压力对共培养上皮细胞下的气道平滑肌细胞影响的研究
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摘要
气道是连接咽和肺之间的呼气和吸气的通道,主要由气道上皮细胞、成纤维细胞、平滑肌细胞、肥大细胞等组成。气道的有节律性的收缩,可以保证呼吸作用的正常进行。当哮喘发生时,会使气道壁频繁痉挛,呼吸困难。哮喘的反复发作会引起气道壁的结构发生巨大变化:气道平滑肌增殖和变厚、细胞外基质沉淀等,导致气流阻塞以及非特异性的气道高反应性[1-2]。
哮喘病现在仍然是一个亟待解决的世界性难题,目前我们对哮喘病的发病机理还存在着争议,还需要我们做更多的基础性研究来更深层次的了解哮喘病。在本课题研究中,我们基于哮喘病患者的气道平滑肌增厚和收缩能力下降这些症状,模拟身体内的力学环境,研究正常的气道平滑肌细胞和上皮细胞在受到外来压力刺激的时候,是否这些正常的平滑肌细胞的生物学特性会发生改变。
上皮细胞位于气道的最外层,在它的下面是平滑肌细胞。当上皮细胞受到刺激后,可以分泌一些细胞因子。这些细胞因子扩散到平滑肌细胞上面以后,会引起平滑肌细胞的某些生物学特性的改变。我们的主要工作就是设计一个细胞共培养模型,把上皮细胞和平滑肌细胞培养在一起,然后对这个系统施加压力,最后检测平滑肌细胞的生物学特性是否改变。主要研究工作和结果如下:
①大鼠气道平滑肌细胞(SMC)、上皮细胞(RTE)的原代培养和鉴定
利用组织贴块法对大鼠气道平滑肌细胞进行原代培养,利用胰蛋白酶消化法对上皮细胞进行原代培养。经过摸索,组织贴块法能够获得纯度很高的平滑肌细胞。在无菌环境下解剖大鼠,将获得的气管剪成小块,平铺在细胞培养瓶中,用DMEM/F12培养基培养六天左右,可见有细胞爬出来,呈现平滑肌细胞特有的外观。同样,利用气管灌注法,0.05%的胰蛋白酶能够消化得到很多上皮细胞,经过一些纯化处理以后,即可以得到纯度较高的上皮细胞。等到两种细胞长满细胞培养瓶底部80%左右时,即可以用0.25%的胰蛋白酶进行消化传代。
将两种细胞进行鉴定。首先,在形态学上,两种细胞的符合各自的标准形态。然后对平滑肌细胞特异的α-肌动蛋白进行免疫荧光染色,得出结论所获得的确实是平滑肌细胞。再根据上皮细胞特征性地表达角蛋白,利用细胞免疫组化(SABC)法,可以判定成功获得上皮细胞。
②受力情况下与上皮细胞共培养的气道平滑肌的增殖
本课题中,利用MTT法,对四种不同培养情况下的平滑肌细胞进行增殖测定,最后绘制出不同培养条件下的气道平滑肌细胞(ASMCs)的生长曲线。结果显示:与上皮细胞共培养,并且受到压力刺激的情况下,平滑肌细胞增殖速度要比其他培养条件要更快一些。并且通过对比分析,说明在压力条件下,上皮细胞会分泌一些细胞因子,这些因子与平滑肌细胞结合,促进平滑肌细胞的增殖。
③受力情况下与上皮细胞共培养的气道平滑肌的刚度
本课题中,利用原子力显微镜(AFM),对四种不同培养情况下的平滑肌细胞的杨氏模量(即刚度)进行测定,通过比较,得到下面结果:和其他几种培养条件相比,与上皮细胞共培养,并且受到压力刺激的情况下,平滑肌细胞的刚度要明显变大。这可以说明,通过共培养系统模拟气道,对其施加压力,平滑肌细胞的刚度会变大,这也许和哮喘患者的气道潜在收缩能力变强。
所有本课题研究的结果表明,在施加压力条件下,平滑肌细胞和上皮细胞的共培养很好的模拟了哮喘患者的气道环境,出现了平滑肌细胞增殖、刚度变大的现象。这样,在以后的研究中,科研人员可以利用这个共培养模型,通过分析这个过程中的培养液中的细胞因子,来证明在受到压力刺激情况下,上皮细胞究竟是通过哪种细胞因子来影响平滑肌细胞的生物特性。
Airway is the channel of aspiration and inspiration connecting throat and lung, and is mainly consisted of Airway epithelial cells, Fibroblast, Smooth muscle cells, Mast cells and so on. The normal respiration could be guaranteed by the rhythmic contraction of airway. Whereas, asthma can lead to the frequent convulsion of the airway wall and result in the scant of breath. What’s worse, the repeat episodes of asthma will cause the great structural change of the airway wall, such as the proliferation and thickening of the airway smooth muscle, precipitation of the extracellular matrix etc., which will lead to the Airflow obstruction as well as the Non-specific Airway hyperresponsiveness.
