Establishment And Bioligical Characteristics Of Kidney Fibroblast Cell Line For Hezuo Swine

As Hezuo swine is one of national genetic protected livestock species from Gansu province, it was selected as the objects of the study. Using animal cell culture technology, by differential adhesion and digestion,15 primary cell lines of Hezuo swine kidney fibroblast cells were constructed successfully. According to the requirements of ATCC for cell lines, their culture conditions and biological characteristics were analyzed. The result showed that:the cells were shaped with spindle or willow leaf shape, vigorous growth. After cultured for 48～72h, cells covered with a layer, radial or spiral-like growth trend by inverted phase contrast microscope. Transmission electron microscopy revealed cytoplasmic fibrous structures. Immunohistochemistry showed vimentin positive and cytokeratin negative. The four choices of DMEM (H), MEM, RPMI1640 medium and HamF12 for cultivation each plus 10% FBS, the results showed that DMEM (H) medium with 10% FBS is most suitable. Intermittent cooling cryopreservation method for frozen cells, after recovery cells grew well, growth curve showed "S" type with the period of incubation, logarithmic growth and stagnation, and consistent with cell growth pattern, average survival rate were 95%, before cryopreservation were 97%, indicates good effect from cryopreservation method. Karyotype analysis showed Hezuo swine kidney fibroblast chromosome is number of 2n= 38, including 18 pairs of autosomes and one pair of sex chromosome, y chromosome is the smallest metacentric chromosome. Statistics of handred metaphase cells which were derived espectively from the cells of male and female individuals, the result is 2n= 38 for female 88%, for male 90%, which indicates the cell line is diploid cells in the main and genetic performance is stability. SDS gel electrophoresis showed five zones for lactate dehydrogenase isozymes, the result is same of repeated from the cells of different individual, indicates the cell line cross-contamination did not occur. Liposome-mediated green, red fluorescent protein reporter plasmid transfected, respectively 45%,35% of the cell transfection efficiency, indicating that Hezuo swine kidney fibroblasts could be transfected with the expression of foreign genes. The test results for bacteria, fungi, mycoplasma and virus infection of exogenous factor were negative. Conclusion:Hezuo swine kidney fibroblast cell lines were corresponded with ATCC cell line established requirements. They reserved ideal biological materials for genomic library construction, cloning and somatic cell genetic diversity, and reserved cell carrier for engineering vaccine construction. It also provide seed cells for tissue engineered kidney.