胞壁酰二肽—抗CD10偶联物免疫导向治疗儿童急性B淋巴细胞性白血病的研究

发布时间：2018-05-08 05:16

本文选题：胞壁酰二肽 + 抗CD10单克隆抗体　；参考：《青岛大学》2012年博士论文

【摘要】：目的靶向性治疗是一种潜在的有效抗白血病治疗方法,尤其在清除微小残留病、防止肿瘤复发方面发挥重要作用。本文将CD10单克隆抗体(anti-CD10 MAb)与胞壁酰二肽(MDP)连接合成新的免疫偶联物(MDP-Ab),观察免疫偶联物中MAb及MDP各自的生物活性。方法(1)MDP-Ab制备：采用化学合成方法将CD10单克隆抗体(anti-CD10 MAb)与胞壁酰二肽(MDP)连接合成新的免疫偶联物(MDP-Ab),硅胶柱及Sephacryl S-100凝胶柱分离纯化合成过程产生的各产物(PDP-arm-Boc、PDP-arm、PDP-MDP、IT-Ab及MDP-Ab);氢离子质谱仪、电喷雾电离质谱仪与紫外分光光度计分析合成中各产物的分子量(结果表达方式为MW+[H])及MDP-Ab中各分子比。以半胱氨酸标准曲线为准,采用Ellman's试剂分析IT-Ab中巯基数量；(2)MDP-Ab中抗体活性检测：分离收集CDl0+急性淋巴细胞性白血病细胞(阳性率≥95%),加入MDP-Ab共同孵育,采用间接流式细胞术检测MDP-Ab与CD10+细胞结合能力,以未偶联的抗CD10抗体作对照。(3) MDP-Ab中MDP活性检测：采用Ficoll-Hypaque法分离急性淋巴细胞性白血病患儿外周血单个核细胞,贴壁3h,悬浮细胞经尼龙膜柱筛选获得T淋巴细胞,冻存备用。取贴壁细胞分为6组,分别为：对照组(rhGM-CSF+rhIL-4)、未偶联的抗CD10抗体组(rhGM-CSF+rhIL-4+anti-CD10)、未偶联的MDP组(rhGM-CSF+rhIL-4+MDP)、MDP-Ab组(rhGM-CSF+rhIL-4+MDP-Ab)、脂多糖(LPS)组(rhGM-CSF+rhIL-4+LPS)及MDP-Ab+LPS组(rhGM-CSF+rhIL-4+MDP-Ab+LPS),培养8天,收集各组树突状细胞(DC),检测细胞免疫表型、内吞作用及细胞上清液中白介素12(IL-12)水平。将丝裂霉素C处理过的DC与急性淋巴细胞性白血病患儿同体的淋巴细胞按1：10比例混合培养72h,CFSE染色法检测各组淋巴细胞增殖情况,ELISA方法检测上清液IFN-Y水平。结果(1)氢离子质谱显示PDP-arm-Boc、PDP-arm、PDP-MDP的分子量结果(MW+[H])分别为372.1,272.1,746.4;根据半胱氨酸标准曲线,显示IT-Ab中巯基数量为4.5；电喷雾电离质谱与紫外线吸光度结果显示免疫偶联物中MDP-Ab分子量约为101KD,其中MDP与Ab分子比为2：1。(2)间接流式细胞术结果示MDP-Ab结合CD10抗原的阳性率为94.69%,与未偶联的抗CD10抗体相比,无显著性差异(p0.05)。(3)DC细胞免疫表型：MDP-Ab促进白血病患儿外周血DC表达HLA-DR、共刺激分子(CD80、CD86)及成熟分子(CD83)的阳性细胞均明显高于对照组、未偶联的抗CD10抗体组及未偶联的MDP组,但低于LPS组及MDP-Ab+LPS组(F=629.619,p=0.000)；(4)IL-12水平：对照组、未偶联的抗CD10抗体组及未偶联的MDP组分别为(42.37±5.83)pg/ml,(54.41±4.35)pg/ml,(60.75±5.06)pg/ml,而MDP-Ab组(80.63±3.73)pg/ml,LPS组(160.15±11.43)pg/ml,MDP-Ab+LPS组(190.33±6.61)pg/ml,明显高于对照组、未偶联的抗CD10抗体组及未偶联的MDP组(F=857.872,p=0.000)。MDP-Ab组与LPS组、MDP-Ab+LPS组相比,亦有显著性差异(p0.05)；(5)DC吞噬功能：对照组(即未成熟DC)为(81.3±10.1)%,而未偶联的抗CD10抗体组、未偶联的MDP组、MDP-Ab组、LPS及MDP-Ab+LPS组分别为(66.7±9.78)%,(62.5±6.72)%,(41.6±5.67)%,(33.9±3.42)%,(25.6±3.68)%,与对照组相比,有显著性统计学意义(F=383.04,p=0.000)。(6)混合淋巴细胞反应：与对照组、未偶联的抗CD10抗体组及未偶联的MDP组相比,MDP-Ab组、LPS组及MDP-Ab+LPS组阳性细胞明显增高,以MDP-Ab+LPS组为最高(F=393.36,p=0.000);(7)IFN-Y水平：对照组、未偶联的抗CD10抗体组及未偶联的MDP组分别为(67.72±3.94)pg/ml、(84.65±4.41)pg/ml、(89.69±3.02)pg/ml,而MDP-Ab组为(173.06±8.14)pg/ml,LPS组为(209.24±9.43)pg/ml,MDP-Ab+LPS组为(356.17±9.48)pg/ml。