本文選題：NK細(xì)胞　切入點：RRV　出處：《華中科技大學(xué)》2014年博士論文

【摘要】：第一部分RRV感染不同年齡小鼠后,肝臟NK細(xì)胞活化的差異 摘要 目的：明確不同年齡小鼠感染了RRV后,小鼠肝臟NK細(xì)胞活化能力的差異； 方法：不同年齡小鼠腹腔注射RRV24小時后,磁珠分選法提取肝臟NK細(xì)胞,通過流式細(xì)胞儀檢測NK細(xì)胞活化后表達(dá)細(xì)胞因子CD69、TFN-a和IFN-y的能力,并進(jìn)行統(tǒng)計學(xué)分析。 結(jié)果：與未感染RRV的新生小鼠相比,感染RRV的新生小鼠肝臟NK細(xì)胞活化后表達(dá)細(xì)胞因子CD69、TFN-a和IFN-y的能力明顯升高,P0.05,有統(tǒng)計學(xué)意義；同樣,感染了輪狀病毒的成年小鼠肝臟NK細(xì)胞活化后分泌細(xì)胞因子CD69、TFN-a和IFN--γ的能力也明顯上調(diào),P0.05,有統(tǒng)計學(xué)意義。但是,與感染了RRV的成年小鼠肝臟NK細(xì)胞相比,感染RRV的新生小鼠肝臟NK細(xì)胞活化后分泌細(xì)胞因子CD69、TFN-a和IFN-γ,的能力卻明顯不足,P0.05,有統(tǒng)計學(xué)意義。 結(jié)論：本實驗說明,RRV可誘導(dǎo)小鼠肝臟NK細(xì)胞活化后分泌炎性細(xì)胞因子上調(diào),但新生小鼠肝臟NK細(xì)胞的活化能力明顯低于成年小鼠。然而隨著新生小鼠年齡的增長,新生小鼠肝臟NK細(xì)胞逐漸成熟,其活化后分泌炎癥因子的能力也逐漸增強。 第二部分新生小鼠Kupffer細(xì)胞分離培養(yǎng)及吞噬活性測定 摘要 目的建立一種新生小鼠肝臟庫普弗(Kupffer)細(xì)胞分離、培養(yǎng)的簡便方法,為BA發(fā)病機制的研究提供可靠的實驗細(xì)胞。 方法取6-8個新生小鼠肝臟置于200目研磨網(wǎng)上剪碎并研磨后,將研磨液種植于無菌細(xì)胞培養(yǎng)瓶A中。培養(yǎng)3-4d后將貼壁細(xì)胞再次懸浮并種植于另一無菌培養(yǎng)瓶B中,將培養(yǎng)瓶B置于含5%CO2細(xì)胞培養(yǎng)箱培養(yǎng)45min后1XPBS緩沖液洗細(xì)胞,獲得二次貼壁細(xì)胞并行F4/80免疫熒光鑒定。二次貼壁細(xì)胞與2um熒光小球共培養(yǎng)1h后,1XPBS緩沖液清洗細(xì)胞,處理完后置于激光共聚焦顯微鏡下觀察。 結(jié)果巨噬細(xì)胞標(biāo)志物F4/80免疫熒光法檢測二次貼壁細(xì)胞,陽性率99%,活性99%,細(xì)胞獲得1×107/L。二次貼壁細(xì)胞與2um熒光小球共培養(yǎng),可見胞漿中吞噬有大量熒光小球。 結(jié)論“二次貼壁法”選擇性貼壁從新生小鼠肝臟獲取Kuppfer細(xì)胞,取材容易,操作方便,不需特殊設(shè)備,可滿足新生小鼠膽道閉鎖發(fā)病機制研究的實驗需求,實驗價值高。 第三部分依賴于IL-4、IFN-γ不同年齡小鼠肝臟NK對MΦ調(diào)控的差異 摘要 目的：以前的研究發(fā)現(xiàn)新生小鼠肝臟NK細(xì)胞的活化能力不足,而活化的NK細(xì)胞是細(xì)胞因子IFN-γ和IL-4的主要來源,希望通過明確MΦ細(xì)胞活化對細(xì)胞因子IFN-y和IL-4劑量依賴性,進(jìn)而明確新生小鼠肝臟NK細(xì)胞對MΦ細(xì)胞的調(diào)控存有無異常。 方法：細(xì)胞因子IFN-γ可輔助MΦ細(xì)胞活化并表達(dá)細(xì)胞因子iNOS和TNF-a,而IL-4可輔助MΦ細(xì)胞活化分泌細(xì)胞因子IL-10。