At present, asthma is still the worldwide difficulty which urgently needs to be solved. However, we haven’t reached an agreement about the pathogenesis of asthma yet, so there are more basic researches to be done in order to deepen our understanding of asthma. Based on the asthma patients’symptoms like thickening of the airway smooth muscle and weakening of the contraction ability, the current study simulate the mechanical environment of body to investigate whether the biological features of normal airway smooth muscle cells and epithelial cells will be changed if they are exposed to external pressure stimulus.
Epithelial cells are in the outermost layer of airway, under which are the smooth muscle cells. Epithelial cells can secrete some cytokines when they receive certain stimulation. Then these cytokines will make the smooth muscle cells change their biological features when they spread over the smooth muscle cells. In the current study, we first design a cell co-culture model and culture the epithelial cells and smooth muscle cells together; then we exert pressure to the system. Finally, the biological features of smooth muscle cells are examined to see if they are changed. The key researches and findings are as follows.
①The primary culture and identification of epithelium cells and Smooth muscle cells of rat airway.
We employ the method of tissue explant to culture smooth muscle cell of rat air duct, and then use the trypsin to digest the epithelium cells. After exploration of cell culture, tissue explant method can extract highly pure smooth muscle cells. In the sterile environments, we anatomise the rat to get the air duct, and then cut it into pieces. Next, these small parts of air duct are tiled in the cell culture bottle and cultured by DMEM/F12 culture medium for 6 days or so. Finally, we can get the target cells demonstrating the particular appearance of smooth muscle cell. With the help of tracheal perfusion, 0.05% trypsin can also digest more epithelium cells. After certain purification, we can get highly pure epithelium cells. When these two kinds of cells cover 80% of cell flask, we can employ 0.25% trpsin for digestion and cell passage.
Then the two kinds of cells are identified. Firstly, in morphology, the two kinds of cells cater to the standard norm of each type respectively. Secondly, the specificα-actin of smooth muscle cells is tested by immuno-fluorescence staining, and we conclude that the kind of cell indeed is smooth muscle cells. Next, based on the epithelium cells’characteristic expression of Keratin as well as adopting cell immuno-histochemistry, it proves that we get the the epithelium cells successfully.
②Proliferation of the Airway smooth muscle cell co-cultured with epithelial cell under physical forces.
In this research, through MTT, we detect the proliferation of smooth muscle cell under 4 different culture circumstances, and draw the curve of growth for airway smooth muscle cell. The results indicate that comparing with other culture conditions, smooth muscle cells grow faster under the stimulation of pressure force and co-culture with epithelial cell. Through comparative analysis, it demonstrates that under physical forces epithelial cells will secrete certain cytokines, which can combine with smooth muscle cells and promote the proliferation of smooth muscle cells.
③Stiffness of the Airway smooth muscle cell co-cultured with epithelial cells under physical forces.
In the research,using Atomic force microscopy(AFM), we detect the young’s modulus, that is the stiffness, of smooth muscle cells under four different cell culture environments. Through comparing, we draw conclusions as follows: comparing with other culture conditions, the co-culture with epithelial cell and pressure force increase the stiffness of smooth muscle cells significantly. It shows that through the virtual airway of co-culture system, the stiffness of smooth muscle cells were increased under the pressure force, it may match with the increase of asthma patients' potential contraction ability of airway.
