人支气管上皮细胞在呼吸道合胞病毒感染后CFTR功能及上皮修复功能研究

发布时间：2018-06-24 14:38

本文选题：RSV + 支气管上皮细胞　；参考：《中南大学》2012年博士论文

【摘要】：目的：呼吸道合胞病毒(Respiratory syncytial virus, RSV)感染是临床最常见的,也是儿科最常遇到的呼吸道病原之一。支气管上皮细胞在RSV感染后,其病理改变取决于某些离子通道的功能活性及表达密度,其是否抑制了其囊性纤维化跨膜转导调节因子(Cystic Fibrosis Transmembrane conductance Regulator, CFTR)的表达水平和功能。我们拟建立RSV感染离体支气管上皮细胞模型,对RSV感染支气管上皮细胞后CFTR的表达及功能进行研究。方法： 1)建立RSV感染离体培养支气管上皮细胞模型将原RSV病毒悬液(TCID50=10-3.82/0.1ml)按1：100000的浓度稀释,得RSV病毒感染人支气管上皮细胞模型的初始病毒浓度,以此浓度的RSV病毒感染人支气管上皮细胞制备离体RSV病毒感染人支气管上皮细胞模型。RT-PCR法验证模型中RSV存在以及其转录表达水平。 2)人支气管上皮细胞感染RSV后CFTR的表达取对数生长期RSV感染人支气管上皮细胞模型中阴性对照组细胞、RSV感染后传代第1代的细胞和可稳定传代的模型组细胞,通过免疫荧光的方法检测RSV稳定感染后细胞CFTR的蛋白水平定位及表达；Western-blot法检测RSV稳定感染后细胞CFTR的蛋白表达；qRT-PCR法检测RSV稳定感染后细胞CFTR的rnRNA表达水平。 3)人支气管上皮细胞感染RSV后CFTR的功能变化取对数生长期RSV稳定感染人支气管上皮细胞模型中阴性对照组细胞、RSV感染后传代第1代细胞和稳定传代的模型组细胞,采用全细胞记录的方法记录RSV持续感染人支气管上皮细胞模型阴性对照组和模型组细胞上CFTR C1-的变化。加入激动剂5μM的forskolin以后,激活cAMP诱导CFTR C1-电流,细胞被钳制在-40mV,给予不同的电压刺激,电压自-100mV逐步增到+100mV,去极化电压时程为200mS,步阶为20mV,间隔为2S。去极化电压结束后恢复至-40mV钳制电压。取各个电压状态下最大电流值,绘制Ⅰ-V关系曲线。用MQAE荧光染料法检测3组细胞的细胞内Cl-浓度,制备标准品,检测标准品Cl-浓度,绘制Cl-浓度标准曲线,根据Cl-浓度标准曲线,计算出待测3组支气管上皮细胞中Cl-浓度,同时MQAE荧光染料法检测3组细胞的细胞内Cl-浓度荧光强度变化率。 4)人支气管上皮细胞感染RSV后上皮修复功能改变制备RSV感染人支气管上皮细胞的损伤模型后,用显微视频分析BI-2000图像／免疫组化分析系统分别于机械损伤后0hrs,8hrs,12hrs,18hrs,24hrs测量缺损面积一次,收集所有时间点的缺损区域图像,勾画缺损区域的边缘,以相关系数r来衡量两者是否存在直线相关关系,绘制时间与修复面积的直线回归方程：Y=a+bX,计算损伤修复指数(RI, repair index),RI值等于回归方程中b的绝对值(RI=│b│),以此反映不同处理组之间不同的修复速度,直线的斜率为损伤修复指数,斜率越大,损伤修复速度越快。结果： 1)RSV感染离体培养支气管上皮细胞模型的建立 RT-PCR法验证模型中RSV存在以及其转录表达水平,发现在RSV感染后的RSV感染人支气管呼吸道细胞模型中,最初几代的RSV的表达水平是逐步增高的,然后维持在较稳定水平,但是从细胞的形态学上来观察,并没有引起肉眼可见的细胞病变,同时也未被人支气管上皮细胞所清除。表示RSV稳定感染离体培养支气管上皮细胞模型建立成功。 2)人支气管上皮细胞感染RSV后CFTR的表达的改变取对数生长期RSV稳定感染人支气管上皮细胞模型中阴性对照组细胞、RSV稳定感染后传代第1代细胞和稳定传代的模型组细胞,通过免疫荧光的方法检测RSV感染细胞CFTR的蛋白水平定位及表达,并随机选取视野,计数荧光染色阳性细胞的百分率。结果显示在RSV感染人支气管上皮细胞模型阴性对照组细胞的细胞膜及胞浆中可见较多的绿色染色的CFTR,并且荧光染色阳性细胞的百分率较高；在RSV稳定感染模型传代第1代细胞就发现其细胞的细胞膜及胞浆中CFTR明显减少,并且荧光染色呈阳性细胞的百分率明显减少；而在RSV稳定感染模型组细胞中细胞膜及胞浆中CFTR表达较阴性对照组细胞明显减少,较RSV感染模型第1代细胞也有略有减少,并且荧光染色阳性细胞的百分率也略有减少。