同种异体NK细胞对人脐静脉内皮细胞的杀伤活性差异及分子机制探讨
发布时间：2019-05-30 09:57
【摘要】： 背景与目的 N K细胞是淋巴细胞中的一类特殊杀伤细胞,其杀伤活性的启动既不需抗原刺激,亦不需抗体参与,具有排斥异己细胞的作用。NK细胞对靶细胞的杀伤作用,受其表面的激活性受体和抑制性受体之间平衡的调节。其中NKG2D传导活化信号,配体包括MICA/MICB,ULBP1-3分子;KIR传导抑制信号,其配体为HLA-I类分子。移植的组织器官是否表达异体NK细胞KIR不能识别的HLA分子,会影响NK细胞对组织细胞的杀伤活性。同种异体NK细胞对靶细胞杀伤与骨髓移植中的肝静脉血管闭塞病(veno-occlusive disease of the liver VOD)及器官移植排斥反应有关系。 血管内皮细胞是供受体循环细胞之间的第一道屏障。循环的宿主淋巴细胞可直接识别异种血管内皮细胞而发动免疫攻击,或与移植物抗原及血管内皮上的抗原递呈细胞相互作用,损伤内皮细胞,从而使移植物血液循环障碍,最终导致移植物被排斥,移植失败。 本研究以人脐静脉内皮细胞系做为靶细胞,探讨同种异体NK细胞对内皮细胞是否有杀伤作用,如果有杀伤,再进一步研究参与杀伤的活化性及抑制性信号系统的分子机制。 方法 第一章检测ECV304细胞HLA-A、B、Cw分型及NKG2D配体MICA/B、ULBP1-3在基因水平的表达 以QIAampDNA抽提试剂提取ECV304细胞的DNA;ECV304细胞HLA分型采用序列特异性引物扩增法(sequence specific primer polymerase chainreproduction,PCR-SSP),RT-PCR检测ECV304细胞NKG2D的配体MICA、MICB、ULBP1、ULBP2、ULBP3的表达情况。 第二章检测不同个体NK细胞KIR2DL1的表达及ECV304细胞、K562细胞NKG2D配体MICA/B、ULBP1-3表达率、HLA-Ⅰ类分子表达率 通过PCR-SSP法及流式细胞仪分别检测8例健康者基因及外周血细胞表面KIR2DL1类分子的表达,用流式细胞仪检测ECV304细胞NKG2D的配体MICA、MICB、ULBP1、ULBP2、ULBP3及HLA-Ⅰ类分子的表达情况。用流式细胞仪检测半数抑制浓度(50%inhibitory concentration,IC50)浓度的环孢素A(cyclosporinA,CSA)及2ug/ml内毒素(Lipopolysaccharide,LPS)作用24小时后的ECV304细胞NKG2D配体的表达率是否有变化。 第三章分离外周血NK细胞、LDH释放法测定NK细胞在效靶比20:1时对ECV304细胞的杀伤活性及anti-KIR2DL1mAb对NK细胞杀伤活性的影响 免疫磁珠法分离纯化8例健康者的NK细胞。自8例健康供者分离外周血NK细胞,流式细胞仪检测KIR2DL1的表达率,LDH释放法测定NK细胞在效靶比20:1时对ECV304细胞、K562细胞的杀伤活性及anti-KIR2DL1mAb对NK细胞杀伤活性的影响。 结果 第一章检测ECV304细胞HLA-A、B、Cw分型及NKG2D配体MICA/B、ULBP1-3在基因水平的表达 1.ECV304细胞HLA基因型:PCR-SSP法检测ECV304细胞HLA-A,B,Cw基因型为A1,-;B18,-;Cw5,-。 2.RT-PCR检测ECV304细胞NKG2D配体的表达:RT-PCR检测结果显示,ECV304细胞在mRNA均表达NKG2D的配体MICA、MICB、ULBP1、ULBP2、ULBP3。 第二章检测不同个体外周血细胞KIR2DL1的表达ECV304细胞、K562细胞NKG2D配体MICA/B、ULBP1-3表达率、HLA-Ⅰ类分子表达率 1.不同个体检测KIR2DL1的表达率:HLA分型表明,ECV304表达KIR2DL1的配体,而不表达KIR2DL2/3、KIR3DL1的配体。PCR-SSP法检测结果显示,8例健康者在基因水平均表达KIR2DL1,并且NK细胞表面KIR2DL1表达率有较大差异,从6.2%-46.2%不等。KIR2DL2/3、KIR3DL1与ECV304细胞表面相应的HLA-A、B类分子间存在错配现象。 2.流式细胞仪检测结果表明ECV304细胞表面不表达NKG2D配体MICA/B、ULBP1-3,HLA-Ⅰ分子表达率为97.5%。 3.流式细胞仪检测结果显示1.63ug/ml CSA及2ug/mlLPS作用24小时后的ECV304细胞表面不表达NKG2D的配体MICA、MICB、ULBP1-3。 4.K562细胞表面MICA/B、ULBP1、ULBP2、ULBP3的表达率分别为61.5%、34.4%、36.7%、21.8%,不表达HLA-Ⅰ类分子。 第三章分离外周血NK细胞、LDH释放法测定NK细胞在效靶比20:1时对ECV304细胞的杀伤活性及anti-KIR2DL1mAb对NK细胞杀伤活性的影响 流式细胞仪检测CD3~-CD16~+CD56~+细胞的纯度大于90.0%。 8例健康供者NK细胞KIR2DL1表达率有较大差异,对ECV304细胞的杀伤活性也有不同,(效靶比20:1)KIR2DL1表达率为6.2%-46.2%时,NK细胞对ECV304细胞的杀伤活性分别为1.2%-28.0%;anti-KIR2DL1 mAb对NK细胞表面相应分子进行封闭,配对样本t检验比较抗体封闭前后NK细胞对ECV304细胞的杀伤活性相比差异有统计学意义(t=-4.860,P=0.002),anti-KIR2DL1 mAb增强NK细胞对ECV304的杀伤活性。双变量相关分析示个体KIR2DL1表达率与NK细胞对ECV304的杀伤率存在负相关(rs=-0.994,P=0.000)。 结论 1.ECV304细胞在mRNA水平表达MICA/B,ULBP1-3,但在蛋白水平未检测到上述分子的表达。流式细胞仪检测结果显示1.63ug/ml CSA及2ug/ml作用24小时后的ECV304细胞表面不表达NKG2D的配体MICA、MICB、ULBP1-3。PCR-SSP法检测ECV304细胞HLA-A,B,Cw基因型为Al,-;B18,-;Cw5,-。HLA分型表明,ECV304表达KIR2DL1的配体,而不表达KIR2DL2/3、KIR3DL1的配体。 2.8例健康供者NK细胞KIR2DL1表达率有较大差异,对ECV304细胞的杀伤活性也有不同,双变量相关分析示个体KIR2DL1表达率与NK细胞对ECV304的杀伤率存在负相关(rs=-0.994,P=0.000)。以anti-KIR2DL1 mAb封闭NK表面KIR2DL1受体,杀伤率均有不同程度的上升,用配对样本t检验,抗体封闭前后杀伤率差异有统计学意义(t=-4.860,P=0.002)。 3.个体KIR2DL1表达率影响NK细胞对ECV304杀伤活性。目前已知的NKG2D配体MICA/B、ULBP1-3不参与NK细胞对ECV304细胞的杀伤。 本研究的创新点 本研究结果显示,同种异体NK细胞对静脉内皮细胞有杀伤作用,HLA-KIR分子错配是杀伤主要分子机制。 研究价值 根据供受者的HLA-KIR分子的表达状态和匹配程度选择供者。
