本文選題：HLA-G1　切入點：NK細(xì)胞　出處：《華中科技大學(xué)》2006年博士論文

【摘要】： 第一部分HLA-G1抑制人自然殺傷細(xì)胞殺傷豬內(nèi)皮細(xì)胞的研究 【目的】研究HLA-G1基因抑制人NK細(xì)胞系NK92和外周血單個核細(xì)胞(hPBMC)對豬血管內(nèi)皮細(xì)胞(PEC)殺傷的作用。 【方法】利用脂質(zhì)體介導(dǎo)的基因轉(zhuǎn)染技術(shù)將pcDNA3-HLA-G1質(zhì)粒轉(zhuǎn)入原代培養(yǎng)的PEC，用流式細(xì)胞儀及間接免疫熒光顯微鏡在蛋白質(zhì)水平上檢測HLA-G1分子在PEC上的表達(dá)；以NK細(xì)胞系(NK92)和hPBMC為效應(yīng)細(xì)胞，用四甲基偶氮唑鹽(MTT)法檢測轉(zhuǎn)染有HLA-G1基因的PEC對NK92和hPBMC殺傷活性的抵御作用。 【結(jié)果】利用脂質(zhì)體轉(zhuǎn)染技術(shù)成功將pcDNA3-HLA-G1轉(zhuǎn)入原代培養(yǎng)的PEC；NK92和hPBMC對轉(zhuǎn)染有HLA-G1的PEC的殺傷效率(41.5％±14.0％；45.4％±12.1％)與對照組(75.3％±10.5％；74.6％±11.2％)相比，均有明顯降低(P＜0.05)。 【結(jié)論】HLA-G1分子可以明顯抑制NK92細(xì)胞以及hPBMC對PEC的殺傷作用。 第二部分建立表達(dá)可溶性HLA-G1真核細(xì)胞系并獲取可溶性HLA-G1 【目的】為了研究可溶性HLA-G1(sHLA-G1)在抑制NK細(xì)胞以及T細(xì)胞介導(dǎo)的異種排斥反應(yīng)中的作用，在本實驗中建立表達(dá)sHLA-G1的真核細(xì)胞系，獲取純化的sHLA-G1蛋白。 【方法】利用核轉(zhuǎn)染技術(shù)將pcDNA3-sHLA-G1質(zhì)粒轉(zhuǎn)入LCL721.221細(xì)胞；流式細(xì)胞儀和熒光顯微鏡對轉(zhuǎn)染率進行判定；G418篩選陽性細(xì)胞；應(yīng)用RT-PCR和Dot-ELISA方法分別在基因水平和蛋白水平對表達(dá)sHLA-G1的LCL721.221細(xì)胞進行鑒定；收集LCL721.221-sHLA-G1細(xì)胞培養(yǎng)液；將HB-95細(xì)胞注入Balb／c小鼠腹腔，產(chǎn)生含抗體的腹水后采用正辛酸-硫酸氨沉淀法純化W6／32抗體，再用免疫親和層析提純sHLA-G1蛋白。 【結(jié)果】核轉(zhuǎn)染技術(shù)可以高效將pcDNA3-sHLA-G1質(zhì)粒轉(zhuǎn)入LCL721.221細(xì)胞，轉(zhuǎn)染率約為14％，RT-PCR和Dot-ELISA的結(jié)果顯示轉(zhuǎn)染和篩選均獲得成功；G418篩選出穩(wěn)定表達(dá)sHLA-G1的LCL721.221細(xì)胞，應(yīng)用免疫親和層析方法提純LCL721.221-sHLA-G1培養(yǎng)液中sHLA-G1純化蛋白約1.3mg。 【結(jié)論】核轉(zhuǎn)染技術(shù)可以高效將pcDNA3-sHLA-G1轉(zhuǎn)入LCL721.221細(xì)胞。免疫親和層析方法成功提純sHLA-G1純化蛋白。 第三部分可溶性HLA-G1抑制NK92的生物學(xué)功能的研究 【目的】研究sHLA-G1對NK細(xì)胞的粘附功能、胞吐、殺傷活性以及釋放細(xì)胞因子等功能的影響作用。 【方法】利用細(xì)胞粘附實驗研究sHLA-G1對NK92細(xì)胞在靜止?fàn)顟B(tài)和滾動狀態(tài)對豬血管內(nèi)皮細(xì)胞系SV-40-PED粘附功能的抑制作用；借助β-hexosaminidase釋放實驗間接反映NK92細(xì)胞殺傷SV-40-PED時所釋放的穿孔素、顆粒酶水平以及sHLA-G1對此的抑制作用；利用ELISA方法檢測sHLA-G1對NK92所釋放的IFN-γ、TNF-α水平的抑制作用；利用MTT方法檢測sHLA-G1抑制NK92對SV-40-PED的殺傷作用。 