Mechanism Of Northeast Wild Boar Fibroblast Cells Response To Cold Stress

Wild boar is one of the terrestrial wildlife under state protection, which are beneficial or of important economic or scientific value. Northeast wild boar belongs to northeast subspecies of Asian wild boar, which is mainly distributed in Greater Khingan Mountain, Lesser Khingan Mountain, Changkuantsailing, Wanda Mountains, Laoyeling, Wanda Mountain, Changbai Mountains, and Heng Ren, Kuandian in the east of Liaoning Province. These regions are characterized by their long and cold winter with the lowest temperature of-40.4℃. Compared to domestic pig, the Northeast wild boar is characterized by strong stress tolerance, disease resistance, wide range of feeding habits, strong resistance ability to bad weather in nature, especially cold environment. The impact of cold stress on the livestock industry is an increasing concern in the world by domestic and foreign farmers and scientists. Although there are many relevant researches, less deeply theoretical study concerns about its influence mechanism. Research on cold stress in China started late, current study on livestock is limited to its impact on blood biochemical parameters, hormones and heat shock protein70(HSP70) expression in peripheral blood lymphocyte. Little research focused on cellular and molecular mechanisms of cold stress. In particular, information about cold-stress related genes is lacking. Changes in the cellular and molecular level are the foundation of a variety of physiological responses.Study on cold stress-related genes in cells helps us to understand the biological mechanisms of the influence of cold stress on body. Wild boar is the ancestor of domestic pig, who have thesimilar genetic bases. While wild boar has not received artificial selction and its current phenotypicproperties are from natural selection. Therefore, it is more close to the real and objective mechanism to study on molecular mechanism of effect of low temperature on its body at the cell level using wild boar as experiment material. This study will peovide theory help to protection and utilization of wild boar much better in the future..Firstly, a culture system of Northeast wild boar fibroblasts was established, a suitable cold treatment was determined, cold inducible RNA binding protein(CIRP) was cloned and analyzed, and by application of several experimental technologies, flow cyometry, RT-PCR, and real-time PCR, the effects of cold stress on cell cycle, apoptosis and cold-stress related genes and its action mechanism were studied. Study on anti-cold mechanism of the Northeast wild boar at the cellular and molecular level provides theoretical reference to establish a cell model for research on molecular mechanism of cold stress, provides strong evidence to reveal the molecular mechanism cold stress using in-vitro cultured cells as the test material, provides theoretical support to cold stress resistance molecular breeding of swine improvement using Northeast wild boar. 1Establishment of fibroblast cell line and its biological characteristic research in Northeast wild boar(1) Under the condition that cell adhesive time was4h, trypsin digestion time was3min and the adhesive time of passage cell was6h, purified fibroblasts could be obtained after the second subculture of tissue and established a simple and flexible fibroblast culture system Northeast wild boar.(2) The fibroblast cell line of Northeast wild boar established grew well with no microbial contamination; the viability of the recovered cells was high, with high growth speed and intact morphology.2Cloning and sequence analysis of cold-induced RNA-binding protein geneTotle RNA was extracted from Northeast wild boar fibroblast cultured at32℃and CIRP cDNA was obtained by RT-PCR, recombinant vector of PMD18-T was constructed and sent to be sequenced. Results suggested that CIRP cDNA of Northeast wild boar was correctly cloned, which has high homology with other species.3The impact of cold stress on the biological character of fibroblast cell line in Northeast wild boar(1) Cell proliferation was inhibited by the substained low temperature culture, which arrested the cells at G0/G1phase, induced cell apoptosis, enhanced its effect with temperature getting lower and induced cold tolerance in cells with time.(2) Treatment with low-temperature arrested the rewarming cultured cells at the G2/M phase and induced apoptosis. Apoptosis induced by low temperature is a delayed and gradual process, which was increased with low temperature.4The impact of cold stress on the expression of HSP70, HSP90and CIRP mRNA in fibroblasts(1) Low-temperature culture inhibited mRNA expression of HSP70and HSP90mRNA, induced CIRP mRNA expression and helped cells obtain cold tolerance ability.(2) Although not significant, mild hypothermia (32℃-25℃) improved the mRNA expressions of HSP70and HSP90in cells during rewarming culture compared to those in the control. CIRP mRNA expression was increased significantly when induced in short time, which protected cells from stress damage.(3) Severe hypothermia (15℃-4℃) acutely induced mRNA expressions of HSP70and HSP90during rewarming culture, which was closely related to the intensity of hypothermia. With temperature decreasing, their expression improved and the expression level of HSP70mRNA was much higher than that of HSP90.