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目的 通过重组2型腺相关病毒(rAAV2)和基因转导,探讨转化生长因子(TGF)β 3对人鼠肝纤维化的影响. 方法构建并验证rAAV2-TGF 3.实验人鼠随机分为正常对照组、模型组、阴性对照组和TGF β 3干预组.40%的四氯化碳复制大鼠肝纤维化模型.在四氯化碳诱导前l周,将rAAV2 TGF β 3 注入大鼠尾静脉,造模8周后处死人鼠,取肝脏组织做HE染色观察肝脏组织学改变,Masson染色观察胶原纤维分布,免疫组织化学染色观察Ⅰ型胶原的表达,对胶原纤维的阳性面积比和Ⅰ型胶原的平均吸光度值进行半定量分析.多组数据问比较采用单因素的方差分析,若各组总体方差齐同,采用SNK-q检验,若方差不齐,采用Durmett'S T3检验. 结果 HE染色结果显示,rAAV2-TGF β3干预后,肝组织纤维间隔增生减少;Masson染色结果显示,人鼠胶原纤维主要分布在血管和汇管区及狄氏间隙,TGF β 3干预组大鼠肝内的胶原纤维阳性面积比为7.7%±1.5%,明显低于模型组的13.2%±2.2%和阴性对照组的12.3%±1.5%(q值分别为9.456和8.217,P值均<0.01).免疫组织化学染色结果显示,Ⅰ型胶原主要表达在血管和汇管区及狄氏间隙,TGF β 3干预组大鼠肝内的Ⅰ型胶原的表达为0.185±0.033,明显低于模型对照组的0.252±0.042和阴性对照组的0.230±0.029(口值分别为6.228和4.346,P值均<0.01).结论 TGF β 3可明显减轻实验人鼠的肝纤维化程度和组织学损伤,降低Ⅰ型胶原的表达. 相似文献
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Objective To investgate the effects of TGF β 3 on rat hepatic fibrosis. Methods The TGF β 3 cDNA was cloned into rAAV2 vector. Rats were randomly divided into four groups: normal control group, model group, negative control group and TGF β 3 group. Hepatic fibrosis was induced by hypodermic injection of 40% CCI4. Recombinant AAV2-TGF β 3 viral particles were injected via the vena caudalis one week before CCh treatment. Rats were executed 8 weeks after CCI4 treatment, global histological change was observed after HE staining, the distribution of the collagen fibers was observed after masson staining, his-tochemistry was done to observe the expression of collagen Ⅰ; The positive area rate of the collagen fibers and the average optical rate of collagen Ⅰ were quantified. Results HE staining indicated that collagen fibers were reduced in the TGF β 3 group. Masson staining shown that the collagen fibers were distributed around the blood vessel, in the portal area and disse space. Compared to the model group (13.2%±2.2%) and negative control group (12.3%±1.5%), the collagen fibers in liver tissues of TGF β3 group (7.7% ± 1.5%) were significantly decreased (q = 9.456, P < 0.01; q = 8.217, P < 0.01). Histochemistry indicated that the collagen fibers of liver tissues of TGF 15 3 group (0.185±0.033) were significantly higher than those in the model group (0.252±0.042) and the negative control group (0.230±0.029), (q = 6.228, P < 0.01; q = 4.346, P < 0.01). Conclusion TGF β 3 alleviates the damage to hepatic cell and the level offibrosis in CCI4 treated rats and inhibits the expression of collagen Ⅰ. 相似文献
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重组转化生长因子β3基因对大鼠肝纤维化细胞模型胶原合成及沉积的影响 总被引:10,自引:0,他引:10
目的 观察重组转化生长因子β3(TGF-β3)基因对大鼠肝纤维化细胞模型胶原合成及沉积的影响.方法 (1)构建质粒pcDNA3.1(+)-TGF-β3和pcDNA3.1(+)-TGF-β1.(2)将pcDNA3.1(+)-TGF-β1转染大鼠肝星状细胞(HSC)-T6,经G418筛选建立稳定高表达TGF-β1的HSC-T6细胞阳性克隆.(3)pcDNA3.1(+)-TGF-B3转染HSC-T6阳性细胞克隆,荧光实时定量PCR法检测TGF-β3mRNA的表达;荧光实时定量PCR法及Western印迹法分别检测TGF-β3、Ⅰ型胶原、基质金属蛋白酶(MMP)-2、MMP-9及TIMP-1的mRNA和蛋白表达情况.结果 (1)pcDNA3.1(+)-TGF-β3和pcDNA3.1(+)-TGF-β3质粒均可转染HSC-T6细胞,以转染48 h时转染效率最高,达28.2%.(2)pcDNA3.1(+)-TGF-β3转染HSC-T6细胞阳性克隆后,与单纯阳性克隆组相比,TGF-β1mRNA表达无明显改变,而蛋白表达明显低(0.48±0.07 vs 0.66±0.14,P=0.023);Ⅰ型胶原mRNA及蛋白的表达明显低(mRNA:7.2±1.0 vs 13.7±0.4,P=0.010,蛋白:0.45±0.21 vs 0.82±0.13,P=0.041),MMP-2 mRNA及蛋白较低,但差异无统计学意义(均P>0.05),MMP-9 mRNA及蛋白表达明显多(均P<0.05),TIMP-1 mRNA及蛋白表达明显低(均P<0.05).结论 (1)重组TGF-β3真核表达载体构建正确,在转染阳性克隆细胞48 h后获得高效表达;(2)稳定高表达TGF-B1的HSC-T6细胞阳性克隆可作为肝纤维化细胞模型;(3)pcDNA3.1(+)-TGF-β3转染阳性克隆能减少胶原合成,并通过调节基质金属蛋白酶及其抑制剂的表达对胶原沉积起抑制作用. 相似文献
