The Developmental Cycle Of Chlamydia Pneumonia In HEp-2 Cells And The Effect Of Chlamydia Pneumonia Infection On The Migration Of Cells

ObjectiveTo investigate the proliferative feature and ultrastructural changes of Chlamydia pneumonia(C.pn) in vitro,and to study the propagation of C.pn in vitro;to observe the effect of C.pn infection on the migration of vascular smooth muscle cell(VSMC) and Human Epidermoid Carcinoma-2(HEp-2) cell respectively.Methods1.Monolayer HEp-2 cells were infected by C.pn.Cytopathic effect was observed with a microscope.Detection of nucleic acid was performed with PCR.2.At 24h,48h and 72h post-infection,the morphological changes of C.pn in HEp-2 cells were observed with fluorescence microscope by acridine orange staining.3.At 24h,48h and 72h post-infection,the ultrastructural characteristics of C.pn in HEp-2 cells were observed with transmission electron microscope(TEM).4.The cytopathic level of HEp-2 cells caused by C.pn infection was evaluated with 50%tissue culture infectious doses.5.In vitro,VSMC and HEp-2 cells were cultured.The post-infection viability of the two different cells was observed by MTT method.6.The post-infection adhesion ratio to collagenⅠof the cultured VSMC and HEp-2 cells was observed by MTT method.7.The changes of post-infection migration of VSMC and HEp-2 cells were observed by Wound-healing assay and Transwell assay.Results1.HEp-2 cells were swollen and desquamated during 24h～72h post-infection. Inclusions appeared in cytoplasm of HEp-2 cells at 24h post-infection and grew fully at 72h post-infection,which were verified by 437bp amplification products of Chlamydia pneumonia-specific sequences.2.The growth of C.pn inclusions was observed at different time points in the infected HEp-2 cells with fluorescence microscope.The process of transmutation was from elementary bodies differentiated into reticulate bodies and then differentiated back to elementary bodies. 3.The similar results were showed with TEM.In addition,the ultrastructural characteristics of C.pn,such as the pear shape of elementary body and miniature body in progeny elementary body,were also displayed with TEM.4.The progeny C.pn gained from HEp-2 cells at 72h post-infection had the highest infectious capability compared with 84h,96h post-infection.5.C.pn infection significantly strengthened the viability of VSMC and HEp-2 cells in vitro.The highest enhancement rates were 171.54%and 43.63%respectively.6.C.pn infection significantly promoted the VSMC and HEp-2 cells adhesion to collagenⅠin vitro.The highest adhesion ratio was 34.4%and 19.9%respectively.7.C.pn infection promoted the migration of VSMC and HEp-2 cells significantly in vitro.ConclusionC.pn completion of a developmental cycle took 72h,and the highest infectious capability of progeny C.pn could be achieved at 72h in vitro for continuous propagation;VSMC and HEp-2 cells migration were promoted by C.pn infection.