【結(jié)果】sHLA-G1無論是在靜止?fàn)顟B(tài)下還是滾動狀態(tài)下都能顯著抑制NK92的粘附功能(P＜0.01)；β-hexosaminidase釋放實驗結(jié)果顯示，NK細(xì)胞與靶細(xì)胞SV-40-PED相互作用2h，，即有明顯的胞吐效應(yīng)，酶釋放率達(dá)14.5％，隨著時間延長，酶釋放率增加，6h時達(dá)高峰64.5％，sHLA-G1組中NK92細(xì)胞的胞吐維持在較低水平，二者間有顯著差異(P＜0.05)；ELISA實驗結(jié)果顯示對照組中NK92所釋放的IFN-γ、TNF-α因子維持在一個較高的水平，sHLA-G1組中NK92所釋放的IFN-γ、TNF-α因子較對照組有顯著降低(P＜0.05)；MTT殺傷實驗結(jié)果顯示sHLA-G1組NK92對SV-40-PED的殺傷效率(25.5±2.1％)顯著低于對照組(71.2±2.6％，P＜0.01)。 【結(jié)論】sHLA-G1可以顯著抑制NK92細(xì)胞的生物學(xué)功能，從而抑制NK92介導(dǎo)的異種移植排斥反應(yīng)。 第四部分人外周血單個核細(xì)胞移植給SCID小鼠異種GVHD模型的建立 【目的】建立一種能直接觀測異種器官移植給人的異種移植所發(fā)生的各種免疫反應(yīng)的動物模型。 【方法】陽性對照組：SCID小鼠經(jīng)尾靜脈注射5×10~7 C57BL／6小鼠脾細(xì)胞；人外周血單核細(xì)胞(hPBMC)組：SCID小鼠在實驗前1d腹腔注射30μl anti-asialo GM1抗體，實驗當(dāng)天給予Co~(60)照射(3.5Gy)、尾靜脈注射5×10~7hPBMC；NK92組：根據(jù)預(yù)處理因素分為3組，A組SCID小鼠在實驗前1d腹腔注射30μl生理鹽水，實驗當(dāng)天給予Co~(60)照射(3.5Gy)和尾靜脈注射5×10~7NK92細(xì)胞；B組SCID小鼠在實驗前1d腹腔注射30μl anti-asialo GM1抗體，實驗當(dāng)天給予Co~(60)照射(3.5Gy)和尾靜脈注射5×10~7 NK92細(xì)胞；C組SCID小鼠在實驗前1d腹腔注射30μl anti-asialo GM1抗體，實驗當(dāng)天給予Co~(60)照射(3.5Gy)和尾靜脈注射5×10~7NK92細(xì)胞(NK92細(xì)胞懸液加入終濃度為200u／ml的rhlL-2)。觀察各組SCID小鼠體重、體型、體位、毛發(fā)、腹瀉和死亡時間；ELISA方法檢測SCID小鼠(PBMC組和NK92組)IFN-γ、TNF-α的分泌水平；對死亡SCID小鼠行常規(guī)病理學(xué)檢查。 【結(jié)果】SCID小鼠在輸注5×10~7 C57BL／6脾細(xì)胞5d后出現(xiàn)弓背、消瘦、皮毛紊亂無光澤、體重減輕等GVHD癥狀，7只SCID小鼠在5～8d內(nèi)死亡，肝臟出現(xiàn)大面積肝細(xì)胞壞死，肝臟失去正常的結(jié)構(gòu)，肝竇中有大量的淋巴細(xì)胞浸潤；hPBMC組SCID小鼠在2周后開始出現(xiàn)GVHD癥狀，肝臟出現(xiàn)大面積肝細(xì)胞壞死，肝竇中有大量的淋巴細(xì)胞浸潤，分別在15～25d內(nèi)死亡；NK92組中A、B、C組SCID小鼠均未出現(xiàn)GVHD；ELISA檢測結(jié)果顯示hPBMC組中在發(fā)生異種GVHD時TNF-α的分泌水平明顯升高，IFN-γ的水平也有一定程度上的升高，而在NK92組，IFN-γ、TNF-α的分泌水平較低，并且顯現(xiàn)出持續(xù)降低趨勢。 【結(jié)論】成功建立人外周血單個核細(xì)胞移植給SCID小鼠異種GVHD模型，為研究異種移植排斥反應(yīng)以及今后建立人移植物對豬的異種GVHD提供一定的啟示。 第五部分可溶性HLA-G1抑制外周血單個核細(xì)胞移植給SCID小鼠的異種GVHD 【目的】研究sHLA-G1在體內(nèi)環(huán)境中對T細(xì)胞活性的影響作用，并且抑制人外周血單個核細(xì)胞(hPBMC)移植給SCID小鼠發(fā)生的異種GVHD。 