MDP-Ab组明显高于对照组、未偶联的抗CD10抗体组及未偶联的MDP组,而低于LPS组及MDP-Ab+LPS组(F=2497.18,p=0.000),且以MDP-Ab+LPS组水平最高。结论1、成功采用化学方法合成新免疫偶联物MDP-Ab 2、MDP-Ab保持抗体特异结合抗原的活性,提示MDP-Ab具有靶向结合CD10+细胞能力。 3、MDP-Ab能促进白血病患儿外周血树突状细胞表达高水平HLA-DR、共刺激分子(CD80/CD86)及CD83,分泌高水平IL-12,与细胞因子、脂多糖联合作用最强。 4、MDP-Ab诱导的树突状细胞能促进同体淋巴细胞增殖,分泌高水平IFN-Y分子,与细胞因子、脂多糖联合作用最强,提示MDP-Ab保持促进树突状细胞增殖及成熟活性。目的探讨胞壁酰二肽-抗CD10偶联物激活淋巴细胞的功能及其对裸鼠白血病移植瘤的杀伤作用。方法采用Ficoll-Hypaque法分离急性淋巴细胞性白血病(ALL)患儿外周血单个核细胞,贴壁3h,取贴壁细胞分为6组,分别为：对照组(rhGM-CSF+rhIL-4)、未偶联的抗CD10抗体组(rhGM-CSF+rhIL-4+anti-CD10)、未偶联的MDP组(rhGM-CSF+rhIL-4+MDP)、MDP-Ab组(rhGM-CSF+rhIL-4+MDP-Ab)、LPS组(rhGM-CSF+rhIL-4+LPS)及MDP-Ab+LPS组(rhGM-CSF+rhIL-4+MDP-Ab+LPS)。采用反复冻融方法制备Nalm-6白血病细胞抗原,于DC诱导培养第4天加入且共同培养,隔日换液,继续培养。第8天,收集各组DC。经Nalm-6抗原致敏、丝裂霉素C处理过的DC与培养第7天的ALL患儿同体T淋巴细胞按1：10比例混合培养48h,加入Nalm-6细胞(效应细胞：靶细胞=10：1)共培育12h,乳酸脱氢酶(LDH)释放法检测各组T淋巴细胞的杀伤活性。将36只4周龄的雄性BALB/C裸鼠皮下接种1×107个/0.2毫升Nalm-6白血病细胞,第7-10天可形成皮下结节的白血病裸鼠模型,随机分为6组(每组6只),移植瘤内分别接种上述各组淋巴细胞,每周1次,共2次,于给药第14天处死裸鼠。观察各组肿瘤体积变化,绘制肿瘤生长曲线,计算肿瘤体积变化率；HE染色观察肿瘤、肝脾组织变化。结果(1)T淋巴细胞杀伤活性：MDP-Ab组杀伤率为(35.66±5.81)%,明显高于对照组(7.29±2.42)%、未偶联的抗CD10抗体组(13.83±2.90)%及未偶联的MDP组(15.16±3.10)%,而低于LPS组(51.33±6.41)%及MDP-Ab+LPS组(63.33±6.92)%,以MDP-Ab+LPS组杀伤活性最强；(2)移植瘤生长：裸鼠皮下成瘤率可达100%,给药前,所有裸鼠肿瘤体积无显著性差异；给药后,MDP-Ab组肿瘤体积大小均明显小于对照组、未偶联的抗CD10抗体组及未偶联的MDP组,但大于LPS组及MDP-Ab+LPS组,且以MDP-Ab+LPS组体积变化最小；(3)各组肿瘤体积变化率：对照组、未偶联的抗CD10抗体组及未偶联的MDP组第14天肿瘤体积变化分别为147.75,114.90,117.36,而MDP-Ab组为47.66,LPS组为32.31及MDP-Ab+LPS组为12.43,明显低于对照组、未偶联的抗CD10抗体组及未偶联的MDP组；(4)肿瘤肉眼及镜下形态学变化：肿瘤早期表现为皮下瘤节,呈圆形或椭圆形,后期表面凹凸不平呈多个结节融合,对照组3只裸鼠出现肿瘤非药物注射部分破溃,甚至糜烂。剥离瘤体时,与皮下组织分界比较清楚,粘连较少,质地坚硬,瘤体剖面血管丰富；光学显微镜下观察,对照组肿瘤细胞大小不一,排列紊乱,以多角形或圆形多见,排列紊乱,多见分裂相；而MDP-Ab组、LPS组及MDP-Ab+LPS组显微镜下可见不同程度的核固缩、碎裂及溶解、胞浆空泡现象,以MDP-Ab+LPS组最明显。。所有组裸鼠的肝脾均未见Nalm-6细胞浸润。结论 1、MDP-抗CD10偶联物诱导的致敏DC体外能提高同体T淋巴细胞的杀伤活性,与LPS共同作用时效应最强。 2、MDP-抗CD10偶联物诱导的致敏DC能促进同体T淋巴细胞发挥体内抑制肿瘤生长作用,且与LPS具有协同作用。 3、成功建立Nalm-6白血病裸鼠模型,无全身转移情况。
[Abstract]:Objective targeted therapy is a potential effective anti leukemia therapy, especially in eliminating small residual disease and preventing tumor recurrence. This paper combines CD10 monoclonal antibody (anti-CD10 MAb) and cell wall acyl two peptide (MDP) to synthesize a new immune coupling agent (MDP-Ab), and observe the respective growth of MAb and MDP in the immune coupling. Substance activity.
