新生小鼠NK细胞对Mφ细胞调控不足致胆道闭锁的机理研究

发布时间：2018-03-23 22:09

本文选题：NK细胞　切入点：RRV　出处：《华中科技大学》2014年博士论文

【摘要】：第一部分RRV感染不同年龄小鼠后,肝脏NK细胞活化的差异摘要目的：明确不同年龄小鼠感染了RRV后,小鼠肝脏NK细胞活化能力的差异；方法：不同年龄小鼠腹腔注射RRV24小时后,磁珠分选法提取肝脏NK细胞,通过流式细胞仪检测NK细胞活化后表达细胞因子CD69、TFN-a和IFN-y的能力,并进行统计学分析。结果：与未感染RRV的新生小鼠相比,感染RRV的新生小鼠肝脏NK细胞活化后表达细胞因子CD69、TFN-a和IFN-y的能力明显升高,P0.05,有统计学意义；同样,感染了轮状病毒的成年小鼠肝脏NK细胞活化后分泌细胞因子CD69、TFN-a和IFN--γ的能力也明显上调,P0.05,有统计学意义。但是,与感染了RRV的成年小鼠肝脏NK细胞相比,感染RRV的新生小鼠肝脏NK细胞活化后分泌细胞因子CD69、TFN-a和IFN-γ,的能力却明显不足,P0.05,有统计学意义。结论：本实验说明,RRV可诱导小鼠肝脏NK细胞活化后分泌炎性细胞因子上调,但新生小鼠肝脏NK细胞的活化能力明显低于成年小鼠。然而随着新生小鼠年龄的增长,新生小鼠肝脏NK细胞逐渐成熟,其活化后分泌炎症因子的能力也逐渐增强。第二部分新生小鼠Kupffer细胞分离培养及吞噬活性测定摘要目的建立一种新生小鼠肝脏库普弗(Kupffer)细胞分离、培养的简便方法,为BA发病机制的研究提供可靠的实验细胞。方法取6-8个新生小鼠肝脏置于200目研磨网上剪碎并研磨后,将研磨液种植于无菌细胞培养瓶A中。培养3-4d后将贴壁细胞再次悬浮并种植于另一无菌培养瓶B中,将培养瓶B置于含5%CO2细胞培养箱培养45min后1XPBS缓冲液洗细胞,获得二次贴壁细胞并行F4/80免疫荧光鉴定。二次贴壁细胞与2um荧光小球共培养1h后,1XPBS缓冲液清洗细胞,处理完后置于激光共聚焦显微镜下观察。结果巨噬细胞标志物F4/80免疫荧光法检测二次贴壁细胞,阳性率99%,活性99%,细胞获得1×107/L。二次贴壁细胞与2um荧光小球共培养,可见胞浆中吞噬有大量荧光小球。结论“二次贴壁法”选择性贴壁从新生小鼠肝脏获取Kuppfer细胞,取材容易,操作方便,不需特殊设备,可满足新生小鼠胆道闭锁发病机制研究的实验需求,实验价值高。第三部分依赖于IL-4、IFN-γ不同年龄小鼠肝脏NK对MΦ调控的差异摘要目的：以前的研究发现新生小鼠肝脏NK细胞的活化能力不足,而活化的NK细胞是细胞因子IFN-γ和IL-4的主要来源,希望通过明确MΦ细胞活化对细胞因子IFN-y和IL-4剂量依赖性,进而明确新生小鼠肝脏NK细胞对MΦ细胞的调控存有无异常。方法：细胞因子IFN-γ可辅助MΦ细胞活化并表达细胞因子iNOS和TNF-a,而IL-4可辅助MΦ细胞活化分泌细胞因子IL-10。MΦ细胞与不同剂量细胞因子IFN--γ共孵育12小时后,通过实时PCR(real time PCR,RT-PCR)和印迹实验(westblot,WB)两种检测方法分别检测细胞因子iNOS和TNF-a基因转录和蛋白表达与IFN-y剂量关系；通过酶联免疫吸附法(ELISA)及RT-PCR法检测MΦ细胞与不同剂量IL-4共孵育12小时后IL-I0基因转录和蛋白表达与IL-4剂量关系。MΦ细胞分别来源于新生小鼠肝脏和成年小鼠肝脏。结果：MΦ细胞与不同剂量IFN-γ共孵育12小时后,其胞内细胞因子iNOS和TNF-a的基因转录和蛋白表达量均随着IFN-y剂量的增加而呈递增趋势；同样,随着IL-4浓度的增加,MΦ细胞胞内IL-I0基因转录和蛋白表达量也逐渐递增,但当IL-4浓度达到100u/L时,IL-10基因转录和蛋白表达量达到峰值,随后逐渐降低。而且,无论新生小鼠肝脏MΦ细胞或成年小鼠肝脏MΦ细胞,它们均有类似表现。结论：MΦ细胞的活化程度对细胞因子IFN-γ和IL-4具有剂量依赖性,在一定范围内,随着细胞因子IFN-γ和IL-4剂量的增加,其分泌炎性细胞因子的能力逐渐增强。而活化的NK细胞是细胞因子IFN-γ和IL-4的主要来源,已明确新生小鼠肝脏NK细胞的活化能力不足,分泌IFN-γ和IL-4等细胞因子能力下降,新生小鼠肝脏NK细胞对MΦ细胞的调控因而存在异常。第四部分不同年龄小鼠肝脏NK细胞对吞噬RRV的MΦ细胞杀伤差异摘要目的：通过比较不同年龄小鼠肝脏NK细胞对吞噬了RRV的MΦ细胞的杀伤活性差异,从而进一步确定了新生小鼠肝脏NK对MΦ细胞调控能力不足。方法：通过磁珠分选法及差别贴壁法分别获取野生型B6成年小鼠和新生小鼠肝脏NK细胞与MΦ细胞。把来源于不同年龄段小鼠肝脏NK细胞及是否事先经过HMGB1活化过分为A、B、C、D四组,然后将不同组NK细胞分别与吞噬了RRV的不同年龄段小鼠肝脏MΦ细胞共培养4小时后,采用非放射性细胞毒试验检测不同组肝脏NK细胞对吞噬RRV后的肝脏MΦ杀伤活性。结果：新生小鼠肝脏NK细胞经细胞因子HMGB1活化后,其对吞噬RRV的不同年龄段小鼠肝脏MΦ细胞杀伤能力均明显增强,P0.05,有统计学意义；同样,经细胞因子HMGB1活化后的成年小鼠肝脏NK细胞,其对吞噬了RRV的不同年龄段小鼠肝脏MΦ细胞杀伤能力也明显增强,P0.05,有统计学意义。但与成年小鼠肝脏NK细胞相比,新生小鼠肝脏NK细胞对吞噬RRV的MΦ细胞杀伤能力明显明显不足,P0.05,有统计学意义。然而随着小鼠年龄的增加,NK细胞逐渐成熟,其杀伤活性可逐渐增强。结论：新生小鼠肝脏NK细胞对吞噬了RRV的MΦ细胞杀伤能力不足,不能有效清除吞噬RRV的MΦ细胞,导致RRV在体内持续存、繁殖并扩散至周围组织,从而造成胆管上皮细胞大量受损及慢性炎症的形成,继而形成BA。
