同種異體NK細胞對人臍靜脈內皮細胞的殺傷活性差異及分子機制探討
發(fā)布時間：2019-05-30 09:57
【摘要】： 背景與目的 N K細胞是淋巴細胞中的一類特殊殺傷細胞,其殺傷活性的啟動既不需抗原刺激,亦不需抗體參與,具有排斥異己細胞的作用。NK細胞對靶細胞的殺傷作用,受其表面的激活性受體和抑制性受體之間平衡的調節(jié)。其中NKG2D傳導活化信號,配體包括MICA/MICB,ULBP1-3分子;KIR傳導抑制信號,其配體為HLA-I類分子。移植的組織器官是否表達異體NK細胞KIR不能識別的HLA分子,會影響NK細胞對組織細胞的殺傷活性。同種異體NK細胞對靶細胞殺傷與骨髓移植中的肝靜脈血管閉塞病(veno-occlusive disease of the liver VOD)及器官移植排斥反應有關系。 血管內皮細胞是供受體循環(huán)細胞之間的第一道屏障。循環(huán)的宿主淋巴細胞可直接識別異種血管內皮細胞而發(fā)動免疫攻擊,或與移植物抗原及血管內皮上的抗原遞呈細胞相互作用,損傷內皮細胞,從而使移植物血液循環(huán)障礙,最終導致移植物被排斥,移植失敗。 本研究以人臍靜脈內皮細胞系做為靶細胞,探討同種異體NK細胞對內皮細胞是否有殺傷作用,如果有殺傷,再進一步研究參與殺傷的活化性及抑制性信號系統(tǒng)的分子機制。 方法 第一章檢測ECV304細胞HLA-A、B、Cw分型及NKG2D配體MICA/B、ULBP1-3在基因水平的表達 以QIAampDNA抽提試劑提取ECV304細胞的DNA;ECV304細胞HLA分型采用序列特異性引物擴增法(sequence specific primer polymerase chainreproduction,PCR-SSP),RT-PCR檢測ECV304細胞NKG2D的配體MICA、MICB、ULBP1、ULBP2、ULBP3的表達情況。 第二章檢測不同個體NK細胞KIR2DL1的表達及ECV304細胞、K562細胞NKG2D配體MICA/B、ULBP1-3表達率、HLA-Ⅰ類分子表達率 通過PCR-SSP法及流式細胞儀分別檢測8例健康者基因及外周血細胞表面KIR2DL1類分子的表達,用流式細胞儀檢測ECV304細胞NKG2D的配體MICA、MICB、ULBP1、ULBP2、ULBP3及HLA-Ⅰ類分子的表達情況。用流式細胞儀檢測半數抑制濃度(50%inhibitory concentration,IC50)濃度的環(huán)孢素A(cyclosporinA,CSA)及2ug/ml內毒素(Lipopolysaccharide,LPS)作用24小時后的ECV304細胞NKG2D配體的表達率是否有變化。 第三章分離外周血NK細胞、LDH釋放法測定NK細胞在效靶比20:1時對ECV304細胞的殺傷活性及anti-KIR2DL1mAb對NK細胞殺傷活性的影響 免疫磁珠法分離純化8例健康者的NK細胞。自8例健康供者分離外周血NK細胞,流式細胞儀檢測KIR2DL1的表達率,LDH釋放法測定NK細胞在效靶比20:1時對ECV304細胞、K562細胞的殺傷活性及anti-KIR2DL1mAb對NK細胞殺傷活性的影響。 結果 第一章檢測ECV304細胞HLA-A、B、Cw分型及NKG2D配體MICA/B、ULBP1-3在基因水平的表達 1.ECV304細胞HLA基因型:PCR-SSP法檢測ECV304細胞HLA-A,B,Cw基因型為A1,-;B18,-;Cw5,-。 2.RT-PCR檢測ECV304細胞NKG2D配體的表達:RT-PCR檢測結果顯示,ECV304細胞在mRNA均表達NKG2D的配體MICA、MICB、ULBP1、ULBP2、ULBP3。 第二章檢測不同個體外周血細胞KIR2DL1的表達ECV304細胞、K562細胞NKG2D配體MICA/B、ULBP1-3表達率、HLA-Ⅰ類分子表達率 1.不同個體檢測KIR2DL1的表達率:HLA分型表明,ECV304表達KIR2DL1的配體,而不表達KIR2DL2/3、KIR3DL1的配體。PCR-SSP法檢測結果顯示,8例健康者在基因水平均表達KIR2DL1,并且NK細胞表面KIR2DL1表達率有較大差異,從6.2%-46.2%不等。KIR2DL2/3、KIR3DL1與ECV304細胞表面相應的HLA-A、B類分子間存在錯配現象。 