摘要
支气管哮喘(简称哮喘)是一种严重影响人类健康的慢性呼吸道疾病。研究显示,全球哮喘病人约3亿,我国哮喘患者数量庞大,且有不断增加的趋势。哮喘这一基因与环境因素共同导致的复杂疾病,即使在今天生物-环境-心理的疾病评估模式中,其发病、发展等环节仍不清楚,故此,目前对哮喘患者的治疗以症状控制为主。多项临床研究都表明,尽管大部分诊断明确的患者在规范化治疗后症状能够得到有效控制,却无法逆转或阻止哮喘的自然病史,最终产生不可逆的气道结构性改变。可以看到,哮喘的发病机制不明,使得哮喘的预防和根治成为了不可能的任务。因此探讨哮喘发病机制、寻找新的治疗乃至预防靶点仍然是呼吸专业关注的重大课题。
随着研究的不断拓展和深入,关于哮喘发病的假说不断增多,其中气道局部环境结构、功能受损,免疫作用异常成为研究热点,气道在哮喘发病中的地位被大大提升。支气管上皮细胞(HBE),作为气道抵抗外来侵害的第一道防线,其物理屏障作用非常明显,气道上皮通透性增高是致喘物质接触上皮下免疫细胞的第一环。同时HBE在气道固有免疫中也发挥着重要的作用。近年来也证实,HBE是哮喘固有免疫与获得性免疫联系的桥梁,在多种触发因素作用下,HBE释放多种炎性介质如近年来证实与哮喘炎症相关的TSLP, VEGF,与免疫、炎性细胞发生广泛的联系,还能够通过上皮间充质转化成为气道结构细胞参与气道重建过程,可见HBE从各个环节参与了哮喘的发生、发展。
哮喘中多种细胞因子表达、释放存在异常,这些因子参与了气道免疫失衡的发生、发展,维持着哮喘气道炎症,促使气道改建。因此,探讨引发炎症失衡的关键细胞因子会给哮喘的防治带来新的曙光。有研究通过转基因动物实验表明,阻断血管内皮生长因子(VEGF),能使哮喘的气道炎症、气道重塑、气道高反应性等病理生理变化趋于正常化。本课题组前期研究已经证实VEGF在职业哮喘诱发因素甲苯二异氰酸盐(TDI),屋尘螨以及过氧化氢等能够导致HBE通透性增高的过程中发挥重要作用。上述均提示VEGF在哮喘的发病中占有一席之地,VEGF可能成为哮喘防治的重要靶点。
1,25-二羟维生素D3(1,25(OH)2D3)是维生素D3的生物活性代谢产物,现在学界将其归为激素一种,而不仅仅是维生素。它的生物学效应是由一种核受体超家族成员-维生素D受体(Vitamin D receptor, VDR)介导,同时还有一部分效应是通过与胞膜的VDR结合,经由非基因组信号转导实现的。1,25-二羟维生素D3除了调节钙和骨代谢,还发挥重要的免疫调节作用。APC和T细胞是它在免疫系统中发挥作用的主要靶细胞。1,25-二羟维生素D3及其类似物可直接作用于T细胞,或通过调节APC的功能间接影响T细胞的功能。1,25-二羟维生素D3及其类似物如：25羟基维生素D3,都对APC和T细胞的作用,这种作用有赖于这两种细胞内VDR的表达,以及它们信号转导通路中的共同靶分子。已有许多实验证实,1,25-二羟维生素D。下调T细胞和APC内核因子-κB(nuclear factor-κB, NF-κB)的表达。众所周知,哮喘的发生与免疫失调密切相关,维生素D对APC和T细胞的作用,是否是它在哮喘中进行调节的途径,尚不得而知,但已经有实验发现维生素D能够通过影响免疫细胞的活化、功能达到影响免疫相关疾病如：糖尿病,炎症性肠病的作用,那么它对同样由T细胞参与介导的哮喘又有怎样的作用,是个引人深思的课题。
近期通过人类基因芯片的研究比对,研究者们发现：编码维生素D通路,包括维生素D的转活、代谢、作用途径的关键蛋白(CYP27A1,CYP27B1,CYP2R1,CYP24A1)以及一些可由维生素D调控转录的因子,它们的编码基因经统计分析与哮喘相关(P<0.05),这显示了维生素D与哮喘发病根本、直接的联系。
近年来,环境因素和人们的生活模式在哮喘发病中的作用越来越明确。而一些人群健康问题,例如：普遍的人群维生素D低水平状况是否与一些疾病发生相关被研究者们关注。关于维生素D与过敏性疾病的研究层出不穷。在VDR敲除小鼠中诱发哮喘模型中发现,虽然IgE浓度增高和Th2型细胞因子增加,但这种小鼠并没有发生气道炎症、嗜酸粒细胞浸润或气道高反应性等哮喘样症状。上述研究证据说明了维生素D在哮喘发病中的地位。但维生素D调节哮喘的具体环节尚鲜见报道,本研究是在前期证明TDI促进气道上皮通透性部分经由VEGF途径的基础上,通过建立维生素D作用细胞的模型,试图观察这一过程,了解维生素D对正常16HBE和TDI刺激的16HBE的通透性及VEGF表达的影响,在体外探讨维生素D对气道上皮炎症介质的释放及屏障功能的影响及可能机制。
方法：
1、制备甲苯二异氰酸酯-人血清白蛋白复合物(TDI-HSA)。
2、购买分析纯的25羟维生素D3。
3、人支气管上皮细胞系16HBE的传代,培养。
