本文選題：高滲 + 輻射敏感性��； 參考：《第二軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：滲透壓是機(jī)體內(nèi)環(huán)境的重要組成部分，滲透壓的平衡對(duì)于維持機(jī)體正常生理功能至關(guān)重要，因?yàn)闈B透壓是維持細(xì)胞體積的重要因素，同時(shí)還能影響機(jī)體的內(nèi)環(huán)境的離子濃度[4]。正常情況下，機(jī)體滲透壓維持在300mOsm左右，該滲透濃度稱為等滲，低于300mOsm稱為低滲，而高于300mOsm則稱為高滲。雖然等滲對(duì)維持機(jī)體生理功能非常重要，但是滲透壓失衡，尤其是發(fā)生高滲的暴露也是生物界中一種常見(jiàn)的現(xiàn)象[4]。例如許多的組織器官如腎小管，其周圍滲透壓可到達(dá)400mOsmol[5]；人員在高溫高濕條件下進(jìn)行高強(qiáng)度作業(yè)可發(fā)生高滲性的脫水[11]；還有自然界中許多生物生活在高鹽高滲的海洋環(huán)境中。這些都說(shuō)明滲透壓作為一種常見(jiàn)的環(huán)境因素與地球上的生命活動(dòng)息息相關(guān)。 電離輻射敏感性（radiation sensitivity）可用來(lái)描述輻射效應(yīng)發(fā)生的速度和嚴(yán)重程度。影響輻射敏感性的環(huán)境因素很多，比如氧含量[1]、溫度[2]、NO濃度[3]等等，這些因素的改變可直接或間接的影響電離輻射對(duì)機(jī)體的效應(yīng)，而隨著研究的深入，也有越來(lái)越多的環(huán)境因素被發(fā)現(xiàn)與電離輻射敏感性相關(guān)。但到目前為止，卻未見(jiàn)有關(guān)滲透壓與電離輻射敏感性關(guān)系的報(bào)道。那么，滲透壓作為一種常見(jiàn)的環(huán)境因素，它的改變是否也能影響電離輻射對(duì)機(jī)體的效應(yīng)呢？ 事實(shí)上，滲透壓的改變對(duì)細(xì)胞的影響已經(jīng)有了很多的研究。滲透壓的改變可導(dǎo)致細(xì)胞的體積發(fā)生變化，而細(xì)胞的許多信號(hào)分子位于細(xì)胞膜上，細(xì)胞的體積變化可擾動(dòng)膜上的信號(hào)分子，導(dǎo)致細(xì)胞內(nèi)下游的信號(hào)通路發(fā)生改變。這些信號(hào)通路中很多是與細(xì)胞凋亡、存活相關(guān)（圖1）。而電離輻射對(duì)細(xì)胞的最重要的效應(yīng)是引起凋亡，這就提示了細(xì)胞周圍滲透壓環(huán)境的改變與電離輻射敏感性之間存在相關(guān)分子基礎(chǔ)的可能性。2010年的《JBC》雜志上報(bào)道一個(gè)研究，發(fā)現(xiàn)阻滯細(xì)胞在凋亡過(guò)程中體積的減小可阻止凋亡的發(fā)生。這說(shuō)明細(xì)胞體積的改變與凋亡具有某種內(nèi)在聯(lián)系。而更進(jìn)一步的證據(jù)則來(lái)自于2008年《Apoptosis》上的一篇報(bào)道[9]。該研究發(fā)現(xiàn)提前高滲預(yù)處理細(xì)胞可減輕STS對(duì)細(xì)胞的毒性作用，降低細(xì)胞的凋亡率。這一發(fā)現(xiàn)更加提示我們滲透壓的改變影響電離輻射敏感性的可能性。 圖1：滲透壓改變對(duì)細(xì)胞信號(hào)通路的影響 基于以上的假設(shè)，我們開(kāi)展了滲透壓對(duì)于細(xì)胞電離輻射敏感性的研究。因?yàn)闈B透壓的改變最常見(jiàn)的是高滲的發(fā)生，因此本課題主要圍繞著高滲環(huán)境對(duì)細(xì)胞的電離輻射敏感性影響而開(kāi)展。本課題主體上分為兩個(gè)部分（圖2），一部分為效應(yīng)研究，另一部分為機(jī)制研究。