嵌合体外周血细胞异源性补体调节蛋白检测的意义

Evaluation of allogenic complement-regulating proteins in the peripheral blood cells of chimeric rat models harboring human cord blood stem cells

目的通过流式细胞术检测人源性补体调节蛋白(hCD55和hCD59)在人脐血干细胞嵌合体大鼠外周血细胞中的分布,分析探讨嵌合体供者诱导异种移植免疫耐受的可能机制。方法采用宫内胎鼠移植加新生鼠肝内注射人脐血干细胞的方法,制作人脐血干细胞嵌合体大鼠模型。4周后利用流式细胞三色标记法,对hCD45/SSC设门时,不同区域内hCD55和hCD59抗原阳性细胞的分布进行分析。利用补体依赖性淋巴细胞毒性反应,评估嵌合体淋巴细胞的免疫适应性。结果hCD45/SSC设门时,在hCD45阳性细胞区域中,hCD55和hCD59阳性率分别高达(53.69±18.23)%和(31.8±27.5)%,但仅占全细胞总数的(2.0±1.32)%和(0.76±0.56)%,与全细胞区域中hCD55和hCD59阳性细胞的百分率(4.11%±2.83)%和(2.82±1.38%)相比,差异具有显著性意义(t=2.71,P=0.043;t=3.64,P=0.015)。嵌合体大鼠补体依赖性淋巴细胞毒性反应为(22.32±15.10)%,低于非嵌合体组大鼠的(57.29±22.65)%,差异具有非常显著性意义(t=4.16,P<0.001)。相关分析显示,嵌合体大鼠补体依赖性淋巴细胞毒性反应与外周血hCD55阳性细胞百分率呈负相关(r=-0.679,P=0.031);外周血hCD55阳性细胞百分率和hCD45阳性细胞百分率呈正相关(r=0.658,P=0.038)。结论人脐血干细胞嵌合体内人源性细胞能够表达人补体调节蛋白。人造血细胞嵌合体淋巴细胞具有抵抗人补体对细胞融解、破坏的作用。随着嵌合体嵌合比例的增加,人源性补体调节蛋白表达增多,嵌合体外周血淋巴细胞抵抗人补体的融解、破坏作用增强。嵌合体内异种补体调节蛋白的存在,可能是造血细胞嵌合体供者诱导异种移植免疫耐受的基础。

Objective To analyze the mechanisms of surrogate tolerogenesis induced by chimeric donors. Methods Hematopoietic stem cells （HSCs） from human cord blood were transplanted into fetal rats via intrauterine injection and infused into the liver of the neonatal rats to establish chimeric rat models with human HSCs. Four weeks after birth, flow cytometry was performed to analyze the percentages of human CD45 （hCD45）, CD55 （hCD55） and CD59 （hCD59）-positive cells in the peripheral blood cells of the chimeric rats. The distributions of hCD55- and hCD59-positive cells in different hCD45/SSC gating regions were observed. The resistance of the peripheral blood lymphocytes to complements-mediated cytolysis was assessed by complement-dependent cytotoxicity （CDC） test in the chimeric rats and compared with that in control rats. The correlation between CDC and the human complement-regulating proteins in the chimeric rats were analyzed statistically. Results On hCD45/SSC gating, the percentages of hCD55- and hCD59-positive cells in hCD45-positives region were （53.69±18.23）% and （31.8±27.5）%, accounting for （2.0±1.32）% and （0.76±0.56）% of the total cell population, respectively, which were significantly lower than the cell percentages in the extensive region （t=2.71, P=0.043 and t-3.64, P=0.015）. The cytolytic rate of PBLs incubated with normal human serum was （22.32±15.10）% in the chimeric rats, significantly lower than that in the non-chimeric rats [（60.7±22.65）%, t=4.16, P〈0.001）. In the chimeric rats, hCD55-positive cell percentage was inversely correlated in the peripheral blood karyocytes the cytolysis rate in CDC （r =-0.679, P=0.031）, and positively correlated to hCD45-positive cell percentage （r=0.658, P=0.038）. Conclusion The hCD45-positives region is the cluster of chimeric human cells expressing human complement-regulating proteins. The peripheral blood lymphocytes from chimeric donor can resist the cytolysis mediated by human complement. The presence of allogenic CD55 and CD59 antigens in chimeric donors may be the basis of surrogate tolerogenesis for xenotransplantation.