摘要
目的:观察从脐血中提取的树突状细胞负载热休克蛋白70-肿瘤抗原肽复合物的细胞表面表型特点及其所激活的细胞毒T淋巴细胞的杀伤活性。方法:实验于2003-10/2005-12在解放军军事医学院兽医研究所完成。①脐血来源于长春市妇产医院足月顺产的非高危妊娠产妇,要求无急慢性感染及血液系统疾病,产妇及其家属均签署知情同意书。热休克蛋白70由本室制备纯化。人骨肉瘤细胞Saos-2(北京肿瘤研究所,批号222RXB013538)。②封闭式采集脐带血,体外分离脐血单个核细胞,调整浓度为1×109L-1,接种于24孔细胞培养板,每孔均加入100μg/L人重组粒-巨噬细胞集落刺激因子、50μg/L白细胞介素4、10μg/L肿瘤坏死因子α。培养14d后,鉴定细胞表面CD1a、HLA-DR的百分率。③用含胎牛血清的IMDM培养Saos-2骨肉瘤细胞至对数生长期,胰酶消化后调整细胞数为5×107L-1。集传20代生长的Saos-2,冻融2次,低渗震荡,离心,上清用盐酸调pH至7.0,过滤后即为肿瘤抗原肽,与热休克蛋白70体外结合。④另取脐血40mL,分离T淋巴细胞,诱导分化至第4天,加入热休克蛋白70-肿瘤抗原肽100μL,促使T淋巴细胞活化为细胞毒T淋巴细胞。⑤效应细胞(细胞毒T淋巴细胞)与靶细胞(Saos-2)按效靶比50∶1,25∶1,12.5∶1,6.25∶1,3.125∶1梯度,四甲基偶氮唑盐法检测细胞毒T淋巴细胞杀伤活性。结果:①细胞形态学观察:细胞因子诱导后第3天,镜下脐血细胞呈均匀散布的细胞聚体,胞体拉长;第7天可见不明显突起;第14天出现具有典型树枝状突起的树突状细胞。②细胞因子诱导后脐血单核细胞表面表型鉴定结果:经细胞因子诱导后,脐血单核细胞CD1a与HLA-DR表达率均较诱导前明显升高[0,(18.43±3.26)%;(4.03±1.66)%,(59.69±8.47)%;P均<0.01]。③细胞毒T淋巴细胞杀伤活性检测结果:培养72h后,细胞毒T淋巴细胞与靶细胞Saos-2效靶比为50∶1时可杀伤决大部分肿瘤细胞,杀伤率为(97.50±11.24)%,效靶比从50∶1至3.125∶1可使靶细胞Saos-2生长呈梯度受到不同程度的抑制。单纯的树突状细胞及未经激活的T淋巴细胞对Saos-2细胞仅有微弱的生长抑制作用。结论:脐血来源的树突状细胞经过热休克蛋白70-肿瘤抗原肽复合物负载致敏后,激活的细胞毒T淋巴细胞对人骨肉瘤细胞Saos-2具有极强的杀伤能力。
AIM： To observe the phenotype characteristics of dendritic cells extracted from cord blood loaded with heat shock protein （HSP） 70-peptide complex, and the killing activity of cytotoxic T lymphocytes. METHODS： The experiment was conducted in the Veterinary Research Institute of Military Medical Science Academy from October 2003 to December 2005. ①The cord blood was collected under sterile condition from the non-high risk parturient without acute or chronic infection or hematological system diseases in Changchun Gynaecology and Obstetrics Hospital. The informed consent was obtained from the parturient and relatives. HSP70 was prepared and purified in our department. Human osteosarcoma Saos-2 was bought from Beijing Tumor Institute （No. 222RXB013538）. ②Mononuclear cells （MNCs） were isolated from cord blood collected closely in vitro, whose concentration was adjusted to 1 ×10^9 L^-1, and then inoculated into a 24-pore culture plate; every pore was added by 100 μg/L granulocyte-macrophage colony stimulating factor, 50 μg/L interleukin 4 and 10 μg/L tumor necrosis factor. After 14 days culture, CDla and HLA-DR percentage from cell surface were tested.③Ostersarcoma Saos-2 was cultured with IMDM containing fetal bovine serum to the exponential phase of growth, and adjusted cell number to 5×10^7 L^-1 after trypsinization. Saos-2 cells of the 20^th passage were treated with freeze thawing for twice, commotion and centrifugalization. Supematant was adjusted by acid hydroc until pH=7.0 and filtered to get antigen peptide, which was combined with HSP70 in vitro. ④ Another 40 mL cord blood was harvested to isolate the T lymphocytes. After 4 days induction, 100 μL HSP70 peptide was added to activate T cells become specific cytotoxic T lymphocytes （CTL）. ⑤The effective cell （CTL） was added to the target cell （Saos-2） by ratio of 50：1, 25：1, 12.5：1, 6.25：1, and 3.125：1. Ml-r assay was employed to detect the inhibition rate of CTL. RESULTS： ①Cell morphology observation： At day 3 after cell factor induction, the gathered cord blood cell dispersed well and the cell body was prolonged under microscope; unobvious prominence was observed after 7 days; typical dendritic cells was found after 14 days. ②Identification result of MNC cell surface type： The induced cord blood MNC had higher expression of CD1a and HLA-DR than that before induction [0, （18.43±3.26）%; （4.03±1.66）%, （59.69±8.47）%; P 〈 0.01]. ③Test result of CTL： After 72 hours cultivation, when the ratio of CTL and target cell osteosarcoma Saos-2 reached to 50：1, most of the tumor cells were killed with a killing rate of （97.50±11.24）%, while the growth of target cell Saos-2 was inhibited in different degrees when the ratio was 50：1 to 3.125：1. Simple dendritic cells and inactivated T lymphocytes had weak inhibiting effect on the growth of Saos-2. CONCLUSION： After the cord blood-derived dendritic cells loading with HSP70-peptide complex, the activated CTL could inhibit osteosarcoma Saos-2 cells significantly.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第11期2017-2020,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
