斛芪浸膏对小鼠胸腺细胞辐射损伤的保护作用
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摘要
研究目的：通过实验探讨中药复方制剂斛芪浸膏是否具有维持60Co辐照后的离体小鼠胸腺细胞的细胞活力，以及抑制细胞凋亡和减轻电离辐射所致细胞氧化损伤的作用。
研究方法：取4-8周健康雄性昆明小鼠，分离胸腺淋巴细胞作原代培养，孵育4小时后，分为对照组(CG)、辐照组(IG)、辐照加低剂量斛芪浸膏组(ILHEG)、辐照加中剂量斛芪浸膏组(IMHEG)及辐照加高剂量斛芪浸膏组(IHHEG)。对照组及辐照组添加不含斛芪浸膏药物的RPMI1640培养液，辐照加低剂量斛芪浸膏组、辐照加中剂量斛芪浸膏组、辐照加高剂量斛芪浸膏组分别添加等量相应浓度的斛芪浸膏溶液，作用10小时。除对照组外，其余各组均采用60CO γ射线5Gy单次辐照。（1）于辐照后12、24、36、48小时，分别用四唑盐比色法检测各组细胞吸光度A值。辐照后第10、24小时，分别用显微镜观察细胞形态变化。辐照后10小时，用PI和Annexin-V双标记，流式细胞仪检测各组细胞早期凋亡比例；辐照后24小时，提取各组细胞总DNA，用琼脂糖凝胶电泳法检测细胞凋亡，观察DNA损伤情况。（2）辐照后30分钟内分别用流式细胞仪及超氧化物检测试剂盒检测各组细胞内活性氧荧光强度及超氧化物含量。
结果：（1）辐照后12-24小时，辐照组细胞活力呈下降趋势，对照组细胞活力水平高于辐照组，辐照加低、中、高斛芪浸膏各组细胞活力均呈现上升趋势，24小时以后，各组细胞活力均开始下降，但辐照加低、中、高剂量斛芪浸膏各组细胞活力水平明显高于对照组及辐照组，且呈现剂量依赖性，辐照后36小时，辐照加低、中、高剂量组与辐照组比较，细胞活力差异有统计学意义（p＜0.05）。辐照后第10、24小时，显微镜下观察，辐照加斛芪浸膏各组细胞变性、固缩变小等细胞坏死、凋亡等表现与辐照组比较相对较轻。辐照后10小时，辐照加低、中、高剂量斛芪浸膏各组细胞早期凋亡比例均低于辐照组，其中高剂量组与辐照组比较，差异有统计学意义（p＜0.05）。辐照后24小时，辐照组及辐照加低、中、高剂量斛芪浸膏各组细胞总DNA经电泳后均出现了典型的DNA阶梯状断裂条带，但辐照加低、中、高剂量斛芪浸膏各组DNA损伤相对较轻，辐照加高剂量斛芪浸膏组作用最明显。（2）与辐照组比较，辐照加中、高剂量斛芪浸膏组细胞内活性氧强度（P＜0.01）及超氧化物水平（P＜0.01）有不同程度下降，差异有统计学意义。辐照加中剂量斛芪浸膏组与辐照加高剂量斛芪浸膏组作用比较无明显差别（P＞0.05）。
结论:斛芪浸膏对60Co辐照后早期小鼠胸腺细胞的细胞活力有保护作用，且呈现剂量依赖性的特点。同时斛芪浸膏能降低细胞的早期及晚期凋亡水平，并能在一定程度上降低电离辐射后早期小鼠胸腺细胞内活性氧水平，具有减轻电离辐射所致的氧化损伤的作用。
Objective: To explore the influence of Chinese herb medicine Huqiextractum on viability and apoptosis in mouse thymocytes against60Co and todetect whether Huqi extractum has effect to alleviate the oxidative injuryinduced by60Co radiation in mice thymocytes.
