Significance of autocrine mechanism of VEGF/VEGF receptor in ATL cell proliferation.

在本研究中，在MS-5共培养体系中，小鼠骨髓源性基质细胞、MS-5、htlv - i转化的T细胞系和ATL细胞临床样品与基质层紧密接触生长，形成由10到100多个细胞组成的所谓“鹅卵石区”（car鹅卵石area， CA）。形态学、免疫表型和southern blotting分析表明，ca组成细胞与ATL细胞兼容，与ATL原代细胞为同一克隆。免疫染色和RNA原位杂交显示5例CA细胞中病毒蛋白、p40tax和p19gag明显降低。在半固体培养条件下，7例ATL的接种细胞数与CA数呈线性关系。因此，我们的共培养系统首次提供了ATL原代细胞的粘附依赖性生长，而不产生htlv - i相关蛋白，这模拟了ATL细胞的体内生长。我们检测到Flt-1 mRNA和受体在三个ATL…更多细胞系（KK1， SO4和st1）中的表达，而没有检测到KDR mRNA。对于来自患者的原代ATL细胞，11个样本中有8个（73%）和11个样本中有1个（9%）分别表达Flt-1和KDR rRNA。此外，在3个细胞系和11个（100%）ATL患者样本中检测到VEGF mRNA的表达。抗VEGF抗体可以抑制血管内皮生长因子及其受体与ATL细胞的结合，但对三种ATL细胞系液体培养的细胞增殖无显著影响。MTT法检测重组人VEGF (rhVEGF) （0 ~ 400 ng/ml）对细胞增殖无影响。另一方面，在我们的共培养系统中，与缺乏抗vegf抗体的情况相比，CA的数量和CA组成细胞的细胞计数没有受到影响。此外，添加rhVEGF对ATL-CA数和细胞计数无影响。接下来，共培养2、5和10天后，通过胰蛋白酶化从共培养体系中收获KKI。在CA形成实验中观察到，虽然添加rhVEGF后收获的细胞数量有增加的趋势，但不同条件之间没有显著的统计学差异。以上结果表明，目前在MS-5共培养体系中，VEGF对ATL细胞生长无明显影响。VEGF/VEGF受体自分泌系统被认为是ATL细胞生物学中与体内微环境相互作用的重要因素，其作用需要更精确地阐明，目前正在进一步的研究中。少

In the present study, in the co=culture system with MS-5, mouse-bone marrow derived stromal cells, MS-5, HTLV-I-transformed T cell lines and clinical samples of ATL cells grew in close contact with stromal layer and formed-so-called "cobblestone areas (CA)" composing 10 to over 100 cells. Morphology, immunophenotypirig, and southern blotting analysis indicated that CA-composing cells were compatible with ATL cells which were identical clone with primary ATL cells. Immunostaining and RNA, in situ hybridization indicated that viral protein, p40tax and p19gag was markedly decreased in CA cells in five cases. There was a linear relationship between inoculated cell number and CA numbers in seven ATL cases in semi-solid cultyure condition. Thus, our co-culture system provides for the first time the adhesion-dependent growth of primary ATL cells without production of HTLV-I-related proteins, which mimics in vivo growth of ATL cells. We detected Flt-1 mRNA and receptor expression in three ATL … More cell lines examined (KK1, SO4, and ST 1), whereas no KDR mRNA was detected. Concerning primary ATL cells from patients, expression of Flt-1 and KDR rRNA was seen in eight of 11 (73%) and one of 1l (9%) samples, respectively. Furthermore, mall three cell lines and 11 of 11 (100%) samples from ATL patients, expression of VEGF mRNA was detected. Anti-VEGF antibody, which could inhibit binding of VEGF and its receptors on ATL cells, had no significant effect on the cell proliferation of three ATL cell lines in liquid culture. Furthermore, the cell proliferation was not affected by recombinant human VEGF (rhVEGF) treatment (0-400 ng/ml) by MTT assay. On the other hand, in CA formation assay with our co-culture system, number of CA and cellular count of CA-composing cells were not affected compared with the condition of absence of anti-VEGF antibody. Furthermore, addition of rhVEGF had no effect of ATL-CA number or celluar count. Next, KKI was harvested from co-culture system after co-culturing at day 2,5, and 10 by trypsinization. As observed in CA formation assay, although harvested cellular numbers tended to be higher by addition of rhVEGF, there was no significant statistical difference between various condition. These observation indicated that VEGF has no significant effect on ATL cell growth in MS-5 co-culture system, for the present. The effect of VEGF/VEGF receptor-autocrine system, which is considered to be important on ATL cell biology in context of interaction with in vivo microenvironment, have to be elucidated more precisely, and further investigations are now in progress. Less