胞壁酰二肽—抗CD10偶聯(lián)物體外刺激記憶T淋巴細胞活化的研究
發(fā)布時間：2018-07-23 14:46
【摘要】：目的:探討免疫偶聯(lián)物胞壁酰二肽(muramyl dipeptide,MDP)-抗CD10偶聯(lián)物(MDP-Antibody,MDP-Ab)對已接種卡介苗的健康兒童外周血中記憶性T淋巴細胞活化的影響。方法:1、采用Ficoll-Hypaque法分離獲得健康兒童外周血單個核細胞(Peripheral blood mononuclear cell,PBMC),10%胎牛血清的RPMI-1640培養(yǎng)液培養(yǎng),分為3組:對照組(PBMC 1×106/m L+抗人CD28單克隆抗體0.5μg/m L)、MDP-Ab組(抗人CD28單克隆抗體+MDP-Ab 20μg/m L)、卡介苗(bacillus calmette-guerin,BCG)組(抗人CD28單克隆抗體+BCG 20μg/m L),光學顯微鏡下觀察細胞生長情況及細胞形態(tài);分別于0、12、24、48、72小時收集細胞上清液,ELISA方法檢測上清液中干擾素γ(interferon-γ,IFN-γ)水平。2、采集健康兒童外周血與RPMI-1640培養(yǎng)液等體積稀釋,分為4組:對照組、MDP-Ab組、BCG組及PHA組(抗人CD28單克隆抗體+PHA 20μg/m L),48小時后收集細胞,流式細胞儀檢測細胞表面CD3、CD4、CD45RA、CD69及胞內(nèi)IFN-γ的表達。結(jié)果:1、細胞生長及細胞形態(tài):與對照組相比,培養(yǎng)24小時后MDP-Ab組及BCG組細胞出現(xiàn)細胞聚集,細胞團中細胞形態(tài)可辨別。隨培養(yǎng)時間延長細胞團的數(shù)量均增多,以BCG組最為明顯。2、IFN-γ水平:對照組在0、12、24、48、72小時細胞上清液中IFN-γ水平分別為(401.76±17.10)pg/ml、(374.64±24.30)pg/ml、(376.53±28.96)pg/ml、(380.02±29.15)pg/ml、(390.17±33.64)pg/ml,MDP-Ab組在不同培養(yǎng)時間下細胞上清液中IFN-γ水平分別為(402.04±23.22)pg/ml、(421.49±24.09)pg/ml、(456.76±41.99)pg/ml、(479.81±21.43)pg/ml、(449.10±13.66)pg/ml。兩因素重復測量資料的方差分析顯示,MDP-Ab刺激后IFN-γ的水平明顯高于對照組(F刺激=13.19,p=0.02),而培養(yǎng)時間對IFN-γ的水平無顯著影響(F時間=1.65,p=0.21);處理因素與培養(yǎng)時間之間存在交互作用(F交互=3.51,p=0.03);對不同時間點兩組間IFN-γ的水平進行t檢驗,MDP-Ab組IFN-γ水平在12h、24h、48h均顯著高于對照組(p均0.05)。BCG組在不同培養(yǎng)時間下細胞上清液中IFN-γ水平分別為(405.34±30.48)pg/ml、(404.98.70±37.08)pg/ml、(444.33±22.94)pg/ml、(502.20±15.52)pg/ml、(517.84±21.54)pg/ml,兩因素重復測量資料的方差分析顯示,BCG組IFN-γ的水平明顯高于對照組(F刺激=5.49,p=0.00),且培養(yǎng)時間對IFN-γ水的水平有顯著影響(F時間=26.60,p=0.00),處理因素與培養(yǎng)時間之間存在交互作用(F交互=5.49,p=0.00);對不同時間點兩組間IFN-γ的水平進行t檢驗,BCG組IFN-γ水平在24h、48h、72h均顯著高于對照組(p均0.05)。3、記憶T淋巴細胞胞內(nèi)IFN-γ表達情況:CD3+CD4+CD45RA-IFN-γ+細胞比例:對照組、MDP-Ab組、BCG組、PHA組分別為(0.010±0.017)%、(0.61±0.20)%、(1.05±0.45)%、(0.27±0.19)%。與對照組相比,MDP-Ab組的CD3+CD4+CD45RAIFN-γ+細胞比例明顯升高(t=5.183,p=0.034),而BCG組及PHA組與對照組相比無統(tǒng)計學差異(p均0.05)。各組表型為CD3+CD4-CD45RA-IFN-γ+的T淋巴細胞均很少甚至不表達。4、記憶T淋巴細胞表面CD69表達情況:⑴CD3+CD4+CD45RA-CD69+細胞比例:對照組、MDP-Ab組、BCG組、PHA組分別為(4.70±1.16)%、(8.99±2.75)%、(13.69±2.79)%、(8.44±3.25)%,MDP-Ab組及BCG組CD3+CD4+CD45RA-CD69+細胞比例均明顯高于對照組(p均0.05),而PHA組與對照組相比無統(tǒng)計學差異(t=1.938,p=0.179);⑵CD3+CD4-CD45RA-CD69+細胞比例:對照組、MDP-Ab組、BCG組、PHA組分別為(1.41±0.31)%、(3.91±1.73)%、(6.76±3.28)%、(5.15±1.79)%,MDP-Ab組及BCG組CD3+CD4-CD45RA-CD69+細胞比例均明顯高于對照組(p均0.05),而PHA組與對照組相比無統(tǒng)計學差異(t=3.601,p=0.067)。⑶CD3+CD4+CD45RA-CD69+與CD3+CD4-CD45RA-CD69+細胞比例比較:MDP-Ab、BCG兩組CD3+CD4+CD45RA-CD69+細胞比例分別為(8.99±2.75)%、(13.69±2.79)%,而CD3+CD4-CD45RA-CD69+細胞比例為(3.91±1.73)%、(6.76±3,28)%,兩組CD4+活化的記憶性T細胞比例均明顯高于CD4-活化的記憶性T細胞比例(p均0.05)。結(jié)論:MDP-Ab能夠體外刺激已接種卡介苗的健康兒童記憶性T淋巴細胞分泌IFN-γ,誘導其表達早期活化標志CD69,提示MDP-Ab可以在體外誘導記憶T淋巴細胞的活化,且細胞活化在CD4+細胞中表現(xiàn)更為顯著。這種活化效應和BCG刺激后發(fā)生的免疫反應相似。這為該偶聯(lián)物應用于白血病的免疫治療提供進一步的理論支持。
[Abstract]:Objective: To investigate the effect of muramyl dipeptide (MDP) - anti CD10 conjugate (MDP-Antibody, MDP-Ab) on the activation of memory T lymphocyte in peripheral blood of BCG healthy children. Methods: 1, to separate the peripheral blood mononuclear cells of healthy children by Ficoll-Hypaque method (Peripheral blood mononuclear). Cell, PBMC), the culture of RPMI-1640 culture of 10% fetal bovine serum was divided into 3 groups: the control group (PBMC 1 x 106/m L+ anti human CD28 monoclonal antibody 0.5 u g/m L), MDP-Ab group (anti human CD28 monoclonal antibody +MDP-Ab 20 micron), and the optical microscope observation of cells under optical microscope Growth and cell morphology, cell supernatant was collected at 0,12,24,48,72 hours, and ELISA method was used to detect.2 of interferon gamma (interferon- gamma, IFN- gamma) in the supernatant. The volume dilution