推测RSV感染细胞后,可破坏气道上皮结构的完整性,致使局部气道微环境呈失稳态的状态,使得气道功能失调,改变了气道上皮应激应答机制,导致气道的功能和结构重塑,形成慢性气道炎症和气道病理改变。分别提取RSV感染人支气管上皮细胞模型阴性对照组细胞、RSV感染后传代第1代细胞和稳定的RSV感染模型组细胞的膜蛋白,Western-blot法检测RSV感染后细胞CFTR的蛋白表达,胶片曝光结果使用Quantity one灰度扫描软件处理数据,其表达强度用CFTR灰度值/β-actin灰度值表示,重复3次,统计结果,绘制直方图。实验结果显示,相对于阴性对照组细胞,RSV感染后第1代细胞和稳定RSV持续感染的模型组细胞CFTR表达明显降低,结果具有明显差异,*P0.05. 分别提取RSV感染人支气管上皮细胞模型阴性对照组细胞、RSV感染后传代第1代细胞和稳定RSV感染模型组细胞的RNA,逆转录cDNA, RT-PCR法检测RSV感染后细胞CFTR的mRNA表达水平,结果mRNA表达=2(_ΔΔτ)。结果显示,相对于阴性对照组细胞,RSV感染后第1代细胞和稳定RSV感染模型组细胞CFTR的mRNA表达明显降低,结果具有明显差异,*P0.05. 3)人支气管上皮细胞感染RSV后CFTR的功能变化全细胞记录的方法记录RSV感染人支气管上皮细胞模型阴性对照组和模型组细胞上CFTR Cl-的变化。结果显示,在RSV感染的作用下,CFTR Cl-电流密度增加,并具有浓度依赖性,在10-3mM为最大半数有效剂量。CFTR Cl-阻断剂glibenclamide能阻断该电流,而非CFTR Cl-阻断剂DIDS不能阻断该电流。MQAE荧光染料法检测3组细胞的细胞内Cl-荧光强度变化率,结果显示RSV持续感染后,细胞内Cl-荧光强度变化率的变化相对于阴性对照组明显减少,表示RSV感染后,调节Cl-通道的通道蛋白调节功能被抑制,Cl-外流降低,细胞内Cl-浓度变化率降低。 4)人支气管上皮细胞感染RSV后上皮修复功能改变结果显示,RSV感染阴性对照组斜率绝对值较大,修复速度较快；RSV稳定感染模型组斜率绝对值较小,修复速度较慢。提示RSV感染HBECs后,HBECs的自身增殖、迁移及损伤修复能力较RSV感染前,有所下降,其损伤修复功能发生重塑,有可能形成反复发作的气道炎症和气道高反应倾向。结论：人支气管上皮细胞在RSV感染后,跨膜蛋白CFTR的表达及功能被明显抑制,减弱CFTR对气道上皮细胞功能稳态的调控,致使气道局部微环境呈现失稳态。进而影响气道粘液从腺管向外分泌,形成气道粘液栓,导致气道阻力增高,诱导气道高反应性疾病的发生发展。
[Abstract]:Objective:
Respiratory syncytial virus (Respiratory syncytial virus, RSV) infection is the most common clinical and one of the most common respiratory pathogens in pediatrics. After RSV infection, the pathological changes of the bronchial epithelial cells depend on the functional activity and expression density of some ion channels, and whether it inhibits the regulatory cause of the transmembrane transduction of cystic fibrosis. The expression level and function of Cystic Fibrosis Transmembrane conductance Regulator (CFTR). We intend to establish an isolated bronchial epithelial cell model of RSV infection in vitro and study the expression and function of CFTR after RSV infection of bronchial epithelial cells.