4、四唑盐(MTT)比色法检测(不同浓度)25羟维生素D3,TDI-HSA对16-HBE活力的影响。分组情况：(1)25羟维生素D3作用：对照组,2×10-9mol/L 25羟维生素D3组,4×10-9mol/L 25羟维生素D3组,6×10-9mol/L 25羟维生素D3组,8×10-9mol/L 25羟维生素D3组,10×10-9mol/L 25羟维生素D3组,12×10-9mol/L25羟维生素D3组,15×10-9mol/L 25羟维生素D3组,20×10-9mol/L25羟维生素D3组；(2)TDI-HSA刺激：对照组,20μg/mlTDI-HSA组,40μg/ml TDI-HSA组,60μg/ml TDI-HSA组,80μg/ml TDI-HSA组,100μg/ml TDI-HSA组,150μg/ml TDI-HSA组。
5、测定不同浓度25羟维生素D3和TDI-HSA对16-HBE细胞单层电阻的影响。分组情况：(1)25羟维生素D3处理：阴性对照组,5×10-9mol/L 25羟维生素D3组,8×10-9mol/L 25羟维生素D3组,10×10"9mol/L 25羟维生素D3组；(2)TDI-HSA刺激：对照组,40μg/mlTDI-HSA组,80μg/ml TDI-HSA组,100μg/ml TDI-HSA组。上述作用因素作用细胞24h后,以膜电阻测定仪测量TRANSWELL小室的细胞单层,取平均值,计算标准化跨膜电阻。
6、分别观察不同浓度25羟维生素D3和TDI-HSA对16-HBE VEGF基因表达的影响,分组情况：(1)25羟维生素D3：阴性对照组,5×10-9nol/L 25羟维生素D3组,8×10-9mol/L25羟维生素D3组,10×10-9mol/L 25羟维生素D3组;(2)TDI-HSA刺激：对照组,40μg/mlTDI-HSA组,80μg/ml TDI-HSA组,100μg/ml TDI-HSA组。按上述刺激24h后,收集细胞,试剂盒一步法提取总RNA,应用Real time-PCR检测16-HBE的VEGF基因表达情况。
7、选取对VEGF抑制最明显的25羟维生素D3浓度5×10-9mol/L预先处理细胞,分别作用3h、6h、9h、12h、24h,吸出含有25羟维生素D3的培养基,换新鲜培养基,然后以终浓度为100μg/ml TDI-HSA刺激各组25羟维生素D3预处理细胞,24h后收集细胞,提取总RNA,应用Real time-PCR检测16-HBE的VEGF基因表达情况。依照步骤5,同样测定上述各组的跨细胞电阻,求得标准化电阻值。
8、应用酶联免疫吸附实验(ELISA)法检测上述细胞培养上清中VEGF蛋白表达情况。
9、统计学方法：采用SPSS13.0统计软件分析,计量资料用均数±标准差表示,方差齐时,总体均数的比较采用单向方差分析(one-way ANOVA),各组间多重比较采用LSD法,方差不齐时总体均数的比较采用Welkch法,各组间多重比较采用Dunnett's T3法检验,以P<0.05具有统计学意义。
结果：
1、不同浓度25羟维生素D3对16-HBE细胞活力影响：25羟维生素D3在12×10-9mol/L浓度以下时对细胞活力无显著影响(与对照组相比,P=0.321)。
2、不同浓度的TDI-HSA对16-HBE细胞活力影响：当TDI-HSA浓度达到150μg/ml后,与对照组相比细胞活力显著下降,具有统计学差异(P<0.001)。
3、TDI-HSA刺激可导致单层16HBE跨膜电阻减低(n=3,P=0.007),即增高单层16HBE通透性,同时可使VEGF表达显著升高(基因水平与对照组比较,P<0.001；蛋白水平与对照组比较,P=0.002,n=5)。
4、10-8M的25羟基维生素D3直接作用于细胞,会减低跨膜电阻(与对照组比较,P=0.002),即增高细胞层通透性,同时可从mRNA水平(与对照组比较,P<0.001)和蛋白水平(与对照组比较,P<0.001)降低气道上皮细胞16HBE的VEGF表达量,
5、25羟基维生素D3处理16HBE 12小时,可以显著降低TDI-HSA刺激引起的VEGF升高(与TDI-HSA刺激组比较,P<0.001)。
6、25羟基维生素D3预处理细胞12h,再给以TDI-HSA刺激,TEER低于TDI直接刺激组(P=0.046),即25羟基维生素D3和TDI-HSA共刺激增高了气道上皮通透性。
结论：
1.25羟维生素D3可以降低16HBE VEGF的表达,对TDI诱导的16HBE VEGF的表达和释放同样具有抑制作用。
2.25羟维生素D3能够增加16HBE单层通透性,也可协同促进TDI诱导的16HBE通透性的增加。
3. VEGF是上皮通透性的重要调节因子,但气道上皮通透性除受VEGF调控外,还存在其他调节途径。
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.
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