Full-length cDNA gene of sodium-dependent dicarboxylate co-transporter protein 1 (SDCT1) is cloned from normal human kidney tissue and inserted into EGFP (enhanced green fluorescent protein) expression vector along with N-terminal and C-terminal truncated SDCT1 genes, so to construct the eukaryotic expression vectors of EGFP/SDCT1 fusion proteins, which are transfected into human renal tubular epithelial cells (HKC). Subcellular localizations of these fusion proteins are observed by laser confocal microscope to determine the localization signal of the SDCT1 protein. Duplex PCR analysis validates that the fusion protein genes have been in- tegrated into the genome of HKC. Western blot indicates that the fusion proteins have been ex- pressed in HKC. Confocal microscopy analysis shows that human SDCT1 predominantly locates on the plasma membrane, which is consistent with the results predicted by bioinformatics ap- proach; in HKC transfected with N-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed on the plasma membrane; in HKC transfected with C-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed in the cytoplasm. EGFP/SDCT1 mRNAs obtained by in vitro transcription are microinjected into Xenopus laevis oocytes for ex- pression and the trans-membrane currents are measured by using two-microelectrode volt- age-clamp technique. Na+ inward currents are present on cellular membrane of the injected oo- cytes. Immunohistochemical staining shows that human SDCT1 proteins are expressed on lu- men membrane of the renal proximal tubule, but are negative in distal tubule, collecting duct, renal interstitium and glomerulus. The above-mentioned studies suggest that human SDCT1 protein is located on the lumen membrane of the renal proximal tubule, the C-terminal sequence of the SDCT1 is required for delivery and targeting localization, and the plasma membrane lo- calization signal of the SDCT1 protein maybe locate in the C-terminal sequence.
BAI XueyuanCHEN XiangmeiFEN ZheWU DiHOU KaiCHENG GenyangPENG Lixia
目的探讨人基质金属蛋白酶组织抑制物-1(tissue inhibitor of metalloproteinase-1,TIMP-1)对肾脏器官衰老过程血管生成的影响。方法对3、12、24月龄人TIMP-1转基因小鼠和野生型小鼠肾组织石蜡切片行PASM染色,观察肾组织内微血管密度变化;Western免疫印迹检测TIMP-1、TIMP-2、基质金属蛋白酶(matrix metalloproteinase,MMP)-2、MMP-9、血管内皮细胞生长因子(VEGF)、Ⅲ型和Ⅳ型胶原蛋白质表达;明胶酶谱和反向酶谱分别检测明胶酶和TIMP-1的活性。结果24月龄时与野生型小鼠相比,转基因小鼠肾小球和肾小管周围的微血管密度降低(P<0.05);TIMP-1、Ⅲ型和Ⅳ型胶原蛋白质表达增多(P<0.05),而MMP-2、MMP-9和VEGF的表达降低(P<0.05);明胶酶谱和反向酶谱结果显示,明胶酶的活性下调(P<0.05),而TIMP-1的活性则上调(P<0.05)。结论TIMP-1可能通过影响血管生成而促进肾脏器官衰老。
组蛋白去乙酰化酶2（silent information regulator2．Sir2）是Sirtuins家族中烟酰胺腺嘌呤二核苷酸（NAD）依赖的去乙酰化酶之一。Sirtuins家族是一种从细菌到复杂的真核生物都高度保守的蛋白质家族．此家族成员都含有一个约由250个氨基酸组成的中心区域，成员之间大约有25％～60％的序列同源性。