效應(yīng)研究上通過(guò)檢測(cè)細(xì)胞活力、增殖、凋亡、DNA損傷等指標(biāo)系統(tǒng)評(píng)價(jià)高滲對(duì)于細(xì)胞電離輻射敏感性的影響，并在多種細(xì)胞中和不同溶質(zhì)類型的高滲溶液下確證這一效應(yīng)的普遍性。機(jī)制研究主要著眼于下游相關(guān)的信號(hào)通路和ROS水平的檢測(cè)。通過(guò)以上的研究，我們發(fā)現(xiàn)高滲溶液的預(yù)處理可增加細(xì)胞照后的活力，減輕照射對(duì)細(xì)胞造成的增殖抑制、凋亡、DNA損傷；高滲可引起細(xì)胞發(fā)生調(diào)節(jié)性體積（RVI）導(dǎo)致K+內(nèi)流增加，促進(jìn)Akt1磷酸化，促使IκB-α降解，同時(shí)還可減少細(xì)胞內(nèi)ROS含量。綜上，我們得出結(jié)論高滲可減輕細(xì)胞電離輻射敏感性，這種效應(yīng)是通過(guò)高滲引起細(xì)胞RVI，促進(jìn)K+內(nèi)流進(jìn)而激活A(yù)kt1，使得其下游的NF-κB釋放入核，最終促進(jìn)細(xì)胞的照后存活而實(shí)現(xiàn)的。 圖2：技術(shù)路線 研究?jī)?nèi)容： 1、L-O2細(xì)胞、HVUEC細(xì)胞、AHH-1細(xì)胞照射劑量與細(xì)胞活力關(guān)系曲線 （1）選取L-O2細(xì)胞，HUVEC細(xì)胞，AHH-1細(xì)胞通過(guò)檢測(cè)細(xì)胞活力和凋亡率繪制不同照射劑量下細(xì)胞輻射敏感性的關(guān)系曲線為下一步實(shí)驗(yàn)選取合適照射劑量提供依據(jù)。 2、高滲對(duì)細(xì)胞輻射敏感性的影響 （1）用CCK-8法檢測(cè)不同滲透壓NaCl溶液經(jīng)不同預(yù)處理時(shí)間對(duì)L-O2細(xì)胞輻射后細(xì)胞活力影響。 （2）CCK-8法檢測(cè)高滲NaCl溶液對(duì)不同細(xì)胞（L-O2細(xì)胞、HVUEC細(xì)胞）電離輻射后細(xì)胞活力影響。 （3）克隆形成實(shí)驗(yàn)檢測(cè)高滲NaCl溶液對(duì)L-O2細(xì)胞電離輻射后細(xì)胞增殖的影響。 （4）流式細(xì)胞儀和Hoechst33342檢測(cè)高滲NaCl溶液對(duì)AHH-1細(xì)胞電離輻射后凋亡的影響。 （5）彗星實(shí)驗(yàn)檢測(cè)高滲NaCl溶液對(duì)AHH-1細(xì)胞電離輻射后DNA損傷的影響。 （6）CCK-8法檢測(cè)不同溶質(zhì)類型高滲溶液對(duì)L-O2細(xì)胞照后細(xì)胞活力的影響。 3、高滲對(duì)Akt1激活的影響 （1）等滲/高滲NaCl溶液預(yù)處理AHH-1細(xì)胞，不同時(shí)間點(diǎn)提取蛋白，Western blot檢測(cè)其中Akt1/p-Akt表達(dá)的情況 4、高滲導(dǎo)致的細(xì)胞調(diào)節(jié)性體積增加（RVI）與Akt1激活的關(guān)系 （1）將AHH-1細(xì)胞用高滲NaCl溶液處理，記錄1h之內(nèi)其體積的變化，并繪制變化曲線。 （2）用陽(yáng)離子通道(HICC)阻滯劑氟芬那酸（FFA）作用于細(xì)胞，然后檢測(cè)細(xì)胞體積的變化和Akt1的激活情況。 （3）用FFA處理細(xì)胞，檢測(cè)其FFA是否能阻斷高滲減輕細(xì)胞輻射敏感性的作用。 5、K+內(nèi)流與細(xì)胞輻射敏感性之間的關(guān)系 （1）運(yùn)用K+通道阻滯劑格列苯脲和開(kāi)放劑吡那地爾作用于L-O2、AHH-1細(xì)胞，通過(guò)檢測(cè)照后細(xì)胞活力、凋亡、增殖、DNA損傷等情況研究K+內(nèi)流對(duì)細(xì)胞輻射敏感性的影響。 （2）檢測(cè)K+通道阻滯劑格列苯脲和開(kāi)放劑吡那地爾對(duì)AHH-1細(xì)胞Akt1激活的影響。 