原癌基因Bmi-1在人胶质母细胞瘤细胞U87 MG放射后衰老过程中的作用机制

发布时间：2018-06-15 23:28

本文选题：胶质瘤 + 衰老　；参考：《山东大学》2014年博士论文

【摘要】：目的胶质瘤是中枢神经系统最常见的肿瘤。WHO分为I-IV级,其中Ⅲ、Ⅳ级胶质瘤可占所有胶质瘤的77.5%。通常WHO Ⅲ级胶质瘤的中位生存期2-3年,胶质母细胞瘤(WHO Ⅳ级)中位生存期仅1年。胶质瘤的预后取决于多种因素和诊治措施,虽然目前恶性胶质瘤的治疗采用手术治疗、放疗、化疗等多种疗法的综合治疗,但仍有相当多的患者在1年内复发。由于胶质瘤生长的特殊部位,使得手术彻底切除具有相当大的难度,并且极易产生后遗症,降低生活质量。虽然以替莫唑胺为代表的新型化疗药物提高了胶质瘤的化疗疗效,但是由于血脑屏障的存在,化疗的效果极其有限。放射治疗在胶质瘤的治疗中具有极其重要的地位,术后放疗可以降低胶质瘤的复发率,杀灭或抑制残余肿瘤细胞,提高治愈机会。对于无法手术切除的胶质瘤,也能采取放射治疗的方法控制肿瘤生长,延长生存期,提高生活质量。但由于胶质瘤具有抗凋亡和放射抵抗的特点,常使得胶质瘤放射治疗效果不理想、易于复发。如何提高胶质瘤的放射敏感性,一直是胶质瘤治疗领域的研究重点之一。近年来的研究发现,某些胶质瘤细胞在接受辐射后,并不发生细胞凋亡,出现的是细胞衰老现象。细胞衰老是指细胞脱离细胞周期不可逆的停滞于细胞周期的某阶段(一般为G0/G1期),细胞仍然存在一些代谢活性,但却不能再进行增殖,这也是一种重要的肿瘤抑制机制。逃逸衰老效应可能在胶质瘤的放射抵抗性中有重要地位。Bmi-1在恶性胶质瘤中呈高表达,通过多种信号途径参与细胞衰老过程,可能是肿瘤细胞逃逸衰老、抗凋亡效应和放化疗抵抗性的关键基因之一。研究Bmi。1与胶质瘤特别是高级别胶质瘤辐射抵抗性之间的关系,不仅具有生物学基础理论研究上的意义,亦具有重要的临床意义。MicroRNA-128a在正常脑组织中含量丰富,表达水平在胶质瘤组织样本中显著下调,并且与神经胶质瘤的增殖能力及神经胶质瘤干细胞的自我更新能力相关。MicroRNA-128a能抑制Bmi-1表达,进而影响其作为癌基因所发挥的表观遗传调节,促进细胞增殖和干细胞自我更新等生物学功能。在本研究中,我们选用了人胶质母细胞瘤细胞株U87MG,进行了这项研究,验证胶质瘤的辐射抵抗性及抗凋亡能力,辐射后细胞衰老的发生,以及辐射对Bmi-1及MicroRNA-128a表达的影响,探索通过调节MicroRNA-128a的表达来调控Bmi-1的表达,是否有可能改变胶质瘤细胞的放射敏感性。以期为MicroRNA-128a及Bmi-1作为胶质瘤放射增敏新靶点的科学研究提供参考。材料与方法 1.人胶质母细胞瘤细胞株U87MG购自中国科学院细胞库,利用常规细胞培养方法进行培养、传代、冻存、复苏。 2.培养U87MG胶质瘤细胞,利用直线加速器对实验用细胞进行放射干预。采用源皮距照射技术,将放射相关参数及所需放射剂量及分割方式报告给有经验的物理师并设计放射计划,在实验前对放射计划进行精确的剂量验证。 3.给予不同剂量的X射线干预U87MG细胞后,采用PI-Annexin V FITC双染色标记U87MG细胞,流式细胞术进行细胞凋亡检测。 4.给予不同剂量的X射线干预U87MG细胞。衰老细胞通常体积变大,表达pH6.0时有高酶活性的p-半乳糖苷酶。