【方法】采用單向混合淋巴細(xì)胞培養(yǎng)實驗檢測sHLA-G1對T細(xì)胞增殖能力的抑制作用；SCID小鼠在實驗前1d腹腔注射30μl anti-asialo GM1抗體，實驗當(dāng)天給予Co~(60)照射(3.5Gy)、尾靜脈注射5×10~7 hPBMC細(xì)胞，其中實驗組1、2中SCID小鼠分別經(jīng)尾靜脈注射2ng和4ng的sHLA-G1治療(實驗當(dāng)天、3、6、9、12和15d)，對照組SCID小鼠注射等量的生理鹽水；ELISA方法檢測各組SCID小鼠IFN-γ、TNF-α的分泌水平；取死亡SCID小鼠行常規(guī)病理學(xué)檢查。 【結(jié)果】對照組中hPBMC的增殖強度比實驗組1和實驗組2均有顯著性增強(P＜0.05)；實驗組1和實驗組2中SCID小鼠存活時間比對照組有顯著延長(P＜0.01)；對照組中SCID小鼠血清中的IFN-γ、TNF-α水平隨著GVHD癥狀的出現(xiàn)，IFN-γ、TNF-α水平顯著升高，實驗組1，2中SCID小鼠血清中的IFN-γ、TNF-α水平一直維持在較低的水平；病理學(xué)檢查對照組SCID小鼠肝臟出現(xiàn)大面積肝細(xì)胞壞死，肝臟失去正常的結(jié)構(gòu)，肝竇中有大量的淋巴細(xì)胞浸潤，而實驗組中SCID小鼠肝臟病理學(xué)檢查均為肝臟輕微可逆性病變，肝竇中僅有散在淋巴細(xì)胞浸潤。 【結(jié)論】sHLA-G1可以在體內(nèi)環(huán)境中抑制T細(xì)胞的生物學(xué)功能，并且可以明顯抑制hPBMC移植給SCID小鼠發(fā)生的異種GVHD。
[Abstract]:Part one HLA-G1 inhibition of human natural killer cells to kill porcine endothelial cells
[Objective] to study the effect of HLA-G1 gene inhibiting human NK cell line NK92 and peripheral blood mononuclear cells (hPBMC) on the killing of porcine vascular endothelial cells (PEC).
[method] using liposome mediated gene transfection technology, the recombinant plasmid pcDNA3-HLA-G1 was transfected into primary cultured PEC, the expression of HLA-G1 molecule on PEC by flow cytometry and immunofluorescence microscopy at the protein level; NK cell line (NK92) and hPBMC as effector cells, using four methyl thiazolyl tetrazolium salt (MTT) method was used to detect the transfection of HLA-G1 gene of PEC cytotoxicity against the effects of NK92 and hPBMC.
[results] using liposome transfection technique successfully pcDNA3-HLA-G1 into primary culture of PEC NK92 and hPBMC HLA-G1; the PEC killing efficiency of transfection (41.5% + 14%; 45.4% + 12.1%) and control group (75.3% + 10.5%; 74.6% + 11.2%) compared were significantly lower (P < 0.05).