6、高滲對(duì)細(xì)胞內(nèi)ROS水平的影響 （1）用DHE探針檢測(cè)等滲/高滲以及格列苯脲對(duì)細(xì)胞照后ROS水平的影響。 實(shí)驗(yàn)結(jié)果： 1、L-O2細(xì)胞、HVUEC細(xì)胞、AHH-1細(xì)胞照射劑量與輻射敏感性關(guān)系 （1）通過(guò)檢測(cè)L-O2細(xì)胞、HUVEC細(xì)胞和AHH-1細(xì)胞不同照射劑量下細(xì)胞活力及凋亡的情況，繪制出照射劑量-活力關(guān)系曲線，選取出合適的實(shí)驗(yàn)照射劑量（L-O2細(xì)胞為8Gy；AHH-1細(xì)胞為6Gy） 2、高滲減輕細(xì)胞輻射敏感性 （1）用CCK-8法檢測(cè)不同滲透壓NaCl溶液經(jīng)不同預(yù)處理時(shí)間對(duì)L-O2細(xì)胞照射后細(xì)胞活力影響，發(fā)現(xiàn)短時(shí)間的高滲預(yù)處理（60min以內(nèi)）與等滲組比較，可增加照后細(xì)胞活力，該效應(yīng)在其他細(xì)胞（HUVEC、A549）中也存在。 （2）克隆形成實(shí)驗(yàn)發(fā)現(xiàn)高滲組比等滲組照后克隆形成率高，說(shuō)明高滲NaCl溶液預(yù)處理能減輕輻射對(duì)L-O2細(xì)胞的增殖抑制作用。 （3）流式細(xì)胞檢測(cè)和Hoechst33342檢測(cè)AHH-1細(xì)胞6Gy照后24小時(shí)凋亡情況，結(jié)果顯示高滲預(yù)處理相比等滲處理組，能減少細(xì)胞照射后的凋亡。 （6）彗星實(shí)驗(yàn)結(jié)果顯示：高滲處理可減輕細(xì)胞照射后DNA的損傷。 （7）CCK-8法檢測(cè)不同溶質(zhì)類型高滲對(duì)細(xì)胞輻射敏感性影響，結(jié)果顯示NaCl、KCl、蔗糖配制的高滲溶液均具有降低L-O2細(xì)胞輻射敏感性的作用。 3、高滲激活A(yù)kt1，增加p-Akt的表達(dá) （1）用高滲NaCl溶液（500mOsm）孵育AHH-1細(xì)胞，分別于0、10、20、30、60min時(shí)間點(diǎn)收集細(xì)胞，提取蛋白，最通過(guò)Wertern blot檢測(cè)Akt1、p-Akt（308）、IκB-α的表達(dá)差異。結(jié)果顯示p-Akt(308)在60min內(nèi)隨時(shí)間變化而呈現(xiàn)表達(dá)增加的趨勢(shì)，到60min時(shí)表達(dá)量最高；IκB-α則隨時(shí)間點(diǎn)增加呈表達(dá)降低的趨勢(shì)，60min時(shí)達(dá)到最低；Akt1表達(dá)量沒(méi)有明顯變化 4、高滲溶液通過(guò)細(xì)胞調(diào)節(jié)性體積增加（RVI）機(jī)制激活A(yù)kt1 （1）CASY-TT計(jì)數(shù)儀檢測(cè)AHH-1細(xì)胞在等滲/高滲下細(xì)胞體積的變化，繪制出變化曲線，顯示等滲NaCl處理?xiàng)l件下，AHH-1細(xì)胞體積沒(méi)有發(fā)生明顯的改變，而在高滲NaCl溶液中細(xì)胞體積立即減小，在1min時(shí)減到最小值，此后隨著時(shí)間增加體積逐漸恢復(fù)，到20min時(shí)恢復(fù)到接近等滲狀態(tài)下體積，證明高滲引起細(xì)胞RVI。 （2）檢測(cè)細(xì)胞體積變化與Akt1激活的關(guān)系，結(jié)果顯示隨時(shí)間推移p-Akt（308）表達(dá)呈增加趨勢(shì)。運(yùn)用HICC阻滯劑FFA，其阻滯高滲條件下AHH-1細(xì)胞RVI的過(guò)程，同時(shí)也阻滯了高滲激活A(yù)kt1的作用。 （3）用FFA合并高滲處理細(xì)胞，發(fā)現(xiàn)FFA可阻滯高滲減輕細(xì)胞輻射敏感性的作用。 5、RVI引起K+內(nèi)流進(jìn)而激活A(yù)kt1，從而減輕細(xì)胞電離輻射敏感性 （1）K+通道阻滯劑格列苯脲阻滯了K+通道，減輕細(xì)胞電離輻射敏感性，而K+通道開(kāi)放劑增加了電離輻射敏感性。 （2）K+通道阻滯劑格列苯脲可激活A(yù)HH-1細(xì)胞Akt1，，導(dǎo)致p-Akt(308)表達(dá)增多，而開(kāi)放劑吡那地爾則阻滯了Akt1的激活。 6、高滲減少細(xì)胞內(nèi)ROS水平 （1）用DHE探針檢測(cè)等滲/高滲以及格列苯脲對(duì)細(xì)胞照后ROS水平的影響。結(jié)果顯示等滲未照射組，細(xì)胞紅色熒光下可見(jiàn)較清晰細(xì)胞染色形態(tài)；照射組紅色熒光下則未能顯示細(xì)胞形態(tài)，且熒光多集中于細(xì)胞核，說(shuō)明照射后細(xì)胞ROS增多，但高滲與格列苯脲處理組較等滲組熒光強(qiáng)度小，說(shuō)明高滲預(yù)處理和格列苯脲預(yù)處理可使照射后L-O2細(xì)胞內(nèi)ROS減少。 討論分析： 穩(wěn)定的滲透壓是維持機(jī)體內(nèi)環(huán)境平衡的重要條件[1]。機(jī)體的滲透壓常維持在290mOsm~300mOsm之間，當(dāng)中很少發(fā)生較大的變動(dòng)。但是機(jī)體有一部分組織器官可常暴露于高滲條件之下，比如腎髓質(zhì)腎小管，因?yàn)橐S持濃縮尿液的作用，腎小管周圍的尿素、NaCl濃度隨著向髓質(zhì)的靠近而不斷增高[5]。因此，髓質(zhì)腎小管的內(nèi)皮細(xì)胞可長(zhǎng)期處于高滲之中。此外，一些哺乳動(dòng)物可因適應(yīng)環(huán)境的需要，體內(nèi)滲透壓并不一定維持在等滲，比如駱駝[6]其血漿的滲透壓可高達(dá)400mOsm。還有一些生物，尤其是海洋生物[7]，其長(zhǎng)期生活在深海環(huán)境，經(jīng)過(guò)億萬(wàn)年的進(jìn)化，已經(jīng)很好適應(yīng)了海水的高滲條件。從這些情況可見(jiàn)，高滲環(huán)境的暴露是生物界常見(jiàn)的現(xiàn)象，也是機(jī)體內(nèi)滲透壓變化的常見(jiàn)結(jié)果。 電離輻射敏感性的影響因素多年來(lái)已經(jīng)有很多研究，包括乏氧[1]、溫度[2]、NO濃度[3]等等。但滲透壓對(duì)于電離輻射的影響卻鮮有報(bào)道。事實(shí)上，高滲作為生物界一種常見(jiàn)的理化環(huán)境，其合并電離輻射的情況也時(shí)有發(fā)生，比如人員在勞動(dòng)作業(yè)時(shí)，受環(huán)境溫度以及勞動(dòng)強(qiáng)度影響可發(fā)生高滲性脫水[11]，導(dǎo)致體內(nèi)細(xì)胞外液滲透壓大于310mOsmol/kg，而這在核潛艇中人員作業(yè)時(shí)更易發(fā)生。在病理情況下，許多疾病患者如肝癌患者可因?yàn)楦闻K功能障礙從而使得體內(nèi)滲透壓產(chǎn)生失衡，這種情況下患者如合并放療會(huì)產(chǎn)生何種影響，也是臨床治療中需待解決的問(wèn)題。除此之外，電離輻射對(duì)可造成細(xì)胞DNA斷裂、細(xì)胞凋亡、周期阻滯。而高滲作為一種強(qiáng)烈的應(yīng)激對(duì)細(xì)胞的影響與電離輻射類似，因此，兩者之間是否有聯(lián)系，尤其是對(duì)細(xì)胞凋亡是否能相互影響或調(diào)控，也是值得深入探討的問(wèn)題。很有意義的一個(gè)工作是Tomohiro Numata等[9]2008年報(bào)道高滲預(yù)處理細(xì)胞，可減少十字孢堿引起的凋亡。這種效應(yīng)是通過(guò)陽(yáng)離子通道（HICC）激活實(shí)現(xiàn)的。該通道激活可以引起陽(yáng)離子的內(nèi)流（主要是K+離子），而內(nèi)流的陽(yáng)離子可能參與到細(xì)胞的凋亡調(diào)控中，激活一些促進(jìn)細(xì)胞存活的蛋白如Hsp70等[13]，從而能降低十字孢堿的細(xì)胞毒作用。