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大鼠肝星状细胞TGF-β3/TGF-β1mRNA比值与TGF-β1、MMP-9、TIMP-1表达的关系 总被引:1,自引:0,他引:1
目的 探讨大鼠肝星状细胞(HSC-T6)中转化生长因子-β3(TGF-β3)和转化生长因子-β1(TGF-β1)mRNA比值变化与TGF-β1、MMP-9、TIMP-1表达的关系.方法 构建质粒pcDNA 3.1(+)-TGF-β3和pcDNA 3.1(+)-TGF-β1.将pcDNA 3.1(+)-TGF-1β1转染HSC-T6细胞株,经筛选建立高表达TGF-β1的HSC-T6细胞阳性克隆.pcDNA 3.1(+)-TGF-β3转染该阳性克隆,48 h后荧光定量PCR法和Western blot法分别检测TGF-β3、TGF-β1、MMP-9和TIMP-1 mRNA和蛋白的变化.结果 空白组、对照组、阳性克隆组及TGF-β3干预组中,TGF-β3/TGF-β1mRNA比值分别为0.286±0.070、0.874±0.141、0.448±0.327和1.277±0.244;阳性克隆组与空白组和对照组相比,TGF-β1和TIMP-1的mRNA及蛋白表达明显增高(P<0.05),MMP-9的mRNA及蛋白表达明显减少(P<0.05);TGF-β3干预组与阳性克隆组相比,TGF-β1蛋白和TIMP-1 mRNA及蛋白表达明显下降(P<0.05),MMP-9 mRNA及蛋白表达明显增加(P<0.05).结论 TGF-β3能下调TGF-β1蛋白表达;当TGF-β3/TGF-β1mRNA比值>1时,TGF-β1和TIMP-1表达减少,MMP-9表达增加;当TGF-β3/TGF-β1mRNA比值<1时,TGF-β1表达减少,TIMP-1和MMP-9表达无变化. 相似文献
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目的探讨重组转化生长因子-β3(TGF-β3)基因对大鼠肝星状细胞(HSC)内、外蛋白合成及分泌的影响。方法构建质粒pcDNA3.1(+)-TGF-β3和pcDNA3.1(+)-TGF-β1。稳定转染:通过脂质体介导的方法,将pcDNA3.1(+)-TGF-β1转染HSC-T6,经G418筛选建立稳定高表达pcDNA3.1(+)-TGF-β1的HSC-T6细胞阳性克隆。再将pcDNA3.1(+)-TGF-β3转染HSC-T6克隆细胞,用W estern b lot法和酶联免疫吸附法(ELISA)分别检测细胞内外TGF-β1、Ⅰ型胶原、基质金属蛋白酶(MMP)-2、MMP-9及金属蛋白酶组织抑制剂(TIMP)-1的蛋白表达量。结果pcDNA3.1(+)-TGF-β3转染HSC-T6阳性细胞克隆后,与单纯阳性克隆组相比,TGF-β1、Ⅰ型胶原、TIMP-1细胞内蛋白表达及培养上清中的分泌水平均明显降低(P〈0.05),MMP-2的表达也降低,但两者间差异无统计学意义(P〉0.05),而MMP-9的表达明显增高(P〈0.05)。结论重组质粒pcDNA3.1(+)-TGF-β3能减少细胞内外Ⅰ型胶原的合成及分泌;通过调节MMP-9及TIMP-1的表达,可抑制细胞外基质的合成,促进其降解。 相似文献
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Objective To investgate the effects of TGF β 3 on rat hepatic fibrosis. Methods The TGF β 3 cDNA was cloned into rAAV2 vector. Rats were randomly divided into four groups: normal control group, model group, negative control group and TGF β 3 group. Hepatic fibrosis was induced by hypodermic injection of 40% CCI4. Recombinant AAV2-TGF β 3 viral particles were injected via the vena caudalis one week before CCh treatment. Rats were executed 8 weeks after CCI4 treatment, global histological change was observed after HE staining, the distribution of the collagen fibers was observed after masson staining, his-tochemistry was done to observe the expression of collagen Ⅰ; The positive area rate of the collagen fibers and the average optical rate of collagen Ⅰ were quantified. Results HE staining indicated that collagen fibers were reduced in the TGF β 3 group. Masson staining shown that the collagen fibers were distributed around the blood vessel, in the portal area and disse space. Compared to the model group (13.2%±2.2%) and negative control group (12.3%±1.5%), the collagen fibers in liver tissues of TGF β3 group (7.7% ± 1.5%) were significantly decreased (q = 9.456, P < 0.01; q = 8.217, P < 0.01). Histochemistry indicated that the collagen fibers of liver tissues of TGF 15 3 group (0.185±0.033) were significantly higher than those in the model group (0.252±0.042) and the negative control group (0.230±0.029), (q = 6.228, P < 0.01; q = 4.346, P < 0.01). Conclusion TGF β 3 alleviates the damage to hepatic cell and the level offibrosis in CCI4 treated rats and inhibits the expression of collagen Ⅰ. 相似文献
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建立了一种快速检测阴道毛滴虫抗体的斑点酶联免疫吸附试验.用国产试剂及材料制备出诊断膜,直接浸入阴道分泌物中反应,在3h内不需任何仪器,凭肉眼就能判读结果.用该法检测了55例经湿片法检查原虫阳性者标本,其抗体阳性率为92.7%(51/55).提示斑点酶联免疫吸附试验是一种灵敏度高、重复性好、特异性强并易于推广的快速诊断阴道毛滴虫抗体的方法. 相似文献