Immortalization Of Swine Trachea Epithelium Cells (STECs) And The Adhesion Ability Of Haemophilus Parasuis On STECs

Tracheal epithelial cells(Trachea epithelium cells,TECs) in mammals are the role of many pathogens target cells, and are also an important barrier to block pathogen invasion. TECs TECs can remove harmful substances from the outside world, such as toxic gases, dust, bacteria, viruses, etc., to make the body from these harmful substances. However,we not seen literature about isoloation STECs.Establishment culture of STECs in vitro can provide material basis for studying STECs.Haemophilus parasuis, (HPS) belongs to the family of Pastteuerllaeae,the genus Haemophilus.HPS is the etiologieal agent of porcine Glsser's Disease,which can invade and cause severe systemic disease,characterized by fibrinous polyserositis,arthritis and meningitis. The higher prevalence of HPS intensive pig farms has become the main reason for the high mortality of young pigs and led to the huge economic losses. In recent years, foreign scholars use a variety of pig cell line models to explore the pathogenesis of the HPS. However, there is not relevant reports in China.This experiment transducted hTERT into STECs to expand their life span. At the same time, the adhering capacity to STECs of Haemophilus Parasuis was observed.The cells were laid the foundation for further stuay the pathogenic mechanism of HPS. Experimental results obtained as follows:(1)Swine trachea epithelium cells were isolated from newborn piglets sterile trachea. 0.1% pronase XIV was poured into trachea and digested.Trachea epithelium cells were collected by sieve filtration and gradient centrifugation. Separation down STECs just as bright spherical, they showed mostly single presence. Most cells had cilia clearly visible which were swing by inverted phase contrast microscope.Cell activity was 90%. These STECs which were collected by this method can provide a good seed cells to next STECs culture.(2)The primary STECs were cultured at 5% and 37℃CO2. The results showed a confluent monolayer was former by 3 days, The cells were cultured in monolayers, the growth of polygonal cobblestone and were stained positively for cytokeratin 8 antigen. The cells purified by differential attachment and the got high purity STECs. In the culture of about 6～8 passages, Cell growth began to stagnate, cells can not subculture again.(3)Primary culture STECs were transfected with hTERT STECs by lipofectamine. The cells were inputed G418 after transfecting 48h and screened 1 month when the positive clones resisted to G418.The cells was continued to expand training and then purified by infinite dilution.hTERT mRNA was detected by RT-PCR in transfected cells.Western Blot was used to detect the protein expression of hTERT.The results showed that hTERT was expressed in transfected cells,while in untransfected STECs was negative.The activity of telomerase in STECs were activated through this method.The cells were cultured to 55 passages after introduction hTERT.(4)Immortalized STECs cultured in vitro for a long-term,adherent cells grew as paving stone.There was clear outline between every cells.The cells maintained contact inhibition.We found that the immortalized STECs had physiological characteristics of normal cells through growth curve.We also find the proliferation activivity of immortalized STECs was higher than primary STECs.(5)The telomerase activated STECs and swine umbilical vein endothelical cell (SUVECs) were infected with different concentration HPS respectivity.The cell adhesion was examined by Gram staining in STECs after infection 1 h,2 h,3 h,4 h.The results indicated that HPS can attach to STECs and SUVECs;HPS has a toxic effect and can cause cell death or apoptosis on STECs.The cell adhesion of HPS was associated with the bacterial concentration and infection time.The HPS could be screened on the infected cell surface after infection 4 h with 1.0×108CFU/mL bacterial concentration.More HPS were examined on the surface of STECs and just few on SUVECs under high power microscope,it suggested that the adhesion ability of HPS was higher to STECs than SUVECs.The pathogenic mechanism of HPS can be studied using this cell adhesion model in future research.Overall results suggested that the hTERT was transfected into STECs successfully,the cell proliferation life span was elongated to 55th in vitro.The result showed that the adhering capacity to STECs of Haemophilus parasuis was higher than SUVECs.Therefore,immortated STECs can be used as an cell adhesion model for HPS.