本文選題：胞壁酰二肽 + 抗CD10單克隆抗體　； 參考：《青島大學(xué)》2012年博士論文

【摘要】：目的靶向性治療是一種潛在的有效抗白血病治療方法,尤其在清除微小殘留病、防止腫瘤復(fù)發(fā)方面發(fā)揮重要作用。本文將CD10單克隆抗體(anti-CD10 MAb)與胞壁酰二肽(MDP)連接合成新的免疫偶聯(lián)物(MDP-Ab),觀察免疫偶聯(lián)物中MAb及MDP各自的生物活性。 方法(1)MDP-Ab制備：采用化學(xué)合成方法將CD10單克隆抗體(anti-CD10 MAb)與胞壁酰二肽(MDP)連接合成新的免疫偶聯(lián)物(MDP-Ab),硅膠柱及Sephacryl S-100凝膠柱分離純化合成過程產(chǎn)生的各產(chǎn)物(PDP-arm-Boc、PDP-arm、PDP-MDP、IT-Ab及MDP-Ab);氫離子質(zhì)譜儀、電噴霧電離質(zhì)譜儀與紫外分光光度計分析合成中各產(chǎn)物的分子量(結(jié)果表達(dá)方式為MW+[H])及MDP-Ab中各分子比。以半胱氨酸標(biāo)準(zhǔn)曲線為準(zhǔn),采用Ellman's試劑分析IT-Ab中巰基數(shù)量；(2)MDP-Ab中抗體活性檢測：分離收集CDl0+急性淋巴細(xì)胞性白血病細(xì)胞(陽性率≥95%),加入MDP-Ab共同孵育,采用間接流式細(xì)胞術(shù)檢測MDP-Ab與CD10+細(xì)胞結(jié)合能力,以未偶聯(lián)的抗CD10抗體作對照。(3) MDP-Ab中MDP活性檢測：采用Ficoll-Hypaque法分離急性淋巴細(xì)胞性白血病患兒外周血單個核細(xì)胞,貼壁3h,懸浮細(xì)胞經(jīng)尼龍膜柱篩選獲得T淋巴細(xì)胞,凍存?zhèn)溆�。取貼壁細(xì)胞分為6組,分別為：對照組(rhGM-CSF+rhIL-4)、未偶聯(lián)的抗CD10抗體組(rhGM-CSF+rhIL-4+anti-CD10)、未偶聯(lián)的MDP組(rhGM-CSF+rhIL-4+MDP)、MDP-Ab組(rhGM-CSF+rhIL-4+MDP-Ab)、脂多糖(LPS)組(rhGM-CSF+rhIL-4+LPS)及MDP-Ab+LPS組(rhGM-CSF+rhIL-4+MDP-Ab+LPS),培養(yǎng)8天,收集各組樹突狀細(xì)胞(DC),檢測細(xì)胞免疫表型、內(nèi)吞作用及細(xì)胞上清液中白介素12(IL-12)水平。將絲裂霉素C處理過的DC與急性淋巴細(xì)胞性白血病患兒同體的淋巴細(xì)胞按1：10比例混合培養(yǎng)72h,CFSE染色法檢測各組淋巴細(xì)胞增殖情況,ELISA方法檢測上清液IFN-Y水平。 結(jié)果(1)氫離子質(zhì)譜顯示PDP-arm-Boc、PDP-arm、PDP-MDP的分子量結(jié)果(MW+[H])分別為372.1,272.1,746.4;根據(jù)半胱氨酸標(biāo)準(zhǔn)曲線,顯示IT-Ab中巰基數(shù)量為4.5；電噴霧電離質(zhì)譜與紫外線吸光度結(jié)果顯示免疫偶聯(lián)物中MDP-Ab分子量約為101KD,其中MDP與Ab分子比為2：1。(2)間接流式細(xì)胞術(shù)結(jié)果示MDP-Ab結(jié)合CD10抗原的陽性率為94.69%,與未偶聯(lián)的抗CD10抗體相比,無顯著性差異(p0.05)。