Methods (1) MDP-Ab preparation: CD10 monoclonal antibody (anti-CD10 MAb) and cell wall acyl two peptide (MDP) were connected to a new immune coupling agent (MDP-Ab), and silica gel column and Sephacryl S-100 gel column were separated and purified for each product (PDP-arm-Boc, PDP-arm, PDP-MDP, IT-Ab, etc.); hydrogen ion mass spectrometer, and electrospray The molecular weight of each product (MW+[H]) and the ratio of each molecule in MDP-Ab were analyzed by the fog ionization mass spectrometer and UV spectrophotometer. The cysteine standard curve was used to analyze the number of sulfhydryl groups in IT-Ab by Ellman's reagent; (2) the detection of antibody activity in MDP-Ab: separation and collection of CDl0+ acute lymphoblastic leukemia Cells (positive rate > 95%) were incubated with MDP-Ab, the binding ability of MDP-Ab and CD10+ cells was detected by indirect flow cytometry, and the uncoupled anti CD10 antibody was used as control. (3) detection of MDP activity in MDP-Ab: the separation of peripheral blood mononuclear cells from children with acute lymphoblastic leukemia by Ficoll-Hypaque method, adherent 3h, and suspended cells via Nepal T lymphocytes were screened by the Dragon membrane column, and the adherent cells were divided into 6 groups: the control group (rhGM-CSF+rhIL-4), the uncoupled anti CD10 antibody group (rhGM-CSF+rhIL-4+anti-CD10), the uncoupled MDP group (rhGM-CSF+rhIL-4+MDP), the MDP-Ab group (rhGM-CSF+ rhIL-4+MDP-Ab), the lipopolysaccharide (LPS) group (rhGM-CSF+rhIL-4+LPS) and MDP-Ab+LPS group. HGM-CSF+rhIL-4+MDP-Ab+LPS), for 8 days, each group of dendritic cells (DC) was collected to detect cell immunophenotype, endocytosis and interleukin 12 (IL-12) level in cell supernatant. Lymphoblastic cells treated with mitomycin C treated with DC were mixed with 72h at 1:10 ratio and CFSE staining method was used to detect each group. Lymphocyte proliferation and IFN-Y level in supernatant were detected by ELISA.