2.流式細胞儀檢測結果表明ECV304細胞表面不表達NKG2D配體MICA/B、ULBP1-3,HLA-Ⅰ分子表達率為97.5%。 3.流式細胞儀檢測結果顯示1.63ug/ml CSA及2ug/mlLPS作用24小時后的ECV304細胞表面不表達NKG2D的配體MICA、MICB、ULBP1-3。 4.K562細胞表面MICA/B、ULBP1、ULBP2、ULBP3的表達率分別為61.5%、34.4%、36.7%、21.8%,不表達HLA-Ⅰ類分子。 第三章分離外周血NK細胞、LDH釋放法測定NK細胞在效靶比20:1時對ECV304細胞的殺傷活性及anti-KIR2DL1mAb對NK細胞殺傷活性的影響 流式細胞儀檢測CD3~-CD16~+CD56~+細胞的純度大于90.0%。 8例健康供者NK細胞KIR2DL1表達率有較大差異,對ECV304細胞的殺傷活性也有不同,(效靶比20:1)KIR2DL1表達率為6.2%-46.2%時,NK細胞對ECV304細胞的殺傷活性分別為1.2%-28.0%;anti-KIR2DL1 mAb對NK細胞表面相應分子進行封閉,配對樣本t檢驗比較抗體封閉前后NK細胞對ECV304細胞的殺傷活性相比差異有統(tǒng)計學意義(t=-4.860,P=0.002),anti-KIR2DL1 mAb增強NK細胞對ECV304的殺傷活性。雙變量相關分析示個體KIR2DL1表達率與NK細胞對ECV304的殺傷率存在負相關(rs=-0.994,P=0.000)。 結論 1.ECV304細胞在mRNA水平表達MICA/B,ULBP1-3,但在蛋白水平未檢測到上述分子的表達。流式細胞儀檢測結果顯示1.63ug/ml CSA及2ug/ml作用24小時后的ECV304細胞表面不表達NKG2D的配體MICA、MICB、ULBP1-3。PCR-SSP法檢測ECV304細胞HLA-A,B,Cw基因型為Al,-;B18,-;Cw5,-。HLA分型表明,ECV304表達KIR2DL1的配體,而不表達KIR2DL2/3、KIR3DL1的配體。 2.8例健康供者NK細胞KIR2DL1表達率有較大差異,對ECV304細胞的殺傷活性也有不同,雙變量相關分析示個體KIR2DL1表達率與NK細胞對ECV304的殺傷率存在負相關(rs=-0.994,P=0.000)。以anti-KIR2DL1 mAb封閉NK表面KIR2DL1受體,殺傷率均有不同程度的上升,用配對樣本t檢驗,抗體封閉前后殺傷率差異有統(tǒng)計學意義(t=-4.860,P=0.002)。 3.個體KIR2DL1表達率影響NK細胞對ECV304殺傷活性。目前已知的NKG2D配體MICA/B、ULBP1-3不參與NK細胞對ECV304細胞的殺傷。 本研究的創(chuàng)新點 本研究結果顯示,同種異體NK細胞對靜脈內皮細胞有殺傷作用,HLA-KIR分子錯配是殺傷主要分子機制。 研究價值 根據供受者的HLA-KIR分子的表達狀態(tài)和匹配程度選擇供者。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392
本文編號：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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392
【參考文獻】
相關期刊論文 前5條
1 龔衛(wèi)娟;KIR研究進展[J];國外醫(yī)學(免疫學分冊);2003年02期
2 肖宇宏;白云;宋敏;張利永;黃鋼;;內毒素刺激人臍靜脈內皮細胞免疫相關分子的表達[J];免疫學雜志;2006年02期
3 周廣臣;內皮細胞與移植排斥反應[J];腎臟病與透析腎移植雜志;2002年04期
4 張彩娥,韓軍艷,楊惠軍,吳雄文,黃亞非,梁智輝,龔非力;血管內皮細胞(ECV304)表達的HLA-G1對NK細胞殺傷活性的抑制作用[J];細胞與分子免疫學雜志;2005年06期
5 梅家轉;郭坤元;;自然殺傷細胞受體及抗腫瘤研究進展[J];醫(yī)學綜述;2006年16期
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