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

目前我们研究NK4通过抗血管生成的作用。我们发现，尽管肿瘤中NK4浓度较低，腹膜内注射NK4比肿瘤内注射NK4更有效地抑制肿瘤生长。我们评估了NK4腺病毒转化后骨髓细胞向异种移植肿瘤的募集情况。方法：建立骨髓移植模型。C57BL6小鼠采用12Gy致死照射和移植的GFP小鼠造血干细胞。通过这种方法，在骨髓移植2周后可获得约80%的嵌合体小鼠。接下来，将嵌合体小鼠接种NK4腺病毒（AdNK4）或Null腺病毒（AdNull）感染的LLC肿瘤。采用激光扫描显微镜观察经zennon TM三色标记试剂盒（分子探针）染色的小鼠骨髓源性细胞和细胞结构血管的生长情况。结果：在体外实验中，感染了NK4腺病毒的肿瘤细胞生长无明显差异，而体内感染了AdNK4腺病毒的肿瘤细胞生长受到明显抑制。虽然肿瘤中有相当数量的GFP阳性细胞浸润，但大多数GFP阳性细胞不与细胞结构血管融合。NK4组切除肿瘤后，肿瘤血管和骨髓源性细胞均明显减少。许多骨髓来源的细胞用血细胞标记物染色（CD45阳性细胞/ GFP阳性细胞AdNK4 71.3%, AdNull 66.0%）。有趣的是，与AdNull感染的肿瘤相比，AdNK4感染的肿瘤中VEGF阳性骨髓来源细胞显著减少（GFP、VEGF双阳性/总GFP阳性细胞分别为36.3%和8.2%）。VEGF阳性骨髓源性细胞的来源和分化尚不清楚。结论：在本研究中，我们没有发现抗血管生成作用与骨髓源性细胞之间的直接关系，但在NK4感染的肿瘤中，骨髓源性细胞减少。这些细胞可能以旁分泌方式对肿瘤血管生成有一定影响。在NK4抑制的肿瘤中，血管生成和骨髓来源细胞之间的关系需要进一步的研究。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