The results from this study all indicate that the co-culture of smooth muscle cells and epithelial cells can perfectly simulate the asthma patients’airway environment when they are exerted to pressure. And in this case, the smooth muscle cells are proliferated and stiffness becomes stronger. Thus, researchers can make use of this co-culture model in the further study. Specifically, through analyzing the cytokines in the culture solution, researchers can demonstrate which kind of cytokines is adopted by epithelial cells to affect the biological features of smooth muscle cells under pressure stimulus.
哮喘病现在仍然是一个亟待解决的世界性难题,目前我们对哮喘病的发病机理还存在着争议,还需要我们做更多的基础性研究来更深层次的了解哮喘病。在本课题研究中,我们基于哮喘病患者的气道平滑肌增厚和收缩能力下降这些症状,模拟身体内的力学环境,研究正常的气道平滑肌细胞和上皮细胞在受到外来压力刺激的时候,是否这些正常的平滑肌细胞的生物学特性会发生改变。
上皮细胞位于气道的最外层,在它的下面是平滑肌细胞。当上皮细胞受到刺激后,可以分泌一些细胞因子。这些细胞因子扩散到平滑肌细胞上面以后,会引起平滑肌细胞的某些生物学特性的改变。我们的主要工作就是设计一个细胞共培养模型,把上皮细胞和平滑肌细胞培养在一起,然后对这个系统施加压力,最后检测平滑肌细胞的生物学特性是否改变。主要研究工作和结果如下:
①大鼠气道平滑肌细胞(SMC)、上皮细胞(RTE)的原代培养和鉴定
利用组织贴块法对大鼠气道平滑肌细胞进行原代培养,利用胰蛋白酶消化法对上皮细胞进行原代培养。经过摸索,组织贴块法能够获得纯度很高的平滑肌细胞。在无菌环境下解剖大鼠,将获得的气管剪成小块,平铺在细胞培养瓶中,用DMEM/F12培养基培养六天左右,可见有细胞爬出来,呈现平滑肌细胞特有的外观。同样,利用气管灌注法,0.05%的胰蛋白酶能够消化得到很多上皮细胞,经过一些纯化处理以后,即可以得到纯度较高的上皮细胞。等到两种细胞长满细胞培养瓶底部80%左右时,即可以用0.25%的胰蛋白酶进行消化传代。
将两种细胞进行鉴定。首先,在形态学上,两种细胞的符合各自的标准形态。然后对平滑肌细胞特异的α-肌动蛋白进行免疫荧光染色,得出结论所获得的确实是平滑肌细胞。再根据上皮细胞特征性地表达角蛋白,利用细胞免疫组化(SABC)法,可以判定成功获得上皮细胞。
②受力情况下与上皮细胞共培养的气道平滑肌的增殖
本课题中,利用MTT法,对四种不同培养情况下的平滑肌细胞进行增殖测定,最后绘制出不同培养条件下的气道平滑肌细胞(ASMCs)的生长曲线。结果显示:与上皮细胞共培养,并且受到压力刺激的情况下,平滑肌细胞增殖速度要比其他培养条件要更快一些。并且通过对比分析,说明在压力条件下,上皮细胞会分泌一些细胞因子,这些因子与平滑肌细胞结合,促进平滑肌细胞的增殖。
③受力情况下与上皮细胞共培养的气道平滑肌的刚度
本课题中,利用原子力显微镜(AFM),对四种不同培养情况下的平滑肌细胞的杨氏模量(即刚度)进行测定,通过比较,得到下面结果:和其他几种培养条件相比,与上皮细胞共培养,并且受到压力刺激的情况下,平滑肌细胞的刚度要明显变大。这可以说明,通过共培养系统模拟气道,对其施加压力,平滑肌细胞的刚度会变大,这也许和哮喘患者的气道潜在收缩能力变强。
所有本课题研究的结果表明,在施加压力条件下,平滑肌细胞和上皮细胞的共培养很好的模拟了哮喘患者的气道环境,出现了平滑肌细胞增殖、刚度变大的现象。这样,在以后的研究中,科研人员可以利用这个共培养模型,通过分析这个过程中的培养液中的细胞因子,来证明在受到压力刺激情况下,上皮细胞究竟是通过哪种细胞因子来影响平滑肌细胞的生物特性。
Airway is the channel of aspiration and inspiration connecting throat and lung, and is mainly consisted of Airway epithelial cells, Fibroblast, Smooth muscle cells, Mast cells and so on. The normal respiration could be guaranteed by the rhythmic contraction of airway. Whereas, asthma can lead to the frequent convulsion of the airway wall and result in the scant of breath. What’s worse, the repeat episodes of asthma will cause the great structural change of the airway wall, such as the proliferation and thickening of the airway smooth muscle, precipitation of the extracellular matrix etc., which will lead to the Airflow obstruction as well as the Non-specific Airway hyperresponsiveness.