[Abstract]:Background and Purpose The N-K cells are a class of special killer cells in the lymphocytes. The activation of the anti-kill activity is not required to be stimulated, nor is the antibody involved. The effect of NK cells on the target cell, which is balanced between the activated receptor and the inhibitory receptor on its surface. wherein the NKG2D conductive activation signal, the ligand comprises a MICA/ MICB, a ULBP1-3 molecule, a KIR conduction inhibition signal, the ligand is an HLA-I class, Whether the transplanted tissue organ expresses the HLA molecule that is not recognized by the NK cell KIR, which can affect the killing of the NK cells on the tissue cells. The effect of allogenic NK cells on the anti-killing of target cells and the hepatic vein occlusion in bone marrow transplantation and the rejection of organ transplantation The vascular endothelial cells are between the donor-circulating cells. The first barrier. The circulating host cell can directly identify the xenogenic vascular endothelial cells to initiate an immune attack, or interact with the antigen presenting cell on the graft antigen and the vascular endothelial cell to damage the endothelial cells, thereby causing the graft to circulate a barrier, resulting in a shift the plants are repelled In this study, the human umbilical vein endothelial cell system was used as the target cell, and the anti-killing effect of the allogenic NK cells on the endothelial cells was discussed. number system The first chapter is to detect the HLA-A, B, Cw and NKG2D ligands MICA/ B in the ECV304 cells. The expression of ULBP1-3 in the gene level was used to extract the DNA of the ECV304 cell with the QIAampDNA extraction reagent. The HLA typing of the ECV304 cell was detected by a sequence-specific primer amplification method (PCR-SSP), and the ECV was detected by RT-PCR. Ligands MICA, MICB, ULBP1 of cell NKG2D The expression of NK cells KIR2DL1 in different individuals and the expression of ECV304 cells, K562 cells NKG2D ligand MICA/ B and U were detected in the second chapter. The expression rate of LBP1-3 and the expression of HLA-I were detected by PCR-SSP and flow cytometry. Expression of KIR2DL1 molecules on the surface of the gene and peripheral blood cells, and the ligands MICA, MICB, ULBP1, and UL of the ECV304 cell NKG2D were detected by flow cytometry. The expression of BP2, ULBP3 and HLA-I molecules was detected by flow cytometry. The expression rate of NKG2D ligand in CV304 cells was changed. In the third chapter, NK cells and LDH release in peripheral blood were isolated and the killing activity of NK cells at the target ratio of 20:1 to ECV304 cells was determined. NK cell with anti-KIR2DL1mAb The NK cells of 8 healthy individuals were isolated and purified by the immunomagnetic bead method. NK cells were isolated from the peripheral blood of 8 healthy donors. The expression rate of KIR2DL1 was detected by flow cytometry. The cytotoxicity of NK cells to the cells of ECV304 and K562 cells was determined by means of the LDH release method. and The effect of anti-KIR2DL1mAb on the killing activity of NK cells was investigated. B, Cw classification and the expression of NKG2D ligand MICA/ B and ULBP1-3 in the gene level 1. ECV304 cell HLA genotype: PCR-SSP method detection ECV304 cells HLA-A, B, Cw genotype A1,-; B18,-; Cw5,-. 