這一研究結(jié)果為我們提供了很好的借鑒，既然高滲預(yù)處理能減少藥物對(duì)細(xì)胞的毒性作用，那是否也能減輕電離輻射對(duì)于細(xì)胞的損傷？因此，本研究以高滲對(duì)細(xì)胞輻射敏感性的影響為著眼點(diǎn)，系統(tǒng)（不同細(xì)胞、不同溶質(zhì)類型高滲溶液）研究?jī)烧咧g的相互關(guān)系及其機(jī)制。 研究結(jié)果顯示：高滲溶液短時(shí)間預(yù)處理細(xì)胞可減輕電離輻射對(duì)細(xì)胞的損傷。其作用機(jī)制是通過(guò)高滲引起細(xì)胞調(diào)節(jié)性細(xì)胞體積增加（RVI），進(jìn)而引起K+內(nèi)流，而內(nèi)流的K+激活了Akt1，IkB-α降解，Nf-kB釋放入核，轉(zhuǎn)錄相關(guān)基因，促進(jìn)細(xì)胞存活。同時(shí)，內(nèi)流的K+還可以減輕ROS水平，減少其對(duì)細(xì)胞的損傷。
[Abstract]:osmotic pressure is an important part of the internal environment of the organism , and the balance of osmotic pressure is essential for maintaining the normal physiological function of the organism , because the osmotic pressure is an important factor to maintain the cell volume , and can also influence the ionic concentration of the internal environment of the organism . Under normal conditions , the osmotic pressure of the organism is maintained around 300mOsm , which is called isosmotic , below 300mOsm is called hypotonic , while higher than 300mOsm is called hyperosmotic . Although isosmosis is very important to maintain physiological function of organism , osmotic pressure imbalance , especially the exposure to hyperosmosis , is a common phenomenon in the biological community . For example , many tissue organs , such as renal tubules , have a peripheral osmotic pressure of up to 400 mlt ; l & gt ; l & lt ; 1 & gt ; l & lt ; 1 & gt ; l ;
Under high temperature and high humidity conditions , the high - permeability dehydration of water can occur in 11 DEG C .
There are also many living organisms living in high - salt and high - permeability marine environments . These indicate that osmotic pressure is a common environmental factor that is closely linked to life activities on Earth .

