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AIM:To investigate the role of caveolin-1 on epithelial-mesenchymal transition (EMT) in human bronchial epithelial (HBE) cells induced by transforming growth factor beta 1 (TGF-β1).METHODS:Immunofluorescence, real-time PCR and Western blot were applied to detect the mRNA and the protein expression of caveolin-1 in the 16HBE cells during EMT.The influence of siRNA-mediated silencing of caveolin-1 on EMT in the 16HBE cells was detected by Western blot.RESULTS:Caveolin-1 was widely present on the cell membrane of the 16HBE cells.The expression of caveolin-1 at mRNA and protein levels was significantly decreased in a time-dependent manner in the 16HBE cells compared with control group (P<0.05) after stimulation with TGF-β1.The morphologic changes of the 16HBE cells induced by TGF-β1 were promoted by caveolin-1 silencing compared with TGF-β1 group.The protein expression of E-cadherin and α-SMA induced by TGF-β1 was promoted by caveolin-1 silencing compared with TGF-β1 group (P<0.05).The phosphorylation levels of AKT and Smad3 were the highest at 30 min and increased significantly compared with control group (P<0.05) after stimulated with TGF-β1.Treatment of the 16HBE cells with TGF-β1 for 30 min after silencing caveolin-1 gene for 24 h significantly increased the phosphorylation levels of AKT and Smad3 compared with TGF-β1 group (P<0.05).CONCLUSION:TGF-β1 down-regulates the expression of caveolin-1 in the 16HBE cells.Caveolin-1 may participate in TGF-β1/Smad pathway and PI3K-AKT pathway, which are the signal transduction pathways for TGF-β1 inducing EMT.
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AIM:To investigate the role of canonical transient receptor potential channel 1 ( TRPC1 ) in the migration of human bronchial epithelial cells (16HBE) induced by transforming growth factor-β1 ( TGF-β1).METH-ODS:Silencing of TRPC1 gene expression was performed by siRNA.The cell activity and apoptosis were measured by CCK-8 assay and flow cytometry, respectively.The migration and invasion abilities of the 16HBE cells were detected by wound-healing assay and Transwell assay.The protein expression of E-cadherin and vimentin was determined by Western blot.RESULTS:TGF-β1 treatment significantly enhanced the cell migration distance compared with control groups ( P0.05).The results of wound-healing and Tr-answell assays showed that migration and invasion abilities in TRPC1 siRNA +TGF-β1 group were markedly suppressed compared with TGF-β1 group (P<0.01).The protein expression of E-cadherin and vimentin induced by TGF-β1 was in-hibited by TRPC1 silencing compared with TGF-β1 group (P<0.05).CONCLUSION:TRPC1 is involved in the migra-tion of human bronchial epithelial cells induced by TGF-β1 through regulating the protein expression of E-cadherin and vim-entin.
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[ ABSTRACT] AIM:To investigate the role of canonical transient receptor potential channel 1 ( TRPC1 ) in the epithelial-mesenchymal transition ( EMT) of human bronchial epithelial ( HBE) cells induced by transforming growth fac-tor-β1 (TGF-β1).METHODS:EMT of 16HBE cells induced by TGF-β1 were identified by microscopy, immunofluores-cence and Western blotting.Immunofluorescence, real-time PCR and Western blotting were applied to detect the mRNA and the protein expression of TRPC1 in the 16HBE cells.The influence of SKF96365 (a TRPC1 blocker) and siRNA-me-diated silencing of TRPC1 on the EMT of the 16HBE cells were detected by microscopy and Western blotting.RESULTS:Treatment with TGF-β1 induced significant morphological changes of the 16HBE cells.Exposure to TGF-β1 decreased the expression of E-cadherin protein (P<0.01) and increased the expression of α-SMA protein (P<0.05) in the 16HBE cells.Immunofluorescence observation indicated that TRPC1 expression in the 16HBE cells was positive.The expression of TRPC1 at mRNA and protein levels was significantly increased in the 16HBE cells after stimulation with TGF-β1 ( P<0.05).The morphological changes of the 16HBE cells induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silen-cing compared with TGF-β1 group.The protein expression of E-cadherin andα-SMA induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silencing compared with TGF-β1 group (P<0.05).CONCLUSION:TGF-β1 induces EMT with the mechanism of up-regulating TRPC1 in human bronchial epithelial cells.
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Teaching healthy lifestyle in combination with physiology is hardly practiced in schools of medicine. Individual lifestyle factor accounts for 60% among the factors that lead to dis-eases. Physiology contains the basic theories for health knowledge. Therefore, during physiology teach-ing , healthy lifestyle was introduced by combining such contents of physiology as digestion and absorption , energy metabolism balance and emotional physiological reaction with four “health corner-stones” such as health diet, regular physical activity, psychological balance, quitting smoking and drinking less alcohol. This teaching mode has widened the teaching ideas, which has not only in-creased the students' interest and significance in physiology learning, but also promoted them to de-velop good healthy lifestyle.