[Abstract]:Part 1 difference in activation of liver NK cells after RRV infection in mice of different ages
abstract
Objective: to determine the difference of activation ability of NK cells in mice liver after RRV infection of different age mice.
Methods: after RRV24 hours of intraperitoneal injection, NK cells from liver were extracted by magnetic beads sorting. The expression of cytokines CD69, TFN-a and IFN-y after NK cell activation was detected by flow cytometry, and analyzed statistically.
Results: compared with the newborn mice without RRV infection, the expression of cytokines in CD69 infected RRV neonatal mouse liver NK cell activation, TFN-a and IFN-y increased significantly, P0.05, have statistical significance; likewise, the secretion of CD69 infected rotavirus adult mouse liver NK cell activation, TFN-a and IFN-- gamma was also up-regulated, P0.05, have statistical significance. However, compared with RRV infected adult mouse liver NK cells, cytokine secretion of CD69 RRV infection in newborn mouse liver after NK cell activation, TFN-a and IFN- gamma, the capacity is obviously insufficient, P0.05, have statistical significance.
Conclusion: This study shows that RRV can secrete inflammatory cytokines induced upregulation of NK in mouse liver cells after activation, but the activation ability of neonatal mouse liver NK cells was significantly lower than that of adult mice. However, along with the age growth of newborn mice, NK cells from neonatal mouse liver gradually mature, the ability of secretion of inflammatory cytokines after activation also increased gradually.
Isolation and culture of Kupffer cells and determination of phagocytic activity in the second part of newborn mice
abstract
Objective to establish a simple method for the isolation and culture of Kupfer (Kupffer) cells in the newborn mice liver, and to provide a reliable experimental cell for the study of the pathogenesis of BA.
Methods 6-8 newborn mouse liver in 200 meshabrasive online cutting and grinding, the grinding liquid grown in sterile cell culture bottle of A. After 3-4d culture, the adherent cells were resuspended in sterile culture and planted another bottle of B, B in the flask containing 5%CO2 cell culture box 1XPBS buffer wash after 45min cell culture, two adherent cells were identified by immunofluorescence. F4/80 parallel two adherent cells with 2um fluorescent beads after 1h co cultured cells were washed with 1XPBS buffer, after processing in laser scanning confocal microscopy.