Methods: Thymocytes which were isolated from4-8week health maleKunming mice were incubated for4hours and then were divided into controlgroup (CG), irradiation group (IG), irradiation plus low-dose Huqi extractumgroup (ILHEG), irradiation plus medium-dose Huqi extractum group(IMHEG) and irradiation plus high-dose Huqi extractum group(IHHEG).Other three experimental groups were added same volume Huqiextractum solution in accord with their respective groups except for controland irradiation groups. In addition to control, other groups were exposedradiation with a single dose of5Gy γ-rays delivered by60CO sources afterincubation for10hours.(1).At the12th,24th,36th and48th hour afterirradiation, changes of thymocytes viability of each group was detected withAbsorbance A and measured by MTT colorimetric assay. Changes ofthymocytes morphology were observed by light microscope at the10th and24th hour after irradiation. And thymocytes apoptosis were assessd by PI andAnnexin-V staining with flow cytometry (FCM) at the10th hour and DNAdamage were measured by agarose gel electrophoresis at the24th hour.(2).After irradiation intra-cellular ROS intensity and contents of superoxide ineach group was measured by FCS and superoxide assay kit within30minutes.
Results:(1).Viability in control and irradiation group were in declineduring the12th to24th hour after irradiation, while viability in irradiationplus low-dose、 medium-does and high-does groups all went up. Thethymocytes viability level in CG was higher than the IG. After the24th hourthymocytes viability in all groups showed an downward trend, but cellviability in irradiation plus low-dose、medium-does and high-does Huqiextractum groups were higher than the CG and IG. The effect of Huqiextractum showed a does-dependent manner. At the36th hour the differenceof cell viability among groups was statistically significant (p＜0.05). Both apoptotic and necrotic morphological changes were observed under the lightmicroscope among experimental groups after irradiation, especially in IG. Atthe10th hour after irradiation the early apoptosis rate in ILHEG、IMHEG andIHHEG were lower than irradiation group, difference between CG andIHHEG was statistically significant (p＜0.05). At the24th hour afterirradiation clear DNA ladder fragments were found in electrophoresis in allgroups except for control. But ILHEG、IMHEG and IHHEG were lessapparent than IG, especially in IHHEG.(2). intra-cellular ROS intensity andcontents of superoxide in ILHEG、IMHEG and IHHEG all descend atdifferent level compare to IG. Difference between IG and IMHEG, IG andIHHEG were statistically significant（p＜0.01）.
Conclusions: Chinese herb medicine Huqi extractum is helpful tomaintain viability and reduce apoptosis of mouse thymocytes exposed to the60CO radiation. At the same time Huqi extractum is useful to lower theintra-cellular ROS and reduce the oxidative injury induced by irradiation.
研究方法：取4-8周健康雄性昆明小鼠，分离胸腺淋巴细胞作原代培养，孵育4小时后，分为对照组(CG)、辐照组(IG)、辐照加低剂量斛芪浸膏组(ILHEG)、辐照加中剂量斛芪浸膏组(IMHEG)及辐照加高剂量斛芪浸膏组(IHHEG)。对照组及辐照组添加不含斛芪浸膏药物的RPMI1640培养液，辐照加低剂量斛芪浸膏组、辐照加中剂量斛芪浸膏组、辐照加高剂量斛芪浸膏组分别添加等量相应浓度的斛芪浸膏溶液，作用10小时。除对照组外，其余各组均采用60CO γ射线5Gy单次辐照。（1）于辐照后12、24、36、48小时，分别用四唑盐比色法检测各组细胞吸光度A值。辐照后第10、24小时，分别用显微镜观察细胞形态变化。辐照后10小时，用PI和Annexin-V双标记，流式细胞仪检测各组细胞早期凋亡比例；辐照后24小时，提取各组细胞总DNA，用琼脂糖凝胶电泳法检测细胞凋亡，观察DNA损伤情况。（2）辐照后30分钟内分别用流式细胞仪及超氧化物检测试剂盒检测各组细胞内活性氧荧光强度及超氧化物含量。
