버섯成分이 Hela 培養細胞增殖에 미치는 影響 = (The) effects of mushroom components on the proliferation of HeLa cell line in vitro

다국어 초록 (Multilingual Abstract)

Introduction
Qualitative analysis of amino acid components contained in 15 species of edible mushrooms were first reported by Kim(1958) and similar studies were successively reported by Huh(1960), Ro and Pyo(1975) and Chung(1976). But nutritional effects of the amino acids of edible mushrooms were not yet investigated. Therefore it is necessary to determine whether the amino acids of mushrooms act as good nutrients for tissue cells.
In this paper an attempt was made to determine the effects of the alcohol extracts and the acid hydrolysate of 11 different edible mushrooms on the proliferaton of HeLa cell line in vitro, considering that there might be certain nutritional effects of the various amino acids and their constituents on the growth and development of the tissue cells.
Materials and Methods
HeLa cells were used which were derived from the epithelium of the human carcinoma and subcultured by successive passages for many generations.
Earle's balanced salt solution(Earle's BSS) and TC-199 tissue culture medium(Difco, Detroit, Michigan) were used as control cell culture medium. Predetermined optimal concentrations of the alcohol extracts of 11 different mushrooms were added to the basal medium which consists of Earle's BSS containing 100㎍/ml streptomycin, 100 units/ml sodium penicillin G and 5% fetal calf serum. Then these mixtures(pH 7.2) were sterilizied by filtration through millipore filter. HeLa cells were detached from the surface of culture vessels with trypsin-versene solution(Difco 1:250) and the concentration was adjusted to 2×10^5 cells/ml in culture medium, and then 1.0ml of cell suspension was placed in each Leighton tube and incubated 37℃ for three days.
Appearance of the proliferating cells was observed under an inverted microscope twice a day and the density of the cells in the culture media was counted by means of hemocytometer at 24, 48 and 72 hours of incubation by discarding the culture medium, dispersing the cells by 1 ml of trypsin-versene solution and resuspending the cells into appropriate amounts. To minimize the experimental errors, the each procedure was run in triplicate.
Results and Discussion
The morphology of the cultured cells in the Earle's BSS mixtures was as normal as that of the cells in the TC-199 medium, forming the normal epithelial cell monolayer along the wall of the culture vessels.
The cells in the Earle's BSS degenerated gradually as time passed and the morphology of cells changed into round shapes until all the cells were destroyed.
The appearance and density of the cells cultured for 24, 48 and 72 hours in the media containing the alcohol extracts and the acid hydrolysates fo 11 different mushrooms are shown in Plate 1. The alcohol extracts of Coprinus comatus, Agaricus campestris, Psaliota campestris, Lentinus edodes and Tricholoma matsutake and the acid hydrolysates of these five mushrooms had favorable effects on the cell proliferation both in their appearance and density as compared with those of the control groups in TC-199 medium. When the alcohol extracts and the acid hydrolysates of Pleurotus ostreatus, Famaria botrytis and Pholiota nameko were added to the media, the cell proliferation was well maintained until 48 hours and thereafter cellular degeneration appeared. The alcohol extracts and the acid hydrolysates of Auricularia auricula-judae, Tremella fuciformis Berk.and Gyrophora esculenta affected the cell proliferation to such degree that they kept on proliferating longer than 24 hours, and all the cells underwent degeneration.
Conclusion
In order to find out nutritional effects of edible mushrooms on the multiplication of tissue cells, the alchol extracts and acid hydrolysates of eleven species of mushrooms were added to solution B(Earle's BSS containing 5% fetal calf serum). A comparison of the respective multiplications of HeLa cells in this solution and in control solution A(TC-199) solution containing 5% fetal calf serum) yielded the following conclusion:
1. The alcohol extracts and acid hydrolysates of Coprinus comatus, Agaricus campestirs, Psalliota campestiris, Lentinus edodes, Tricholoma matsutake, Pleurotus ostreatus, Ramaria botrytis and Pholiota nameko influenced favorably the maintenance of the normal form and monolayer of HeLa cells.
2. The growth curves of HeLa cells in the cultures containing, respectively, the alcohol extracts and acid hydrolysates of these eight mushrooms showed that five species, i.e., Coprinus comatus, Agaricus campestris, Psalliota campestris, Lentinus edodes and Tricholoma matsutake effected an excellent multiplication and that the other three species were less effective than those five species.
3. As to the effects on the cell multiplication, no marked difference was observed between the alcohol extracts and the acid hydrolysates of the mushrooms tested.

목차 (Table of Contents)

• 목차
• 1. 緖論 = 3
• 2. 材料 및 方法 = 3
• 2·1. 버섯 成分 = 3
• 2·2. 細胞培養液 = 4
• 2·3. 細胞株 = 8
• 2·4. 細胞培養 = 8
• 2·5. 培養細胞 觀察 = 8
• 3. 實驗結果 = 8
• 3·1. 培養細胞의 形態 = 8
• 3·2. 細胞 增殖曲線 = 9
• 4. 考察 = 11
• 5. 結論 = 12
• 參考文獻 = 14
• 英文抄錄 = 15
• 寫眞附圖 = 17