HLA-G抑制人NK、T细胞介导的异种移植排斥反应的研究

发布时间：2018-03-28 14:04

  本文选题：HLA-G1　切入点：NK细胞　出处：《华中科技大学》2006年博士论文

【摘要】： 第一部分HLA-G1抑制人自然杀伤细胞杀伤猪内皮细胞的研究 【目的】研究HLA-G1基因抑制人NK细胞系NK92和外周血单个核细胞(hPBMC)对猪血管内皮细胞(PEC)杀伤的作用。 【方法】利用脂质体介导的基因转染技术将pcDNA3-HLA-G1质粒转入原代培养的PEC，用流式细胞仪及间接免疫荧光显微镜在蛋白质水平上检测HLA-G1分子在PEC上的表达；以NK细胞系(NK92)和hPBMC为效应细胞，用四甲基偶氮唑盐(MTT)法检测转染有HLA-G1基因的PEC对NK92和hPBMC杀伤活性的抵御作用。 【结果】利用脂质体转染技术成功将pcDNA3-HLA-G1转入原代培养的PEC；NK92和hPBMC对转染有HLA-G1的PEC的杀伤效率(41.5％±14.0％；45.4％±12.1％)与对照组(75.3％±10.5％；74.6％±11.2％)相比，均有明显降低(P＜0.05)。 【结论】HLA-G1分子可以明显抑制NK92细胞以及hPBMC对PEC的杀伤作用。 第二部分建立表达可溶性HLA-G1真核细胞系并获取可溶性HLA-G1 【目的】为了研究可溶性HLA-G1(sHLA-G1)在抑制NK细胞以及T细胞介导的异种排斥反应中的作用，在本实验中建立表达sHLA-G1的真核细胞系，获取纯化的sHLA-G1蛋白。 【方法】利用核转染技术将pcDNA3-sHLA-G1质粒转入LCL721.221细胞；流式细胞仪和荧光显微镜对转染率进行判定；G418筛选阳性细胞；应用RT-PCR和Dot-ELISA方法分别在基因水平和蛋白水平对表达sHLA-G1的LCL721.221细胞进行鉴定；收集LCL721.221-sHLA-G1细胞培养液；将HB-95细胞注入Balb／c小鼠腹腔，产生含抗体的腹水后采用正辛酸-硫酸氨沉淀法纯化W6／32抗体，再用免疫亲和层析提纯sHLA-G1蛋白。 【结果】核转染技术可以高效将pcDNA3-sHLA-G1质粒转入LCL721.221细胞，转染率约为14％，RT-PCR和Dot-ELISA的结果显示转染和筛选均获得成功；G418筛选出稳定表达sHLA-G1的LCL721.221细胞，应用免疫亲和层析方法提纯LCL721.221-sHLA-G1培养液中sHLA-G1纯化蛋白约1.3mg。 【结论】核转染技术可以高效将pcDNA3-sHLA-G1转入LCL721.221细胞。免疫亲和层析方法成功提纯sHLA-G1纯化蛋白。 第三部分可溶性HLA-G1抑制NK92的生物学功能的研究 【目的】研究sHLA-G1对NK细胞的粘附功能、胞吐、杀伤活性以及释放细胞因子等功能的影响作用。 【方法】利用细胞粘附实验研究sHLA-G1对NK92细胞在静止状态和滚动状态对猪血管内皮细胞系SV-40-PED粘附功能的抑制作用；借助β-hexosaminidase释放实验间接反映NK92细胞杀伤SV-40-PED时所释放的穿孔素、颗粒酶水平以及sHLA-G1对此的抑制作用；利用ELISA方法检测sHLA-G1对NK92所释放的IFN-γ、TNF-α水平的抑制作用；利用MTT方法检测sHLA-G1抑制NK92对SV-40-PED的杀伤作用。 【结果】sHLA-G1无论是在静止状态下还是滚动状态下都能显著抑制NK92的粘附功能(P＜0.01)；β-hexosaminidase释放实验结果显示，NK细胞与靶细胞SV-40-PED相互作用2h，，即有明显的胞吐效应，酶释放率达14.5％，随着时间延长，酶释放率增加，6h时达高峰64.5％，sHLA-G1组中NK92细胞的胞吐维持在较低水平，二者间有显著差异(P＜0.05)；ELISA实验结果显示对照组中NK92所释放的IFN-γ、TNF-α因子维持在一个较高的水平，sHLA-G1组中NK92所释放的IFN-γ、TNF-α因子较对照组有显著降低(P＜0.05)；MTT杀伤实验结果显示sHLA-G1组NK92对SV-40-PED的杀伤效率(25.5±2.1％)显著低于对照组(71.2±2.6％，P＜0.01)。 【结论】sHLA-G1可以显著抑制NK92细胞的生物学功能，从而抑制NK92介导的异种移植排斥反应。 