Results the macrophage marker F4/80 immunofluorescence assay was used to detect two adherent cells. The positive rate was 99% and the activity 99%. The cells obtained 1 * 107/L. two times, and the adherent cells co cultured with 2um fluorescent beads.
Conclusion the "two adherence" selective attachment of Kuppfer cells from newborn mice liver is easy and easy to operate, without special equipment, which can meet the experimental needs of the pathogenesis of biliary atresia in neonatal mice, and the experimental value is high.
The third part depends on the difference in the regulation of NK in the liver of mice in different ages of IL-4, IFN- gamma and M
abstract
Objective: Previous studies have found that activation ability of neonatal mouse liver NK cells, activated NK cells are the main source of cytokines IFN- and IL-4, to depend on the cytokines IFN-y and IL-4 dose through clear cell activation with M, and then clear the regulation of liver NK cells of newborn mice with M cells have no abnormal.
Methods: the cytokines IFN- can assist with M cell activation and cytokine expression of iNOS and TNF-a, and IL-4 can activate the cytokine secretion of IL-10.M cells with different doses of phi IFN-- cytokine was incubated for 12 hours with auxiliary M cells by real-time PCR (real time, PCR, RT-PCR) and (westblot, Western blot WB) two kinds of detection methods were used to detect the cytokines iNOS and TNF-a gene transcription and protein expression of IFN-y and dose relationship; by enzyme-linked immunosorbent assay (ELISA) and RT-PCR method with M cell detection and different doses of IL-4 were incubated for 12 hours after IL-I0 gene transcription and protein expression of IL-4 and relationship between the dose of.M with cell respectively. From the liver of neonatal mouse liver and adult mice.
Results: M cells with different doses of gamma phi IFN- were incubated 12 hours after gene transcription and protein expression of cytokines iNOS and TNF-a in the cells were increased with the dose of IFN-y showed increasing trend; also, with the increase of IL-4 concentration, M with intracellular IL-I0 gene transcription and protein expression is gradually increasing, but when the concentration of IL-4 reached 100u/L, IL-10 gene transcription and protein expression reached the peak, then gradually decreased. Moreover, both liver cells of newborn mice with M or adult mouse liver cells with M, they have similar performance.
Conclusion: the degree of activation of M cells with a dose dependent on the cytokines IFN- and IL-4, in a certain range, with the increase of cytokines IFN- and IL-4 dose, the secretion of inflammatory cytokines increased gradually. And the activation of NK cells is the main source of cytokines IFN- and IL-4 have. Clear the activation ability of neonatal mouse liver NK cells, decreased IFN- secretion and IL-4 cell factor, regulation of NK cells in liver of newborn mice with M cells and abnormal.
Fourth parts of the liver NK cells in mice of different ages to kill the M phagocytosis of RRV
abstract
Objective: by comparing the killing activity difference of liver NK cells from different age mice to phagocytosis of RRV M cells, we further confirmed that the NK of liver of newborn mice was not enough to control M cells.
Methods: the acquisition of wild type B6 adult and neonatal mice liver NK cells and M cells by MACS with adherent method. The differences from different age groups of mice liver NK cells and whether in advance through activation of HMGB1 too much for the A, B, C, D four groups, then NK cells in different groups and the phagocytosis of mice of different ages with liver M RRV cells were cultured for 4 hours, using non radioactive cytotoxicity assay cytotoxicity of liver NK cells after phagocytosis of RRV of different groups of liver with M activity.
Results: the liver of newborn mice NK cells by cytokine activated HMGB1, the killing of different age groups of mice liver with M phagocytosis ability of RRV increased significantly and P0.05, there was statistical significance; similarly, the cytokine activated HMGB1 in adult mouse liver NK cells, which also significantly enhance the phagocytic of different age mouse liver M cell killing ability of P0.05 phi RRV, with statistical significance. But compared with NK cells in the livers of adult mice, NK cells of newborn mice liver on phagocytosis of RRV M cells with obvious ability is obviously insufficient, P0.05, have statistical significance. However, with the increase of age of mice, NK cells gradually mature, and the killing activity can be gradually increased.
Conclusion: NK cells of liver deficiency of newborn mice consumed RRV with M cell killing ability, can effectively remove the phagocytosis of RRV M cells with RRV in vivo, resulting in persistent, breed and spread to the surrounding tissue, resulting in the formation of a large number of damaged bile duct epithelial cells and chronic inflammation, and the formation of BA.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R722.1

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