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Objective To investigate the effect of the serum obtained from rat with hepatopuimonary syndrome (HPS) on Akt mRNA and protein expression in rat pulmonary microvascular endotheliai cells (PMVECs) and the role of Akt signaling pathway in the proliferation of PMVECs in the HPS. Methods Healthy 3-4-month-old SD rats of both sexes were used in this study. HPS was produced by chronic ligation of common bile duct according to the method described by Fallon. liver cirrhosis and pulmonary microvascular proliferation were verified by microscopic examination of the liver and lung tissue 2 weeks after bile duct ligation. Serum was obtained from blood taken from aorta of HPS rats. Primary PMVECs were cultured and randomly divided into 2 groups: control group and HPS group. In HPS group serum was added to cultured PMVECs (final concentration was 10%) and incubated. Akt mRNA and protein expression was determined at 24, 48 and 72 h of incubation by RT-PCR and Western blot. The proliferation of PMVECs was detected by MTT and ~3H-TdR. Results The proliferation of PMVECs was significantly enhanced and the expression of Akt mRNA and protein was significantly increased in HPS group as compared with control group. Conclusion The Akt signaling pathway might play an important role in proliferation of PMVECs in the HPS.
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Objective To investigate the role of methylated DNA mismatch repair gene MLH1 and MSH2 in the acquired multidrug-resistance of human small cell lung cancer cells H446.Methods The reverse transcription polymerase chain reaction(RT-PCR)and Western blot were applied to measure MLH1 and MSH2 mRNA and protein expressions of the multidrug-resistant cells H446/DDP and its parental cells H446.The promoter methylation status of the genes was assessed by methylation-specific PCR(MSP).Results The expressions of MLH1 and MSH2 significantly decreased both in mRNA level and protein level.Promoter methylation of MLH1 was observed in H446/DDP cells but not in H446 cells.Promoter semi-methylation of MSH2 in H446 cells was transformed to methylation in H446/DDP cells.Conclusion The downregulation of DNA mismatch repair gene MLH1 and MSH2 induced by its promoter methylation may play an important role in the acquired multidrug resistance of human small cell lung cancer.
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Objective To investigate whether sialic acid disaccharides, specificity agglutinins and neuramidinases could inhibit Pseudomonas aeruginose infection in rat trachea vulnerated by hydrochloric acid. Methods One hundred and forty male Wistar rats were vulnerated by hydrochloric acid through intratracheal instillation and then inoculated by Pseudomonas aeruginose admixed Sial ?2,3-Gal, Sial ?2,6-GalNAc, Maackia amurensis agglutinin, Sambucus nigra agglutinin, specificity ?2,3-neuramidinase or non-specificity neuramidinase respectively. Thirty minutes and 48 h after inoculation, whole trachea of 8 rats in each group was weighed, homogenated, and quantitative bacterial culture was performed while whole trachea of another 2 was fixed in 10% methanol, embedded with paraffin and HE stained. Results Sial ?2,3-related disaccharide, agglutinin and neuramidinases could decrease colony count of trachea 30 min after inoculation remarkably. But only Sial ?2,3-Gal and neuramidinases could obviously decrease colony count 48 h later. Sial ?2,6-related substrances have not analogical effects. Conclusion Sial ?2,3-related substrances could interfere Pseudomonas aeruginose binding/multiplication in the trachea, especially Sial ?2,3-Gal and neuramidinase.
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Objective To establish a comprehensive characterization protocol of rat pulmonary microvascular endothelial cells(RPMVECs) cultured by tissue-sticking method.Methods RPMVECss were cultured with peripheral lung tissue-sticking method.Histological sections from peripheral lung tissue pieces used for cell culture were examined.With rat pulmonary artery smooth muscle cells and human umbilical vein endothelial cells as controls,the cultured cells were identified by immunocytochemical staining using CD34 antibody,lectin from Bandeiraea simplicifolia and factor Ⅷ related antigen.The cell morphology and ultrastructure were observed with inverted light microscope and transmission electron microscope respectively.Results Histological sections showed that tissue pieces were scissored from peripheral lung lobes accurately.The cultured cells had characterization of binding lectin from Bandeiraea simplicifolia and positive immunocytochemical staining with CD34 antibody,but negative for factor Ⅷ related antigen.Weibel-Palade bodies were not found in cells.Conclusion Factor Ⅷ related antigen and Weibel-Palade body are not ideal indexes for RPMVECs identification.The combination of peripheral lung tissue section,CD34 immunocytochemical staining and lectin from Bandeiraea simplicifolia binding assay provides a simple and reasonable comprehensive characterization protocol for RPMVECs.