(3)DC細(xì)胞免疫表型：MDP-Ab促進(jìn)白血病患兒外周血DC表達(dá)HLA-DR、共刺激分子(CD80、CD86)及成熟分子(CD83)的陽性細(xì)胞均明顯高于對照組、未偶聯(lián)的抗CD10抗體組及未偶聯(lián)的MDP組,但低于LPS組及MDP-Ab+LPS組(F=629.619,p=0.000)；(4)IL-12水平：對照組、未偶聯(lián)的抗CD10抗體組及未偶聯(lián)的MDP組分別為(42.37±5.83)pg/ml,(54.41±4.35)pg/ml,(60.75±5.06)pg/ml,而MDP-Ab組(80.63±3.73)pg/ml,LPS組(160.15±11.43)pg/ml,MDP-Ab+LPS組(190.33±6.61)pg/ml,明顯高于對照組、未偶聯(lián)的抗CD10抗體組及未偶聯(lián)的MDP組(F=857.872,p=0.000)。MDP-Ab組與LPS組、MDP-Ab+LPS組相比,亦有顯著性差異(p0.05)；(5)DC吞噬功能：對照組(即未成熟DC)為(81.3±10.1)%,而未偶聯(lián)的抗CD10抗體組、未偶聯(lián)的MDP組、MDP-Ab組、LPS及MDP-Ab+LPS組分別為(66.7±9.78)%,(62.5±6.72)%,(41.6±5.67)%,(33.9±3.42)%,(25.6±3.68)%,與對照組相比,有顯著性統(tǒng)計學(xué)意義(F=383.04,p=0.000)。(6)混合淋巴細(xì)胞反應(yīng)：與對照組、未偶聯(lián)的抗CD10抗體組及未偶聯(lián)的MDP組相比,MDP-Ab組、LPS組及MDP-Ab+LPS組陽性細(xì)胞明顯增高,以MDP-Ab+LPS組為最高(F=393.36,p=0.000);(7)IFN-Y水平：對照組、未偶聯(lián)的抗CD10抗體組及未偶聯(lián)的MDP組分別為(67.72±3.94)pg/ml、(84.65±4.41)pg/ml、(89.69±3.02)pg/ml,而MDP-Ab組為(173.06±8.14)pg/ml,LPS組為(209.24±9.43)pg/ml,MDP-Ab+LPS組為(356.17±9.48)pg/ml。MDP-Ab組明顯高于對照組、未偶聯(lián)的抗CD10抗體組及未偶聯(lián)的MDP組,而低于LPS組及MDP-Ab+LPS組(F=2497.18,p=0.000),且以MDP-Ab+LPS組水平最高。 結(jié)論1、成功采用化學(xué)方法合成新免疫偶聯(lián)物MDP-Ab 2、MDP-Ab保持抗體特異結(jié)合抗原的活性,提示MDP-Ab具有靶向結(jié)合CD10+細(xì)胞能力。 3、MDP-Ab能促進(jìn)白血病患兒外周血樹突狀細(xì)胞表達(dá)高水平HLA-DR、共刺激分子(CD80/CD86)及CD83,分泌高水平IL-12,與細(xì)胞因子、脂多糖聯(lián)合作用最強(qiáng)。 4、MDP-Ab誘導(dǎo)的樹突狀細(xì)胞能促進(jìn)同體淋巴細(xì)胞增殖,分泌高水平IFN-Y分子,與細(xì)胞因子、脂多糖聯(lián)合作用最強(qiáng),提示MDP-Ab保持促進(jìn)樹突狀細(xì)胞增殖及成熟活性。 目的探討胞壁酰二肽-抗CD10偶聯(lián)物激活淋巴細(xì)胞的功能及其對裸鼠白血病移植瘤的殺傷作用。 方法采用Ficoll-Hypaque法分離急性淋巴細(xì)胞性白血病(ALL)患兒外周血單個核細(xì)胞,貼壁3h,取貼壁細(xì)胞分為6組,分別為：對照組(rhGM-CSF+rhIL-4)、未偶聯(lián)的抗CD10抗體組(rhGM-CSF+rhIL-4+anti-CD10)、未偶聯(lián)的MDP組(rhGM-CSF+rhIL-4+MDP)、MDP-Ab組(rhGM-CSF+rhIL-4+MDP-Ab)、LPS組(rhGM-CSF+rhIL-4+LPS)及MDP-Ab+LPS組(rhGM-CSF+rhIL-4+MDP-Ab+LPS)。采用反復(fù)凍融方法制備Nalm-6白血病細(xì)胞抗原,于DC誘導(dǎo)培養(yǎng)第4天加入且共同培養(yǎng),隔日換液,繼續(xù)培養(yǎng)。