| Hyriopsis cumingii is one of the most important freshwater mussels in China.It plays an important strategic and economic role in freshwater culture.There is no systematic study on the ability of cell proliferation and nacre formation of mantle cells of different years of Hyriopsis cumingii.In this paper,we first studied the proliferation and biomineralization of the mantle cells of Hyriopsis cumingii at different years,and determined that Hyriopsis cumingii with fast cell proliferation and strong biomineralization was the donor of pearl culture.Then we cultured the mantle,gill,visceral mass tissues of the most suitable year of Hyriopsis cumingii in vitro to explore the proliferation and biomineralization activity of different tissue cells,as well as the effect of helium mutation on cell growth,and determined the best tissue cells as donors.Finally,the mantle cells and gill cells were transplanted into the living mantle to explore the growth status of cells after transplantation and the influence of living environment on cell proliferation,in order to provide basic data for further study of cell proliferation of Hyriopsis cumingii and selection of donor and the best tissue in artificial pearl cultivation.The main research contents and results are as follows:1.Proliferation and biomineralization of mantle cells of different clam years in Hyriopsis cumingiiIn this study,five groups of Hyriopsis cumingii of different years(0.5,1,2,3,4)were selected.The proliferation index(Pr I =(S+ G2 / M)/(G0 / G1 + S + G2 / M)and intracellular Ca2+ concentration of the mantle cell cycle were analyzed by flow cytometry and real-time fluorescence quantitative PCR was analyzed CA,ALP gene expression and CA and ALP activities were detected.The results showed that the concentrations of Pr I and Ca2 + in the mantle cells of the 2 year mantle were significantly higher than those of other years mantle(P < 0.05);ALP and CA of biomineralization related factors were expressed in the mantle tissues of different years of Hyriopsis cumingii,and they were highly expressed in the lower years(0.5,1),but the expression gradually decreased with the increase of year.The 2 year Hyriopsis cumingii was only inferior to the lower year mussel,showing a sustained high expression.Mineralization factors were mainly used to regulate the growth of Hyriopsis cumingii shell in the low year stage.The mineralization activity of the 2 year was significantly higher than that of the 3 and the 4 year Hyriopsis cumingii(P < 0.05),suggesting that the year of the Hyriopsis cumingii had a better pearl accumulation advantage.In conclusion,the 2 year mantle cells have strong proliferation ability,high intracellular calcium concentration,and relatively high activity of biomineralization related genes and enzymes.Therefore,it is concluded that the 2 year Hyriopsis cumingii is suitable for both donor and cell culture.In this study,the mantle of Hyriopsis cumingii(0.5,1,2,3,4)was made into tissue slices and inserted into the inner and outer epidermis of the mantle of Hyriopsis cumingii(2).On the 5th,20 th and 30 th day after the operation,the samples were taken to observe the morphological changes of the development of pearl sac.The results showed that the development of the pearl sac of the 2 year Hyriopsis cumingii was significantly faster than that of the other years Hyriopsis cumingii(P < 0.05).It is further proved that the 2year Hyriopsis cumingii has a significant advantage as a donor.2.Cell culture in vitro of different tissues of 2 years Hyriopsis cumingiiThe mantle,gill and visceral mass of 2-year-old Hyriopsis cumingii were cultured.After 240 hours of continuous culture,the cell morphology and viability were observed,the Pr I and Ca2+ concentration of cell cycle were analyzed by flow cytometry,and real-time fluorescence quantitative PCR,and the expression of CA and ALP genes and protease activity related to biomineralization were discussed.The results showed that gill cells and mantle cells had significant consistency in cell proliferation and biomineralization.The microscopic observation showed that gill cells had more advantages than mantle cells and visceral cells,but under the same culture conditions,especially in the optimal culture stage(96h-120h),the proliferation ability of visceral mass cells was lower than that of mantle cells and gill cells(P <0.05).In this chapter,we also introduced a new mutation technology ARTP.The results showed that the activities of mantle cells,gill cells and visceral cells in ARTP group were significantly higher than those in NC group at 24 h(P < 0.05),and the activities of cells in ARTP group were slightly higher than those in NC group at 120 h(P > 0.05)At h,the activity of cells in ARTP group was the highest(P < 0.05),and at 140 h,the activity of cells also showed a high level(P < 0.05).The results showed that he mutation could shorten cell recovery time and increase cell proliferation activity to a certain extent.This study has opened up a new direction for cell culture of Hyriopsis cumingii.It is found that gill cells and mantle cells have similar functions,and even the ability of proliferation and biomineralization of gill cells is stronger than that of mantle cells.This experiment laid a foundation for cell culture of Hyriopsis cumingii and established a new idea for the selection of incubation time point of large-scale nucleated beads.3.The effect of cultured mantle cells and gill cells implanted in vivo on cell viabilityThe mantle cell and gill cells of the 2 year Hyriopsis cumingii were cultured in vitro to 24 h,72 h,120 h,and 240 h,and then transplanted to the inner and outer epidermis of the mantle of the living Hyriopsis cumingii.The viability of the cells was measured at 24 h,72 h,120 h,192 h and 240 h after transplantation.The results showed that the cells cultured in vitro to 24 h,72 h,and 120 h had a higher cell viability in the transplanted body than the pre-transplantation within a period of time after transplantation.However,when the cells were cultured to 240 h in vitro,the cells showed a downward trend after being transplanted into the living body.The experimental results show that gill cells and mantle membrane cells cultured to 24 h,72 h,and 120 h in vitro can survive in vivo and the living environment has a significant effect on promoting cell proliferation,but for cells with poor viability in vitro,even The viability of cells implanted into living cells cannot be significantly improved. |
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