Method:
1) establish RSV infection in vitro cultured bronchial epithelial cell model.
The original RSV virus suspension (TCID50=10-3.82/0.1ml) was diluted by the concentration of 1:100000, and the initial virus concentration of the human bronchial epithelial cell model of RSV virus infection was obtained. The concentration of RSV virus infected human bronchial epithelial cells to prepare the RSV virus infected human bronchial epithelial cell model in the.RT-PCR method to verify the existence of RSV and its transformation. Record the level of expression.
2) the expression of CFTR after human bronchial epithelial cells infected with RSV.
The negative control group cells in the RSV infected human bronchial epithelial cell model of the logarithmic growth period were taken. After RSV infection, the cells of first generation and the stable passage model group cells were passed, and the protein level and expression of CFTR after RSV infection were detected by immunofluorescence, and the Western-blot method was used to detect CFTR in the cells after RSV infection. Protein expression; qRT-PCR method was used to detect the rnRNA expression level of CFTR in RSV after stable infection.
3) changes in CFTR function after human bronchial epithelial cells infected with RSV.
The negative control group cells in the RSV stable infected human bronchial epithelial cell model during the logarithmic growth period were obtained. After RSV infection, the first generation cells and the model groups of the stable passages were passed. The changes of CFTR C1- on the negative control group and the model group of the RSV continuous infection of the human bronchial epithelial cell model were recorded by the whole cell recording method. After 5 mu M of forskolin, activate cAMP to induce CFTR C1- current, cells are clamped in -40mV, give different voltage stimuli, voltage increases gradually from -100mV to +100mV, depolarizing voltage time range is 200mS, step step is 20mV, the interval is back to -40mV clamp voltage after the end of 2S. depolarization voltage. Take the maximum current value under each voltage state, draw the maximum current value under each voltage state, draw I -V relation curve. The intracellular Cl- concentration of 3 groups of cells was detected by MQAE fluorescent dye method, standard products were prepared, Cl- concentration was detected, Cl- concentration standard curve was plotted. According to the Cl- concentration standard curve, the concentration of Cl- in the 3 groups of bronchial epithelial cells was calculated, and the concentration of Cl- concentration in 3 groups of cells was detected by the MQAE fluorescein dye method. Degree change rate.
4) changes in epithelial repair function after human bronchial epithelial cells infected with RSV.
After the damage model of RSV infected human bronchial epithelial cells was prepared, the BI-2000 image / immunohistochemical analysis system was used to measure the defect area of 0hrs, 8hrs, 12hrs, 18hrs, 24hrs after the mechanical damage, and the defect area images were collected at all time points, and the edge of the defect area was drawn, and the correlation coefficient r was used to measure both the two. Whether there is a linear correlation, draw the linear regression equation of time and repair area: Y=a+bX, calculate the damage repair index (RI, repair index), RI value is equal to the absolute value of B in the regression equation (RI= b), in order to reflect the different repair speed between different treatment groups, the slope of the line is the damage repair index, the greater the slope, damage repair The faster the speed of recovery.
Result:
1) establishment of RSV infected bronchial epithelial cell model in vitro
RT-PCR was used to verify the presence of RSV and its transcriptional expression level. It was found that the expression level of RSV in the first generations of RSV infected human bronchial respiratory cell model after RSV infection was gradually increased, and then maintained at a relatively stable level, but it was observed from the cell morphology, and did not cause cellular lesions visible to the naked eye, At the same time, it was not removed by human bronchial epithelial cells, indicating that RSV was successfully established for stable infection of bronchial epithelial cells in vitro.
2) changes in CFTR expression after human bronchial epithelial cells infected with RSV.
