| Co-Investigator(Kenkyū-buntansha) |
JINNAI Itsuroh Nagasaki University, Graduate School of Biomedical Science, Assistant Professor, 大学院・医歯薬学総合研究科, 助教授 (70162823)
TSUKASAKI Kunihiro Nagasaki University Hospital, Lecurer, 医学部附属病院, 講師 (40274659)
HAYASHIBARA Toshihisa Nagasaki University, Graduate School of Biomedical Science, Lecturer, 大学院・医歯薬学総合研究科, 講師 (40325634)
NAGAI Kasuhiro Nagasaki University Hospital, Lecturer, 医学部附属病院, 講師 (30304942)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
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
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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
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