【摘要】： [目的]本研究旨在細(xì)胞水平，用過(guò)氧化氫模擬電離輻射來(lái)探討熱休克蛋白作為輻射防護(hù)劑的可行性，包括熱休克蛋白是否能提高細(xì)胞活力，是否能減少細(xì)胞基因突變率兩個(gè)方面。由于熱休克(預(yù))處理是誘導(dǎo)熱休克蛋白過(guò)表達(dá)最簡(jiǎn)單有效的物理方法，因此本實(shí)驗(yàn)重點(diǎn)研究V79細(xì)胞(成體)和NIH3T3細(xì)胞(胚胎)的最佳熱休克(預(yù))處理?xiàng)l件，研究熱克預(yù)處理對(duì)過(guò)氧化氫誘導(dǎo)產(chǎn)生細(xì)胞活力損傷的影響以及熱休克(預(yù))處理對(duì)過(guò)氧化氫誘導(dǎo)細(xì)胞發(fā)生基因突變的影響，為本課題今后進(jìn)一步的研究工作打下基礎(chǔ)。 [方法]主要包括三個(gè)方面： 1．用正交試驗(yàn)法分析了V79細(xì)胞、NIH3T3細(xì)胞最佳的熱休克(預(yù))處理?xiàng)l件(細(xì)胞存活率采用MTT法檢測(cè))。 2．分別采用克隆法(細(xì)胞克隆形成率)和臺(tái)盼藍(lán)染色法(細(xì)胞染色率)分析細(xì)胞的活力(增殖能力和存活能力)，從而分析熱休克(預(yù))處理對(duì)過(guò)氧化氫誘導(dǎo)細(xì)胞產(chǎn)生活力損傷的影響。 3．用6-巰基鳥(niǎo)嘌呤(6-TG)篩選HPRT基因突變細(xì)胞并形成細(xì)胞克隆，應(yīng)用Giemsa染色法計(jì)數(shù)細(xì)胞克隆數(shù)，從而分析熱休克(預(yù))處理對(duì)過(guò)氧化氫誘導(dǎo)細(xì)胞發(fā)生基因突變的影響。 [結(jié)果] 1．對(duì)于V79細(xì)胞來(lái)說(shuō)，熱休克(預(yù))處理溫度42℃，時(shí)間1h，熱休克后恢復(fù)4-8小時(shí)的條件為最佳。對(duì)于NIH3T3細(xì)胞來(lái)說(shuō)，熱休克(預(yù))處理溫度在41℃，熱休克處理時(shí)間0.5h-2h，熱休克后恢復(fù)時(shí)間2-8小時(shí)均可。 2．對(duì)于V79細(xì)胞來(lái)說(shuō)，最佳的熱休克(預(yù))處理?xiàng)l件下最大可以提高存活率達(dá)20％，對(duì)于NIH3T3細(xì)胞來(lái)說(shuō)，最佳的熱休克(預(yù))處理?xiàng)l件下最大可以提高存活率達(dá)40％。 3．低細(xì)胞密度下，熱休克(預(yù))處理對(duì)低濃度過(guò)氧化氫(0.06mmol／L-0.2mmol／L)誘導(dǎo)產(chǎn)生的細(xì)胞克隆能力損傷(不發(fā)生細(xì)胞凋亡)沒(méi)有明顯的保護(hù)作用(p＞0.05)，對(duì)中高濃度過(guò)氧化氫(0.5mmol／L-4mmol／L)誘導(dǎo)產(chǎn)生的細(xì)胞存活能力的損傷(發(fā)生細(xì)胞凋亡)亦沒(méi)有明顯的保護(hù)作用(p＞0.05)。 4．高細(xì)胞密度下，42℃以上，1.0h的熱處理會(huì)誘導(dǎo)V79細(xì)胞活力產(chǎn)生明顯的損傷(P＜0.05)。而42℃及以下，1.0h熱處理不會(huì)誘導(dǎo)V79細(xì)胞活力產(chǎn)生明顯的損傷(P＞0.05)，且42℃，1.0h熱處理對(duì)中高濃度過(guò)氧化氫(0.5 mmol／L-4mmol／L)誘導(dǎo)產(chǎn)生的細(xì)胞克隆能力損傷和細(xì)胞存活能力的損傷都具有明顯的保護(hù)作用(P＜0.01)。 5．42℃以上，1.0h的熱處理會(huì)誘導(dǎo)V79細(xì)胞產(chǎn)生明顯的HPRT基因突變(p＜0.01)，細(xì)胞存活率明顯降低。42℃及以下，，1.0h的熱處理不會(huì)誘導(dǎo)產(chǎn)生明顯的HPRT基因突變(p＞0.05)。且42℃，1.0h熱休克(預(yù))處理可以明顯降低過(guò)氧化氫誘導(dǎo)V79產(chǎn)生的HPRT基因突變率，起到明顯的基因保護(hù)作用(P＜0.05)。并且在過(guò)氧化氫濃度為＜2mmol／L范圍內(nèi)(中濃度范圍內(nèi))，保護(hù)效應(yīng)比過(guò)氧化氫濃度＞2mmol／L(高濃度范圍內(nèi))更明顯。 [結(jié)論] 1．42℃及以下的熱處理不會(huì)誘導(dǎo)高密度V79細(xì)胞產(chǎn)生明顯的細(xì)胞活力損傷。