| Objective To establish Beijing duck nephric mesenchymal stem cells(NMSCs) in vitro culture system and research their biological characteristics. To explore the effection of pravastatin on nephric mesenchymal stem cells proliferation.Methods(1) The isolation and culture of NMSCs: The 15 day old Beijing duck embryo kidney for research material, mixed enzyme digestion to obtain NMSCs, then passage, cryopreservation and resuscitation.(2) The study on the biological characteristics of NMSCs: Morphological observation; drawing cell growth curve; calculating the doubling time; specific surface marker RT-PCR and immunofluorescence detection; multi differentiation potential identification: NMSCs could be induced directly into adipocytes, islet cells, hepatocytes and epithelial cells, after induced, the cells were dealt with specific staining, RTPCR and immunofluorescence.(3) The study of pravastatin effection on NMSCs proliferation; NMSCs were randomly divided into the control group and the experimental groups, the experimental groups were added to the culture medium for 0.001, 0.01, 0.1, 1, 10 μmol?L-1 five concentrations of pravastatin, morphological changes of cells were observed after drug action; MTS cell proliferation assay; fluorescence quantitative PCR detection of cells proliferation gene, flow cytometry positive analysis of specific markers antigen expression and of proliferating cell nuclear antigen.Results(1) Cells spread to the 23 generation, long spindle shaped, has the strong proliferation ability.(2) Cell growth curve showed a typical "S" type, the incubation period, the logarithmic phase, the stationary phase and the decline phase, in line with the general law of cell growth in vitro, the population doubling time were 31.85 h, 35.07 h, 40.98 h.(3) RT-PCR detected that Vimentin, Fibronectin, CD29, CD71, CD44 and CD73 were positive, CD34 and CD45 were negative. Immunofluorescence detected that Vimentin, CD29, CD71 and CD44 were positive.(4) NMSCs were successfully induced into islet cells, adipocytes, hepatocytes and epithelial cells.(5) Pravastatin action after 76 h, cells showed long spindle, no significant changes in morphology.(6) Compared with control group, concentrations of was 0.001, 0.01, 0.1, 1 μmol?L-1pravastatin group could promote NMSCs proliferation, 0.001 μmol?L-1 and 10 μmol?L-1 pravastatin group had no effect on promoting proliferation of NMSCs.(7) Compared with the control group, the 0.001, 0.01,0.1 μmol?L-1 pravastatin group relative expression of genes proliferation Ki67 increased, 0.01, 0.1, 1μmol?L-1 relative expression of proliferation genes PCNA increased, other concentration groups compared with the control group had no significant difference.(8) After pravastatin effect 1 w, no significant difference between the expression quantity of cell surface marker antigens. Vimentin positive rate was 98.51% in the control group, the experimental group was 97.43%; CD44 positive rate was 97.21% in the control group, the experimental group was 97.78%; the expression quantity of proliferating cell nuclear antigens increased, Ki67 positive rate was 2.39% in the control group, the experimental group was 12.3%; PCNA positive rate was 11.07% in the control group, the experimental group was 26.14%.Conclusions This study successfully established Beijing duck embryo nephric mesenchy –mal stem cells culture system in vitro, NMSCs self proliferation and multilineage differenti-ation potential were identified, confirmed that in vitro cultured NMSCs derived from duck embryonic nephric mesenchymal stem cells, and had similar biological characteristics with mesenchymal stem cells. Pravastatin function did not affect NMSCs morphology and biological characteristics, pravastatin concentration of 0.001~1 μmol?L-1 could promote NMSCs proliferation, pravastatin concentration of 10 μmol?L-1 had no effect on NMSCs proliferation. The research that pravastatin act on NMSCs should provide theoretical basis and experimental evidences for clinical medication. |
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