Results (1) the hydrogen ion mass spectrometry showed that the molecular weight results (MW+[H]) of PDP-arm-Boc, PDP-arm and PDP-MDP were 372.1272.1746.4, respectively. According to the cysteine standard curve, the number of sulfhydryl groups in IT-Ab was 4.5, and the result of electrospray ionization mass spectrometry and ultraviolet absorbance showed that the MDP-Ab molecular weight of the immuno couple was 101KD, MDP and Ab molecular ratio. The results of 2:1. (2) indirect flow cytometry showed that the positive rate of MDP-Ab binding CD10 antigen was 94.69%, and there was no significant difference (P0.05) compared with the uncoupled anti CD10 antibody. (3) DC cell immunophenotype: MDP-Ab promoted the HLA-DR of DC expression in peripheral blood of children with leukemia, and the positive cells of CO stimulator (CD80, CD86) and mature molecule (CD83) were all significantly higher In the control group, the uncoupled anti CD10 antibody group and the uncoupled MDP group were lower than the LPS group and the MDP-Ab+LPS group (F=629.619, p=0.000); (4) the level of IL-12: the control group, the uncoupled anti CD10 antibody group and the uncoupled MDP group were (42.37 + 5.83) pg/ml, (54.41 + 4.35) pg/ml, (60.75 + 5.06) pg/ml, and (80.63 + 3.73), 160 .15 + 11.43) pg/ml, group MDP-Ab+LPS (190.33 + 6.61) pg/ml, obviously higher than the control group, the uncoupled anti CD10 antibody group and the uncoupled MDP group (F=857.872, p=0.000).MDP-Ab group and LPS group, MDP-Ab+LPS group also have significant difference (P0.05); (5) the control group (81.3 + 10.1)%, and uncoupled anti - Anti The body group, uncoupled MDP group, MDP-Ab group, LPS and MDP-Ab+LPS group were (66.7 + 9.78)%, (62.5 + 6.72)%, (41.6 + 5.67)%, (33.9 + 3.42)%, (25.6 + 3.68)%. Compared with the control group, there was significant statistical significance (F=383.04, p=0.000). (6) mixed lymphocyte reaction: compared with the control group, uncoupled anti CD10 antibody group and uncoupled MDP group, M The positive cells in group DP-Ab, group LPS and group MDP-Ab+LPS were significantly higher than group MDP-Ab+LPS (F=393.36, p=0.000); (7) the level of IFN-Y: the control group, the uncoupled anti CD10 antibody group and the uncoupled MDP group were (67.72 + 3.94) pg/ml, (84.65 + 4.41) pg/ml, (89.69 + 3.02) pg/ml, while the group was (173.06 + 8.14), and 209.24 + 9.43 Pg/ml, MDP-Ab+LPS group (356.17 + 9.48) pg/ml.MDP-Ab group was significantly higher than the control group, uncoupled anti CD10 antibody group and uncoupled MDP group, but lower than the LPS group and MDP-Ab+LPS group (F=2497.18, p=0.000), and the highest level in the MDP-Ab+LPS group.
Conclusion 1. The new immune conjugate MDP-Ab was successfully synthesized by chemical method.
2, MDP-Ab maintains the specific binding activity of antibodies, suggesting that MDP-Ab has the ability to target CD10+ cells.
3, MDP-Ab can promote the expression of high level HLA-DR in peripheral blood dendritic cells in peripheral blood of children with leukemia, CO stimulatory molecules (CD80/CD86) and CD83, and secrete high level IL-12. The combination of lipopolysaccharide and cytokine is the strongest.
4, MDP-Ab induced dendritic cells can promote the proliferation of hermaphroditic lymphocytes and secrete high level IFN-Y molecules. It is the strongest combination with cytokine and lipopolysaccharide, suggesting that MDP-Ab can promote the proliferation and maturation of dendritic cells.
Objective to investigate the activation of lymphocytes and its killing effect on nude mice transplanted with leukemia by the two wall peptide CD10 conjugate.
