FUKASAWA Manabu Yamagata Univ., Sch. of Med., Instructor, 医学部, 助手 (90218876)
SHIMANUKI Takao Yamagata Univ., Sch. of Med., Instructor, 医学部, 助手 (90211284)
WASHIO Masahiko Yamagata Univ., Sch. of Med., Prof., 医学部, 教授 (20018310)
|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1992 : ¥200,000 (Direct Cost : ¥200,000)
Fiscal Year 1991 : ¥1,800,000 (Direct Cost : ¥1,800,000)
Neonatal cardiac myocytes (CMs) and fibroblasts (CFs) have various potential capacities including cell proliferation, growth, and differentiation. In addition, CMs interact with and are modified by the presence of CFs. In this study, we evaluated the modulation of CM beating, growing and hypothermic injury by CFs. CMs and CFs were isolated from neonatal rat ventricles and cultures of myocyte only or in co-culture with CFs were established. A CM and CF concentration of 2.5 x 10^B cells/ml was chosen and the proportion of CF was determined to be 33% (ml CM: ml CF) as follows: 4:0 (group C), 4:2 (group M-F). In the normothermic study, CMs and CFs were cultured for 21 days,and myocyte beating rate and hypertrophy were evaluated. In the hypothermic study, cells were incubated at 4 ﾟC for 12, 18, 24, 36 and 48 hrs on the 4th day of culture,, and the enzyme release (CPK and LDH) and the recovery of CM beating rate were measured.
CM beating of group C increased rapidly with peak value on day 5
(265 beats/min), which continued for 5 days, and then decreased. In group M-F, the increase of CM beating was markedly suppressed, however, the enlargement of CM volume was stimulated (133% of control group myocyte). In the hypothermic study, the recovery ratio of CM beating rate in group C decreased at 18 hrs (55.6% of control), reaching null levels at 48 hrs. However, group M-F showed a significantly increased recovery (at 18 hours: 95.7%; at 48 hours: 27.6%). Release of CPK and LDH in group C increased gradually by 24 hours, and showed a marked increase at 48 hours (CPK:816 mIU/flask, LDH:1186 mIU/flask). However, the levels of CPK and LDH in group M-F were significantly lower at 18 hours, and the greatest difference was observed at 48 hours (CPK:216, LDH:473). Thus, cardiac fibroblasts suppressed the myocyte beating, stimulated the hypertrophy and protected the hypothermic injury. These results indicated that fibroblasts controlled cardiac myocyte viability and growth.
研究結果:4℃、48時間までの低温状態では、線維芽細胞の存在により、低温による心筋細胞障害の進行は著明に抑制された。線維芽細胞は、非生理的状態(例えば低温、虚血、再灌流など)において生ずる心筋細胞障害に対し保護的に作用するものと考えられた。一方、保存液に関しては、細胞外液型保存液に比して、細胞内液型では細胞障害性が高く、また、細胞外液型保存液としては、細胞培養用基礎培地が最も適切なものと考えられた。緩衝剤ではHEPES bufferは心筋細胞保存に対し有効であったが、燐酸及び重炭酸では細胞毒性が認められた。 Less