目的研究SHARP-2（rat enhancer of split-and hairy-related protein-2）特异性基因沉默后对白细胞介素2（IL-2）和1干扰素（IFN-γ）基因表达及肾移植受者大鼠存活时间的影响。方法采用基因重组、转染、共转染等分子生物学方法将针对S...目的研究SHARP-2（rat enhancer of split-and hairy-related protein-2）特异性基因沉默后对白细胞介素2（IL-2）和1干扰素（IFN-γ）基因表达及肾移植受者大鼠存活时间的影响。方法采用基因重组、转染、共转染等分子生物学方法将针对SHARP-2的短发夹式RNA干扰序列导入到第3代自失活慢病毒载体Vira Power packaging mix，以构建重组慢病毒；有限稀释法确定病毒滴度；实时定量PCR法研究基因表达变化；原位大鼠肾移植模型研究SHARP-2基因沉默后受者存活时间变化。结果在正常大鼠肾脏细胞中，重组慢病毒LV-SHARP-2iC对SHARP-2的基因沉默效率达84％。在复感染指数（MOI）=20时，用spinoeulation法可使57％的T细胞受转染。在活化T细胞中，SHARP．2基因沉默后对IL-2和IFN-γ的基因表达抑制率分别达61．3％和68．7％。和空白对照以及随意序列对照组相比，5×10^7TUSHARP。2特异的RNA干扰重组慢病毒LV-SHARP-2iC灌注供肾后，肾移植受者大鼠的中位存活时间延长4～5d。移植肾局部组织分析发现SHARP-2基因沉默效果达61％，IL-2和IFN-γ的基因表达则分别下降了47．3％和58．2％。结论成功构建了以第3代自失活慢病毒为载体的、针对SHARP-2基因的短发夹式RNA干扰重组病毒LV—SHARP-2iC，该体系能使SHARP-2基因表达有效沉默，进而抑制活化T细胞中与排斥相关的IL-2和IFN-γ的基因表达，在肾移植模型中延长受者大鼠存活时间。展开更多
We aim to investigate the effect of transforming growth factor （TGF）-β1 on the expression of enhancer of split- and hairy-related protein-2 （SHARP-2） messenger RNA （mRNA） and its signaling pathway. In this stud...We aim to investigate the effect of transforming growth factor （TGF）-β1 on the expression of enhancer of split- and hairy-related protein-2 （SHARP-2） messenger RNA （mRNA） and its signaling pathway. In this study, several cell lines including LLC-PK1 （a porcine kidney tubular epithelial cell line）, MDCK （Madin-Darby canine kidney） and CTLL-2 （cytotoxic T-lymphocyte line） were treated with recombinant human TGF-131, and a series of experiments were carried out, involving Northern blot analysis of total RNA from these cells. Further, several specific chemical inhibitors were applied before TGF-β1 treatment to probe the signaling pathway. The results showed that TGF-β1 can significadtly up-regulate SHARP-2 mRNA expression in the LLC-PK1 cell line. The peak level of induction was found 2 h after TGF-β1 stimulation. While one phospho- inositide 3-kinases （PI-3） kinase inhibitor, LY294002, completely blocked the effect of TGF-131 on SHARP-2 mRNA expression in LLC-PK1 cells at a low concentration, other inhibitors, including PD98059, staurosporine, AG490, wortmannin, okadaic acid and rapamycin, had no effect. The effect of LY294002 was dose-dependent. We conclude that, in LLC-PK1 cells at least, TGF-β1 can effectively induce the SHARP-2 mRNA expression and that the PI-3 kinase pathway can mediate this effect.展开更多
