γH2AX对胶质瘤放射敏感性预测效果的实验研究

发布时间：2019-01-05 11:25

【摘要】：目的：神经胶质瘤为颅内常见肿瘤,为预后较差的颅内原发性恶性肿瘤,具有浸润性生长和边界不清等生物学特点,手术难以彻底切除。术后放射治疗是胶质瘤综合治疗中较为重要的手段。但是恶性胶质瘤具有放射抗性致其放射治疗效果欠佳,近来多个研究证实γH2AX逐渐成为检测DNA双链断裂的一项新型生物学标志。在本研究中,我们通过体外培养高级别胶质瘤细胞系(U87、U251和LN229),研究其放射敏感性以及经X射线照射后三种细胞株γH2AX蛋白的表达变化,并分析γH2AX蛋白表达与胶质瘤细胞放射敏感性之间的相互关系。方法： 1.选择高级别人脑胶质瘤U87、U251和LN229细胞株,分成0Gy组、2Gy组、4Gy组、6Gy组、8Gy组和10Gy组,经常规细胞培养、细胞计数后,行X线照射,继续细胞培养2周左后,经固定、染色后镜下检测细胞集落数,计算细胞克隆形成率、细胞存活分数,并绘制细胞存活曲线、由生物学参数比较细胞系间放射敏感性； 2.选择高级别人脑胶质瘤U87、U251和LN229细胞株,按照经X线照射后培养的时间分为0h组、0.5h组、1h组、2h组、6h组、12h组、24h组、36h组和48h组,常规细胞培养至细胞贴壁并铺满整个培养瓶约75%体积时,行2Gy照射,然后放置于细胞培养箱中分别培养0h、0.5h、1h、2h、6h、2h、24h、36h和 48h,依次分别提取制备细胞总蛋白,经蛋白变性、聚丙烯酰胺凝胶电泳分离、蛋白质转移至PVDF膜、封闭、Ⅰ抗反应、Ⅱ抗孵育后,ECL化学发光试剂盒显影。采用Alpha Innotech凝胶成像仪检测结果,Image J计算各电泳条带平均光密度值,从而检测其γH2AX蛋白表达变化。目的蛋白质相对表达量=目的条带灰度值／同一样本内参灰度值。 3.统计学分析γH2AX蛋白表达与胶质瘤细胞放射敏感性之间的相关性。以Western blotting条带中6h组与0.5h组的蛋白表达量差值代表相对衰减速度,以目的蛋白质峰值与初始值之间差值代表目的蛋白升高程度,分别与放射增敏比行相关性分析。结果： 1.细胞克隆形成实验显示,随着X线照射剂量的增加,胶质瘤细胞存活分数逐渐降低,细胞克隆形成率减少,U87、LN229、U251细胞的放射增敏比(SER)依次为1.31±0.04、1.14±0.02、1.00±0.00,并采用单因素方差分析得出：放射增敏比(SER)自高至低依次为U87、LN229、U251(均P=0.000)； 2.Western blotting法显示,随着经X线照射后培养时间的延长,各胶质瘤细胞系γH2AX蛋白表达呈现出先上升后下降的时间效应曲线,U87、LN229和U251细胞株出现峰值时间依次为2h、1h和1h(P=0.000、0.000、0.015)。 3.yH2AX蛋白相对衰减速度和γH2AX升高程度均与放射增敏比呈正相关。(r=0.733,p=0.025；r=0.672,P=0.047)。结论： 1.三种高级别人脑胶质瘤细胞株放射敏感性自高至低依次为U87、LN229、U251; 2.电离辐射产生的yH2AX蛋白在放射性较抵抗的胶质瘤细胞株中达到峰值的时间相对更早； 3.电离辐射产生的γH2AX蛋白在辐射较敏感的胶质瘤细胞株中升高程度以及衰减速度均相对较高； 4.yH2AX蛋白有望成为高级别胶质瘤的分子生物学标志物,并作为预测胶质瘤放射敏感性的重要指标。
[Abstract]:Objective: The glioma is a common type of intracranial tumor. It is a kind of intracranial primary malignant tumor with poor prognosis. It has the biological characteristics of infiltrative growth and non-clear boundary, and the operation is difficult to be completely cut off. Postoperative radiation therapy is an important means in the comprehensive treatment of glioma. However, the radiation resistance of the malignant glioma is not good, and the recent researches have shown that HH2AX is a new biological marker for detecting the double-strand break of DNA. In this study, we studied the radiosensitivity of the high-level glioma cell line (U87, U251, and LN229) in vitro, and the expression of the HH2AX protein in the three cell lines after X-ray irradiation. The relationship between the expression of HH2AX and the radiosensitivity of glioma cells was analyzed. square Methods: 1. High-grade human glioma U87, U251 and LN229 cell lines were selected, divided into 0Gy group, 2Gy group, 4Gy group, 6Gy group, 8Gy group and 10Gy group. After cell count, X-ray irradiation was performed and the cell culture was continued for 2 weeks. The cell colony number, the cell clone formation rate, the cell survival score, and the cell survival curve were measured under a fixed and stained mirror, and the cell line-to-cell radiation was compared by the biological parameters. Sensitivity; 2. High-level human glioma U87, U251 and LN2 The cell line was divided into 0h group, 0.5h group, 1h group, 2h group, 6h group, 12h group, 24h group, 36h group and 48h group according to the time after the X-ray irradiation. 