Construction Of CTECs Co-culture System And Study Of Cytopathogenic Effects On CTECs By IBV

Trachea is one of the most important gateways for pathogen to infect the host bodies. Trachea epithelium cells (TECs) are not only the target cells attacked by pathogen, but also a significantly important barrier of host bodies inhibiting the invasion of pathogen. In the recent ten years, many progresses on isolation and cultivation of TECs have been achieved. In vitro culture of human, mammal, and rodent TECs has been used to evaluate the characteristics and activities of these cells. Especially, air-liquid interface culture of TECs has been widely used in the research of occurrence mechanism of respiratory diseases, mechanism of cytopathogenic effect on TECs, and drug delivery and screening. However, there was no report on isolation and cultivation of chicken trachea epithelium cells (CTECs), only reports on human, dog, mouse, rabbit and guinea pig were documented. Up to the present day the usual culture condition of the above several kinds of TECs is not obviously useful for the growth of CTECs in vitro, so we explore CTECs culture condition in order to establish in vitro culture system of CTECs, which would provide materials to the research based on CTECs cellular model.Due to the variation of Infectious Bronchitis Virus (IBV), different serotypes of virus occurred. Therefore, cross protection was deficient among different serotypes, making it difficult to prevent diseases transmitting by the normal immunization measurements. To provide theoretical basis to prevent and control Infectious Bronchitis Disease, scholars all over the world have been making efforts trying to explain the molecular mechanism of infection, duplicating, and variation of IBV, as well as the virulence and tissue tropism and host range mechanism. Reports revealed that during the infection, IBV firstly duplicated on CTECs in vivo, which could be theoretically demonstrated by proliferation characteristics of IBV on CTECs in vitro. Furthermore, many local isolates are inadaptable to be cultured in cells. Therefore, exploration of culture characteristics of IBV on CTECs could provide ideal host cells to the isolation, identification and application research of local epidemic strains.For the purposes above, research of establishing a CTECs co-culture system and growing characteristics of IBV on CTECs was carried out. And findings were obtained as follows:1. Chicken trachea was perfused with streptomyces proteinase, digested over night, and epithelium cells were collected, followed by three steps purification. Thus highly purified chicken trachea epithelium primary cells were obtained. Observed with microscope, separated cells were globular, luminous, single existed and larger than red blood cells. Cilia were clearly seen on most cells, swinging at the culture medium. Cells activity reached 90%, providing fine seeded cells to the further study.2. To explore an optimized culture system of CTECs, TECs of pig, rabbit and mouse were separated and cultured. Results showed that, with the same culture system, adherence rates of pig, rabbit and mouse TECs were higher than that of chicken. Cells of the formers fully covered the culture dishes 48 h after inoculation, and arranged in a typical shape of"cobble-stone". Pig TECs were even continuously passaged to the 8th generation or above. Whereas, adherence rate of CTECs was low and cells grew in a scattered pattern, scarcely converged. The survival course was short, and cells could not be passaged. From the results above, some possible assumptions were made. Receptors of cytokines were scarcely homologous between chicken and mammal. While cytokines which promoted cells to adhere and proliferate were mainly derived from human or mammal bodies. So the promoting effects on chicken cells were not as significant as that on mammal cells by mammal cytokines, indicating the limit of chicken cells adherence and proliferation in conventional culture system.3. To solve the in vitro culture problem of CTECs, co-culture system comprised of CTECs culture and chicken embryo fibroblast (CEF) culture was applied. With this co-culture system, growing conditions of CTECs were observed, and the preferred formula of culture medium was determined firstly. In this test, Millicell plug-in nest dishes containing special filter membrane were used. CTECs were inoculated on the dish bottom, and CEF were cultured on the filter membrane. Aperture of filter membrane was 0.4μm, permitting the transmitting and exchanging of cytokines secreted by the two types of cells, thus boosting the growth of CTECs. Moreover, the two types of cells were separated by the filter membrane, so each of them could be solely observed and analyzed. Results showed that co-cultured CTECs overspread the dish bottom 72 h after inoculation, and cells arranged in a typical shape of"cobble-stone". Cells increased and vitality strengthened than that solely cultured. The findings preliminarily demonstrated that establishing of CTECs and CEF co-culture system dramatically ameliorated the CTECs growing condition, providing basic technical insurance to the exploration and application of CTECs in vitro culture.4. To imitate the natural physiological environment of chicken trachea, air-liquid interface co-culture system was applied. In this culture system, nest dish contained a supporting membrane covered with human embryo collagen, on which CTECs were interface cultured, CEF were cultured on bottom of nest dish, and low concentration of sera were needed. Compared to the conventional solely cultured cells, interface cultured CTECs stratified on the supporting membrane, and cells survival course prolonged obviously. This system made CTECs form some kind of structure that was close to the natural chicken trachea epithelium, inducing differentiation, maturation and functional expression of CTECs in vitro. Additionally, it might offer some basis to construct ideal cellular model of drug screening, respiratory disease pathogenesis, and interaction of pathogen and epithelial cells.5. To study the culture characteristics of IBV on CTECs, 4 isolates of standard strains and local strains of IBV were inoculated to the monolayer CTECs. Results suggested that the 4 isolates of IBV strains all needed no adaptation on CTECs. After the first infection, pathological changes characterized by cilia shedding, organelles damage, nucleolus decomposition, and membrane disintegration were developed on cells within 72 h. Cytotoxicity harvested from CTECs infected with IBV was inoculated to chicken embryos, passaged for 1 to 3 generations, and then characteristic changes were observed. Development of chicken embryos was hampered, and some embryos even died. In the control group, Marc 145 cells infected with IBV were continuously blind passged to 10th generation, but cytopathy did not occurred. 72h after infection, virus concentration of different groups was detected. Results showed that the virus concentration of the CTECs group was significantly higher than that of Marc 145 group, indicating that CTECs were more sensitive to IBV than Marc 145 cells, and IBV proliferated stably on cultured CTECs. The 4 low passaged IBV strains harvested from chicken embryos could grow and propagate on cells, and quickly induced CTECs to develop cytopathy. To sum up, CTECs is an ideal host cell for IBV, can offer an excellent cellular model for the study of pathogenic mechanism, and provide technical support to isolation, characterization and serological examination of IBV.In conclusion, in vitro culture technique of CTECs was not only successfully established, but also applied to the exploration of IBV culture characteristics, supplying an ideal host cell for the cultivation of IBV in vitro. Additionally, CTECs interface co-culture system which made it possible to imitate the natural trachea physiological environment was constructed, laying a foundation to the further study of CTECs. Results above were all reported for the first time in and abroad.