Radiation sensitivity can be used to describe the speed and severity of radiation effect . The environmental factors affecting radiation sensitivity are many , such as oxygen content , temperature , NO concentration , and so on . The changes of these factors can directly or indirectly affect the effect of ionizing radiation on the organism . However , there are more and more environmental factors that have been found to be related to the sensitivity of ionizing radiation .

In fact , changes in osmotic pressure have made a lot of studies on the effect of osmotic pressure on cells . Changes in osmotic pressure can cause changes in the volume of cells . Many of these signaling molecules are located on the cell membranes . The most important effect of ionizing radiation on cells is the possibility of a correlation between changes in cell volume and susceptibility to ionizing radiation . This finding suggests that early hyperosmotic pretreatment cells can reduce the toxicity of STS to cells and decrease the apoptosis rate of cells . This finding suggests that the change of osmotic pressure affects the susceptibility to ionizing radiation .

Figure 1 : Effect of osmotic pressure changes on cell signaling pathways

Based on the above assumptions , we conducted a study on the susceptibility of osmotic pressure to ionizing radiation of cells . Because of the most common changes in osmotic pressure , the effects of hyperosmosis on the susceptibility to ionizing radiation of cells were studied .
In conclusion , it is concluded that hyperosmotic can reduce the susceptibility to ionizing radiation in cells , which can reduce the intracellular ROS content . In conclusion , we conclude that high permeability can reduce the susceptibility to ionizing radiation in cells . This effect is achieved by high permeability to induce cell RVI , promote K + influx and activate Akt1 , so that the downstream NF - 魏B is released into the nucleus and finally promotes the survival of the cells .

Figure 2 : Technical Route

Study Content :

1 , L - O2 cell , HVUEC cell , AHH - 1 cell irradiation dose and cell viability relationship curve

( 1 ) selecting L - O2 cells , HUVEC cells and AHH - 1 cells to draw the relation curve of cell radiation sensitivity at different irradiation doses by detecting cell viability and apoptosis rate , and selecting the appropriate irradiation dose according to the next step .

2 . Effect of Hypertonic on Cell Radiation Sensitivity

( 1 ) Using CCK - 8 method to detect the effect of different osmotic pressure NaCl solution on the cell viability after irradiation with different pretreatment time on L - O2 cells .

( 2 ) CCK - 8 method was used to detect the effect of NaCl solution on cell viability after ionizing radiation in different cells ( L - O2 cells , HVUEC cells ) .

( 3 ) The effect of high osmotic NaCl solution on the proliferation of L - O2 cells after ionizing radiation was investigated .

( 4 ) Flow cytometry and hoechst 33342 were used to detect the effect of NaCl solution on the apoptosis of AHH - 1 cells after ionizing radiation .

( 5 ) The effect of high osmotic NaCl solution on DNA damage after AHH - 1 cell ionizing radiation was detected by comet assay .

( 6 ) CCK - 8 method was used to detect the effect of different solute types on cell viability after irradiation with L - O2 .

3 . Effect of Hypertonic on Akt1 Activation

( 1 ) The AHH - 1 cells were pretreated with isosmotic / hyperosmotic NaCl solution , and protein was extracted at different time points . Western blot was used to detect the expression of Akt1 / p -

4 . Relationship between cell regulatory volume increase ( RVI ) and Akt1 activation induced by hyperosmosis

( 1 ) The AHH - 1 cells were treated with a highly osmotic NaCl solution and the volume changes were recorded within 1 h , and the curve was plotted .

( 2 ) A cationic channel ( HICC ) blocker flufenamic acid ( FFA ) was used to act on the cell , and then the change of cell volume and the activation of Akt1 were detected .

( 3 ) FFA was used to treat the cells and detect whether FFA could block the radiosensitivity of the cells .

5 . Relationship between K + influx and cell radiation sensitivity

( 1 ) The effect of K + influx on cell radiation sensitivity was studied by means of K + channel blocker and open dose of piraridil on L - O2 and AHH - 1 cells .

( 2 ) To examine the effect of K + channel blocker glyburide and open dose piazir on the activation of AHH - 1 cell Akt1 .

6 . Effect of Hypertonic on the Level of ROS in Cells

( 1 ) Using DHE probe to detect the effects of isosmotic / hyperosmotic and glimepiride on post - cell ROS level .

Experimental results :

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本文編號(hào)：2054897