Analyses of inhibitory effect of tumor angiogenesis, targeting the dynamism of bone marrow derived cells

到目前为止，我们已经研究了NK4通过抑制血管生成的作用。我们认识到，尽管NK4在肿瘤中的浓度较低，但NK4仍然比肿瘤内注射更有效地抑制肿瘤生长。我们评价了NK4腺病毒转化后骨髓细胞向移植瘤的募集情况。C57BL6小鼠接受12Gy射线致死照射，移植GFP小鼠的造血干细胞。骨髓移植2周后，可获得约80%的嵌合体小鼠。然后，用NK4腺病毒(AdNK4)或空腺病毒(AdNull)感染嵌合体小鼠，接种LLC肿瘤。于接种后第14天处死小鼠，测量肿瘤长度，切除肿瘤，激光扫描显微镜下观察骨髓来源细胞及Zennon TM三色标记试剂盒(分子探针)染色的细胞结构血管。结果：体外t…NK4腺病毒和空腺病毒对肿瘤细胞的生长无明显影响，但在体内，AdNK4对肿瘤生长有明显的抑制作用。虽然在肿瘤中有相当数量的GFP阳性细胞浸润，但大多数GFP阳性细胞没有与细胞结构血管融合。在NK4组切除的肿瘤中，肿瘤血管和骨髓来源细胞均显著减少。CD45阳性细胞/GFP阳性细胞AdNK4占71.3%，AdNull占66.0%。值得注意的是，感染AdNK4的肿瘤与感染AdNull的肿瘤相比，VEGF阳性的骨髓来源细胞明显减少(GFP、VEGF双阳性/总GFP阳性细胞分别为36.3%、8.2%)。结论：本研究未发现抗血管生成作用与骨髓来源细胞的直接关系，但在NK4感染的肿瘤中，骨髓来源细胞减少，这些细胞可能通过旁分泌方式对肿瘤血管生成起作用。在被NK4抑制的肿瘤中，血管生成和骨髓来源细胞之间的关系还需要进一步的研究。较少

We have studied effect of NK4 through anti-angiogenesis so far. We realized that NK4 suppressed tumor growth more effective in spite of low concentration of NK4 in tumor by intra-peritoneal injection than by intra-tumoral injection. We evaluated the recruitment of cells from bone marrow to xenograft tumor after transforming of NK4 adenovirus.Method : Bone marrow transplantation was established below. The C57BL6 mice were exposed by lethal irradiation of 12Gy and transplanted hematopoietic stem cells from GFP mice. By this procedure, about 80% chimera mice can be obtained 2 weeks after bone marrow transplantation. Next, the chimera mice were inoculated LLC tumor infected by NK4 adenovirus (AdNK4)or Null adenovirus (AdNull). The tumor implanted in mice were measured in length and resected on day14 after inoculation and evaluated bone marrow derived cell and cells structured vessels dyed with zennon TM Tricolor Labeling Kit (Molecular probe) by laser scanning microscopyResult : In vitro t … More here is no difference in growth of tumor cells infected with between NK4 adenovirus and Null adenovirus however, in vivo AdNK4 infected tumor were suppressed significantly in tumor growth. Though there are considerable amount of GFP positive cells infiltrated in tumor, most GFP positive cells did not merge with cells structured vessels. In resected tumor of NK4 groups, both tumor vessels and bone marrow derived cells decreased significantly. Many bone marrow derived cells were dyed with hematocyte markers (CD45 positive cells / GFP positive cells AdNK4 71.3%, AdNull 66.0% ). Interestingly, VEGF positive bone marrow derived cells decreaced considerably in tumor infected with AdNK4 compared to tumor infected with AdNull (GFP, VEGF double positive / total GFP positive cells: 36.3%, 8.2% respectively). The origin and differentiation of VEGF positive bone marrow derived cells has not unknown yet.Conclusion : In this study, we could not find direct relation between anti-angiogenic effect and bone marrow derived cells, however, bone marrow derived cells decreased in tumor infected with NK4., These cells possibly have some effect on tumor angiogenesis in the paracrine manner. Further examination on the association among angiogenesis and bone marrow derived cells in the tumor suppressed by NK4 is needed. Less