배양에 따른 심장근육세포의 박동과 세포사이결합의 변화

Abstract

This experiment was designed for the elucidation of relationships between the cell adhesion molecules and synchronous beating rates of cardiomyocytes isolated from ventricles of 3-day-old rats. These cells were grown on the culture vessel coated or non-coated with cardiogel for 1, 3, 5, and 7 days. The synchronous beating rate and morphologic changes of cells were investigated under the inverted microscope. Those changes of cardiomyocytes were observed by transmission elcectron microscopy (TEM). Connexin43, pan-cadherin, and alpha-sarcomeric actin were stained by indirect immunofluorescent (IF) technique. Western blotting were used for identifying unique bands of connexin43 and pan-cadherin in the regions of specific size. The synchronous beating numbers of cardiomyocytes in each group on the dish coated or non-coated with cardiogel were significantly different for 3, 5, and 7 days in culture. The maximum values of synchronized beating in the cells appeared on the day 5. The beating numbers of cells grown on coated dish comparing with non-coated dish were significantly increased on the day 5 and day 7. The proliferation of cardiomyocytes increased markedly in the cardiogel-coated dish on the day 5, while the number of fibroblasts in non-coated dish were obviously increased on the day 7. In indirect IF studies, a normal redistribution of connexin43, pan-cadherin, and alpha-sarcomeric actin of the cells was expressed in day 3 throughout day 5. The reaction intensity of those proteins were increased or decreased in the proportion to the beating numbers. The lack of reaction was appeared in the fibroblasts consisting of monolayer on the day 7. Unique bands of connexin43 and pan-cadherin having a specific size were marked on Western Blotting. The structures such as gap junction, fascia adherence and desmosome compatible with intercellular adhesion in cardiac muscle were abundant on day 3 to 7. In conclusion, the amount or reaction intensity of connexin43 and pan-cadherin in cardiomyocytes were stronger simultaneously with the increase of synchronous beating numbers, particularly for 3 and 5 days. The maximum beating rates of cardiomyocytes reached on the day 5, while the maximum beating rates of them were markedly decreased owing to the proliferation of fibroblasts on day 7. TEM findings consisting of intercalated discs might be explained regardless of coating with cardiogel on why the close relationships between the beating rates and the connexin43 and pan-cadherin of those cells in culture exist.

생후 3일된 흰쥐의 심장근육세포들을 배양하면서 배양시간에 따른 동시박동 수를 측정하였고, 세포사이결합의 분포양 상을 조사하였다. 이를 통해 동시박동 수의 변화와 세포사이결합과의 관계를 이해하고자 한다. 심장근육세포를 분리한 후, cardiogel이 처리 또는 처리되지 않은 배양용기에서 1, 3, 5, 7일 동안 배양하였다. 도립위상차현미경 하에서 세포들의 동시 박동 수를 측정하였고, 투과전자현미경과 함께 형태학적 변화들을 관찰하였다. Connexin43, pan-cadherin, alpha-sarcomeric actin을 간접면역형광 염색하였고, Western blotting으로는 connexin43과 pan-cadherin의 특정한 띠들을 확인하였다. Cardiogel 처리군 및 비처리군 모두에서 배양 3, 5, 7일에 동시박동 수는 유의한 차이를 보였고, 처리군의 5일에 최고 수 치를 기록하였다. Cardiogel 처리군에서 비처리군에 비해 동시박동 수가 배양 5일과 7일에 유의하게 증가하였다. Cardiogel 처리군에서 비처리군에 비해 배양 5일에 심장근육세포들의 증식이 현저하였지만, 배양 7일에는 비처리군에서 상대적으로 많은 섬유모세포들이 나타났다. Connexin43, pan-cadherin 및 alpha-sarcomeric actin은 배양 1일의 세포들에 비해 3, 5, 7일에 정상적으로 재분포되었고, 동시박동 수에 따라 그 반응도 증감하였다. 배양 7일에 단층을 형성한 부분에 염색되지 않는 섬유모세포들이 증가하였다. 특정한 부위에서 connexin43과 pan-cadherin의 띠를 확인하였다. 배양 3, 5, 7일에 교통반점, 부 착막, 부착반점이 많이 나타났다. 결론적으로 심장근육세포의 배양 3일과 5일에는 동시박동 수의 증가에 따라 connexin43과 pan-cadherin의 양도 증가하 였다. 배양 5일에는 동시박동 수치가 최고에 이르렀으나, 7일에는 섬유모세포의 증식으로 동시박동 수가 떨어졌다. 따라서 사이원반을 구성하는 미세구조적 소견들은 cardiogel 처리군과 비처리군 모두에서 동시박동 수와 connexin43과 pancadherin과의 관계를 해석하는데 도움이 되었다.