Experimental Study Of The Infection Of Peulcimum Marneffei On Tupaia Belangeris And Brochial Epithlial Cells

Part 1 Pathological observation of Tree shrew infected with Penicillium marneffeiObjective To establish Penicillium marneffei infection model in Tupaia belangeri, observe the histopathological changes, and discuss the pathogenic mechanisms of Penicillium marneffei.Methods Twenty male Tupaiabelangeri chinensis were randomly divided into five groups (one control group and four experimental groups divided into four different periods:two weeks, three weeks, four weeks and six weeks). P.marneffei produce a large number of conidia which were prepared as suspension at the concentrtion of 2×108/ml, and 500ul suspension was dripped into the nose of Tupaia belangeri every week. After the certain time, the Tupaia belangeri were executed, the lung tissue was taken to fungal culture and the histopathological changes of lung, live, spleen and lymphonodus were examined after hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) stain. The Broncho-alveolar lavage fluid (BALF) was taken and centrifuged, and the resuspended cell pellet was examined after Switzerland’s-Jim dyeing. The total leukocyte were calculated and classified into neutrophil, lymphocytes and macrophages. All data were analyzed by using SPSS software (version 16.0).Result The lung tissue of all the experimental group were cultured on the PDA medium and P.marneffei was identificated by the microscopic observation. Visible histopathological changes in organizations of Tupaia belangeri, including lung, spleen and lymph node were obsered. The main histopathology of lung was inflammation surrounding the small tracheal lumen, and increased macrophages and neutrophils were observed in alveolar cavity. The leukocyte of BALF in experimental group was greater than that in control group, and the classification result showed that neutrophil and lymphocyte were the main significantly increased cells in Tupaia belangeri infected for 2,3 and 4 weeks, and lymphocyte was the main significantly increased cell in Tupaia belangeri infected for 6 weeks (P<0.05)Conclution (1) The Tupaia belangeri infected with PM showed obvious inflammatory change in lung, spleen and lymph node; (2) In the BALF of the Tupaia belangeri infected with PM, the neutrophil was the main significantly increased cells whin 4 weeks, while the lymphocyte was the main significantly increased cells after 4 weeks,Part 2 Experimental study on the adherence and phagocytosis of BEAS-2B to Penicilium marneffei conidia and the secretion of IL-6 and IL-8 in BEAS-2B cell stimulated by conidiaObjective To explore the adherence and phagocytosis of BEAS-2B to Penicilium marneffei conidia and the secretion of IL-6 and IL-8 in BEAS-2B cell stimulated by conidiaMethod Human bronchial epithelial cell lines BEAS-2B was cultured with PM conidia on a glass slide for 6 hours, then the adherence was examined after the periodic acid-Schiff (PAS) stain. The phagocytosis of PM conidia by BEAS-2B was examinated by transmission electron microscopy every one houre after culture within 5 hours. BEAS-2B cells were divided into ten groups (control group, LPS group, HIV-negative isolates group, HIV-negative isolates+LPS group, HIV-positive isolates group, HIV-positive isolates+LPS group, FRR2161 group, FRR2161+LPS group, wild isolates group and wild isolates+LPS group). The stimulators were cultured with BEAS-2B for 24,48 and 72 hours. The levels of IL-6 and IL-8 in supernatant were examined with enzyme-linked immunosorbent assay (ELISA). All data were analyzed by using SPSS software (version 16.0).Result It was observed that the PM conidia were adhered closely to BEAS-2B after BEAS-2B was cultured with PM conidia for 6 hours. Under the transmission electron microscopy, PM conidia were observed to be phagocytosed in 2 hours, and lysosome in cell increted. The vacuoles and mild swelling of mitochondria were observed in BEAS-2B cells. And different degrees of damage of BEAS-2B to PM conidia were observed within 5 hours. The result of ELISA indicated that all of the level of IL-6 and IL-8 secretion of BEAS-2B was significantly increased by the four PM isolates (the HIV-negative isolates, HIV-positive isolates, FRR2161, and wild isolates group), and HTV-negative isolates+LPS showed a weaker stimulation than HIV-positive isolates+LPS, FRR2161+LPS, and wild isolates+LPS (P<0.05).Conclusion (1) PM conidia could adhere to bronchial epithelial cell lines BEAS-2B; (2) BEAS-2B showed phagocytosis to PM conidia within 5 hours, the vacuoles and mild swelling of mitochondria in BEAS-2B cells and different degrees of damage of BEAS-2B to PM conidia were observed;(3) PM conidia could stimulate the increase of IL-6 and EL-8 secretion in BEAS-2B, and the HTV-negative isolates+LPS showed a weaker stimulation than HIV-positive isolates+LPS, FRR2161+LPS, and wild isolates+LPS.