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目的探讨凋亡和炎症在人组织型激肽释放酶基因(hum an tissue kallire in,HK)肾脏保护中的作用和机制。方法健康雄性Wistar大鼠32只,随机分为假手术组(sham)、手术组(operation),手术+绿色荧光蛋白(GFP)尾静脉注射组(GFP组)以及手术+人组织型激肽释放酶基因组(HK组)。尾静脉注射病毒后3周,采取5/6肾切除术构建动物模型。术后第12周处死动物,肾组织石蜡切片行HE染色和M asson染色检测肾脏结构和胶原沉积。Tunnel染色和Caspase-3活性测定以检测凋亡细胞。Western blot技术检测肾组织Bcl-2、Bc l-xl、Bax、VCAM-1和MCP-1表达水平。结果 HE染色显示HK过表达明显抑制了5/6肾切除大鼠肾小管扩张和组织节段性硬化。Masson染色表明HK明显抑制了5/6肾切除大鼠肾脏胶原沉积(P<0.05)。Tunnel染色和Caspase-3活性测定结果显示HK显著减少肾脏细胞的凋亡(P<0.05)。W estern b lot显示HK明显降低了促凋亡蛋白Bax和炎症相关蛋白MCP-1和VCAM-1的表达,上调了凋亡抑制蛋白Bc l-2和Bc l-xl的表达(P<0.05)。结论 HK基因的肾脏保护作用至少部分是通过抑制细胞凋亡和炎症。机制至少包括下调促凋亡蛋白Bax和上调凋亡抑制蛋白Bcl-2和Bcl-xl的表达,抑制炎症相关蛋白MCP-1和VCAM-1的表达。
Objective To investigate the role and mechanism of apoptosis and inflammation in renal protection of human tissue kallikrein (HK). Methods Thirty-two male Wistar rats were randomly divided into sham operation group, operation group and GFP tail vein injection group (GFP group) and operation + human tissue-type kinin release Enzyme genome (HK group). Three weeks after the tail vein injection of the virus, a 5/6 nephrectomy was used to construct the animal model. Animals were sacrificed 12 weeks after operation, and paraffin sections of kidney tissue were examined by HE staining and M asson staining for renal structure and collagen deposition. Tunnel staining and Caspase-3 activity assay to detect apoptotic cells. Western blot was used to detect the expression of Bcl-2, Bcl-xl, Bax, VCAM-1 and MCP-1 in renal tissue. Results HE staining showed that overexpression of HK significantly inhibited tubular dilatation and segmental sclerosis in 5/6 nephrectomized rats. Masson staining showed that HK significantly inhibited renal collagen deposition in 5/6 nephrectomized rats (P <0.05). TUNEL staining and Caspase-3 activity assay showed that HK significantly reduced the apoptosis of renal cells (P <0.05). Western Blot showed that HK significantly decreased the expression of pro-apoptotic protein Bax and inflammatory proteins MCP-1 and VCAM-1, up-regulated the expressions of Bcl-2 and Bcl-xl (P <0.05) . Conclusions The renal protective effect of HK gene is at least partly through inhibiting apoptosis and inflammation. Mechanisms include at least downregulation of pro-apoptotic protein Bax and up-regulation of the expression of the apoptosis-inhibitory proteins Bcl-2 and Bcl-xl, and suppression of the expression of inflammatory-related proteins MCP-1 and VCAM-1.