The negative control group cells in the RSV stable infection of human bronchial epithelial cell model during the logarithmic growth period were taken. After RSV, the first generation cells and the model group cells were stabilized after the infection. The protein level and expression of CFTR in RSV infected cells were detected by immunofluorescence, and the fluorescent staining positive cells were counted with the field of vision. The results showed that more green stained CFTR was found in the cell membrane and cytoplasm of the negative control group of the RSV infected human bronchial epithelial cell model, and the percentage of the fluorescent staining positive cells was higher. The cell membrane and the cytoplasm of the cells in the first generations of the RSV stable infection model found that the cell membrane and the CFTR in the cytoplasm were significantly reduced. The percentage of positive cells with fluorescent staining decreased significantly, while the expression of CFTR in the cell membrane and cytoplasm in the RSV stable infection model group decreased significantly than that in the negative control group, and the percentage of the first generation cells in the RSV infection model decreased slightly, and the percentage of the fluorescent staining positive cells decreased slightly. Speculates after the RSV infected cells. It can destroy the integrity of the airway epithelium and cause the unstable state of the local airway microenvironment, which makes the airway dysfunction and changes the mechanism of airway epithelial stress response, resulting in the function and structural remodeling of the airway, and the formation of chronic airway inflammation and airway pathological changes.
RSV infected human bronchial epithelial cell model negative control group cells, RSV infection after first generation of cells and stable RSV infection model group cells membrane protein, Western-blot method to detect the protein expression of CFTR after RSV infection, the film exposure results using Quantity one gray scale scanning software to deal with the data, its expression intensity is CF TR gray value / beta -actin gray value was expressed, repeated 3 times, statistical results, drawing histogram. Experimental results showed that compared with negative control group cells, the expression of CFTR expression in the first generation cells after RSV infection and the model group with stable RSV infection was obviously reduced, and the results were significantly different, *P0.05.
RSV infected human bronchial epithelial cell model negative control group cells, RSV infection after first generation of cells and stable RSV infection model group RNA, reverse transcription cDNA, RT-PCR method to detect the mRNA expression of CFTR in RSV infected cells, mRNA expression =2 (delta delta delta). Results showed that relative to negative control group cells, RSV infection The mRNA expression of CFTR in the first generation cells and the stable RSV infection model group was significantly lower than that in the control group, and the results showed significant difference. *P0.05.
3) changes in CFTR function after human bronchial epithelial cells infected with RSV.
The whole cell recording method recorded the changes in CFTR Cl- on the cells of the RSV infected human bronchial epithelial cell model negative control group and the model group. The results showed that the CFTR Cl- current density increased and had a concentration dependence under the effect of RSV infection, and the.CFTR Cl- blocker glibenclamide could block the current in 10-3mM as the maximum half effective dose of.CFTR Cl- blocker glibenclamide. Non CFTR Cl- blocker DIDS could not block the intracellular Cl- fluorescence intensity change of 3 groups of cells detected by the current.MQAE fluorescent dye method. The results showed that the change rate of Cl- fluorescence intensity in the cells decreased significantly after RSV continuous infection, indicating that after RSV infection, the modulation function of the channel protein regulating the Cl- channel was suppressed. The Cl- outflow decreased and the intracellular Cl- concentration decreased.
4) changes in epithelial repair function after human bronchial epithelial cells infected with RSV.
The results showed that the absolute value of the slope of the negative control group of RSV infection was larger and the repair speed was faster. The absolute value of the slope of the RSV stable infection model group was smaller and the repair speed was slower. It was suggested that after RSV infection HBECs, the ability of HBECs to proliferate, transfer and repair the injury was lower than that before RSV infection, and the repair function of the HBECs was reshaped, and it might form the reverse. Recurrent airway inflammation and airway hyperresponsiveness.
Conclusion:
After RSV infection, the expression and function of the transmembrane protein CFTR were obviously inhibited, and the regulation of the functional homeostasis of the airway epithelial cells was weakened by CFTR, which resulted in the instability of the local microenvironment of the airway, which would affect the secretion of airway mucus outward from the gland tube and form the airway mucus suppository, leading to the increase of airway resistance and the induction of airway high reaction. The occurrence and development of the disease.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R725.1

【参考文献】