本文選題：RSV + 支氣管上皮細胞�。� 參考：《中南大學》2012年博士論文

【摘要】：目的： 呼吸道合胞病毒(Respiratory syncytial virus, RSV)感染是臨床最常見的,也是兒科最常遇到的呼吸道病原之一。支氣管上皮細胞在RSV感染后,其病理改變?nèi)Q于某些離子通道的功能活性及表達密度,其是否抑制了其囊性纖維化跨膜轉(zhuǎn)導調(diào)節(jié)因子(Cystic Fibrosis Transmembrane conductance Regulator, CFTR)的表達水平和功能。我們擬建立RSV感染離體支氣管上皮細胞模型,對RSV感染支氣管上皮細胞后CFTR的表達及功能進行研究。 方法： 1)建立RSV感染離體培養(yǎng)支氣管上皮細胞模型 將原RSV病毒懸液(TCID50=10-3.82/0.1ml)按1：100000的濃度稀釋,得RSV病毒感染人支氣管上皮細胞模型的初始病毒濃度,以此濃度的RSV病毒感染人支氣管上皮細胞制備離體RSV病毒感染人支氣管上皮細胞模型。RT-PCR法驗證模型中RSV存在以及其轉(zhuǎn)錄表達水平。 2)人支氣管上皮細胞感染RSV后CFTR的表達 取對數(shù)生長期RSV感染人支氣管上皮細胞模型中陰性對照組細胞、RSV感染后傳代第1代的細胞和可穩(wěn)定傳代的模型組細胞,通過免疫熒光的方法檢測RSV穩(wěn)定感染后細胞CFTR的蛋白水平定位及表達；Western-blot法檢測RSV穩(wěn)定感染后細胞CFTR的蛋白表達；qRT-PCR法檢測RSV穩(wěn)定感染后細胞CFTR的rnRNA表達水平。 3)人支氣管上皮細胞感染RSV后CFTR的功能變化 取對數(shù)生長期RSV穩(wěn)定感染人支氣管上皮細胞模型中陰性對照組細胞、RSV感染后傳代第1代細胞和穩(wěn)定傳代的模型組細胞,采用全細胞記錄的方法記錄RSV持續(xù)感染人支氣管上皮細胞模型陰性對照組和模型組細胞上CFTR C1-的變化。加入激動劑5μM的forskolin以后,激活cAMP誘導CFTR C1-電流,細胞被鉗制在-40mV,給予不同的電壓刺激,電壓自-100mV逐步增到+100mV,去極化電壓時程為200mS,步階為20mV,間隔為2S。去極化電壓結(jié)束后恢復至-40mV鉗制電壓。取各個電壓狀態(tài)下最大電流值,繪制Ⅰ-V關(guān)系曲線。用MQAE熒光染料法檢測3組細胞的細胞內(nèi)Cl-濃度,制備標準品,檢測標準品Cl-濃度,繪制Cl-濃度標準曲線,根據(jù)Cl-濃度標準曲線,計算出待測3組支氣管上皮細胞中Cl-濃度,同時MQAE熒光染料法檢測3組細胞的細胞內(nèi)Cl-濃度熒光強度變化率。 4)人支氣管上皮細胞感染RSV后上皮修復功能改變 制備RSV感染人支氣管上皮細胞的損傷模型后,用顯微視頻分析BI-2000圖像／免疫組化分析系統(tǒng)分別于機械損傷后0hrs,8hrs,12hrs,18hrs,24hrs測量缺損面積一次,收集所有時間點的缺損區(qū)域圖像,勾畫缺損區(qū)域的邊緣,以相關(guān)系數(shù)r來衡量兩者是否存在直線相關(guān)關(guān)系,繪制時間與修復面積的直線回歸方程：Y=a+bX,計算損傷修復指數(shù)(RI, repair index),RI值等于回歸方程中b的絕對值(RI=│b│),以此反映不同處理組之間不同的修復速度,直線的斜率為損傷修復指數(shù),斜率越大,損傷修復速度越快。 結(jié)果： 1)RSV感染離體培養(yǎng)支氣管上皮細胞模型的建立 RT-PCR法驗證模型中RSV存在以及其轉(zhuǎn)錄表達水平,發(fā)現(xiàn)在RSV感染后的RSV感染人支氣管呼吸道細胞模型中,最初幾代的RSV的表達水平是逐步增高的,然后維持在較穩(wěn)定水平,但是從細胞的形態(tài)學上來觀察,并沒有引起肉眼可見的細胞病變,同時也未被人支氣管上皮細胞所清除。表示RSV穩(wěn)定感染離體培養(yǎng)支氣管上皮細胞模型建立成功。 2)人支氣管上皮細胞感染RSV后CFTR的表達的改變 取對數(shù)生長期RSV穩(wěn)定感染人支氣管上皮細胞模型中陰性對照組細胞、RSV穩(wěn)定感染后傳代第1代細胞和穩(wěn)定傳代的模型組細胞,通過免疫熒光的方法檢測RSV感染細胞CFTR的蛋白水平定位及表達,并隨機選取視野,計數(shù)熒光染色陽性細胞的百分率。