肺炎衣原体在HEp-2细胞中增殖的观察

The proliferation of Chlamydia pneumonia in HEp-2 cells

目的研究肺炎衣原体AR-39株在体外培养中的增殖规律，进一步了解其超微结构的变化，并探讨AR-39株在体外连续培养的方法。方法AR-39株感染人喉癌细胞（human epider-moid carcinoma-2，HEp-2），光镜下观察细胞病变；PCR技术鉴定其核酸；荧光显微镜和电镜下观察感染24h、48h和72h后AR-39株在HEp-2细胞内的形态变化。结果光镜下可见HEp-2细胞内形成包涵体；PCR扩增出肺炎衣原体437bp的特异性片段；荧光显微镜和电镜显示AR-39株在HEp-2细胞内包涵体从幼稚型到成熟型的衍变过程，并观察到梨形原体及微型小体等AR-39株特征性的超微结构。结论AR-39株在HEp-2细胞内经72h完成一个发育周期，此时可获得高感染性的肺炎衣原体用于连续传代。

Objective To investigate the proliferative feature and ultrastructural changes of Chlamydia pneumonia strain AR-39 in vitro , and to study the propagation of AR-39 in vitro . Methods Monolayer HEp-2 cells were infected by AR-39 with a multiplicity of infection 0.5. Cytopathic effect was observed with a microscope. Detection of nucleic acid was performed by PCR. At 24 h, 48 h and 72 h post-infection, the morphological changes of AR-39 in HEp-2 cells were observed with fluorescence microscope by acridine orange staining and transmission electron microscope （TEM）. Results HEp-2 cells were swollen and desquamated during 24-72 h post-infection. Inclusions appeared in cytoplasm of HEp-2 cells at 24 h post-infection and grew fully at 72 h post-infection, which were verified by 437 bp amplification products of Chlamydia pneumonia-specific sequences. The growth of AR-39 inclusions was observed at different time points in the infected HEp-2 cells with fluorescence microscope. The similar results that a development cycle was from elementary bodies differentiated into reticulate bodies and then differentiated back to elementary bodies were showed with TEM; In addition, the characteristics of AR-39 uhrastructure, such as the pear shape of elementary body and miniature body in progeny elementary body, were also displayed with TEM. Conclusion AR-39 completion of a development cycle takes 72 h, and the highest infectious capability of progeny AR-39 could be achieved at 72 h in vitro for continuous propagation.