[Conclusion] HLA-G1 can obviously inhibit the killing effect of NK92 cells and hPBMC on PEC.
The second part established the expression of soluble HLA-G1 eukaryotic cell line and obtained soluble HLA-G1
[Objective] in order to study the role of soluble HLA-G1 (sHLA-G1) in inhibiting NK cells and T cell mediated xenograft rejection, a eukaryotic cell line expressing sHLA-G1 was established in this experiment, and purified sHLA-G1 protein was obtained.
[method] using nucleofection pcDNA3-sHLA-G1 plasmid into LCL721.221 cells; the transfection rate was determined by flow cytometry and fluorescence microscope; G418 positive cells were selected; using RT-PCR and Dot-ELISA method respectively at the mRNA level and protein level on the expression of sHLA-G1 LCL721.221 cells were identified; collected LCL721.221-sHLA-G1 cells cultured with HB-95 cell Balb; C / mice produce antibody containing ascites by caprylic acid ammonium sulfate precipitation and purification of W6 / 32 antibody, purification of sHLA-G1 protein by immuno affinity chromatography.
[results] nuclear transfection technology improves the efficiency of pcDNA3-sHLA-G1 plasmid was transfected into LCL721.221 cells, the transfection rate is about 14%, RT-PCR and Dot-ELISA results showed that the transfection and screening were successfully screened; G418 stable expression of sHLA-G1 LCL721.221 cells by immunoaffinity chromatography method for purification of LCL721.221-sHLA-G1 medium purified sHLA-G1 fusion protein was about 1.3mg.
[Conclusion] nuclear transfection technique can efficiently transfer pcDNA3-sHLA-G1 into LCL721.221 cells. The purified protein of sHLA-G1 is purified by immuno affinity chromatography.
Study on the biological function of third part soluble HLA-G1 in inhibiting NK92
[Objective] to study the effect of sHLA-G1 on the function of adhesion, exocytosis, killing activity and releasing cytokine in NK cells.
[method] using cell adhesion experimental study on inhibitory effect of sHLA-G1 on NK92 cells of SV-40-PED adhesion function of porcine endothelial cell line in the stationary state and rolling state; by means of -hexosaminidase beta release experiments indirectly reflect NK92 cell killing SV-40-PED perforin, Granzyme levels and the inhibitory effect of sHLA-G1 in IFN- by ELISA gamma; methods to detect the sHLA-G1 release of NK92, inhibition of TNF- alpha level; using MTT method to detect sHLA-G1 inhibit the killing effect of NK92 on SV-40-PED.
[results] sHLA-G1 or rolling adhesion function state can significantly inhibit NK92 in resting state (P < 0.01); the -hexosaminidase beta release experiments showed that NK cells and target cells SV-40-PED 2H interaction, which has obvious effect of exocytosis, enzyme release rate reached 14.5%, with the prolongation of time, enzyme the release rate increased, reached a peak at 6h 64.5%, NK92 cell group sHLA-G1 exocytosis remained at a low level, there are significant differences between the two (P < 0.05); ELISA experimental results show that the IFN- gamma release in control group NK92, TNF- alpha factor is maintained at a high level, the IFN- gamma the release of NK92 in the sHLA-G1 group, the TNF- factor was significantly lower than the control group (P < 0.05); MTT cell experiment results show that the killing efficiency of sHLA-G1 group NK92 of SV-40-PED (25.5 + 2.1%) was significantly lower than the control group (71.2 + 2.6%, P < 0.01).
[Conclusion] sHLA-G1 can inhibit the biological function of NK92 cells and inhibit the rejection of xenotransplantation mediated by NK92.
The establishment of fourth parts of human peripheral blood mononuclear cells transplantation to SCID mouse GVHD model
[Objective] to establish an animal model that can directly observe the various immune responses in xenotransplantation of xenotransplantation.