結(jié)果顯示在RSV感染人支氣管上皮細胞模型陰性對照組細胞的細胞膜及胞漿中可見較多的綠色染色的CFTR,并且熒光染色陽性細胞的百分率較高；在RSV穩(wěn)定感染模型傳代第1代細胞就發(fā)現(xiàn)其細胞的細胞膜及胞漿中CFTR明顯減少,并且熒光染色呈陽性細胞的百分率明顯減少；而在RSV穩(wěn)定感染模型組細胞中細胞膜及胞漿中CFTR表達較陰性對照組細胞明顯減少,較RSV感染模型第1代細胞也有略有減少,并且熒光染色陽性細胞的百分率也略有減少。推測RSV感染細胞后,可破壞氣道上皮結(jié)構(gòu)的完整性,致使局部氣道微環(huán)境呈失穩(wěn)態(tài)的狀態(tài),使得氣道功能失調(diào),改變了氣道上皮應激應答機制,導致氣道的功能和結(jié)構(gòu)重塑,形成慢性氣道炎癥和氣道病理改變。 分別提取RSV感染人支氣管上皮細胞模型陰性對照組細胞、RSV感染后傳代第1代細胞和穩(wěn)定的RSV感染模型組細胞的膜蛋白,Western-blot法檢測RSV感染后細胞CFTR的蛋白表達,膠片曝光結(jié)果使用Quantity one灰度掃描軟件處理數(shù)據(jù),其表達強度用CFTR灰度值/β-actin灰度值表示,重復3次,統(tǒng)計結(jié)果,繪制直方圖。實驗結(jié)果顯示,相對于陰性對照組細胞,RSV感染后第1代細胞和穩(wěn)定RSV持續(xù)感染的模型組細胞CFTR表達明顯降低,結(jié)果具有明顯差異,*P0.05. 分別提取RSV感染人支氣管上皮細胞模型陰性對照組細胞、RSV感染后傳代第1代細胞和穩(wěn)定RSV感染模型組細胞的RNA,逆轉(zhuǎn)錄cDNA, RT-PCR法檢測RSV感染后細胞CFTR的mRNA表達水平,結(jié)果mRNA表達=2(_ΔΔτ)。結(jié)果顯示,相對于陰性對照組細胞,RSV感染后第1代細胞和穩(wěn)定RSV感染模型組細胞CFTR的mRNA表達明顯降低,結(jié)果具有明顯差異,*P0.05. 3)人支氣管上皮細胞感染RSV后CFTR的功能變化 全細胞記錄的方法記錄RSV感染人支氣管上皮細胞模型陰性對照組和模型組細胞上CFTR Cl-的變化。結(jié)果顯示,在RSV感染的作用下,CFTR Cl-電流密度增加,并具有濃度依賴性,在10-3mM為最大半數(shù)有效劑量。CFTR Cl-阻斷劑glibenclamide能阻斷該電流,而非CFTR Cl-阻斷劑DIDS不能阻斷該電流。MQAE熒光染料法檢測3組細胞的細胞內(nèi)Cl-熒光強度變化率,結(jié)果顯示RSV持續(xù)感染后,細胞內(nèi)Cl-熒光強度變化率的變化相對于陰性對照組明顯減少,表示RSV感染后,調(diào)節(jié)Cl-通道的通道蛋白調(diào)節(jié)功能被抑制,Cl-外流降低,細胞內(nèi)Cl-濃度變化率降低。 4)人支氣管上皮細胞感染RSV后上皮修復功能改變 結(jié)果顯示,RSV感染陰性對照組斜率絕對值較大,修復速度較快；RSV穩(wěn)定感染模型組斜率絕對值較小,修復速度較慢。提示RSV感染HBECs后,HBECs的自身增殖、遷移及損傷修復能力較RSV感染前,有所下降,其損傷修復功能發(fā)生重塑,有可能形成反復發(fā)作的氣道炎癥和氣道高反應傾向。 結(jié)論： 人支氣管上皮細胞在RSV感染后,跨膜蛋白CFTR的表達及功能被明顯抑制,減弱CFTR對氣道上皮細胞功能穩(wěn)態(tài)的調(diào)控,致使氣道局部微環(huán)境呈現(xiàn)失穩(wěn)態(tài)。進而影響氣道粘液從腺管向外分泌,形成氣道粘液栓,導致氣道阻力增高,誘導氣道高反應性疾病的發(fā)生發(fā)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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R725.1

【參考文獻】

相關(guān)期刊論文 前2條

1 秦曉群;向陽;劉持;譚宇蓉;屈飛;彭麗花;朱曉琳;秦嶺;;支氣管上皮細胞在氣道高反應中的作用(英文)[J];生理學報;2007年04期

2 黃升海,史百芬,李京培,閻懷士,王明麗,胡勇;病毒感染與成人哮喘發(fā)病關(guān)系的研究[J];中華結(jié)核和呼吸雜志;2000年09期

，

本文編號：2061853