群体反应性抗体干扰脐血CD34＋细胞增殖、分化的实验研究

Effect of the serum panel reactive antibody on proliferation and differentiation of cord blood CD34＋ cells in vitro

目的探讨血清特异性群体反应性抗体（PRA）对脐血CD34＋细胞增殖、分化能力的影响。方法取含PRA（经实验证实）的B地中海贫血患儿血清，与脐血CD34＋细胞、补体孵育，观察PRA对CD34＋细胞增殖、分化的影响，分A组（不加血清组）、B组（PRA血清组）、C组（PRA血清加补体组）、D组（补体组）、E组（PRA阴性血清组）共5组。孵育后以3H—TaR掺入法测定细胞DNA合成及流式细胞仪检测AnnexinV和CD95表达；并进行集落培养，于第10天计数集落。结果A组为（20．71±2．81）U／L，低于B组（64．28±5．12）U／L、C组（84．29±4．99）U／L，B组低于c组；D、E组均为（22．86±2．91）U／L和（22．86±2．91）U／L，均低于B、c组；各组氚每分钟8射线释放量（epm）：A组为（22629±3288），高于B组（4598±2178）和C组（1626±1192），A组和D、E组之间的差异无统计学意义（P〉0．05）；A组的总集落数、粒-巨噬细胞集落形成单位（CFU—GM）、混合系集落形成单位（CFU-GEMM）及爆式红系集落形成单位（BFU—E）数均高于B、c组，B组的总集落数、CFU—GM及CFU—GEMM数均高于C组；D组和E组的各种集落数与A组的差异无统计学意义（P〉0．05）；各组CD34＋细胞AnnexinV及CD95表达百分率差异无统计学意义（P〉0．05）。结论特异性PRA血清对脐血CD34＋细胞的增殖和分化有抑制作用，补体可增强上述作用；特异性PRA血清对脐血CD34＋细胞的凋亡无明显影响。

Objective The low rate of engraftment in children with β-thalassemia has seriously restricted the popularity of the hematopoietic stem cell transplantation （ HSCT）. Panel reactive antibody （PRA） has been regarded as one of the important factors for the success of kidney transplantation. Poly- transfusion before transplantation is associated with the production of PRA. Also PRA is produced in the children with β-thalassemia major who need poly-transfusion for life. PRA might be one of factors inducing the low rate of engraftment in children with β-thalassemia. This study focused on observing the effect of PRA on the proliferation, differentiation, apoptosis and tlecrosis of cord blood CD34＋ cells in vitro by incubating the cord blood CD34＋ cells with serum containing PRA. Method Seven samples of cord blood were collected and the HLA typing for every sample was done. Seven sera positive for PRA and seven negative sera were selected respectively. Mononuclear cells （MNCs） were obtained by Ficoll-Hapaque density gradient centrifugation. CD34＋ cells were isolated from MNCs by positive selection using an immunomagnetic separation （ CD34＋ progenitor cell isolation kit and auto-MACS）. The CD34＋cells of umbilical cord blood were incubated with the serum and complement in the following groups： A （absence of serum）, B （presence of PRA positive serum ）, C （presence of PRA positive serum and complement ）, D （presence of complement）, and E （ presence of PRA negative serum）. After incubation the samples were centrifuged and the supernatant was collected for LDH detection. At the same time the CD34＋ cells were harvested for assessing the expression of Annexin V and CD95 of the CD34＋ ceils by flow cytometry and also for the detection of the DNA synthesis by 3 H-TaR incorporation. Meanwhile the cells were inoculated into the methylcellulose cultural system. The proliferation and hematopoietic potential of the CD34＋ cell of cord blood by the colony formation assay were detected on the day 10. Result The concentration of LDH in group A was （20. 71 ＋2. 81） U/L, which was significantly lower than that in group B （ 64. 28 ＋5.12） U/L and group C （ 84. 29 ＋ 4. 99 ） U/L. The concentration of LDH in group B was significantly lower than that in group C, while there were no significant differences in the concentration of LDH among groups A, D and E （ P 〉 0. 05 ）. The cpm in group A was （ 22 629± 3288 ）, which was significantly higher than that in group B （4598 ±2178） and group C （1626 ± 1192）. And the epm in group B was significantly higher than that in group C. There were no significant differences in the cpm among groups A, D and E （ P 〉 0.05 ）. On day 10 of culture, the total colonies, granulocyte-maerophage clony forming unit （ CFU-GM ） , mixed clony forming unit （CFU-GEMM）and erythroid burst clony forming unit （BFU-E） in group A were significantly higher than that in group B and C. The total colonies, CFU-GM and CFU-GEMM in group B were significantly higher than those in group C. No significant differences were found in the total colonies, CFU-GM, CFU- GEMM and BFU-E among groups A, D and E （ P 〉 0. 05 ）. There were no statistically significant differences in the CD95 and Annexin V expression among all the groups （P 〉 0. 05 ）. Conclusion PRA could impair the membrane, decrease the DNA synthesis, and inhibit the colony formation of CD34＋ cord blood cells, which could be strengthened by the presence of the complement at the given concentration in our study. PRA had no significant influence on the apoptosis of CD34＋ cells in vitro.