热休克（预）处理对过氧化氢诱导细胞损伤的保护作用
发布时间：2019-01-01 10:39
【摘要】： [目的]本研究旨在细胞水平，用过氧化氢模拟电离辐射来探讨热休克蛋白作为辐射防护剂的可行性，包括热休克蛋白是否能提高细胞活力，是否能减少细胞基因突变率两个方面。由于热休克(预)处理是诱导热休克蛋白过表达最简单有效的物理方法，因此本实验重点研究V79细胞(成体)和NIH3T3细胞(胚胎)的最佳热休克(预)处理条件，研究热克预处理对过氧化氢诱导产生细胞活力损伤的影响以及热休克(预)处理对过氧化氢诱导细胞发生基因突变的影响，为本课题今后进一步的研究工作打下基础。 [方法]主要包括三个方面： 1．用正交试验法分析了V79细胞、NIH3T3细胞最佳的热休克(预)处理条件(细胞存活率采用MTT法检测)。 2．分别采用克隆法(细胞克隆形成率)和台盼蓝染色法(细胞染色率)分析细胞的活力(增殖能力和存活能力)，从而分析热休克(预)处理对过氧化氢诱导细胞产生活力损伤的影响。 3．用6-巯基鸟嘌呤(6-TG)筛选HPRT基因突变细胞并形成细胞克隆，应用Giemsa染色法计数细胞克隆数，从而分析热休克(预)处理对过氧化氢诱导细胞发生基因突变的影响。 [结果] 1．对于V79细胞来说，热休克(预)处理温度42℃，时间1h，热休克后恢复4-8小时的条件为最佳。对于NIH3T3细胞来说，热休克(预)处理温度在41℃，热休克处理时间0.5h-2h，热休克后恢复时间2-8小时均可。 2．对于V79细胞来说，最佳的热休克(预)处理条件下最大可以提高存活率达20％，对于NIH3T3细胞来说，最佳的热休克(预)处理条件下最大可以提高存活率达40％。 3．低细胞密度下，热休克(预)处理对低浓度过氧化氢(0.06mmol／L-0.2mmol／L)诱导产生的细胞克隆能力损伤(不发生细胞凋亡)没有明显的保护作用(p＞0.05)，对中高浓度过氧化氢(0.5mmol／L-4mmol／L)诱导产生的细胞存活能力的损伤(发生细胞凋亡)亦没有明显的保护作用(p＞0.05)。 4．高细胞密度下，42℃以上，1.0h的热处理会诱导V79细胞活力产生明显的损伤(P＜0.05)。而42℃及以下，1.0h热处理不会诱导V79细胞活力产生明显的损伤(P＞0.05)，且42℃，1.0h热处理对中高浓度过氧化氢(0.5 mmol／L-4mmol／L)诱导产生的细胞克隆能力损伤和细胞存活能力的损伤都具有明显的保护作用(P＜0.01)。 5．42℃以上，1.0h的热处理会诱导V79细胞产生明显的HPRT基因突变(p＜0.01)，细胞存活率明显降低。42℃及以下，，1.0h的热处理不会诱导产生明显的HPRT基因突变(p＞0.05)。且42℃，1.0h热休克(预)处理可以明显降低过氧化氢诱导V79产生的HPRT基因突变率，起到明显的基因保护作用(P＜0.05)。并且在过氧化氢浓度为＜2mmol／L范围内(中浓度范围内)，保护效应比过氧化氢浓度＞2mmol／L(高浓度范围内)更明显。 [结论] 1．42℃及以下的热处理不会诱导高密度V79细胞产生明显的细胞活力损伤。热休克(预)处理对中高浓度过氧化氢诱导高密度V79细胞产生的细胞活力损伤均具有保护作用，并且42℃，1h的热休克(预)处理条件能较好地起到保护作用。 2．42℃及以下的热处理不会诱导V79细胞产生明显的基因突变。热休克(预)处理能保证较高细胞存活率的同时，减少细胞的基因突变率，并且42℃，1h的热休克(预)处理条件能较好地起到保护作用。 综上所述，热休克(预)处理对过氧化氢诱导的细胞损伤具有保护效应，具有作为辐射防护剂的基本特性之一。
[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
本文编号：2397440
[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
本文编号：2397440
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