| Asthma is a chronic respiratory diseases, and it brought great harm to human life and health. Researches show that there is about 300 million people with asthma worldwide, and in China, the number is huge and growing. Asthma is a complex disease lead by genetic and environmental factors. Even today under the biological-environment-psychological evaluation model of disease, the mechanism of asthma is still unclear, therefore, the current treatment of patients with asthma symptom control based. A number of clinical studies have shown that although most of correctly diagnosed patients after standard treatment can have an effective symptom controll, but this can not reverse or stop the natural history of asthma, and ultimately leads to irreversible airway structural changes. We can see that the pathogenic mechanism of asthma is unknown, making prevention and cure of asthma becomes an impossible mission. So focus on the pathogenesis of asthma, and find new therapeutic targets for prevention of respiratory professional is still a major issue of concern.With the continuous expansion and in-depth study, there are more and more hypothesis on the mechanism of asthma, including airway structure of the local environment, impaired immune function abnormalities and so on, the airway epitheliums palys an important role in asthma. Human bronchial epithelial cell (HBE) is the first line to defense against external stimuli act as an impotant barrier plays an important role in innate immunity. The impaired epitheliums with enhanced permeability can not exclude those asthmogens such as TDI. Recent research suppot HBE is a bridge that links the innate immunity and acquired immunity in asthma.It react with Variety external stimulus and release lots of cytokines like TSLP and VEGF, then contacts with a variety of cells involved asthma, also paticipate MTE influenc the reconstruction of airway. HBE plays a central role in all aspcets of the occurrence of asthma.There is immune imbalance in asthma, Variety cytokines involved in the onset and development of asthma. So it is important to find the key factors, which will bring a new dawn for prevention and treatment of asthma.Some studies have shown that vascular endothelial growth factor (VEGF) plays an important role in the pathophysiology of asthma. Lee CG's research showed that vascular endothelial growth factor(VEGF) played a key role in asthma airway remodeling, Th2 inflammation, airway hyper-responsiveness and was an important indicator of asthma by the transgenic animals Knocked out VEGF. Our previous studies have proved that VEGF plays an important role in occupational toluene diisocyanate (TDI) induced permeability of AEC. The above studies suggest VEGF may be an important target for prevention and treatment of asthma.In recent years, environmental factors and lifestyle of people in the pathogenesis of asthma are defined. And some human health problems, such as:whether the generally low level of vitamin D status in serum is associated with a number of diseases should be investigated. There are pile of researchs on the association of Vitamin D and allergic diseases.1,25-dydroxyvitamin D3 (1,25 (OH)2D3) and 25-dydroxyvitamin D3 is the active form of vitamin D3, it approch the effect by binding Vitamin D receptor (Vitamin D receptor, VDR). The classic function of vitamin D is to regulate calcium homeostasis and thus bone formation and resorption. However,less-traditional functions of vitamin D have been demonstrated and include effects on the immune response. Gene chips found that:gens encoding the key protein pathway CYP27A1, CYP27B1, CYP2R1, CYP24A1 wihch regulate metabolism pathway and activition of vitamin D, and some regulation of transcription by vitamin D factor, the coding genes associated with asthma significantly(p<0.05), which shows the basic vitamin D and asthma, a direct link. The Th2 cell-driven disease experimental asthma failed to develop in VDR KO mic,researchs observed that VDR KO mice did develop antigen-specific Th2 cell responses in the periphery and increased IgE and Th2 cytokines but failed to develop lung inflammation or airway hyperresponsiveness. There are evidence showed that vitamin D status linked to asthma.However, the approach which vitamin D protect experimental animals form developing asthma is still rarely reported, we have established the cell model stimulited by vitamin D attempted to observe the effect on permeability and VEGF expression caused by vitamin D in normal AEC and those treated by TDI. Hope the study may provide a new experimental evidence of pathogenesis and therapeutic targets for asthma.Methods:1. Construction of TDI-HSA.2. Purchase of 25-dydroxyvitamin