针对光学显微镜景深扩展中的多聚焦图像融合问题,提出了一种基于方向特性的新轮廓波域多聚焦图像融合算法。该算法首先对图像进行新轮廓波变换(New Contourlet Transform with Sharp Frequency Localization,NCT-SFL),分解得到不同尺度...针对光学显微镜景深扩展中的多聚焦图像融合问题,提出了一种基于方向特性的新轮廓波域多聚焦图像融合算法。该算法首先对图像进行新轮廓波变换(New Contourlet Transform with Sharp Frequency Localization,NCT-SFL),分解得到不同尺度、不同方向的高低频系数,低频系数融合使用算术平均法,高频系数融合分为两步:先采用改进拉普拉斯能量和(Sum Modified Laplacian,SML)提取特征值;然后定义新的与方向分解一一对应的椭圆方向窗,在确定的椭圆窗参数下,对提取的特征值进行累加并以此为依据对高频系数进行融合,最后通过反新轮廓波变换得到融合图像。在实验部分用定义的新的客观评价指标互结构信息(Mutual Structural Information,MSI)对融合算法进行了评价,结果表明:对多聚焦图像本文所提方法比新轮廓波域方形窗算法MSI提高了2.94%,比Contourlet域方形窗与椭圆窗算法MSI分别提高了10.44%和8.56%。说明本文方法能提取源图像中更多的清晰信息到融合图像,是一种有效的景深扩展手段。展开更多
文摘目的研究SHARP-2（rat enhancer of split-and hairy-related protein-2）特异性基因沉默后对白细胞介素2（IL-2）和1干扰素（IFN-γ）基因表达及肾移植受者大鼠存活时间的影响。方法采用基因重组、转染、共转染等分子生物学方法将针对SHARP-2的短发夹式RNA干扰序列导入到第3代自失活慢病毒载体Vira Power packaging mix，以构建重组慢病毒；有限稀释法确定病毒滴度；实时定量PCR法研究基因表达变化；原位大鼠肾移植模型研究SHARP-2基因沉默后受者存活时间变化。结果在正常大鼠肾脏细胞中，重组慢病毒LV-SHARP-2iC对SHARP-2的基因沉默效率达84％。在复感染指数（MOI）=20时，用spinoeulation法可使57％的T细胞受转染。在活化T细胞中，SHARP．2基因沉默后对IL-2和IFN-γ的基因表达抑制率分别达61．3％和68．7％。和空白对照以及随意序列对照组相比，5×10^7TUSHARP。2特异的RNA干扰重组慢病毒LV-SHARP-2iC灌注供肾后，肾移植受者大鼠的中位存活时间延长4～5d。移植肾局部组织分析发现SHARP-2基因沉默效果达61％，IL-2和IFN-γ的基因表达则分别下降了47．3％和58．2％。结论成功构建了以第3代自失活慢病毒为载体的、针对SHARP-2基因的短发夹式RNA干扰重组病毒LV—SHARP-2iC，该体系能使SHARP-2基因表达有效沉默，进而抑制活化T细胞中与排斥相关的IL-2和IFN-γ的基因表达，在肾移植模型中延长受者大鼠存活时间。
基金supported by the National Natural Science Foundation of China (Nos. 30471641 and 30872389)the Natural Science Foundation of Zhejiang Province, China (No. Y207088)
文摘We aim to investigate the effect of transforming growth factor （TGF）-β1 on the expression of enhancer of split- and hairy-related protein-2 （SHARP-2） messenger RNA （mRNA） and its signaling pathway. In this study, several cell lines including LLC-PK1 （a porcine kidney tubular epithelial cell line）, MDCK （Madin-Darby canine kidney） and CTLL-2 （cytotoxic T-lymphocyte line） were treated with recombinant human TGF-131, and a series of experiments were carried out, involving Northern blot analysis of total RNA from these cells. Further, several specific chemical inhibitors were applied before TGF-β1 treatment to probe the signaling pathway. The results showed that TGF-β1 can significadtly up-regulate SHARP-2 mRNA expression in the LLC-PK1 cell line. The peak level of induction was found 2 h after TGF-β1 stimulation. While one phospho- inositide 3-kinases （PI-3） kinase inhibitor, LY294002, completely blocked the effect of TGF-131 on SHARP-2 mRNA expression in LLC-PK1 cells at a low concentration, other inhibitors, including PD98059, staurosporine, AG490, wortmannin, okadaic acid and rapamycin, had no effect. The effect of LY294002 was dose-dependent. We conclude that, in LLC-PK1 cells at least, TGF-β1 can effectively induce the SHARP-2 mRNA expression and that the PI-3 kinase pathway can mediate this effect.