of peripheral blood and RPMI-1640 medium in healthy children was collected and divided into 4 groups: control group, MDP-Ab group, BCG group and PHA group (anti human CD28 monoclonal antibody +PHA 20 mu g/m) After 48 hours, cells were collected and flow cytometry was used to detect the expression of CD3, CD4, CD45RA, CD69 and intracellular IFN- gamma on cell surface. Results: 1, cell growth and cell morphology: compared with the control group, cells in the MDP-Ab and BCG groups appeared to be aggregated after 24 hours of culture, and the cell morphology could be identified. The number of cell groups increased with the time of culture. The most obvious.2, IFN- gamma level in the BCG group: the level of IFN- gamma in the cell supernatant of the control group was (401.76 + 17.10) pg/ml, (374.64 + 24.30) pg/ml, (376.53 + 28.96) pg/ml, (380.02 + 29.15) pg/ml, (390.17 + 33.64) pg/ml, and MDP-Ab group was (402.04 + 23) in the cell supernatant at different incubation times, respectively. .22) pg/ml, (421.49 + 24.09) pg/ml, (456.76 + 41.99) pg/ml, (479.81 + 21.43) pg/ml, (449.10 + 13.66) pg/ml. two factors of repeated measurements of variance analysis showed that the level of IFN- gamma after MDP-Ab stimulation was significantly higher than that of the control group (F stimulates =13.19, p=0.02), and the incubation time had no significant effect on the level of IFN- gamma. Treatment factors There was a interaction between the culture time and the incubation time (F interaction =3.51, p=0.03); the level of IFN- gamma in two groups at different time points was examined by t. The level of IFN- gamma in group MDP-Ab was significantly higher than that of the control group (P all 0.05) in the cell supernatant of the.BCG group (405.34 + 30.48), respectively (405.34 + 30.48). L, (444.33 + 22.94) pg/ml, (502.20 + 15.52) pg/ml, (517.84 + 21.54) pg/ml, and the variance analysis of the repeated measurements of two factors showed that the level of IFN- gamma in the BCG group was significantly higher than that of the control group (F stimulated =5.49, p=0.00), and the incubation time had a significant influence on the level of IFN- gamma water (F =26.60,), and there was an interaction between the processing factors and the incubation time. The effect (F interaction =5.49, p=0.00), t test on the level of IFN- gamma between two groups at different time points, BCG group IFN- gamma level in 24h, 48h, 72h were significantly higher than that of the control group (P 0.05).3, the proportion of gamma + cells in the memory lymphocyte was (0.010 + 0.017)% respectively (0.010 + 0.017), respectively, (0.61 + 0.). 20)%, (1.05 + 0.45)%, (0.27 + 0.19)%. Compared with the control group, the proportion of CD3+CD4+CD45RAIFN- gamma + cells in the MDP-Ab group was significantly higher (t=5.183, p=0.034), but there was no statistical difference between the BCG group and the PHA group (P 0.05). The T lymphocyte of each group was CD3+CD4-CD45RA-IFN- y + with little or no.4, and the memory T lymphocyte surface CD 69 expression: (1) CD3+CD4+CD45RA-CD69+ cell ratio: the control group, the MDP-Ab group, the BCG group and the PHA group were (4.70 + 1.16)%, (8.99 + 2.75)%, (13.69 + 2.79)%, (8.44 + 3.25)%, and the proportion of CD3+CD4+CD45RA-CD69+ cells in MDP-Ab and BCG groups were significantly higher than those in the control group (P 0.05), but there was no statistical difference between the PHA group and the control group (t=1.938, p=0.179). The proportion of CD3+CD4-CD45RA-CD69+ cells: the control group, the MDP-Ab group, the BCG group and the PHA group were (1.41 + 0.31)%, (3.91 + 1.73)%, (6.76 + 3.28)%, (5.15 + 1.79)%, and the proportion of CD3+CD4-CD45RA-CD69+ cells in MDP-Ab and BCG groups were significantly higher than those in the control group (P 0.05), but there was no statistical difference between the group PHA and the control group (t=3.601, p=0.067). 