结果：（1）辐照后12-24小时，辐照组细胞活力呈下降趋势，对照组细胞活力水平高于辐照组，辐照加低、中、高斛芪浸膏各组细胞活力均呈现上升趋势，24小时以后，各组细胞活力均开始下降，但辐照加低、中、高剂量斛芪浸膏各组细胞活力水平明显高于对照组及辐照组，且呈现剂量依赖性，辐照后36小时，辐照加低、中、高剂量组与辐照组比较，细胞活力差异有统计学意义（p＜0.05）。辐照后第10、24小时，显微镜下观察，辐照加斛芪浸膏各组细胞变性、固缩变小等细胞坏死、凋亡等表现与辐照组比较相对较轻。辐照后10小时，辐照加低、中、高剂量斛芪浸膏各组细胞早期凋亡比例均低于辐照组，其中高剂量组与辐照组比较，差异有统计学意义（p＜0.05）。辐照后24小时，辐照组及辐照加低、中、高剂量斛芪浸膏各组细胞总DNA经电泳后均出现了典型的DNA阶梯状断裂条带，但辐照加低、中、高剂量斛芪浸膏各组DNA损伤相对较轻，辐照加高剂量斛芪浸膏组作用最明显。（2）与辐照组比较，辐照加中、高剂量斛芪浸膏组细胞内活性氧强度（P＜0.01）及超氧化物水平（P＜0.01）有不同程度下降，差异有统计学意义。辐照加中剂量斛芪浸膏组与辐照加高剂量斛芪浸膏组作用比较无明显差别（P＞0.05）。
结论:斛芪浸膏对60Co辐照后早期小鼠胸腺细胞的细胞活力有保护作用，且呈现剂量依赖性的特点。同时斛芪浸膏能降低细胞的早期及晚期凋亡水平，并能在一定程度上降低电离辐射后早期小鼠胸腺细胞内活性氧水平，具有减轻电离辐射所致的氧化损伤的作用。
Objective: To explore the influence of Chinese herb medicine Huqiextractum on viability and apoptosis in mouse thymocytes against60Co and todetect whether Huqi extractum has effect to alleviate the oxidative injuryinduced by60Co radiation in mice thymocytes.
Methods: Thymocytes which were isolated from4-8week health maleKunming mice were incubated for4hours and then were divided into controlgroup (CG), irradiation group (IG), irradiation plus low-dose Huqi extractumgroup (ILHEG), irradiation plus medium-dose Huqi extractum group(IMHEG) and irradiation plus high-dose Huqi extractum group(IHHEG).Other three experimental groups were added same volume Huqiextractum solution in accord with their respective groups except for controland irradiation groups. In addition to control, other groups were exposedradiation with a single dose of5Gy γ-rays delivered by60CO sources afterincubation for10hours.(1).At the12th,24th,36th and48th hour afterirradiation, changes of thymocytes viability of each group was detected withAbsorbance A and measured by MTT colorimetric assay. Changes ofthymocytes morphology were observed by light microscope at the10th and24th hour after irradiation. And thymocytes apoptosis were assessd by PI andAnnexin-V staining with flow cytometry (FCM) at the10th hour and DNAdamage were measured by agarose gel electrophoresis at the24th hour.(2).After irradiation intra-cellular ROS intensity and contents of superoxide ineach group was measured by FCS and superoxide assay kit within30minutes.
Results:(1).Viability in control and irradiation group were in declineduring the12th to24th hour after irradiation, while viability in irradiationplus low-dose、 medium-does and high-does groups all went up. Thethymocytes viability level in CG was higher than the IG. After the24th hourthymocytes viability in all groups showed an downward trend, but cellviability in irradiation plus low-dose、medium-does and high-does Huqiextractum groups were higher than the CG and IG. The effect of Huqiextractum showed a does-dependent manner. At the36th hour the differenceof cell viability among groups was statistically significant (p＜0.05). Both apoptotic and necrotic morphological changes were observed under the lightmicroscope among experimental groups after irradiation, especially in IG. Atthe10th hour after irradiation the early apoptosis rate in ILHEG、IMHEG andIHHEG were lower than irradiation group, difference between CG andIHHEG was statistically significant (p＜0.05). At the24th hour afterirradiation clear DNA ladder fragments were found in electrophoresis in allgroups except for control. But ILHEG、IMHEG and IHHEG were lessapparent than IG, especially in IHHEG.(2). intra-cellular ROS intensity andcontents of superoxide in ILHEG、IMHEG and IHHEG all descend atdifferent level compare to IG. Difference between IG and IMHEG, IG andIHHEG were statistically significant（p＜0.01）.
Conclusions: Chinese herb medicine Huqi extractum is helpful tomaintain viability and reduce apoptosis of mouse thymocytes exposed to the60CO radiation. At the same time Huqi extractum is useful to lower theintra-cellular ROS and reduce the oxidative injury induced by irradiation.
引文
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