骨髓腔内输注人造血干／祖细胞促进NOD-SCID小鼠体内造血功能的恢复

本研究比较经骨髓腔内输注（intra—bone marrow infusion，iBMI）及静脉输注（intravenous infusion，iVI）人脐血（cord blood，CB）造血千／祖细胞（HS／PC）对NOD～SCID受鼠体内造血重建的影响。将纯化的CB CD34^＋细胞移植到受亚致死剂量照射的NOD—SCID受鼠体内。受鼠随机分为3组：①iBMI组：骨髓腔内输注CD34^＋细胞5×10^5／只；②iVl组：尾静脉输注CD34^＋细胞5×10^5／只；③阴性对照组：输注PBS缓冲液。于异种移植后第3、5及第8周通过流式细胞术、聚合酶链式反应、免疫组织化学及造血祖细胞集落分析的方法比较人造血系统各系细胞植入率，通过二次移植实验比较NOD—SCID受鼠体内人HS／PC长期造血重建能力。结果表明：移植后第8周，iB—MI组受鼠骨髓、外周血及脾脏中人CD45^＋细胞比例均显著高于iVI组（P〈0．05）；iBMI组及iVI组移植的HS／PC均具有多向分化能力；移植后第8周，iBMI组受鼠骨髓中CD45^＋CD19^＋细胞、CD45^＋CD33^＋细胞、CD45^＋CD56^＋细胞及CD45^＋CD34^＋细胞比例均显著高于iVI组（P〈0．05），CD45^＋CD14^＋细胞及CD45^＋CD41a^＋细胞比例亦高于iVI组（P〉0．05）。iVI组及iBMI组长期存活受鼠的肝脏、脾脏、肺脏、外周血、骨髓细胞中均可检测到人17号染色体α-卫星特异性片段。应用免疫组织化学法在移植后第8周的iBMI组受鼠的脾脏、肝脏和肺脏中均可检出人CD45抗原的表达.iBMI组受鼠的骨髓细胞各系集落总数于移植后第8周显著高于iVI组（P〈0．05）。二次移植后第6周，iVI组及iBMI组受鼠的各组织脏器中均可检出人17号染色体α-卫星特异性片段的表达。结论：与iVI相比，经iBMI移植人CBCD34^＋细胞至NOD—SCID受鼠可以促进HS／PC的造血重建及多向分化，提高植入率。

Intra-bone Marrow Infusion of Human Cord Blood Hematopoietic Stem/ Progenitor Cells Improves Hematopoietic Reconstitution in NOD-SCID Mice

The purpose of this study was to explore the effect of intra-bone marrow infusion （iBMI） of cord blood （CB）-derived hematopoietic stern/progenitor cells （HS/PCs） on human bematopoietic reconstitution in xenotransplanted NOD-SCID mouse model. Aliquots containing 5 × 10^5 CB CD34 ^＋ cells were transplanted into sublethally irradiated NOD-SCID mouse via intravenous infusion （iVI） or iBMI routes. 64 female NOD-SCID mice were divided randomly into 3 groups ： iBMI group, iVI group and negative control group. The engraftment levels of human hematopoietic cells at 3, 5 and 8 weeks after xenotransplantation were detected by fluorescence-activated cell sorter （FACS）, polymerase chain reaction （PCR）, immunohistochemistry and HPC colony formation assay, and long-term hematopoietic reconstitution capacity of HSC was tested by secondary transplantation. The results showed that the percentages of human CD45 ^＋ cells in bone marrow, peripheral blood and spleen of recipient in iBMI group at 8 weeks after xenotransplantation were significantly higher than those in iVI group （p 〈 0.05 ）. HS/PCs given through both iVI and iBMI methods had the ability of multilineage differentiation, the percentages of CD45 ^＋ CD19^＋ cells, CD45 ^＋ CD33 ^＋ cells, CD45^＋ CD56 ^＋ cells and CD45^＋ CD34^＋ cells of recipient bone marrow in iBMI group at 8 weeks after xenotransplantation were significantly higher than those in iVI group （p 〈 0.05 ）, while other lineages in iBMI group were also higher than that in iVI group （p 〉0.05）. α-satellite-specific fragment of human chromosome 17 could be detected by PCR in liver, spleen, lung, peripheral blood and bone marrow cells of long-term survival recipients in both iVI and iBMI groups. Human CD45 antigen could be detected by immunohistochemical method in spleen, liver and lung of recipients in iBMI group at 8 weeks after xenotransplantation. Total colony count in iBMI group at 8 weeks after xenotransplantation was significantly higher than that in iV/group （p 〈 0.05 ）. α-satellite-specific fragment of human chromosome 17 could be detected in all above organs of both group recipients at 6 weeks after secondary xenotransplantation. It is concluded that iBMI of CB CD34 ^＋ cells improves hematopoietic reconstitution in xenotransplanted NOD-SCID mouse model.