Establishment Of Duolang Sheep Fibroblast Cell Bank And The Effects Of Nobiletin On Its Apoptosis

To explore the application of somatic cells in preservation of genetic resource, the Chinese local breeds Duolang sheep were used as research object in this experiment, and fibroblast cell bank was established by using tissue block adhering method. The established cell line was biologically detected according to the criterion of ATCC, and the effects of nobiletin on apoptosis of Duolang sheep fibroblast were studied in this experiment. The results are as follows:1. Totally 40 Duolang sheep were used in this study, and the cells were serially subcultivated 3 generations. The numbers of frozen tubes were 103, and each tube contained 3.88×106 cell/ml.2. The cultured cells possess typical morphology of fibroblast, and most of them showed the shapes of shuttle or irregular triangle. The cells were presented as radiated, flamy or whirlpool while growing, and were observed healthy with good morphology under the microscope.3. After cell recovery, the growth curve of Duolang sheep cells was looks like typical "S" shape, had experienced the latent phase, exponential growth phase and the stagnation phase. It was consistent with general cell growth rules in vitro. Before freezing and after recovery the survival percentage was 96.8% and 94.4% respectively. The main reason for this descent was the mechanical and chemical damages on cells during the cell freezing and recovery.4. We carried out microbiology testing for bacterium, fungus and mycoplasma of Duolang sheep fibroblast cell line, and the result was negative.5. Karyotype analysis showed that the diploid chromosome number of Duolang sheep cell was 2n=54, the chromosomal aberration rate was lower than 3%, and there was no variation of chromosomal structure. All of this indicated that the cell line we established were stable diploid cells and comparatively stable characteristics of genetic performance in vitro.6. The analysis of isoenzyme pattern of lactic dehydrogenase (LDH) and malic dehydrogenase (MDH) showed unique bands with 5 LDH bands and 5 MDH bands. This was of species specificity and could be one main reference for species and breeds classification. Moreover, the stable genetic property of cells indicated there had no species cross-contamination and the malignant transformation.7. Through the observation of apoptotic cell morphology, and detection of cell cycle, membrane potential, the rate of apoptosis, we came to the conclusions: at 48 h after adding the nobiletin of different concentration, under the optical microscope to observe AO / EB staining cells, 50mg /L, 60 mg /L groups had no significant changes in cell morphology, while 70 mg /L, 80 mg /L, 90 mg /L groups had significant morphological changes, including smaller torn and wrinkled cell bodies, and tipical apoptotic features that the majority dead cells soon collapse and necrosis. The apoptosis rate was increased significantly with increasing concentration of nobiletin detected by Annexin V/PI dual-staining methods. Rhodamine 123 combined with flow cytometry to detect mitochondrial membrane potential which was found decreased significantly in the nobiletin treated group comparing to the control group. Therefoer, the effect of nobiletin has concentration-dependent characteristics.