2.RT-PCR were used to detect the expression of NKG2D ligand in ECV304 cells: the results of RT-PCR showed that ECV304 cells were in m The expression of NKG2D, MICA, MICB, ULBP1, ULBP2 and ULBP3 were all expressed in the RNA, and the expression of KIR2DL1 in the peripheral blood cells of different individuals was detected by the second chapter. Cell, K562 cell NKG2D ligand MICA/ B, ULBP1-3 expression rate, HLA-I type molecule expression rate 1. The expression rate of KIR2DL1 was detected by different individuals: HLA score The results showed that ECV304 expressed KIR2DL1 and did not express the ligand of KIR2DL2/3 and KIR3DL1. KIR2DL1 was expressed and the expression rate of KIR2DL1 on the surface of NK cells was significantly different from 6.2% to 46.2%. There was a mismatch between the HLA-A and B molecules on the surface of IR2DL2/3, KIR3DL1 and ECV304. The expression rate of NKG2D ligand MICA/ B, ULBP1-3 and HLA-I was 97.5% in the surface of CV304 cells. The surface of ECV304 cells after 24 hours of LPS action did not express NKG2D ligand MICA, MICB, ULBP1-3.4. K562 cell surface, MICA/ B, ULB, The expression rates of P1, ULBP2 and ULBP3 were 61.5%, 34.4%, 36.7%, 21.8%, respectively. The anti-killing activity of NK cells against ECV304 cells at the target ratio of 20:1 was determined by the release method. The effect of KIR2DL1mAb on NK cell killing activity was more than 90.0%. The expression rate of NK cell KIR2DL1 in 8 healthy donors was significantly different, and the expression rate of KIR2DL1 was 6.2%-46.2 for ECV304 cells. In%, the cytotoxicity of NK cells to ECV304 cells was 1.2%-28.0%, and anti-KIR2DL1 mAb was used to close the corresponding molecules on the surface of NK cells. The difference in wound activity was statistically significant (t =-4.860, P = 0.002), anti-KIR2DL1 mAb enhanced NK cell response to ECV304. kill There was a negative correlation between the expression rate of KIR2DL1 and the killing rate of NK cells in ECV304 (rs =-0.9). Conclusion 1. ECV304 cells express MICA/ B and ULBP1-3 at the mRNA level, but the expression of the above-mentioned molecules is not detected at the level of the protein. The results of flow cytometry show that the surface of the ECV304 cells after 24 hours after the action of 1.63 ug/ ml of CSA and 2 ug/ ml does not express the NKG. Detection of the HLA-A, B and Cw groups of ECV304 cells by PCR-SSP method with the ligands MICA, MICB and ULBP1-3 of 2D The expression of KIR2DL1 in the expression of KIR2DL1 and the expression of KIR2DL2/3, KIR3DL1, and the expression of KIR2DL1/3 and KIR3DL1 in the ECV304 were significantly different from the expression of KIR2DL1/3 and KIR3DL1. There was a negative correlation between the expression rate of KIR2DL1 and the killing rate of NK cells on ECV304 (rs =-0.994, P = 0.000). B. The KIR2DL1 receptor on the NK surface is closed, and the killing rate is increased to a certain extent, and the NK surface KIR2DL1 receptor is used. The difference of anti-kill rate before and after antibody closure was statistically significant for t-test (t =-4.860, P = 0.002). 3. Individual KIR The expression rate of 2DL1 affects the killing activity of NK cells against ECV304. The known NKG2D ligands, MICA/ B, ULBP1- 3 Not involved The anti-killing of NK cells on ECV304 cells.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2488749
