Role Of TLR2 In Vascular Smooth Muscle Cell Migration Induced By Chlamydia Pneumoniae Infection

Objective To establish the model of Chlamydia pneumonia (C.pn)-infected vascular smooth muscle cell (VSMC) in vitro, to observe the effects of C.pn infection on migration of VSMC and investigate the role of Toll like receptor 2 (TLR2) in VSMC migration induced by C.pn infection, to explore the related molecular mechanisms.Methods1. VSMCs were primary cultured by the explant method from the thoracic aorta of SD rats. VSMCs were identified by immunofluorescence analysis with a murine monoclonal antibody against a-smooth muscle actin (a-SM actin).2. VSMCs were infected with C.pn after the culture and propagation of C.pn AR-39 in Human Epidermoid Carcinoma-2 (HEp-2).3. The specific C.pn DNA fragment was identified by polymerase chain reaction (PCR). The morphological characteristics of C.pn inclusions in VSMCs were examined by Lugol's staining and acridine orange (AO) staining. Successful infection was confirmed by immunofluorescence analysis with a murine monoclonal antibody against C.pn.4. At 24 h,48h and 72 h post-infection, the ultrastructure characteristics of C.pn proliferation and the infected VSMCs were observed under transmission electron microscope (TEM).5. At 24 h post-infection, Wound-healing assay and Transwell assay were performed to investigate the effect of C.pn infection on VSMC migration.6. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the mRNA expression level of TLR2 and TLR4 in C.pn-infected VSMCs at different time potins.7. The intracellular location of C.pn and TLR2 in C.pn-infected VSMC was observed by immunofluorescence with a murine monoclonal antibody against C.pn and a rabbit monoclonal antibody against TLR2 under confocal laser scanning microscopy (CLSM).8. The effect of TLR2 was blocked by a TLR2 neutral antibody, and then RT-PCR was performed to detect the MyD88 mRNA expression in order to determine the block effect of TLR2.9. Wound-healing assay and Transwell assay were performed to explore the role of TLR2 in VSMC migration induced by C.pn infection. migration induced by C.pn infection.