树突状细胞—肾癌786-O细胞株融合瘤苗的制备及其体外抗肾癌效应实验研究

发布时间：2018-06-09 22:55

本文选题：树突状细胞 + 瘤苗　；参考：《重庆医科大学》2008年硕士论文

【摘要】： 目的:制备和筛选纯净的树突状细胞(Dendritic cell, DC)-肾癌786-O细胞株融合瘤苗,探索融合瘤苗的生物学特性及体外抗肾癌效应,为基于DC的肾癌免疫治疗提供理论基础和实验依据。方法:1、树突状细胞-肾癌786-O细胞株融合瘤苗制备及形态学观察。密度梯度离心从健康人外周血分离单个核细胞,联用细胞因子rhGM-CSF、rhIL-4和TNF-a诱导培养7-9天即为成熟DC,流式细胞仪检测细胞表面分子表达。将冻存的肾癌786-O细胞株复苏培养,与成熟DC按5:1比例混合,用聚乙二醇(Polyethylene glycol, PEG)作融合剂诱导细胞融合。加入与DC体外诱导时相同浓度的细胞因子rhGM-CSF、rhIL-4静置培养,24h后弃去悬浮细胞,贴壁细胞继续培养,4d后收集悬浮和半悬浮融合细胞。分别在光镜和电镜下观察融合细胞形态结构。 2、融合瘤苗生物学特性及体外抗肾癌786-O细胞免疫特性。体外培养融合瘤苗并计数,描绘细胞体外生长曲线,比较融合细胞和亲代细胞体外培养分裂增殖的能力;混合淋巴细胞反应(Mixed lymphocytereaction,MLR)检测比较DC-RCC786-O融合瘤苗、单纯培养的DC、与肾癌786-O混合培养相同天数的DC三组细胞刺激同种异体T淋巴细胞增殖的能力;流式细胞术检测融合瘤苗细胞表面分子CD86,HLA-DR的表达,与单纯培养DC表面分子的表达水平比较;联合免疫缺陷(Severe combined immunodeficient,SCID)小鼠背部皮下注射融合瘤苗,检测融合瘤苗体内应用的安全性;MTT比色法检测比较DC-RCC786-O融合瘤苗、单纯培养DC、与肾癌786-O混合培养DC三组细胞分别诱导细胞毒性T淋巴细胞(Cytotoxic T lymphocytes,CTL)体外杀伤肾癌靶细胞的能力;流式细胞术检测融合瘤苗对杀伤后肾癌786-O靶细胞细胞周期的影响。结果: 1、患者外周血单个核细胞经诱导分化后可获得形态典型的树突状细胞,成熟DC表达表面分子CD86, HLA-DR分别为(62.07±2.01)%和(66.30±1.51)%;PEG可有效促使DC与肾癌786-O细胞株融合;用两次贴壁法可分选出较纯的DC-786-O融合瘤苗;融合瘤苗较亲代细胞体积明显增大,在培养基中悬浮生长。 2、融合瘤苗体外生长曲线平缓,无明显细胞倍增期;融合瘤苗在SCID鼠体内注射不生成肿瘤;瘤苗表面共刺激分子CD86, HLA-DR的表达率分别为(81.23±1.01)%和(80.16±1.11)%,明显高于单纯DC表达水平(P0.05);融合瘤苗刺激T淋巴细胞增殖能力明显高于亲代DC和与786-O混合培养的DC,差异均具有显著性(P0.05);融合瘤苗与T细胞在效靶比为1:1时刺激T细胞增殖能力最强;MTT比色检测,融合瘤苗组对肾癌靶细胞的杀伤率为(75.49±5.10)%,与混合培养DC组杀伤率(45.44±2.87)%和单纯T淋巴细胞对照组杀伤率(14.55±0.75)%相比差异均具有显著性(P0.05)。结论: 1、外周血单核细胞经rhGM-CSF、rhIL-4、TNF-α等细胞因子联合应用诱导分化可获得具有典型形态和细胞表型的成熟DC;DC与786-O细胞株经PEG诱导融合和两次贴壁分选纯化后可获得较为纯净的融合瘤苗。 2、融合瘤苗体外悬浮生长,分裂增殖能力弱,在免疫缺陷动物体内不具有成瘤活性;融合瘤苗表达共刺激分子CD86、HLA-DR的水平和体外刺激T淋巴细胞增殖能力明显高于亲代DC和与亲代786-O细胞株混合培养的DC;融合瘤苗具有显著的体外诱导CTL效应细胞特异性抑制和杀伤肾癌786-O细胞的能力。 3、基于DC和肾癌786-O细胞株的融合瘤苗可以作为一种新的有效的肾癌免疫治疗手段。
[Abstract]:Objective: to prepare and screen pure dendritic cells (Dendritic cell, DC) - 786-O cell line fusion vaccine, explore the biological characteristics of the fusion vaccine and the effect of anti renal cancer in vitro, and provide the theoretical basis and experimental basis for the immunotherapy of renal cancer based on DC.
Methods: 1, the preparation and morphological observation of the fusion vaccine of the dendritic cells - the 786-O cell line of renal carcinoma. The density gradient centrifugation was used to separate the mononuclear cells from the healthy human peripheral blood. The cell surface molecular expression was detected by the flow cytometer for 7-9 days with the cytokine rhGM-CSF, rhIL-4 and TNF-a induced culture, and the 786-O cell line of the frozen renal cell carcinoma was recovered. Su culture, mixed with mature DC in proportion to 5:1, using polyethylene glycol (Polyethylene glycol, PEG) as a melting mixture to induce cell fusion, adding the same concentration of cytokine rhGM-CSF with DC in vitro, rhIL-4 static culture, 24h after suspension cells, adherent cells continue to culture, 4D after collecting suspended and semi suspended fusion cells. Respectively in light The morphology and structure of the fused cells were observed under microscope and electron microscope.