第四部分人外周血单个核细胞移植给SCID小鼠异种GVHD模型的建立 【目的】建立一种能直接观测异种器官移植给人的异种移植所发生的各种免疫反应的动物模型。 【方法】阳性对照组：SCID小鼠经尾静脉注射5×10~7 C57BL／6小鼠脾细胞；人外周血单核细胞(hPBMC)组：SCID小鼠在实验前1d腹腔注射30μl anti-asialo GM1抗体，实验当天给予Co~(60)照射(3.5Gy)、尾静脉注射5×10~7hPBMC；NK92组：根据预处理因素分为3组，A组SCID小鼠在实验前1d腹腔注射30μl生理盐水，实验当天给予Co~(60)照射(3.5Gy)和尾静脉注射5×10~7NK92细胞；B组SCID小鼠在实验前1d腹腔注射30μl anti-asialo GM1抗体，实验当天给予Co~(60)照射(3.5Gy)和尾静脉注射5×10~7 NK92细胞；C组SCID小鼠在实验前1d腹腔注射30μl anti-asialo GM1抗体，实验当天给予Co~(60)照射(3.5Gy)和尾静脉注射5×10~7NK92细胞(NK92细胞悬液加入终浓度为200u／ml的rhlL-2)。观察各组SCID小鼠体重、体型、体位、毛发、腹泻和死亡时间；ELISA方法检测SCID小鼠(PBMC组和NK92组)IFN-γ、TNF-α的分泌水平；对死亡SCID小鼠行常规病理学检查。 【结果】SCID小鼠在输注5×10~7 C57BL／6脾细胞5d后出现弓背、消瘦、皮毛紊乱无光泽、体重减轻等GVHD症状，7只SCID小鼠在5～8d内死亡，肝脏出现大面积肝细胞坏死，肝脏失去正常的结构，肝窦中有大量的淋巴细胞浸润；hPBMC组SCID小鼠在2周后开始出现GVHD症状，肝脏出现大面积肝细胞坏死，肝窦中有大量的淋巴细胞浸润，分别在15～25d内死亡；NK92组中A、B、C组SCID小鼠均未出现GVHD；ELISA检测结果显示hPBMC组中在发生异种GVHD时TNF-α的分泌水平明显升高，IFN-γ的水平也有一定程度上的升高，而在NK92组，IFN-γ、TNF-α的分泌水平较低，并且显现出持续降低趋势。 【结论】成功建立人外周血单个核细胞移植给SCID小鼠异种GVHD模型，为研究异种移植排斥反应以及今后建立人移植物对猪的异种GVHD提供一定的启示。 第五部分可溶性HLA-G1抑制外周血单个核细胞移植给SCID小鼠的异种GVHD 【目的】研究sHLA-G1在体内环境中对T细胞活性的影响作用，并且抑制人外周血单个核细胞(hPBMC)移植给SCID小鼠发生的异种GVHD。 【方法】采用单向混合淋巴细胞培养实验检测sHLA-G1对T细胞增殖能力的抑制作用；SCID小鼠在实验前1d腹腔注射30μl anti-asialo GM1抗体，实验当天给予Co~(60)照射(3.5Gy)、尾静脉注射5×10~7 hPBMC细胞，其中实验组1、2中SCID小鼠分别经尾静脉注射2ng和4ng的sHLA-G1治疗(实验当天、3、6、9、12和15d)，对照组SCID小鼠注射等量的生理盐水；ELISA方法检测各组SCID小鼠IFN-γ、TNF-α的分泌水平；取死亡SCID小鼠行常规病理学检查。 【结果】对照组中hPBMC的增殖强度比实验组1和实验组2均有显著性增强(P＜0.05)；实验组1和实验组2中SCID小鼠存活时间比对照组有显著延长(P＜0.01)；对照组中SCID小鼠血清中的IFN-γ、TNF-α水平随着GVHD症状的出现，IFN-γ、TNF-α水平显著升高，实验组1，2中SCID小鼠血清中的IFN-γ、TNF-α水平一直维持在较低的水平；病理学检查对照组SCID小鼠肝脏出现大面积肝细胞坏死，肝脏失去正常的结构，肝窦中有大量的淋巴细胞浸润，而实验组中SCID小鼠肝脏病理学检查均为肝脏轻微可逆性病变，肝窦中仅有散在淋巴细胞浸润。 【结论】sHLA-G1可以在体内环境中抑制T细胞的生物学功能，并且可以明显抑制hPBMC移植给SCID小鼠发生的异种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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392;R617


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