致死剂量γ射线照射小鼠脾淋巴细胞凋亡特征及与Bax和Bcl-X_L表达的关系

Apoptotic characteristics of spleen lymphocytes in mice irradiated by lethal dose and its relationship to the expression of Bax and Bcl-X_L proteins

目的 观察致死剂量 γ射线照射后小鼠脾淋巴细胞凋亡特征及其与 Bax和 Bcl XL 表达的关系。方法 清洁级 C5 7小鼠 2 2 5只 ,随机分为 0、6、9、12、15和 2 0 Gy6个组 ,经 γ射线全身一次照射 ,于照射前和照射后 6 h～ 2 8d活杀取材 ,用原位末端标记法 (TU NEL)和 Annexin V流式细胞术 (FCM)检测淋巴细胞凋亡和坏死情况 ,用碱性磷酸酶免疫组化技术检测 Bax和 Bcl XL 蛋白的表达。结果 1各剂量组小鼠外周血白细胞在照射后 6 h出现一过性升高 ,以后迅速下降。 6 Gy照射后 7d降至最低 ,至照射后 2 8d基本恢复到正常水平。 2不同剂量照射后 6 h脾淋巴细胞凋亡率达高峰 ;照射后 6～ 2 4 h,≤ 12 Gy照射后凋亡率随照射剂量的增加而升高 ,≥ 15 Gy照射后凋亡率下降。 3FCM检测结果证实 ,6～ 12 Gy照射后 6 h,脾淋巴细胞凋亡率呈现出量效关系 ,≥ 15 Gy照射后凋亡率下降 ,而坏死率明显升高。DNA凝胶电泳图谱分析支持上述结果。 4照射后 6 h,6 Gy照射后脾淋巴细胞 Bax蛋白表达增加 ,至 12 Gy达峰值 ,显示出量效关系 ;≥ 15 Gy未显示出量效关系 ;Bcl XL 蛋白随照射剂量增加而持续降低 ,在 6～ 12 Gy也显示出较好的量效关系。结论 6～ 12 Gy照射后脾淋巴细胞死亡方式以凋亡为主 ,≥ 15

Objective To observe the apoptotic characteristics of mouse spleen lymphocyte after lethal dose γirradiation and its relationship to the expression of Bax and BclX L proteins. Methods Two hundred and twentyfive secondgrade C57 mice were randomly divided into six groups of 0, 6, 9, 12, 15 and 20 Gy. They were sacrificed by dislocation and samples were taken on 128 days after whole body single γirradiation. Lymphocyte apoptosis and necrosis were analyzed by TdTmediated dUTP nick end labeling (TUNEL) and flow cytometry (FCM) techniques. The expression of Bax and BclX L proteins were estimated by immunohistochemical method. Results ①The number of peripheral white blood cells of mice increased temporarily at 6 hours after radiation, thereafter, began to decrease rapidly, which reached the minimum on day 7 and recovered normal level basically one month after 6 Gy γirradiation. ②Apoptotic rate of spleen lymphocytes increased significantly, peaking at 6 hours after radiation, which was found to have a doseresponse relationship during 624 hours after ≤12 Gy irradiation, but decreased after ≥15 Gy irradiation . ③It was confirmed by FCM that the apoptotic rate of spleen lymphocytes increased along with the elevation of radiation dose. However, the apoptotic rate began to decrease and the necrotic rate rose distinctively after ≥15 Gy irradiation. The analysis of DNA gel electrophoresis supported abovementioned results. ④The expression of Bax protein in spleen lymphocyte enhanced at 6 hours after 6 Gy γirradiation and peaked by 12 Gyirradiation, showing a dosedependent pattern, but which was not be found after ≥ 15 Gy γirradiation . On the other hand, the expression of BclX L protein reduced persistently with the increase of radiation dose, and also presented a better dosedependent effect after ≤12 Gy irradiation. Conclusion After 612 Gy γirradiation, the apoptosis is the major death way of spleen lymphocyte, while both necrosis and apoptosis are important death pathways after ≥15 Gy irradiation. Proapoptotic Bax and antiapoptotic BclX L play an important role in the apoptotic regulation of spleen lymphocytes induced by lethal dose radiation.