2, the biological characteristics of the fusion vaccine and the immune characteristics of the 786-O cell in vitro. The fusion tumor vaccine was cultured in vitro, the growth curve of the cells in vitro was described, the ability of the fusion cell and the parental cells to be cultured in vitro was compared, and the mixed lymphocyte reaction (Mixed lymphocytereaction, MLR) was used to compare the DC-RCC786-O fusion tumor vaccine and simple The cultured DC, the DC three groups with the same number of days with the kidney cancer 786-O, stimulated the proliferation of allogenic T lymphocytes. Flow cytometry was used to detect the expression of the surface molecules of the fusion tumor cells CD86, HLA-DR, compared with the expression level of the pure DC surface molecules; the joint immunodeficiency (Severe combined immunodeficient, SCID) mice The fusion tumor vaccine was injected subcutaneously on the back to detect the safety of the fusion vaccine in vivo; the MTT colorimetric assay was used to compare the DC-RCC786-O fusion tumor seedlings, the DC was cultured and the DC three groups were cultured with the renal carcinoma 786-O to induce the cytotoxic T lymphocyte (Cytotoxic T lymphocytes, CTL) to kill the target cells of the renal cancer in vitro; flow cytometry Effect of fusion vaccine on cell cycle of 786-O cells after killing renal cell carcinoma.
Result:
1, the peripheral blood mononuclear cells can obtain typical dendritic cells after induction of differentiation, the mature DC expression surface molecule CD86, HLA-DR (62.07 + 2.01)% and (66.30 + 1.51)% respectively. PEG can effectively promote the fusion of DC and renal cancer 786-O cell lines, and the more pure DC-786-O fusion vaccine can be selected by the two adherence method; the fusion vaccine is relative. The volume of the cells increased significantly and suspended in the medium.
2, the growth curve of the fusion vaccine was slow and no obvious cell doubling period; the fusion vaccine was injected in SCID mice without the formation of tumor; the surface CO stimulation of the tumor vaccine was CD86, the expression rate of HLA-DR was (81.23 + 1.01)% and (80.16 + 1.11)% respectively, which was significantly higher than that of pure DC (P0.05), and the proliferation of T lymphocyte stimulated by fusion vaccine was significantly higher. The difference between the parent DC and the 786-O mixed with DC was significant (P0.05), and the fusion vaccine and T cells stimulated T cells to proliferate the strongest when the target ratio was 1:1, and the cytotoxicity of the fusion vaccine group to the target cells of renal cancer was (75.49 + 5.10)%, and the killing rate of the mixed culture DC group (45.44 + 2.87)% and the pure T lymphocyte control. The killing rate of the group (14.55 + 0.75)% was significantly different from that of the control group (P0.05).
Conclusion:
1, mature DC with typical morphology and cell phenotype can be obtained by combined use of rhGM-CSF, rhIL-4, TNF- alpha and other cytokines in peripheral blood mononuclear cells. DC and 786-O cell lines can be purified by PEG induced fusion and two adherent separation and purified to obtain a more pure fusion vaccine.
2, the fusion vaccine was suspended in vitro, the ability of mitosis and proliferation was weak, and there was no tumor activity in the immune deficient animals. The fusion vaccine expressed the co stimulator CD86, the level of HLA-DR and the proliferation of T lymphocyte in vitro were significantly higher than that of the parent DC and the mixed culture of the parent 786-O cell line, and the fusion vaccine had a significant in vitro induction. The CTL effect cells specifically inhibit and kill the ability of renal cell carcinoma 786-O cells.
3, fusion tumor vaccine based on DC and renal cell carcinoma 786-O cells can be used as a new effective immunotherapy for renal cell carcinoma.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R737.11

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