|Budget Amount *help
¥3,600,000 (Direct Cost : ¥3,600,000)
Fiscal Year 1999 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1998 : ¥3,100,000 (Direct Cost : ¥3,100,000)
Intrahepatic cholangioma is characterized by stromal fibrosis and proliferation of intra- and peri-tumoral alpha-smooth muscle actin positive cells which are considered as activated hepatic stellate cells (HSC). These findings suggest that HSC may be influenced by the tumorcells and support tumor invasion. In the present study, we examined the influence of human intrahepatic cholangioma cell line on proliferation, migration and gene expression of matrix metalloproteinases (MMPs) which play important roles in tumore invasion, of human HSC-derived cell line in vitro. METHODS : HSC-derived cell line, LI-90 cells were cultured or co-cultured with intrahepatic cholangioma cell line, HuCCTl cells without cellular contact in a Traswell cell culture chamber. Proliferation of LI-90 cells was examined by WST-1. The invasion and migration toward HuCCTlcells were assayed in a Transwell chamber. Gene expression of MMP-1, 2 and 14 in LI-90 cells were evaluated by semi-quantitative RT-PCR.RESULTS : 1
) Proliferation of LI-90 cells significantly increased in co-culture with HuCCTl cells compared with that in single culture (p < 0.01). 2) The number of invading or migrating LI-90 cells across the filter was significantly higher in co-culture with HuCCTl cells than that in single culture (p < 0.01). 3) Gene expressions of MMP-1, 2 and 14 in LI-90 cells in co-culture with HuCCTlcells were higher than those in single culture. Our in vitro study demonstrated that HuCCTl cells induced proliferation, migration and MMPs gene expressions of LI-90cells. CONCLUSION : These suggest that intrahepatic cholangioma cells might recruit HSC and induce proliferation and MMPs gene expressions of HSC, resulting in supporting invasion of intrahepatic cholangioma in vivo.
2.in vitroにおけるHuCCT1のLI90に及ぼす影響について,transwell chamberを使用し,形態,増殖,浸潤,migration,MMP-1,2,14の遺伝子発現の点から検討した.形態的にco-culture前はhill and valley状でやや細長い星状であったが,co-cultureにより細胞は分散,伸展した.増殖能は,co-cultureにより亢進した.MMP-1,2,14の遺伝子発現は,co-culture後の細胞よりRNAを抽出しRT-PCRで検討し,いずれのMMPも遺伝子発現量が増加した.浸潤わよびmigration実験は,下室にHuCCT1を,上室にLI90をまきtranswellの下面に移動した細胞数で検討したが,下室に癌細胞を培養したほうが細胞数は多かった.