第8天,收集各組DC。經(jīng)Nalm-6抗原致敏、絲裂霉素C處理過的DC與培養(yǎng)第7天的ALL患兒同體T淋巴細(xì)胞按1：10比例混合培養(yǎng)48h,加入Nalm-6細(xì)胞(效應(yīng)細(xì)胞：靶細(xì)胞=10：1)共培育12h,乳酸脫氫酶(LDH)釋放法檢測各組T淋巴細(xì)胞的殺傷活性。將36只4周齡的雄性BALB/C裸鼠皮下接種1×107個/0.2毫升Nalm-6白血病細(xì)胞,第7-10天可形成皮下結(jié)節(jié)的白血病裸鼠模型,隨機(jī)分為6組(每組6只),移植瘤內(nèi)分別接種上述各組淋巴細(xì)胞,每周1次,共2次,于給藥第14天處死裸鼠。觀察各組腫瘤體積變化,繪制腫瘤生長曲線,計算腫瘤體積變化率；HE染色觀察腫瘤、肝脾組織變化。 結(jié)果(1)T淋巴細(xì)胞殺傷活性：MDP-Ab組殺傷率為(35.66±5.81)%,明顯高于對照組(7.29±2.42)%、未偶聯(lián)的抗CD10抗體組(13.83±2.90)%及未偶聯(lián)的MDP組(15.16±3.10)%,而低于LPS組(51.33±6.41)%及MDP-Ab+LPS組(63.33±6.92)%,以MDP-Ab+LPS組殺傷活性最強(qiáng)；(2)移植瘤生長：裸鼠皮下成瘤率可達(dá)100%,給藥前,所有裸鼠腫瘤體積無顯著性差異；給藥后,MDP-Ab組腫瘤體積大小均明顯小于對照組、未偶聯(lián)的抗CD10抗體組及未偶聯(lián)的MDP組,但大于LPS組及MDP-Ab+LPS組,且以MDP-Ab+LPS組體積變化最��；(3)各組腫瘤體積變化率：對照組、未偶聯(lián)的抗CD10抗體組及未偶聯(lián)的MDP組第14天腫瘤體積變化分別為147.75,114.90,117.36,而MDP-Ab組為47.66,LPS組為32.31及MDP-Ab+LPS組為12.43,明顯低于對照組、未偶聯(lián)的抗CD10抗體組及未偶聯(lián)的MDP組；(4)腫瘤肉眼及鏡下形態(tài)學(xué)變化：腫瘤早期表現(xiàn)為皮下瘤節(jié),呈圓形或橢圓形,后期表面凹凸不平呈多個結(jié)節(jié)融合,對照組3只裸鼠出現(xiàn)腫瘤非藥物注射部分破潰,甚至糜爛。剝離瘤體時,與皮下組織分界比較清楚,粘連較少,質(zhì)地堅硬,瘤體剖面血管豐富；光學(xué)顯微鏡下觀察,對照組腫瘤細(xì)胞大小不一,排列紊亂,以多角形或圓形多見,排列紊亂,多見分裂相；而MDP-Ab組、LPS組及MDP-Ab+LPS組顯微鏡下可見不同程度的核固縮、碎裂及溶解、胞漿空泡現(xiàn)象,以MDP-Ab+LPS組最明顯。。所有組裸鼠的肝脾均未見Nalm-6細(xì)胞浸潤。 結(jié)論 1、MDP-抗CD10偶聯(lián)物誘導(dǎo)的致敏DC體外能提高同體T淋巴細(xì)胞的殺傷活性,與LPS共同作用時效應(yīng)最強(qiáng)。 2、MDP-抗CD10偶聯(lián)物誘導(dǎo)的致敏DC能促進(jìn)同體T淋巴細(xì)胞發(fā)揮體內(nèi)抑制腫瘤生長作用,且與LPS具有協(xié)同作用。 3、成功建立Nalm-6白血病裸鼠模型,無全身轉(zhuǎn)移情況。
[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.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R733.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

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