肝脏自然杀伤细胞在轮状病毒致小鼠胆道闭锁中的作用研究
发布时间：2018-11-25 21:39
【摘要】：目的：了解肝脏自然杀伤细胞（natural killer cell，NK）在轮状病毒（rhesusrotavirus，RRV）致小鼠胆道闭锁（biliary atresia，BA）中的作用。 方法：实验动物C57BL/6（B6）；TLR2基因敲除鼠[TLR2(-/-)]；C57BL/10（B10）；TLR4基因敲除鼠[TLR4(-/-)]。以上四种小鼠均购买自南京大学模式动物研究所。其中B6和TLR2(-/-)背景相同，B10和TLR4(-/-)背景相同。 1.动物实验：选择生后1d、7d及成年（6-8w）三个时间的四种小鼠，腹腔注射RRV后24h，用磁珠分选技术分选肝脏NK细胞，，利用流式细胞术检测肝NK细胞膜表面分子CD69表达水平，及NK细胞内肿瘤坏死因子-α（TNF-α）和干扰素-γ（IFN-γ）的表达。分别进行基因敲除小鼠和野生型小鼠的比较和生后1d、7d、成年小鼠的比较。 2.细胞实验：用非放射性细胞杀伤试剂盒检测四种小鼠肝脏NK细胞对小鼠肝外胆管上皮细胞的杀伤活性。 结果：1.腹腔注射RRV后，野生型小鼠NK细胞活化表达水平比同龄的TLR敲除小鼠明显较高； 2.腹腔注射RRV后新生小鼠NK细胞活化较低； 3.野生型小鼠肝脏NK细胞比同龄的TLR基因敲除小鼠杀伤肝外胆管上皮细胞的百分率高； 4.HMGB1活化后小鼠肝NK细胞对小鼠肝外胆管上皮细胞的杀伤百分率较正常组高；小鼠肝NK细胞对感染RRV后小鼠肝外胆管上皮细胞的杀伤百分率较正常组高。 结论：1.新生鼠肝脏NK细胞的功能不足，清除感染RRV的胆管上皮细胞能力低下； 2.HMGB1可以和NK细胞的表面TLR2和TLR4结合，并且激活NK细胞。活化的NK对胆管上皮细胞有较强杀伤作用；RRV感染后的胆管上皮细胞容易被NK细胞杀伤。
[Abstract]:Aim: to investigate the role of hepatic natural killer cells (natural killer cell,NK) in rotavirus (rhesusrotavirus,RRV) -induced biliary atresia (biliary atresia,BA) in mice. Methods: C57BL/6 (B6); TLR2 knockout mice [TLR2 (- / -)]; C57BL/10 (B10); TLR4 gene knockout mice [TLR4 (- / -)]. The above four kinds of mice were purchased from the Institute of Model Animals, Nanjing University. Where B 6 and TLR2 (- / -) have the same background, and B 10 and TLR4 (- / -) have the same background. 1. Animal experiment: four kinds of mice were selected at 1 day after birth and 3 hours after birth (6-8 weeks). 24 hours after intraperitoneal injection of RRV, liver NK cells were sorted by magnetic bead sorting technique. Flow cytometry was used to detect the expression level of CD69 on the surface of liver NK cell membrane. And the expression of tumor necrosis factor- 伪 (TNF- 伪) and interferon- 纬 (IFN- 纬) in NK cells. The gene knockout mice and wild-type mice were compared respectively, and 1 day after birth and 7 days after birth, adult mice were compared. 2. Cell experiment: the cytotoxicity of four kinds of murine liver NK cells to mouse extrahepatic bile duct epithelial cells was detected by non-radioactive cell killing kit. Results: 1. After intraperitoneal injection of RRV, the activation and expression level of NK cells in wild type mice was significantly higher than that in TLR knockout mice of the same age; 2. The activation of NK cells in neonatal mice was lower after intraperitoneal injection of RRV. The rate of killing extrahepatic bile duct epithelial cells by NK cells of wild type mice was higher than that of TLR gene knockout mice of the same age, and the killing rate of NK cells on extrahepatic bile duct epithelial cells of mice after 4.HMGB1 activation was higher than that of normal mice. The killing rate of mouse liver NK cells on extrahepatic bile duct epithelial cells after RRV infection was higher than that of normal group. Conclusion: 1. The function of NK cells in the liver of neonatal rats was insufficient, and the ability of clearing bile duct epithelial cells infected with RRV was low, and 2.HMGB1 could bind to TLR2 and TLR4 on the surface of NK cells and activate NK cells. Activated NK has strong killing effect on bile duct epithelial cells, and bile duct epithelial cells infected with RRV are easy to be killed by NK cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.7
本文编号：2357452
[Abstract]:Aim: to investigate the role of hepatic natural killer cells (natural killer cell,NK) in rotavirus (rhesusrotavirus,RRV) -induced biliary atresia (biliary atresia,BA) in mice. Methods: C57BL/6 (B6); TLR2 knockout mice [TLR2 (- / -)]; C57BL/10 (B10); TLR4 gene knockout mice [TLR4 (- / -)]. The above four kinds of mice were purchased from the Institute of Model Animals, Nanjing University. Where B 6 and TLR2 (- / -) have the same background, and B 10 and TLR4 (- / -) have the same background. 1. Animal experiment: four kinds of mice were selected at 1 day after birth and 3 hours after birth (6-8 weeks). 24 hours after intraperitoneal injection of RRV, liver NK cells were sorted by magnetic bead sorting technique. Flow cytometry was used to detect the expression level of CD69 on the surface of liver NK cell membrane. And the expression of tumor necrosis factor- 伪 (TNF- 伪) and interferon- 纬 (IFN- 纬) in NK cells. The gene knockout mice and wild-type mice were compared respectively, and 1 day after birth and 7 days after birth, adult mice were compared. 2. Cell experiment: the cytotoxicity of four kinds of murine liver NK cells to mouse extrahepatic bile duct epithelial cells was detected by non-radioactive cell killing kit. Results: 1. After intraperitoneal injection of RRV, the activation and expression level of NK cells in wild type mice was significantly higher than that in TLR knockout mice of the same age; 2. The activation of NK cells in neonatal mice was lower after intraperitoneal injection of RRV. The rate of killing extrahepatic bile duct epithelial cells by NK cells of wild type mice was higher than that of TLR gene knockout mice of the same age, and the killing rate of NK cells on extrahepatic bile duct epithelial cells of mice after 4.HMGB1 activation was higher than that of normal mice. The killing rate of mouse liver NK cells on extrahepatic bile duct epithelial cells after RRV infection was higher than that of normal group. Conclusion: 1. The function of NK cells in the liver of neonatal rats was insufficient, and the ability of clearing bile duct epithelial cells infected with RRV was low, and 2.HMGB1 could bind to TLR2 and TLR4 on the surface of NK cells and activate NK cells. Activated NK has strong killing effect on bile duct epithelial cells, and bile duct epithelial cells infected with RRV are easy to be killed by NK cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.7
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