MΦ細(xì)胞與不同劑量細(xì)胞因子IFN--γ共孵育12小時后,通過實時PCR(real time PCR,RT-PCR)和印跡實驗(westblot,WB)兩種檢測方法分別檢測細(xì)胞因子iNOS和TNF-a基因轉(zhuǎn)錄和蛋白表達(dá)與IFN-y劑量關(guān)系；通過酶聯(lián)免疫吸附法(ELISA)及RT-PCR法檢測MΦ細(xì)胞與不同劑量IL-4共孵育12小時后IL-I0基因轉(zhuǎn)錄和蛋白表達(dá)與IL-4劑量關(guān)系。MΦ細(xì)胞分別來源于新生小鼠肝臟和成年小鼠肝臟。 結(jié)果：MΦ細(xì)胞與不同劑量IFN-γ共孵育12小時后,其胞內(nèi)細(xì)胞因子iNOS和TNF-a的基因轉(zhuǎn)錄和蛋白表達(dá)量均隨著IFN-y劑量的增加而呈遞增趨勢；同樣,隨著IL-4濃度的增加,MΦ細(xì)胞胞內(nèi)IL-I0基因轉(zhuǎn)錄和蛋白表達(dá)量也逐漸遞增,但當(dāng)IL-4濃度達(dá)到100u/L時,IL-10基因轉(zhuǎn)錄和蛋白表達(dá)量達(dá)到峰值,隨后逐漸降低。而且,無論新生小鼠肝臟MΦ細(xì)胞或成年小鼠肝臟MΦ細(xì)胞,它們均有類似表現(xiàn)。 結(jié)論：MΦ細(xì)胞的活化程度對細(xì)胞因子IFN-γ和IL-4具有劑量依賴性,在一定范圍內(nèi),隨著細(xì)胞因子IFN-γ和IL-4劑量的增加,其分泌炎性細(xì)胞因子的能力逐漸增強。而活化的NK細(xì)胞是細(xì)胞因子IFN-γ和IL-4的主要來源,已明確新生小鼠肝臟NK細(xì)胞的活化能力不足,分泌IFN-γ和IL-4等細(xì)胞因子能力下降,新生小鼠肝臟NK細(xì)胞對MΦ細(xì)胞的調(diào)控因而存在異常。 第四部分不同年齡小鼠肝臟NK細(xì)胞對吞噬RRV的MΦ細(xì)胞殺傷差異 摘要 目的：通過比較不同年齡小鼠肝臟NK細(xì)胞對吞噬了RRV的MΦ細(xì)胞的殺傷活性差異,從而進(jìn)一步確定了新生小鼠肝臟NK對MΦ細(xì)胞調(diào)控能力不足。 方法：通過磁珠分選法及差別貼壁法分別獲取野生型B6成年小鼠和新生小鼠肝臟NK細(xì)胞與MΦ細(xì)胞。把來源于不同年齡段小鼠肝臟NK細(xì)胞及是否事先經(jīng)過HMGB1活化過分為A、B、C、D四組,然后將不同組NK細(xì)胞分別與吞噬了RRV的不同年齡段小鼠肝臟MΦ細(xì)胞共培養(yǎng)4小時后,采用非放射性細(xì)胞毒試驗檢測不同組肝臟NK細(xì)胞對吞噬RRV后的肝臟MΦ殺傷活性。 結(jié)果：新生小鼠肝臟NK細(xì)胞經(jīng)細(xì)胞因子HMGB1活化后,其對吞噬RRV的不同年齡段小鼠肝臟MΦ細(xì)胞殺傷能力均明顯增強,P0.05,有統(tǒng)計學(xué)意義；同樣,經(jīng)細(xì)胞因子HMGB1活化后的成年小鼠肝臟NK細(xì)胞,其對吞噬了RRV的不同年齡段小鼠肝臟MΦ細(xì)胞殺傷能力也明顯增強,P0.05,有統(tǒng)計學(xué)意義。但與成年小鼠肝臟NK細(xì)胞相比,新生小鼠肝臟NK細(xì)胞對吞噬RRV的MΦ細(xì)胞殺傷能力明顯明顯不足,P0.05,有統(tǒng)計學(xué)意義。然而隨著小鼠年齡的增加,NK細(xì)胞逐漸成熟,其殺傷活性可逐漸增強。 結(jié)論：新生小鼠肝臟NK細(xì)胞對吞噬了RRV的MΦ細(xì)胞殺傷能力不足,不能有效清除吞噬RRV的MΦ細(xì)胞,導(dǎo)致RRV在體內(nèi)持續(xù)存、繁殖并擴散至周圍組織,從而造成膽管上皮細(xì)胞大量受損及慢性炎癥的形成,繼而形成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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R722.1

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