3 The ratio of -CD69+ to CD3+CD4-CD45RA-CD69+ cells: the proportion of CD3+CD4+CD45RA-CD69+ cells in MDP-Ab and BCG two groups was (8.99 + 2.75)%, (13.69 + 2.79)%, and the proportion of CD3+CD4-CD45RA-CD69+ cells was (3.91 + 1.73)%, (6.76 + 3,28)%. The proportion of memory T cells activated by CD4+ in two groups was significantly higher than that of CD4- activated memory T cells (0 P 0) 5). Conclusion: MDP-Ab can stimulate the memory T lymphocytes of healthy children who have been inoculated with BCG in vitro to secrete IFN- gamma and induce the expression of the early activation marker CD69, suggesting that MDP-Ab can induce the activation of memory T lymphocytes in vitro, and the activation of cell activation in CD4+ cells is more significant. This activation effect and the immunization of BCG after stimulation. The disease response is similar, which provides further theoretical support for the application of the conjugate in leukemia immunotherapy.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.7
本文編號：2139737
[Abstract]:Objective: To investigate the effect of muramyl dipeptide (MDP) - anti CD10 conjugate (MDP-Antibody, MDP-Ab) on the activation of memory T lymphocyte in peripheral blood of BCG healthy children. Methods: 1, to separate the peripheral blood mononuclear cells of healthy children by Ficoll-Hypaque method (Peripheral blood mononuclear). Cell, PBMC), the culture of RPMI-1640 culture of 10% fetal bovine serum was divided into 3 groups: the control group (PBMC 1 x 106/m L+ anti human CD28 monoclonal antibody 0.5 u g/m L), MDP-Ab group (anti human CD28 monoclonal antibody +MDP-Ab 20 micron), and the optical microscope observation of cells under optical microscope Growth and cell morphology, cell supernatant was collected at 0,12,24,48,72 hours, and ELISA method was used to detect.2 of interferon gamma (interferon- gamma, IFN- gamma) in the supernatant. The volume dilution of peripheral blood and RPMI-1640 medium in healthy children was collected and divided into 4 groups: control group, MDP-Ab group, BCG group and PHA group (anti human CD28 monoclonal antibody +PHA 20 mu g/m) After 48 hours, cells were collected and flow cytometry was used to detect the expression of CD3, CD4, CD45RA, CD69 and intracellular IFN- gamma on cell surface. Results: 1, cell growth and cell morphology: compared with the control group, cells in the MDP-Ab and BCG groups appeared to be aggregated after 24 hours of culture, and the cell morphology could be identified. The number of cell groups increased with the time of culture. The most obvious.2, IFN- gamma level in the BCG group: the level of IFN- gamma in the cell supernatant of the control group was (401.76 + 17.10) pg/ml, (374.64 + 24.30) pg/ml, (376.53 + 28.96) pg/ml, (380.02 + 29.15) pg/ml, (390.17 + 33.64) pg/ml, and MDP-Ab group was (402.04 + 23) in the cell supernatant at different incubation times, respectively. .22) pg/ml, (421.49 + 24.09) pg/ml, (456.76 + 41.99) pg/ml, (479.81 + 21.43) pg/ml, (449.10 + 13.66) pg/ml. two factors of repeated measurements of variance analysis showed that the level of IFN- gamma after MDP-Ab stimulation was significantly higher than that of the control group (F stimulates =13.19, p=0.02), and the incubation time had no significant effect on the level