[Abstract]:Background and Purpose The N-K cells are a class of special killer cells in the lymphocytes. The activation of the anti-kill activity is not required to be stimulated, nor is the antibody involved. The effect of NK cells on the target cell, which is balanced between the activated receptor and the inhibitory receptor on its surface. wherein the NKG2D conductive activation signal, the ligand comprises a MICA/ MICB, a ULBP1-3 molecule, a KIR conduction inhibition signal, the ligand is an HLA-I class, Whether the transplanted tissue organ expresses the HLA molecule that is not recognized by the NK cell KIR, which can affect the killing of the NK cells on the tissue cells. The effect of allogenic NK cells on the anti-killing of target cells and the hepatic vein occlusion in bone marrow transplantation and the rejection of organ transplantation The vascular endothelial cells are between the donor-circulating cells. The first barrier. The circulating host cell can directly identify the xenogenic vascular endothelial cells to initiate an immune attack, or interact with the antigen presenting cell on the graft antigen and the vascular endothelial cell to damage the endothelial cells, thereby causing the graft to circulate a barrier, resulting in a shift the plants are repelled In this study, the human umbilical vein endothelial cell system was used as the target cell, and the anti-killing effect of the allogenic NK cells on the endothelial cells was discussed. number system The first chapter is to detect the HLA-A, B, Cw and NKG2D ligands MICA/ B in the ECV304 cells. The expression of ULBP1-3 in the gene level was used to extract the DNA of the ECV304 cell with the QIAampDNA extraction reagent. The HLA typing of the ECV304 cell was detected by a sequence-specific primer amplification method (PCR-SSP), and the ECV was detected by RT-PCR. Ligands MICA, MICB, ULBP1 of cell NKG2D The expression of NK cells KIR2DL1 in different individuals and the expression of ECV304 cells, K562 cells NKG2D ligand MICA/ B and U were detected in the second chapter. The expression rate of LBP1-3 and the expression of HLA-I were detected by PCR-SSP and flow cytometry. Expression of KIR2DL1 molecules on the surface of the gene and peripheral blood cells, and the ligands MICA, MICB, ULBP1, and UL of the ECV304 cell NKG2D were detected by flow cytometry. The expression of BP2, ULBP3 and HLA-I molecules was detected by flow cytometry. The expression rate of NKG2D ligand in CV304 cells was changed. In the third chapter, NK cells and LDH release in peripheral blood were isolated and the killing activity of NK cells at the target ratio of 20:1 to ECV304 cells was determined. NK cell with anti-KIR2DL1mAb The NK cells of 8 healthy individuals were isolated and purified by the immunomagnetic bead method. NK cells were isolated from the peripheral blood of 8 healthy donors. The expression rate of KIR2DL1 was detected by flow cytometry. The cytotoxicity of NK cells to the cells of ECV304 and K562 cells was determined by means of the LDH release method. and The effect of anti-KIR2DL1mAb on the killing activity of NK cells was investigated. B, Cw classification and the expression of NKG2D ligand MICA/ B and ULBP1-3 in the gene level 1. ECV304 cell HLA genotype: PCR-SSP method detection ECV304 cells HLA-A, B, Cw genotype A1,-; B18,-; Cw5,-. 