1h,2h,6h,2h,24 h, 36h and 48h, respectively, respectively extracting the total protein of the preparation cell, the protein denaturation, the polytetramine amine gel electrophoresis separation, the protein transfer to the PVDF membrane, the sealing, the first anti-reaction, the second anti-incubation, the ECL chemistry, Development of the light-emitting kit. As a result of the detection using the Alpha Innotech Gel imager, the image J calculates the average optical density values for each of the electrophoretic bands, thereby detecting its BHC2A X-protein expression change. Target protein relative expression quantity = target band gray value/ same Statistical analysis of the expression of HH2AX and the radiation of glioma cells The correlation between the sensitivity and sensitivity was expressed by the difference of the protein expression between the 6 h group and the 0. 5 h group in the Western blotting band. The difference between the peak value of the target protein and the initial value represents the degree of increase of the target protein, and the difference between the peak value of the target protein and the initial value represents the degree of increase of the target protein, respectively. increase-to-sensitivity ratio The results were as follows: 1. The cell clone formation experiment showed that, with the increase of X-ray irradiation dose, the survival score of glioma cells decreased gradually, the formation rate of cell clone decreased, and the radiosensitivity ratio (SER) of U87, LN229 and U251 cells was 1.31, 0.04, 1.14, and 0.. 02, 1. 00-0. 00, and using a single-factor analysis of variance, the radiosensitivity ratio (SER) is from high to low in order of U87, LN229, U, 251 (P = 0.000); With the extension of culture time after X-ray irradiation, the expression of HH2AX protein in glioma cell lines showed a time-effect curve which decreased first, and the peak time of U87, LN229 and U251 cell lines was 2h, 1h and 1h (P = 0. 0). 00, 0. 000, 0. 015). 3. yH2AX protein relative decay rate and FH2 The degree of increase of AX was positively correlated with the radiosensitivity ratio. (r = 0.733, p = 0.025 锛況= Conclusion: 1. The radiosensitivity of three high-grade human glioma cell lines from high to low, U87, LN229, U251; 2. The yH2AX protein produced by ionizing radiation is in radiation. the time to reach the peak in the resistance-resistant glioma cell line is relatively early; 3. the h2ax protein produced by the ionizing radiation is relatively high in radiation, The degree of increase and the rate of attenuation in the sensitive glioma cell line are relatively high; the 4. yH2AX protein is expected to be a high-grade glioma
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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