of IFN- gamma. Treatment factors There was a interaction between the culture time and the incubation time (F interaction =3.51, p=0.03); the level of IFN- gamma in two groups at different time points was examined by t. The level of IFN- gamma in group MDP-Ab was significantly higher than that of the control group (P all 0.05) in the cell supernatant of the.BCG group (405.34 + 30.48), respectively (405.34 + 30.48). L, (444.33 + 22.94) pg/ml, (502.20 + 15.52) pg/ml, (517.84 + 21.54) pg/ml, and the variance analysis of the repeated measurements of two factors showed that the level of IFN- gamma in the BCG group was significantly higher than that of the control group (F stimulated =5.49, p=0.00), and the incubation time had a significant influence on the level of IFN- gamma water (F =26.60,), and there was an interaction between the processing factors and the incubation time. The effect (F interaction =5.49, p=0.00), t test on the level of IFN- gamma between two groups at different time points, BCG group IFN- gamma level in 24h, 48h, 72h were significantly higher than that of the control group (P 0.05).3, the proportion of gamma + cells in the memory lymphocyte was (0.010 + 0.017)% respectively (0.010 + 0.017), respectively, (0.61 + 0.). 20)%, (1.05 + 0.45)%, (0.27 + 0.19)%. Compared with the control group, the proportion of CD3+CD4+CD45RAIFN- gamma + cells in the MDP-Ab group was significantly higher (t=5.183, p=0.034), but there was no statistical difference between the BCG group and the PHA group (P 0.05). The T lymphocyte of each group was CD3+CD4-CD45RA-IFN- y + with little or no.4, and the memory T lymphocyte surface CD 69 expression: (1) CD3+CD4+CD45RA-CD69+ cell ratio: the control group, the MDP-Ab group, the BCG group and the PHA group were (4.70 + 1.16)%, (8.99 + 2.75)%, (13.69 + 2.79)%, (8.44 + 3.25)%, and the proportion of CD3+CD4+CD45RA-CD69+ cells in MDP-Ab and BCG groups were significantly higher than those in the control group (P 0.05), but there was no statistical difference between the PHA group and the control group (t=1.938, p=0.179). The proportion of CD3+CD4-CD45RA-CD69+ cells: the control group, the MDP-Ab group, the BCG group and the PHA group were (1.41 + 0.31)%, (3.91 + 1.73)%, (6.76 + 3.28)%, (5.15 + 1.79)%, and the proportion of CD3+CD4-CD45RA-CD69+ cells in MDP-Ab and BCG groups were significantly higher than those in the control group (P 0.05), but there was no statistical difference between the group PHA and the control group (t=3.601, p=0.067). 3 The ratio of -CD69+ to CD3+CD4-CD45RA-CD69+ cells: the proportion of CD3+CD4+CD45RA-CD69+ cells in MDP-Ab and BCG two groups was (8.99 + 2.75)%, (13.69 + 2.79)%, and the proportion of CD3+CD4-CD45RA-CD69+ cells was (3.91 + 1.73)%, (6.76 + 3,28)%. The proportion of memory T cells activated by CD4+ in two groups was significantly higher than that of CD4- activated memory T cells (0 P 0) 5). Conclusion: MDP-Ab can stimulate the memory T lymphocytes of healthy children who have been inoculated with BCG in vitro to secrete IFN- gamma and induce the expression of the early activation marker CD69, suggesting that MDP-Ab can induce the activation of memory T lymphocytes in vitro, and the activation of cell activation in CD4+ cells is more significant. This activation effect and the immunization of BCG after stimulation. The disease response is similar, which provides further theoretical support for the application of the conjugate in leukemia immunotherapy.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.7
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