2.RT-PCR were used to detect the expression of NKG2D ligand in ECV304 cells: the results of RT-PCR showed that ECV304 cells were in m The expression of NKG2D, MICA, MICB, ULBP1, ULBP2 and ULBP3 were all expressed in the RNA, and the expression of KIR2DL1 in the peripheral blood cells of different individuals was detected by the second chapter. Cell, K562 cell NKG2D ligand MICA/ B, ULBP1-3 expression rate, HLA-I type molecule expression rate 1. The expression rate of KIR2DL1 was detected by different individuals: HLA score The results showed that ECV304 expressed KIR2DL1 and did not express the ligand of KIR2DL2/3 and KIR3DL1. KIR2DL1 was expressed and the expression rate of KIR2DL1 on the surface of NK cells was significantly different from 6.2% to 46.2%. There was a mismatch between the HLA-A and B molecules on the surface of IR2DL2/3, KIR3DL1 and ECV304. The expression rate of NKG2D ligand MICA/ B, ULBP1-3 and HLA-I was 97.5% in the surface of CV304 cells. The surface of ECV304 cells after 24 hours of LPS action did not express NKG2D ligand MICA, MICB, ULBP1-3.4. K562 cell surface, MICA/ B, ULB, The expression rates of P1, ULBP2 and ULBP3 were 61.5%, 34.4%, 36.7%, 21.8%, respectively. The anti-killing activity of NK cells against ECV304 cells at the target ratio of 20:1 was determined by the release method. The effect of KIR2DL1mAb on NK cell killing activity was more than 90.0%. The expression rate of NK cell KIR2DL1 in 8 healthy donors was significantly different, and the expression rate of KIR2DL1 was 6.2%-46.2 for ECV304 cells. In%, the cytotoxicity of NK cells to ECV304 cells was 1.2%-28.0%, and anti-KIR2DL1 mAb was used to close the corresponding molecules on the surface of NK cells. The difference in wound activity was statistically significant (t =-4.860, P = 0.002), anti-KIR2DL1 mAb enhanced NK cell response to ECV304. kill There was a negative correlation between the expression rate of KIR2DL1 and the killing rate of NK cells in ECV304 (rs =-0.9). Conclusion 1. ECV304 cells express MICA/ B and ULBP1-3 at the mRNA level, but the expression of the above-mentioned molecules is not detected at the level of the protein. The results of flow cytometry show that the surface of the ECV304 cells after 24 hours after the action of 1.63 ug/ ml of CSA and 2 ug/ ml does not express the NKG. Detection of the HLA-A, B and Cw groups of ECV304 cells by PCR-SSP method with the ligands MICA, MICB and ULBP1-3 of 2D The expression of KIR2DL1 in the expression of KIR2DL1 and the expression of KIR2DL2/3, KIR3DL1, and the expression of KIR2DL1/3 and KIR3DL1 in the ECV304 were significantly different from the expression of KIR2DL1/3 and KIR3DL1. There was a negative correlation between the expression rate of KIR2DL1 and the killing rate of NK cells on ECV304 (rs =-0.994, P = 0.000). B. The KIR2DL1 receptor on the NK surface is closed, and the killing rate is increased to a certain extent, and the NK surface KIR2DL1 receptor is used. The difference of anti-kill rate before and after antibody closure was statistically significant for t-test (t =-4.860, P = 0.002). 3. Individual KIR The expression rate of 2DL1 affects the killing activity of NK cells against ECV304. The known NKG2D ligands, MICA/ B, ULBP1- 3 Not involved The anti-killing of NK cells on ECV304 cells.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
【参考文献】
相关期刊论文 前5条
1 龚卫娟;KIR研究进展[J];国外医学(免疫学分册);2003年02期
2 肖宇宏;白云;宋敏;张利永;黄钢;;内毒素刺激人脐静脉内皮细胞免疫相关分子的表达[J];免疫学杂志;2006年02期
3 周广臣;内皮细胞与移植排斥反应[J];肾脏病与透析肾移植杂志;2002年04期
4 张彩娥,韩军艳,杨惠军,吴雄文,黄亚非,梁智辉,龚非力;血管内皮细胞(ECV304)表达的HLA-G1对NK细胞杀伤活性的抑制作用[J];细胞与分子免疫学杂志;2005年06期
5 梅家转;郭坤元;;自然杀伤细胞受体及抗肿瘤研究进展[J];医学综述;2006年16期
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