Separation and culture of the connective tissue cells expressing 43 kDa zinc-binding protein from common carp

• Main Authors: Yen-Hua Chen, 陳彥樺
• Other Authors: Sen-Shyong Jeng
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/75473976451628202464

博士 === 國立臺灣海洋大學 === 食品科學系 === 96 === Summary Zinc concentration in the digestive tract of common carp is always 3-10 times higher than most animal tissues. It was known that this high zinc came from a 43 kDa zinc-binding membrane protein. An indirect immunoperoxidase staining method using antibody against the 43 kDa zinc-binding protein was applied to the sections of digestive tract of the fish. It was found that the zinc-binding protein is mainly located in the connective tissue of its lamina propria and submucosal layer. Immunohistochemical staining of the 43 kDa zinc-binding protein also indicated that the 43 kDa zinc-binding protein is located on the connective tissue cells. In order to study the physiological meaning of high zinc in the common carp, in this report, separation and culture of the connective tissue cells expressing the 43 kDa zinc-binding protein from common carp were performed. To release the connective tissue from the digestive tract of the common carp, the mucosa was scraped with a glass slide, and the remaining de-mucosa tissue was treated with various enzymes. It shows that the digestion capacity of collagenase type IV is much higher than collagenase type I , or hyaluronidase . Chemical analysis of the digested solution indicated that the connective tissue was virtually completely digested. The cells collected from the collagenase type IV- digested solution were rather homogeneous, and over 90% of the cells were round with a diameter of �� 6 �慆 ; these are the cells which express the 43 kDa zinc-binding protein as indicated by indirect immunofluorescent staining. The mean zinc concentration in the connective tissue cells of common carp was found to be 2.21 μg zinc/(106 cell) which is about 20-30 times higher than grass carp, silver carp and tilapia. SDS-PAGE and Western blot demonstrated that the detergent extract of the connective tissue cells of common carp showed that a protein of apparent molecular weight 43 kDa was the major component. In this study, the 43 kDa zinc-binding protein accounted approximately 80% of the total proteins in the detergent extract of the connective tissue cells of the common carp. Primary explant culture of the de-mucosa tissue of the digestive tract of common carp was performed under the addition of DMEM/F12 medium supplement with 20% carp serum at 27℃. It was found that the cells harvested from primary explant culture of common carp were round with a diameter of �� 6 �慆. These cells grew rapidly within 4 h. Under time lapse video micrographic examination cell division of the connective tissue cell was observed. Connective tissue cells suspension was separated from the primary explant culture. Under the condition of supplementing with 20% carp serum to the medium and at 27℃, the connective tissue cells proliferated in suspension, and the population doubling time was about 1 h. At h 4, the cell concentration reached its maximum concentration of 4×104 cells/ml, and maintained at this concentration until h 22. Connective tissue cells from common carp did not grow in the medium without adding carp serum or supplement with fetal bovine serum. The connective tissue cells from common carp could be serial subcultured if the medium (and the carp serum) was periodically changed. Proliferation of the connective tissue cells from common carp was enhanced when ZnCl2 was supplemented. In order to know the differentiation of the connective tissue cells from common carp, primary explant culture of the de-mucosa tissue was carried out. It was found that the cells released from the explant proliferated rapidly within 4 h. From h 4 to h 30, the number and morphology of the cells did not change much. However, from h 31 to d 5, the size of the cells changed from its original size of ~6 �慆 to ~16 �慆, and the shape changed from round to triangle cylinder. The ~16 �慆 cells adhered to the cultured wells at d 5. At d 10, the size of the adhered cells became larger to a size of ~20 �慆. Collagen stain indicated that the cells might contain collagen. At d 20, the cells grew to a “mass” and the size was ＞50 �慆. Silver and Hematoxylin and Periodic acid Schiff and Hematoxylin stain indicated that extracellular matrix was released from the cells. Immunofluorescent staining indicated that the “mass” of the cell was reactive to the antibody against the 43 kDa zinc-binding protein. At d 40 of the primary explant culture, the cells differentiated to different type of “tissues”. The most common one was the connective tissue-like. In the connective tissue-like, the cells did not contact each other but surrounded by extracellular matrix. At d 85, it was found that there were “holes” in the extracellular matrix. The cells might be in its aging state. Based on the above results, it is concluded that high zinc in common carp derived from the fibroblast of its connective tissue cells. Fibroblast from common carp could proliferate in suspension and have a population doubling time of 1 h. It appears that the fibroblast could differentiate to “different form of tissues”. The fibroblast from common carp might be a very useful material for studying the biology of animal cells and the physiology of common carp.