【作者】 任秀宝； 于津浦； 曹水； 安秀梅； 辛宁；

【Author】 Ren Xiu-Bao Yu Jin-Pu Cao Shui An Xiu-Mei Xin Ning Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin, 300060

【机构】 天津医科大学附属肿瘤医院免疫室；

【摘要】 目的:探索供受者之间母胎微嵌合状态对活化HLA 半相合异基因造血干细胞抗肿瘤活性的影响。方法:选取30 例进行活化的HLA半相合异基因造血干细胞治疗的实体肿瘤患者,提取供者基因组DNA,利用HLA—DRB通用引物扩增HLA—DRB位点第二外显子,扩增产物稀释100倍后作为模板,采用PCR—SSP方法进行低分辨率的HLA—DR配型。 PCR产物经电泳鉴定是否存在母胎微嵌合。对母胎微嵌合状态不同的患者,利用FISH和STR—PCR方法比较接受治疗后供者细胞在患者体内的停留时间,利用ELISA法比较不同时间点患者血清中Th1／Th2类细胞因子的浓度。结果:提取基因组DNA的纯度可达(1．843±0．136),浓度为(213．7±116) ng／μl。利用HLA—DR通用引物可特异性扩增出272bp的 PCR产物,经2％琼脂糖电泳鉴定无异常杂带。巢式PCR— SSP法灵敏度较高,特异性较好,在母子／母女移植患者中的检出率为40％,而在父女／父子移植和兄弟姐妹移植中的检出率均为0,与文献报道一致。在移植3月后对3例接受活化的HLA半相合异基因移植治疗的患者(其中1例为存在母胎微嵌合的母子移植,1例为不存在母胎微嵌合的母子移植, 1例为不存在母胎微嵌合的兄妹移植)进行FISH杂交检测, 发现除一例母胎微嵌合(+)的男性患者外周血中检测到供者 (女性)来源的XX染色体杂交点外,其余均未观察到阳性结果。同时发现母胎微嵌合(+)患者治疗后Th1类细胞因子 (以IFN-γ为代表)在体内保持较高浓度的时间显著延长, 而Th2类细胞因子的变化趋势无明显变化。结论:供受者之间的母胎微嵌合状态有利干提高活化HLA半相合异基因造血干细胞的抗肿瘤疗效。更多还原

【Abstract】 Objeotive: To study the possible influences of the feto-maternal microchimerism in donors and recipients on antitumor effects of activated HLA-haploidentical allogenic hematopoietic stem cells(haplo-HSCs). Methods: Genomic DNA samples were extracted from 30 pairs of patients and related donors who received activated haplo-HSCs treatment. The feto-maternal microchimerism was analyzed by nested PCR-SSP typing. Then the survival time of donor cells transfused into patients who are feto-maternal microchimerism(+) were compared with those feto-maternal microchimerism(-) by FISH and STR-PCR method. And differences in cytokines secreted in serum were identified by ELISA method. Results: The purity of extracted genomic DNA was (1.843 ± 0.136) with concentration of (213.7 ± 116)ng/ μ l. A distinct 272 bp product was obtained after fist cycle PCR amplification with the general primers of HLA-DRB. The sensitivity and the specificity of nested PCR-SSP typing is relatively higher that tradional PCR-SSP typing that feto-maternal microchimerism could be identified even in the four times diluted genomic DNA sample. The positive rate of feto-maternal microchimerism in maternal/children donation is 40% but none in paternal/children donation. The survival time of donor cells transfused could last about 3 months detected by FISH in a feto-maternal microchimerism(+) patient, and the Th1-type of cytokines remained at the relative high level for more days. Conclusion: The feto-Maternal Microchimerism in Donors and Recipients improves antitumor effect of activated related haplo-HSCs.更多还原