D3.3. The culture and passage of 16HBE.4. Methyltetrazolium (MTT) assay was used to assess the HBE Cell viability under different concentration of 25-dydroxyvitamin D3and TDI-HSA respectly, groups as follows:(1)Treatment with 25-dydroxyvitamin D3:The normal control group, 25-dydroxyvitamin D3 2×10-9mol/L,25-dydroxyvitamin D3 4×10-9mol/L,25-dydroxyvitamin D3 6×10-9mol/L,25-dydroxyvitamin D3 8×10-9mol/L,25-dydroxyvitamin D3 10×10-9mol/L,25-dydroxyvitamin D3 12×10-9mol/L,25-dydroxyvitamin D3 15×10-9mol/L,25-dydroxyvitamin D3 20×10-9mol/L; (2) TDI-HSA stimulation:The normal control group,20μg/ml TDI-HSA,40μg/ml TDI-HSA,60μg/ml TDI-HSA,80μg/ml TDI-HSA,100μg/ml TDI-HSA,150μg/ml TDI-HSA.5. Transepithelial electrical resistance(TER) was measured in real-time using MILLICELL-ERS Voltohmmeter. Cells cultured in transwells were stimulated by 25-dydroxyvitamin D3 and TDI-HSA respectively. Groups as follows:(1) Treatment with 25-dydroxyvitamin D3:The normal control group, 25-dydroxyvitamin D3 5×10-9mol/L,25-dydroxyvitamin D3 8×10-9mol/L,25-dydroxyvitamin D3 10×10-9mol/L; (2) TDI-HSA stimulation:The normal control group,40μg/ml TDI-HSA,80μg/ml TDI-HSA,100μg/ml TDI-HSA. TERX S of transwell formed standard TER.6. Real time-PCR was applied to detect VEGF gene expression of 16-HBE under the different concentrations of 25-dydroxyvitamin D3 and TDI-HSA, groups as follows: (1) Treatment with 25-dydroxyvitamin D3:The normal control group, 25-dydroxyvitamin D3 5×10-9mol/L,25-dydroxyvitamin D3 8×10-9mol/L,25-dydroxyvitamin D3 10×10-9mol/L; (2) TDI-HSA stimulation:The normal control group,40μg/ml TDI-HSA,80μg/ml TDI-HSA,100μg/ml TDI-HSA.After stimulated for 24h, the cell culture supernatant was collected, and extracted the total RNA, Real time-PCR was used to detection VEGF gene expression of 16-HBE. 7. Selected concentration of 25-dydroxyvitamin D310×10-9mol/L which significently inhabit the expression of VEGF pre-treat 16HBE for 3h,6h,9h,12h,24h, and then stimulate with TDI-HSA for 24h, the cell culture supernatant was collected, cells extracted the total RNA, Real time-PCR was used to detection VEGF gene expression of 16-HBE.8. Enzyme-linked immunosorbent assay (ELISA) was applied to detect the cell culture supernatant of VEGF protein expression.9. Statistical methods:SPSS 13.0 analysis statistical software was used for date analysis. Data was expressed as mean±SD, One-way analysis of variance (one-way ANOVA) was used to compare the overall mean when the variance was Homogeneity, and LSD method was used for Multiple comparisons among the groups; when the variance was not Homogeneity, Welkch method was used to compare the overall mean, Dunnett's T3 was used for Multiple comparisons among the groups. Significance was accepted when p< 0.05.Results:1. The effects of different concentrations of 25-dydroxyvitamin D3 on cell viability of normal human bronchial epithelial cell 16-HBE:the concentration of 25-dydroxyvitamin D3 below 12×10-9mol/L for 16-HBE did not significantly affect cell viability, while the concentration of 25-dydroxyvitamin D3 12×10-9mol/L significantly reduced cell viability (P<0.05). The concentration of TDI-HSA is 150μg/ml for 16-HBE significantly reduced cell viability (P<0.001).2. TDI-HSA can enhance the permeability of 16HBE cell layer (P<0.05). Also TDI-HSA increses the expression of VEGF significently(P<0.05).3. The effects of 25-dydroxyvitamin D3 on VEGF gene expression of 16-HBE, results:5×10-9mol/L,8×10-9mol/L,10×10-9mol/L; group decreased VEGF gene expression compared with the control group, with statistical significance (P <0.05). But the observetion of permeability which was negative correlated with TER is increased.(P<0.05)4.25-dydroxyvitamin D3 as pretreatment added into cell cultures for 12h or less(3h,6h,9h) can reduce the expression of VEGF which enhanced by TDI. (P <0.05)5.25-dydroxyvitamin D3 can decrease expression of VEGF in 16HBE,however the declin of TER in the TDI cultures with pertreatment of 25-dydroxyvitamin D3 for 12h is more sever than those cultures without a pretreatment by 25-dydroxyvitamin D3.Conclusion:The combination of TDI-HSA increased the gene and protein expression of VEGF in human bronchiolar epithelium cell(16-HBE) under certain concentrations, which did not influence the Cell viability; 25-dydroxyvitamin D3 directly decreased the gene and protein expression of VEGF in human bronchiolar epithelium cell(16-HBE) under certain concentrations; And 25-dydroxyvitamin D3 pretreatment reduce the expression of VEGF in TDI cultures; But these two stimulins together increased permeability of 16HBE,this may suggested besides VEGF permeability also be regulated via a another pathway. |
PDF Full Text Request