胞壁酰二肽—抗CD10偶联物体外刺激记忆T淋巴细胞活化的研究
发布时间：2018-07-23 14:46
【摘要】：目的:探讨免疫偶联物胞壁酰二肽(muramyl dipeptide,MDP)-抗CD10偶联物(MDP-Antibody,MDP-Ab)对已接种卡介苗的健康儿童外周血中记忆性T淋巴细胞活化的影响。方法:1、采用Ficoll-Hypaque法分离获得健康儿童外周血单个核细胞(Peripheral blood mononuclear cell,PBMC),10%胎牛血清的RPMI-1640培养液培养,分为3组:对照组(PBMC 1×106/m L+抗人CD28单克隆抗体0.5μg/m L)、MDP-Ab组(抗人CD28单克隆抗体+MDP-Ab 20μg/m L)、卡介苗(bacillus calmette-guerin,BCG)组(抗人CD28单克隆抗体+BCG 20μg/m L),光学显微镜下观察细胞生长情况及细胞形态;分别于0、12、24、48、72小时收集细胞上清液,ELISA方法检测上清液中干扰素γ(interferon-γ,IFN-γ)水平。2、采集健康儿童外周血与RPMI-1640培养液等体积稀释,分为4组:对照组、MDP-Ab组、BCG组及PHA组(抗人CD28单克隆抗体+PHA 20μg/m L),48小时后收集细胞,流式细胞仪检测细胞表面CD3、CD4、CD45RA、CD69及胞内IFN-γ的表达。结果:1、细胞生长及细胞形态:与对照组相比,培养24小时后MDP-Ab组及BCG组细胞出现细胞聚集,细胞团中细胞形态可辨别。随培养时间延长细胞团的数量均增多,以BCG组最为明显。2、IFN-γ水平:对照组在0、12、24、48、72小时细胞上清液中IFN-γ水平分别为(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组在不同培养时间下细胞上清液中IFN-γ水平分别为(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。两因素重复测量资料的方差分析显示,MDP-Ab刺激后IFN-γ的水平明显高于对照组(F刺激=13.19,p=0.02),而培养时间对IFN-γ的水平无显著影响(F时间=1.65,p=0.21);处理因素与培养时间之间存在交互作用(F交互=3.51,p=0.03);对不同时间点两组间IFN-γ的水平进行t检验,MDP-Ab组IFN-γ水平在12h、24h、48h均显著高于对照组(p均0.05)。BCG组在不同培养时间下细胞上清液中IFN-γ水平分别为(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,两因素重复测量资料的方差分析显示,BCG组IFN-γ的水平明显高于对照组(F刺激=5.49,p=0.00),且培养时间对IFN-γ水的水平有显著影响(F时间=26.60,p=0.00),处理因素与培养时间之间存在交互作用(F交互=5.49,p=0.00);对不同时间点两组间IFN-γ的水平进行t检验,BCG组IFN-γ水平在24h、48h、72h均显著高于对照组(p均0.05)。3、记忆T淋巴细胞胞内IFN-γ表达情况:CD3+CD4+CD45RA-IFN-γ+细胞比例:对照组、MDP-Ab组、BCG组、PHA组分别为(0.010±0.017)%、(0.61±0.20)%、(1.05±0.45)%、(0.27±0.19)%。与对照组相比,MDP-Ab组的CD3+CD4+CD45RAIFN-γ+细胞比例明显升高(t=5.183,p=0.034),而BCG组及PHA组与对照组相比无统计学差异(p均0.05)。各组表型为CD3+CD4-CD45RA-IFN-γ+的T淋巴细胞均很少甚至不表达。4、记忆T淋巴细胞表面CD69表达情况:⑴CD3+CD4+CD45RA-CD69+细胞比例:对照组、MDP-Ab组、BCG组、PHA组分别为(4.70±1.16)%、(8.99±2.75)%、(13.69±2.79)%、(8.44±3.25)%,MDP-Ab组及BCG组CD3+CD4+CD45RA-CD69+细胞比例均明显高于对照组(p均0.05),而PHA组与对照组相比无统计学差异(t=1.938,p=0.179);⑵CD3+CD4-CD45RA-CD69+细胞比例:对照组、MDP-Ab组、BCG组、PHA组分别为(1.41±0.31)%、(3.91±1.73)%、(6.76±3.28)%、(5.15±1.79)%,MDP-Ab组及BCG组CD3+CD4-CD45RA-CD69+细胞比例均明显高于对照组(p均0.05),而PHA组与对照组相比无统计学差异(t=3.601,p=0.067)。⑶CD3+CD4+CD45RA-CD69+与CD3+CD4-CD45RA-CD69+细胞比例比较:MDP-Ab、BCG两组CD3+CD4+CD45RA-CD69+细胞比例分别为(8.99±2.75)%、(13.69±2.79)%,而CD3+CD4-CD45RA-CD69+细胞比例为(3.91±1.73)%、(6.76±3,28)%,两组CD4+活化的记忆性T细胞比例均明显高于CD4-活化的记忆性T细胞比例(p均0.05)。结论:MDP-Ab能够体外刺激已接种卡介苗的健康儿童记忆性T淋巴细胞分泌IFN-γ,诱导其表达早期活化标志CD69,提示MDP-Ab可以在体外诱导记忆T淋巴细胞的活化,且细胞活化在CD4+细胞中表现更为显著。这种活化效应和BCG刺激后发生的免疫反应相似。这为该偶联物应用于白血病的免疫治疗提供进一步的理论支持。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
本文编号：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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
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