上皮细胞—树突状细胞转分化在失血性休克诱发SIRS中的作用及维生素C的保护机制研究

发布时间：2017-12-28 22:33

本文关键词：上皮细胞—树突状细胞转分化在失血性休克诱发SIRS中的作用及维生素C的保护机制研究　出处：《上海交通大学》2015年博士论文　论文类型：学位论文

【摘要】：目的:探讨上皮细胞-树突状细胞转分化(EDT)在失血性休克诱发全身炎症反应综合征(SIRS)中的作用及可能机制;并进一步研究维生素C是否通过抑制上皮细胞-树突状细胞转分化而抑制失血性休克导致的大鼠多器官损伤。方法:以缺氧培养干预体外培养的大鼠肠粘膜上皮IEC-6细胞,部分给予维生素C干预(20-100uM)。在缺氧培养2、6、24、48小时后提取蛋白质、mRNA及培养上清液。观察树突状细胞特异性细胞间粘附分子非整合素蛋白3(DC-SIGN)、E-钙粘蛋白(E-cadherin)、糖原合成酶激酶-3(GS K-3β)表达情况,及上清液白介素-1β(IL-1β)、白介素-6(IL-6)含量变化;从表型及分泌功能上探讨缺氧对小肠上皮细胞-树突状细胞转分化的影响,及VitC对这一过程所起的作用(n=3);以siRNA干扰IEC-6的E-cadherin表达,比较正常IEC-6和经过siR NA干扰的IEC-6在缺氧培养及Vit C干预后DC-SIGN表达情况的不同;以TDZD-8抑制GSK-3β活性,比较正常IEC-6和经过TDZD-8干预的IEC-6在缺氧培养及VitC干预后,DC-SIGN、E-cadherin表达情况的不同。共同探讨缺氧诱导上皮细胞-树突状细胞转分化的可能机制(n=3)。通过股动脉放血建立大鼠失血性休克模型,部分大鼠在复苏时给予维生素c干预(100mg/kg)。于复苏后2、6、24小时处死大鼠取标本,检测小肠、肾脏组织内dc-sign表达情况及定位,组织病理损伤,血浆和组织内促炎细胞因子含量(肿瘤坏死因子-a、白介素-6),及血清生化指标(尿素氮、肌酐、乳酸脱氢酶及乳酸)(n=6)。探讨失血性休克状态下的上皮细胞-树突状细胞转分化现象与组织炎症反应、脏器病理及功能损害的关系,及vitc干预所起的作用;结果:缺氧培养2小时后iec-6细胞表达dc-sign显著升高,同时e-cadherin表达下降,gsk-3β活性下降(p0.05)。维生素c以剂量依赖的方式抑制缺氧诱导的iec-6细胞dc-sign表达升高,并缓解缺氧导致的e-cadherin表达下降及gsk-3β活性下降(p0.05)。预先使用sirna干扰e-cadherin表达后,维生素c抑制缺氧诱导iec-6细胞dc-sign表达升高的作用被扭转(p0.05)。预先使用特异性gsk-3β活性抑制剂tdzd-8抑制gsk-3β活性后,维生素c抑制缺氧诱导iec-6细胞e-cadherin表达下降的作用被扭转(p0.05)。失血性休克诱导小肠上皮细胞、肾小管上皮细胞表达dc-sign,维生素c(100mg/kg)显著抑制dc-sign的表达(p0.05)。维生素c干预显著抑制小肠、肾脏病理损伤,降低组织及血浆促炎细胞因子水平,改善血清生化指标(p0.05)结论:缺氧培养及失血性休克可以诱导上皮细胞-树突状细胞转分化;维生素c对这一现象有抑制作用,并可通过抑制上皮细胞-树突状细胞转分化有效减轻失血性休克早期多器官炎症反应。
[Abstract]:Objective: To investigate the epithelial cells transdifferentiation of dendritic cells (EDT) in hemorrhagic shock induced systemic inflammatory response syndrome (SIRS) in the role and possible mechanism; and further study whether vitamin C through inhibition of epithelial cells and dendritic cell differentiation and inhibition of hemorrhagic shock induced multiple organ injury of rats. Methods: the rat intestinal mucosal epithelial IEC-6 cells were cultured in vitro, and the vitamin C intervention (20-100uM) was partially given. Protein, mRNA and culture supernatant were extracted after 2, 6, 24 and 48 hours in hypoxia. Observation of dendritic cell specific intercellular adhesion molecule 3 (DC-SIGN) non integrin protein, E- cadherin (E-cadherin) and glycogen synthase kinase -3 (GS K-3) expression, and supernatants of interleukin -1 beta (IL-1 beta) and interleukin -6 (IL-6) content change; from the table type and secretory function on effect hypoxia on the transdifferentiation of intestinal epithelial cells, dendritic cells, and the VitC of the process effect (n=3); siRNA interference IEC-6 expression of E-cadherin, IEC-6 and siR compared to normal after NA interference of IEC-6 intervention DC-SIGN in hypoxic culture and Vit expression of C is different; inhibition of GSK-3 beta activity in TDZD-8 compared with normal IEC-6 and TDZD-8, after the intervention of IEC-6 in hypoxia and the intervention of VitC, DC-SIGN and E-cadherin expression in different situations. To discuss the possible mechanism of hypoxia induced transdifferentiation of epithelial cells - dendritic cells (n=3). The rat model of hemorrhagic shock was established through the blood release of the femoral artery, and some rats were given vitamin C intervention (100mg/kg) during the resuscitation. In 2, after the recovery of 6, 24 hours, the rats were sacrificed, specimens, detection of the small intestine and kidney tissue DC-SIGN expression and localization, tissue damage, inflammatory cytokines in plasma and tissues (promoting tumor necrosis factor interleukin -a, -6), and serum biochemical indexes (blood urea nitrogen, creatinine, lactate dehydrogenase and lactic acid) (n=6). To investigate the relationship between hemorrhagic shock under the condition of epithelial cells and dendritic cell transdifferentiation and tissue inflammation, organ function and pathological damage, and the effect of VitC intervention; results: 2 hours after hypoxia in IEC-6 cells the expression of DC-SIGN was significantly increased, while expression of E-cadherin decreased, decreased GSK-3 activity (P0.05). Vitamin C inhibited the expression of DC-SIGN in hypoxia induced IEC-6 cells in a dose-dependent manner, alleviated the decrease of E-cadherin expression and decreased the activity of GSK-3 beta (P0.05). When siRNA interfered with E-cadherin expression in advance, the effect of vitamin C on the inhibition of hypoxia induced DC-SIGN expression in IEC-6 cells was reversed (P0.05). After the specific GSK-3 beta activity inhibitor tdzd-8 was used to inhibit GSK-3 beta activity, vitamin C inhibited the decrease of E-cadherin expression in IEC-6 cells induced by hypoxia, which was reversed (P0.05). The expression of DC-SIGN in small intestinal epithelial cells and renal tubular epithelial cells was induced by hemorrhagic shock, and vitamin C (100mg/kg) significantly inhibited the expression of DC-SIGN (P0.05). Vitamin C supplementation significantly inhibited intestine and kidney pathological damage, reduce tissue and plasma levels of proinflammatory cytokines, improve the serum biochemical indexes (P0.05) conclusion: hypoxia and hemorrhagic shock can induce epithelial cell dendritic cell transdifferentiation; vitamin C on this phenomenon are inhibited, and through inhibition of epithelial cells dendritic cell transdifferentiation can effectively reduce the blood loss of multiple organ inflammatory reaction at early stage of shock.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R459.7

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