熱休克(預(yù))處理對(duì)中高濃度過(guò)氧化氫誘導(dǎo)高密度V79細(xì)胞產(chǎn)生的細(xì)胞活力損傷均具有保護(hù)作用，并且42℃，1h的熱休克(預(yù))處理?xiàng)l件能較好地起到保護(hù)作用。 2．42℃及以下的熱處理不會(huì)誘導(dǎo)V79細(xì)胞產(chǎn)生明顯的基因突變。熱休克(預(yù))處理能保證較高細(xì)胞存活率的同時(shí)，減少細(xì)胞的基因突變率，并且42℃，1h的熱休克(預(yù))處理?xiàng)l件能較好地起到保護(hù)作用。 綜上所述，熱休克(預(yù))處理對(duì)過(guò)氧化氫誘導(dǎo)的細(xì)胞損傷具有保護(hù)效應(yīng)，具有作為輻射防護(hù)劑的基本特性之一。
[Abstract]:[Objective] The purpose of this study was to study the feasibility of heat shock protein as a radiation protective agent by using hydrogen peroxide to simulate ionizing radiation. Since the heat shock (pre-) treatment is the most simple and effective physical method for inducing the overexpression of the heat shock protein, the experiment focuses on the optimal heat shock (pre-) treatment conditions of the V79 cell (body) and the NIH3T3 cell (embryo), The effect of thermal shock pretreatment on the cell viability injury induced by hydrogen peroxide and the effect of heat shock (pre-treatment) on the gene mutation of hydrogen peroxide-induced cell were studied.[Method] mainly The optimal heat shock (pre) treatment conditions of V79 cells and NIH3T3 cells were analyzed by orthogonal test. The cell viability (cell viability and viability) was analyzed by the cloning method (cell clone formation rate) and the trypan blue staining method (cell staining rate), respectively, to analyze the heat shock (pre-). The effects of treatment on the viability of the cells induced by hydrogen peroxide were studied. 3. The HPRT gene mutation cells were screened by 6-(6-TG) and the cell clones were formed. The number of cell clones was counted by Giemsa staining. heat shock (Pre-) Effect of treatment on the gene mutation of the hydrogen peroxide-inducing cell.[Result] 1. For V79 In the case of cells, the conditions for heat shock (pre-treatment) at 42. degree. C., time 1h, and recovery of 4-8 hours after heat shock were the best. For NIH3T3 cells, heat shock (The pre-treatment temperature was at 41 鈩

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