/ml) (p = 0.018). The same finding was also evident when comparing VDMI subgroup mean serum TIMP1 (18.71 × 103 pc/ml) to control group (p = 0.025). There was no significant difference between mean serum MMP3 levels in cases group (mean = 67.39 × 103) as compared to control group (mean = 61.65 × 103 pc/ml) (p = 0.519). Conclusion: Patients with VD particularly VDMI has lower serum level of TIMP1 as compared to control group.
文摘目的:观察肝爽颗粒对HSC-T6细胞Ⅰ型胶原(CollagenⅠ,ColⅠ)、Ⅲ型胶原(CollagenⅢ,ColⅢ)、基质金属蛋白酶组织抑制因子1(Tissue Inhibitor of Metalloproteinase1,TIMP1)基因及蛋白表达的影响。方法:用浓度0.025、0.05、0.1、0.15、0.2、0.35、0.5、1.0、1.5、2.0、4.0、8.0、16.0mg/ml肝爽颗粒作用于HSC-T6细胞48小时,采用MTT比色法观察其对HSC-T6细胞生长的影响;以0.05、0.1、0.2mg/ml肝爽颗粒作用于HSC-T6细胞48小时,采用逆转录PCR与ELISA方法分别测定其对HSC-T6细胞ColⅠ、ColⅢ、TIMP1基因及蛋白表达的影响。结果:空白对照组ColⅠ、ColⅢ、TIMP1基因表达水平分别为0.91±0.11、1.54±0.09、1.83±0.13辉度值,蛋白表达水平分别为(187.63±4.11)、(7.59±1.04)、(23.85±2.13)ng/ml,以0.05、0.1、0.2mg/ml肝爽颗粒作用于HSC-T6细胞48小时,浓度0.05mg/ml肝爽颗粒仅能降低ColⅠ蛋白的表达;当浓度升为0.10mg/ml时不仅可显著降低ColⅠ蛋白的表达,而且对ColⅢmRNA与蛋白的表达亦产生明显抑制作用;当浓度达到0.20mg/ml时作用更明显,可显著降低ColⅠ、ColⅢmRNA,ColⅠ、ColⅢ、TIMP1蛋白的表达。结论:肝爽颗粒能明显抑制HSC-T6细胞ColⅠ、ColⅢ基因表达,抑制ColⅠ、ColⅢ、TIMP1蛋白表达,从而可抑制Ⅰ、Ⅲ型胶原的合成,减弱TIMP1对基质金属蛋白酶的抑制作用,这可能是其抗纤维化的作用机制之一。
文摘Small hairpin RNA （shRNA） was used to silence the HIF1α gene in human retinal pigment epithelial cells （RPE） under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloproteinase tissue inhibitor 1 （TIMP1）. By using chemical hypoxic inducer CoCl2 to mimic RPE hypoxic environment, shRNA against the targeting region of HIF1α mRNA sequence was synthesized by a method of in vitro transcription, and the HIF1α was interfered in RPE cultured under hypoxia （induced by 150 μmol/L CoCl2 ）. RT-PCR was employed to detect the expression of HIF1α and TIMP1. The expression levels of HIF1α and TIMP1 were measured by using Western blotting. The results showed that after the RPE were transfected with specific shRNA against HIF1α mRNA, RT PCR revealed that under hypoxia, the efficacy of HIF1α gene silencing in RPE was 83.4 %. Western blotting revealed that the expression levels of HIF1α protein was dramatically dropped. In addition, RT-PCR results demonstrated that the expression of TIMP1 mRNA was decreased by 28.9 %, and the expression levels of TIMP1 protein were also significantly reduced by Western blotting. It was suggested that shRNA targeted against HIF1α mRNA could effectively silence the HIF1α gene, subsequently effectively inhibit the hypoxia-induced up-regulation of TIMP1.