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BACKGROUND:How to obtain sufficient autogeneic chondrocytes is a problem which must be answered as soon as possible in both the transplantation of chondrocytes and the development of cartilage engineering.Bone marrow-derived mesenchymal stem cells have the potential of multidirectional differentiation. Under different induced conditions, they can differentiate into multiple tissue cells, such as chondrocyte, osteoblasts , sarcoblast, nerve cells and so on. Insulin-like growth factor-I plays an important role in regulating the formation and development of limb and cartilage.OBJECTIVE:To observe the effect of the insulin-like growth factor-I and culture solution of chondrocyte on inducing bone marrow-derived mesenchymal stem cells to differentiate into chondrocytes in vitro.DESIGN:Open experiment.MATERIALS:This experiment was carried out at the Medical Study Center, Second Hospital Affiliated to Sun Yat-sen University from March to November 2003.Human bone marrow-derived mesenchymal stem cells were harvested from 4 to 6-month old embryos, all of which were from pregnant women who needed to terminate of pregnancy by induction delivery with water bag for health.METHODS:Human embryonic mesenchymal stem cells were isolated with Percoll separating medium. Subsequently, the cells were amplified in vitro,and the expression of surface makers of bone marrow-derived mesenchymal stem cells, such as CD44, CD71, CD34 and CD45 were measured with flow cytometer to identify the cells in our experiment. 100 μg/L insulin-like growth factors and culture solution of chondrocytes were added in the culture medium of bone marrow-derived mesenchymal stem cells of the fourth generation. The morphological changes of induced cells were observed with an inverted microscope. The expression of type Ⅱ cartilage matrix was observed by collogen immunohistochemistry. The proteoglycan level in the cells was detected, too.MAIN OUTCOME MEASURES:Phenotype of bone marrow-derived mesenchymal stem cells was identified through detecting the expressions of CD34, CD44 and CD4.Type Ⅱ collagen immunohistochemistry and the change of cellular ability to secrete proteoglycan after induction were observed to determine whether bone marrow-derived mesenchymal stem cells can differentiate into chondrocytes.RESULTS: ① Being observed under an inverted microscope,the bone marrow-derived mesenchymal stem cells presented a morphology like fibroblasts when they were cultured in vitro. ②Identification of surface antigen of bone marrow-derived mesenchymal stem cells: These cells were detected to have good homogenicity with flow cytometer. The fourth generation of bone marrow-derived stem cells positively expressed CD44, negatively expressed CD34 and CD45, suggesting these cells had the characteristics of bone marrow-derived mesenchymal stem cells. ③Observation of the morphological change of chondrocytes induced by bone marrow-derived mesenchymal stem cells under optical microscope: chondrocyte condition culture solution and insulin-like growth factors-Ⅰ were added to co-culture.During the process of culture, bone marrow-derived mesenchymal stem cells were seen to have a shape of round gradually. Fifteen days later, some cells presented a shape of short-shuttle or polygon with short mutations,which were the shape characteristics of chondrocytes. ④Immunohistochemical staining of Type Ⅱ collagen: In the insulin-like growth factor group,72.5% cells had many brown granules in cytoplasm, which were weakly positive or strongly positive expression of Type Ⅱ collagen. In the control group, bone marrow-derived mesenchymal stem cells was negative expression of type Ⅱ collagen on the 15th day. ⑤ Measurement of proteoglycan level: After co-culture with insulin-like growth factor-Ⅰ and chondrocyte culture solution for 15 days, proteoglycan was higher in the cells of co-culture group [ (8.92±0.91) μg/L ] than in culture group [(2.56±0.26) μg/L,P ＜ 0.05], but lower than in the chondrocyte group[(13.69±1.51) μg/L, P＜ 0.05].CONCLUSION:Insulin-like growth factor-Ⅰ and chondrocyte culture solution can induce bone marrow-derived mesenchymal stem cells to differentiate into chondrocytes.
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AIM: To observe the realgar induced the apoptosis of eosinophils from bronchoalveolar lavage fluid in asthmatic guinea pigs and investigate the mechanism that realgar treated asthma.METHODS: The morphology of apoptosis of eosinophils was observed by Giemsa staining and electron microscope.The rate of apoptosis of eosinophils was assayed by the flow cytometry.RESULTS: The characteristic changes of the apoptosis in both light microscope and electron microscope were shown after 6 hours treatment of realgar.Flow cytometry showed that the rate of apoptosis of the eosinophils was increased with both increasing realgar concentration and prolonging realgar action time to the cells.CONCLUSION: Realgar promotes the apoptosis of eosinophils from bronchoalveolar lavage fluid in asthmatic guinea pig.Realgar induced the apoptosis of eosinophils is one of the causeses for asthmatic treatment.
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Aim To investigate the effect of Tetramethylpyrazine (TMP) on the proliferation of airway smooth muscle cells (ASMCs). Methods Primary ASMCs of rats were cultured. The absorbance (A490) value of ASMCs treatment with platelet-derived growth factor (PDGF) in the presence of TMP was detected by MTTto observe the anti-proliferation of TMP. The levels of ERK1/2 and p-ERK1/2 proteins were determined by Western blot.Results In presence of the TMP with different concentrations (12.5,25,50,100 and 200 ?mol?L-1) at 6,12,24,36 and 48 hours,compared with control groups,the average inhibitory rates of cell proliferation in all groups were increased significantly (P