[method] the positive control group: SCID mice by tail vein injection of 5 * 10~7 C57BL / 6 mouse spleen cells; human peripheral blood mononuclear cells (hPBMC) in the experimental group: SCID mice before intraperitoneal injection of 1D 30 l anti-asialo GM1 antibody, the day of the experiment Co~ (60) radiation (3.5Gy), teleutostatic intravenous injection of 5 * 10~7hPBMC; group NK92: according to the pretreatment factors are divided into 3 groups, A group of SCID mice in the experiment before intraperitoneal injection of 1D 30 l normal saline, the day of the experiment to Co~ (60) radiation (3.5Gy) and intravenous injection of 5 * 10~7NK92 cells; SCID mice in the experimental group B before intraperitoneal injection of 1D 30 l anti-asialo GM1 antibody, the day of the experiment Co~ (60) radiation (3.5Gy) and intravenous injection of 5 * 10~7 NK92 cells; SCID mice in the experimental group C before intraperitoneal injection of 1D 30 l anti-asialo GM1 antibody, the day of the experiment Co~ (60) radiation (3.5Gy) and intravenous injection of 5 * 10~ 7NK92 cell (NK92 cell suspension into The final concentration was rhlL-2 of 200u / ml. The body weight, body shape, body position, hair, diarrhea and death time of SCID mice were observed. ELISA method was used to detect the secretion level of IFN- gamma and TNF- alpha of SCID mice (PBMC group and NK92 group), and the pathological examination of death SCID mice was performed.
[results] SCID mice in the infusion of 5 * 10~7 C57BL / 6 spleen cells after 5D back, thin fur disorder without luster, weight loss and other symptoms of GVHD, the death of 5 ~ 8D in 7 SCID mice liver, there is a large area of necrosis of liver cells, liver lost normal structure, there are a lot of hepatic sinus lymphocyte infiltration; hPBMC group of SCID mice at 2 weeks after the start of GVHD symptoms, liver large area necrosis of liver cells, liver sinus with massive lymphocyte infiltration, were killed in 15 ~ 25d; in group NK92, A, B, GVHD were not found in SCID mice in C group; ELISA results showed that the secretion level TNF- in the GVHD hPBMC group in xenograft was significantly elevated in IFN- gamma levels also increased to a certain extent, while in group NK92, IFN- gamma, the secretion level of TNF- alpha is low, and showed decreased trend.
[Conclusion] the successful establishment of human peripheral blood mononuclear cells transplantation to SCID mice xenogeneic GVHD model will provide some inspiration for the study of xenograft rejection and the establishment of human xenograft GVHD for pigs.
Fifth soluble HLA-G1 inhibits the xenotransplantation of peripheral blood mononuclear cells to SCID mice in GVHD
[Objective] to study the effect of sHLA-G1 on the activity of T cells in vivo, and inhibit the transplantation of human peripheral blood mononuclear cells (hPBMC) to SCID GVHD. mice.
[method] by mixed lymphocyte culture inhibition assay of sHLA-G1 on proliferation of T cells; SCID mice before intraperitoneal injection of 1D 30 l anti-asialo GM1 antibody, the day of the experiment Co~ (60) radiation (3.5Gy), intravenous injection of 5 * 10~7 hPBMC cells in the experimental group, 1, 2 SCID mice were treated by sHLA-G1 2ng and 4ng tail intravenous injection (the day of the experiment, 3,6,9,12 and 15d), the control group normal saline injected SCID mice; ELISA was detected in SCID mice IFN- gamma, the secretion level of TNF- alpha; and death of SCID mice underwent routine pathological examination.
[results] the proliferation of strength in control group hPBMC than in group 1 and group 2 were significantly increased (P < 0.05); the experimental group 1 and 2 SCID in the experimental group than in the control group, the survival time was significantly prolonged (P < 0.01); the control group of IFN- gamma SCID in mice serum, TNF- alpha with the emergence of symptoms of GVHD level, IFN- gamma, alpha TNF- levels were significantly higher in experimental group, IFN- gamma 1, 2 SCID in sera of mice, TNF- levels remained at a low level; pathological examination of SCID mice in control group liver large area necrosis of liver cells, liver lost normal structure in hepatic sinus a large number of lymphocytes, and the liver of SCID mice in the experimental group the pathological examination of liver were mild and reversible lesions, liver sinus only scattered lymphocytes.
[Conclusion] sHLA-G1 can inhibit the biological function of T cells in the body environment, and can obviously inhibit the xenogenic GVHD. of hPBMC transplantation to SCID mice.

【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】：2006
【分類號】：R392;R617


本文編號：1676634