肝细胞有效生成血小板能力的因素分析研究

Factor analysis of effective platelet-producing ability of fetal liver-derived cells

目的探究不同来源的供体造血干细胞(hematopoietic stem cells, HSCs)体内移植后生成血小板(PLT)的差异因素, 进而为解决临床上HSCs移植后PLT重建不良的问题提供新思路。方法通过Ficoll分离技术分别从孕14.5 d(E14.5)胎肝(FL)和8周龄小鼠骨髓(BM)中分离HSCs及其下游髓系和淋巴系细胞, 即单个核细胞(MNCs)总和, 移植入清髓辐照后的受体小鼠胫骨骨髓腔内, 构建造血重建FL-MNCs和BM-MNCs小鼠移植治疗模型。定期检测受体小鼠外周血血常规指标。通过流式细胞方法检测供体细胞在受体内的嵌合率及PLT形成上游细胞。进一步利用磁珠分选FL-MNCs和BM-MNCs供体细胞中CD41+巨核细胞, 并诱导PLT生成。另外, 通过定量RT-PCR检测两种来源的CD41+巨核细胞的PLT生成相关基因的表达情况。结果 FL-MNCs移植组和BM-MNCs移植组的血常规参数包括白细胞(WBC)、红细胞(RBC)、PLT均在移植后第4周恢复正常水平, 受体的血细胞基本由供体细胞替代。FL-MNCs移植组比BM-MNCs移植组受体小鼠的PLT水平上升更快, 移植后第4周FL-MNCs组和BM-MNCs组的PLT水平分别为(1.45±0.37)×1012/L和(1.22±0.24)×1012/L, P<0.05。同时, FL-MNCs中含有更高比例的Lin-Sca-1+c-Kit+造血干细胞(FL-MNCs为7.60%±1.40%, BM-MNCs为1.10%±0.46%, P<0.01)、Lin-Sca-1-c-Kit+CD41+CD150+巨核祖细胞(FL-MNCs为3.05%±0.22%, BM-MNCs为0.15%±0.02%, P<0.01), 以及CD41+CD42b+成熟巨核细胞(FL-MNCs为1.60%±0.06%, BM-MNCs为0.87%±0.11%, P<0.01)。体外功能性研究发现胎肝来源的FL-MNCs-CD41+巨核细胞在体外诱导后可更快地生成前血小板样细胞, 第3天即有前血小板样细胞形成, 第5天有明显PLT形成, 而骨髓来源的BM-MNCs-CD41+巨核细胞第5天仍没有前血小板样细胞形成。FL-MNCs-CD41+能表达更高水平的PLT生成相关基因Mpl(3.25倍)、Fog1(3倍)和Gata1(1.5倍)(P<0.05)。结论 FL-MNCs具有更好的受体血小板植入效果以及更快的体外PLT分化速率, 这可能与FL-MNCs细胞组成中较高比例的巨核细胞数量及其Mpl、Fog1和Gata1等相关基因的表达水平有关。这些供体细胞特征的因素分析研究有望为临床治疗造血干细胞移植后PLT重建不良提供供体细胞参考依据。

Objective To study the different factors affecting platelet production post transplantation of hematopoietic stem cells(HSCs)isolated from different sources in order to explore novel options for treating platelet depletion following HSCs transplantation.Methods HSCs and their downstream derivatives including myeloid and lymphoid cells(i.e.,collective of mononuclear cells(MNCs)),were isolated from E14.5 fetal liver(FL)and bone marrow(BM)of 8-week-old mice by Ficoll separation technique.These cells were subsequently transplanted into the tibia bone marrow cavity of recipient mice post lethal myeloablative treatment in order to construct the FL-MNCs and BM-MNCs transplantation mouse model.Routine blood indices were examined in these recipient mice.The chimeric rate of donor cells in recipient peripheral blood cells were determined by flow cytometry.Different groups of cells involved in platelet reconstruction were analyzed.CD41+megakaryocytes were sorted from fetal liver or bone marrow using magnetic beads,which were then induced to differentiate into platelets in an in vitro assay.Quantitative RT-PCR was used to detect the expression of platelet-related genes in CD41+megakaryocytes from the two sources.Results Both the FL-MNCs and the BM-MNCs transplantation groups resumed normal hematopoiesis at the 4th week after transplantation,and the blood cells of the recipient mice were largely replaced by the donor cells.Compared with the mice transplanted with BM-MNCs,the platelet level of mice transplanted with FL-MNCs recovered faster and were maintained at a higher level.At week 4,the PLT level of the FL-MNCs group was(1.45±0.37)×1012/L,and of the BM-MNCs group was(1.22±0.24)×1012/L,P<0.05.The FL-MNCs contain a higher proportion of hematopoietic stem cells(Lin-Sca-1+c-Kit+)(7.60%±1.40%)compared to the BM-MNCs(1.10%±0.46%),P<0.01;the proportion of the megakaryocyte progenitor cells(Lin-Sca-1-c-Kit+CD41+CD150+)and mature megakaryocyte cells(CD41+CD42b+),also differ significantly between the FL-MNCs(3.05%±0.22%,1.60%±0.06%,respectively)and the BM-MNCs(0.15%±0.02%,0.87%±0.11%,respectively)groups,both P<0.01.In vitro functional studies showed that FL-MNCs-CD41+megakaryocytes could produce proplatelet-like cells more quickly after induction,with proplatelet-like cells formation on day 3 and significant platelet-like particle formation on day 5,in contrast to bone marrow-derived BM-MNCs-CD41+megakaryocytes that failed to form proplatelet-like cell on day 5.In addition,FL-MNCs-CD41+cells expressed higher levels of platelet-related genes,Mpl(3.25-fold),Fog1(3-fold),and Gata1(1.5-fold)(P<0.05).Conclusion Compared with the BM-MNCs group,the FL-MNCs transplantation group appears to have a more efficient platelet implantation effect in the HSCs transplantation recipient in vivo,as well as a higher platelet differentiation rate in vitro.This might be related to a higher proportion of megakaryocytes and higher expression levels of genes such as Mpl,Fog1,and Gata1 that could be important for platelet formation in FL-MNCs-CD41+cells.Further exploration of the specific functions of these genes and the characteristics of the different proportions of the donor cells will provide valuable clues for the future treatment of platelets reconstitution after HSCs transplantation clinically.