Methods the peripheral blood mononuclear cells of children with acute lymphoblastic leukemia (ALL) were separated by Ficoll-Hypaque method, and the adherent 3H was used. The parietal cells were divided into 6 groups: the control group (rhGM-CSF+rhIL-4), the uncoupled anti CD10 antibody group (rhGM-CSF+rhIL-4+anti-CD10), the uncoupled MDP group (rhGM-CSF+rhIL-4+MDP), and the MDP-Ab group (rhGM-CSF+rhIL-4+MD). P-Ab), group LPS (rhGM-CSF+rhIL-4+LPS) and MDP-Ab+LPS group (rhGM-CSF+rhIL-4+MDP-Ab+LPS). Nalm-6 leukemic cell antigen was prepared by repeated freezing and thawing methods. It was induced and cultured in DC for fourth days to join and co culture, exchange liquid for the next day and continue to be cultured. Eighth days, the DC. was sensitized by Nalm-6 antigen, and DC and culture of mitomycin C treated for seventh days The children of ALL were mixed with T lymphocyte at 1:10 ratio to culture 48h, adding Nalm-6 cells (effect cells: target cells =10:1) to co breed 12h, and lactate dehydrogenase (LDH) release method to detect the killing activity of T lymphocytes in each group. 36 4 weeks old male BALB/C nude mice were subcutaneously inoculated with 1 x 107 /0.2 ml Nalm-6 leukemia cells, the 7-10 day could be formed. The nude mice model of subcutaneous nodules was randomly divided into 6 groups (6 rats in each group). The cells in each group were inoculated respectively in the transplanted tumor, 1 times a week, 2 times a week, and were killed in nude mice for fourteenth days. The tumor volume changes were observed, the tumor growth curve was plotted and the tumor volume change rate was calculated; HE staining was used to observe the tumor and liver and spleen tissue changes.
Results (1) the killing activity of T lymphocyte: the killing rate of MDP-Ab group was (35.66 + 5.81)%, obviously higher than that of the control group (7.29 + 2.42)%, the uncoupled anti CD10 antibody group (13.83 + 2.90)% and the uncoupled MDP group (15.16 + 3.10)%, and lower than the LPS group (51.33 +)% and MDP-Ab+ LPS group (63.33 + 6.92)%, with the strongest activity of MDP-Ab+LPS group; (2) xenograft Length: the rate of subcutaneous tumor formation in nude mice was up to 100%. There was no significant difference in tumor volume of all nude mice before administration. After administration, the volume of tumor in group MDP-Ab was significantly smaller than that of the control group, uncoupled anti CD10 antibody group and uncoupled MDP group, but larger than group LPS and MDP-Ab+LPS group, and the volume of MDP-Ab+LPS group was the smallest; (3) volume of tumor in each group Rate of change: in the control group, the fourteenth day tumor volume changes of the uncoupled anti CD10 antibody group and the uncoupled MDP group were 147.75114.90117.36, while the MDP-Ab group was 47.66, the LPS group was 32.31 and the MDP-Ab+LPS group was 12.43, obviously lower than the control group, the uncoupled anti CD10 antibody group and the uncoupled MDP group; (4) the tumor naked eye and the morphological changes under the microscope. In the early stage of the tumor, the tumor appeared as a subcutaneous tumor, circular or oval, and the later surface was uneven with multiple nodules. The 3 nude mice in the control group had a non drug injection part of the tumor and even erosive. When the tumor was stripped, the subcutaneous tissue was separated clearly, the adhesion was less, the texture was hard, and the tumor body was rich in blood vessels; optical microscope was used. In the control group, the size of the tumor cells in the control group was different and arranged in disorder. In the group MDP-Ab, group LPS and MDP-Ab+LPS, there were different degrees of nuclear condensation, fragmentation and dissolution, and cytoplasmic vacuoles, in group MDP-Ab+LPS, the liver and spleen of all groups of nude mice were not Nalm-6 fine. Cell infiltration.
conclusion
1, sensitized DC induced by MDP- anti CD10 conjugates can enhance the killing activity of T lymphocytes in vitro, and the effect is the strongest when combined with LPS.
2, sensitized DC induced by MDP- anti CD10 conjugates can promote the inhibition of tumor growth in vivo by T lymphocytes and play a synergistic role with LPS.
3, the nude mice model of Nalm-6 leukemia was successfully established without systemic metastasis.

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R733.7

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