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目的 研究X射线外照射对体外原代培养的大鼠血管平滑肌细胞 (VSMC)生长、增殖的影响及作用机制。方法 体外培养VSMC ,给予单次 8MVX射线照射 ,源皮距为 10 0cm ,剂量率 40 0cGy/min。按照射剂量分为 2、5、10、15、2 0Gy组 ,以 0Gy为对照组。分别采用细胞计数法、克隆形成实验、四唑盐 (MTT)比色实验、伊红排斥实验、流式细胞术及末端脱氧核苷酸转移酶介导的dUTP缺口末端标记 (TUNEL)技术 ,动态观察对VSMC生长、增殖、死亡率、凋亡的影响。结果 ①照射后 2 4hVSMC数降低 ,72h后明显恢复 (P <0 .0 5 )。②照射后 48h内VSMC吸光度 (A570 )值降低 ,72h 2、5Gy组A570值升高 ,而 10、15、2 0Gy组升高不明显 (P <0 .0 5 )。③ 72h内 10、15、2 0Gy组VSMC死亡率较高 ,6d后 ,各剂量组死亡率下降至较低水平 (P <0 .0 5 )。④克隆形成实验提示X射线照射可直接导致DNA链断裂 ,引起细胞死亡。⑤碘化丙啶 (PI)染色流式细胞仪直方图上可见亚二倍体峰 ,TUNEL法阳性细胞核呈棕黄色 ,荧光镜下可见凋亡小体。结论 X射线外照射可抑制VSMC的生长和增殖 ,可能通过两种不同的机制即致死性和 (或 )亚致死性损伤及诱导凋亡导致VSMC死亡。为应用放射治疗防治血管成形术后再狭窄提供了理论依据
Objective To investigate the effects and possible mechanisms of X-ray irradiation on the growth and proliferation of primary cultured rat vascular smooth muscle cells (VSMCs). Methods VSMCs were cultured in vitro and irradiated with a single 8MV X-ray. The source skin distance was 10 cm and the dose rate was 40 0 cGy / min. According to the injection dose is divided into 2,5,10,15,2 0Gy group, 0Gy as the control group. Cell counting, clonogenic assay, MTT colorimetric assay, Eosin rejection assay, flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) The effects on VSMC growth, proliferation, mortality and apoptosis were observed. Results ① The number of VSMC decreased at 24 hours after irradiation and recovered significantly after 72 hours (P <0.05). (2) The A570 value of VSMC decreased 48h after irradiation, A570 value of 2,5 Gy group increased at 72h, but was not significantly increased at 10, 15, and 20 Gy (P <0.05). (3) The mortality rate of VSMC in 10,15,2 0 Gy group was higher than that in 72 h group. After 6 days, the mortality rate of each dose group decreased to a lower level (P <0.05). Clone formation experiments suggest that X-ray irradiation can directly lead to DNA strand breaks, causing cell death. ⑤ Propidium iodide (PI) staining flow cytometry histogram subduploid peak, TUNEL positive brownish brown nuclei, apoptotic bodies can be seen under fluoroscopy. Conclusion X-ray external irradiation can inhibit the growth and proliferation of VSMC, which may result in the death of VSMC through two different mechanisms, namely, fatal and / or sublethal injury and apoptosis induction. The application of radiation therapy to prevent restenosis after angioplasty provides a theoretical basis