At present, asthma is still the worldwide difficulty which urgently needs to be solved. However, we haven’t reached an agreement about the pathogenesis of asthma yet, so there are more basic researches to be done in order to deepen our understanding of asthma. Based on the asthma patients’symptoms like thickening of the airway smooth muscle and weakening of the contraction ability, the current study simulate the mechanical environment of body to investigate whether the biological features of normal airway smooth muscle cells and epithelial cells will be changed if they are exposed to external pressure stimulus.
Epithelial cells are in the outermost layer of airway, under which are the smooth muscle cells. Epithelial cells can secrete some cytokines when they receive certain stimulation. Then these cytokines will make the smooth muscle cells change their biological features when they spread over the smooth muscle cells. In the current study, we first design a cell co-culture model and culture the epithelial cells and smooth muscle cells together; then we exert pressure to the system. Finally, the biological features of smooth muscle cells are examined to see if they are changed. The key researches and findings are as follows.
①The primary culture and identification of epithelium cells and Smooth muscle cells of rat airway.
We employ the method of tissue explant to culture smooth muscle cell of rat air duct, and then use the trypsin to digest the epithelium cells. After exploration of cell culture, tissue explant method can extract highly pure smooth muscle cells. In the sterile environments, we anatomise the rat to get the air duct, and then cut it into pieces. Next, these small parts of air duct are tiled in the cell culture bottle and cultured by DMEM/F12 culture medium for 6 days or so. Finally, we can get the target cells demonstrating the particular appearance of smooth muscle cell. With the help of tracheal perfusion, 0.05% trypsin can also digest more epithelium cells. After certain purification, we can get highly pure epithelium cells. When these two kinds of cells cover 80% of cell flask, we can employ 0.25% trpsin for digestion and cell passage.
Then the two kinds of cells are identified. Firstly, in morphology, the two kinds of cells cater to the standard norm of each type respectively. Secondly, the specificα-actin of smooth muscle cells is tested by immuno-fluorescence staining, and we conclude that the kind of cell indeed is smooth muscle cells. Next, based on the epithelium cells’characteristic expression of Keratin as well as adopting cell immuno-histochemistry, it proves that we get the the epithelium cells successfully.
②Proliferation of the Airway smooth muscle cell co-cultured with epithelial cell under physical forces.
In this research, through MTT, we detect the proliferation of smooth muscle cell under 4 different culture circumstances, and draw the curve of growth for airway smooth muscle cell. The results indicate that comparing with other culture conditions, smooth muscle cells grow faster under the stimulation of pressure force and co-culture with epithelial cell. Through comparative analysis, it demonstrates that under physical forces epithelial cells will secrete certain cytokines, which can combine with smooth muscle cells and promote the proliferation of smooth muscle cells.
③Stiffness of the Airway smooth muscle cell co-cultured with epithelial cells under physical forces.
In the research,using Atomic force microscopy(AFM), we detect the young’s modulus, that is the stiffness, of smooth muscle cells under four different cell culture environments. Through comparing, we draw conclusions as follows: comparing with other culture conditions, the co-culture with epithelial cell and pressure force increase the stiffness of smooth muscle cells significantly. It shows that through the virtual airway of co-culture system, the stiffness of smooth muscle cells were increased under the pressure force, it may match with the increase of asthma patients' potential contraction ability of airway.
The results from this study all indicate that the co-culture of smooth muscle cells and epithelial cells can perfectly simulate the asthma patients’airway environment when they are exerted to pressure. And in this case, the smooth muscle cells are proliferated and stiffness becomes stronger. Thus, researchers can make use of this co-culture model in the further study. Specifically, through analyzing the cytokines in the culture solution, researchers can demonstrate which kind of cytokines is adopted by epithelial cells to affect the biological features of smooth muscle cells under pressure stimulus.
引文
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