X射线对血管平滑肌细胞作用机制的实验研究

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100cm，剂量率400cGy/min。按照射剂量分为2、5、10、20Gy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（MIT）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24hVSM数降低，72h后明显恢复（P＜0．05）。②照射后48h内VSMC吸光度（A570）值降低，72h2、5Gy组A570值升高，而10、15、20Gy组各高不明显（P＜0．05）。③72h内10、15、20Gy组VSMC死亡率较高，6d后，各剂量组死亡率下降至较低水平（P＜0．05）。④克隆形成实验提示X坶照射可直接导致NA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SMC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。

Effects of X-ray radiation on cultured vascular smooth muscle cells

Objective　 Proliferation of vascular smooth muscle cells (VSMC) represents an important event in vascular lesion formation. The aim of this investigation is to study the effect of X-ray irradiation on the viability of VSMC and the cellular mechanism, depending on which irradiation inhibits VSMC proliferation. Methods　The different 8MV doses ranging from 2～20 Gy were delivered to cultured VSMC with 0 Gy serving as control. Cytometry, clonogenic assay and MTT calorimetric assay, eosin exclusion test, flow cytometry and the terminal deoxynucleotidyltransferase-mediated DUTP nick end labeling (TUNEL) technique were to determine the cell amount, proliferation, growth, death and aptosis rates of VSMC. According to these references, the VSMC irradiated repopulation model can be established. Aptosis cells were identified and quantified by the TUNEL and flow cytometry techniques. Results　 ①The cell amount of VSMC began to decrease 24 hours after irradiation and to recover up to 72 hours（<0.05）.The decrement of VSMC cellularity was proportionally related to the escalated dose effect. ②With MTT assay, the A570 value of VSMC decreased within 48 hours after irradiation and the value began to rise 72 hours later, faster in the 2Gy and 5Gy groups（P＜0.05）. ③ Eosin stain exclusion revealed the very high VSMC's death rate after irradiation of 10,15 and 20 Gy. The mortalities of VSMC in all groups came down to a lower level（P＜0.05） 6 days later. ④ The VSMC survival data from the clonogenic assay were consistent with the mechanism of radiation-induced cell killing. ⑤ DNA hypodiploid peaks were found on the flow cytometry histograms and the TUNEL assay , revealing the presence of VSMC DNA fragmentation. Conclusions　 It can be concluded that there are two mechanisms to inhibit VSMC proliferation and even result in death, lethal and/or sublethal cellular injury and apoptosis. Therefore, X-ray irradiation can effectively prevent restenosis after angioplasty