故采用p-半乳糖苷酶染色进行细胞衰老的检测。 5.给予不同剂量的X射线干预U87MG细胞后,将待测细胞提取总蛋白,BCA法测定蛋白浓度后,利用蛋白免疫印迹方法进行Bmi-1蛋白表达检测。 6.给予不同剂量的X射线干预U87MG细胞后,提取总RNA纯化后进行RNA质量检验和纯度测定。样品检验合格后,采用实时定量PCR方法进行Bmi-1mRNA及MicroRNA-128a表达的检测。 7.给予不同剂量的X射线干预U87MG细胞后,采用荧光探针DCFH-DA标记细胞,利用流式细胞术检测2',7'-二氯荧光黄的荧光,评估细胞内ROS水平。结果 1.从细胞生长曲线可以看出,各组细胞均呈现对数增殖趋势,X线辐射后均未出现明显的细胞数量减少。其中1Gy、2Gy组增殖总趋势与对照组无明显差异。在接受辐射后的24h,1Gy、2Gy组增殖较对照组增加,差异具有统计学意义(P0.05)。4Gy、6Gy、8Gy组在辐射后72h较对照组出现明显的增殖减缓趋势,差异具有统计学意义(P0.05)。这表明U87MG细胞存在一定的辐射抵抗性。 2.从结果抑制率曲线可以明显看出,1Gy、2Gy组在接受辐射后的5d内呈现明显的刺激增殖效应,其结果具有统计学意义(P0.05)；在辐射后第7天呈现出20%左右的抑制率。6Gy、8Gy组可以观察到X射线对U87MG胶质瘤细胞生长具有明显的抑制作用,抑制率随辐射后时间而增加,与辐射剂量成正相关,其结果具有统计学意义。所有组别细胞在辐射后第7天均呈现明显的抑制效应,其抑制率平均值如下：1Gy组20.35%、2Gy组20.53%、4Gy组48.8%、6Gy组68.51%、8Gy组83.15%,其效应强度与辐射剂量有相关性,说明X射线对U87MG细胞的增殖具有抑制效应。 3.不同剂量X线辐射后72h,各组细胞均未出现明显的细胞凋亡。说明X射线不能诱导U87MG胶质瘤细胞凋亡,其辐射抑制效应可能不是通过细胞凋亡机制实现的,应该有其他细胞抑制机制的存在。 4.不同剂量辐射后,在相差显微镜下观察细胞的形态变化,可以观察到衰老细胞体积明显变大,细胞扁平度增加,细胞核增大、染色深、核内可见颗粒状包含物。β-半乳糖苷酶染色检测衰老细胞的比例与辐射强度呈正相关,各组衰老细胞比例与对照组相比较差异具有统计学意义(P0.05)。这表明X射线对U87MG胶质瘤细胞的抑制效应是通过促进细胞衰老实现的。 5.X线辐射后24小时,1Gy、2Gy组S期细胞比例较对照组明显增加,说明细胞在低剂量辐射的刺激下,DNA合成增加。各组G0/G1期细胞比例随放射强度增加而增加,呈正相关性。这也一定程度上说明X射线对U87胶质瘤细胞的抑制有细胞衰老机制的参与。辐射后72h,各辐射组细胞S期细胞均较对照组增加,推测可能的原因是辐射导致细胞周期再分布,而晚S期细胞放射不敏感,造成S期细胞比例增加。G2/M期细胞比例呈现随放射剂量增加而增加的趋势,在6Gy、8Gy组,出现明显的G0/G1期细胞比例减少、G2/M期细胞比例增多的现象,可能存在某种机制,使U87MG细胞发生逃逸衰老的情况,这也是U87MG细胞辐射抵抗性的产生机制之一。 6.辐射后72小时,U87MG胶质瘤细胞的Bmi-1的蛋白表达水平和mRNA表达水平在6Gy和8Gy组明显上调。说明U87MG细胞逃逸衰老的过程中可能有Bmi-1基因的参与。 7.辐射后72h,8Gy组MicroRNA-128a表达明显减少,减少程度具有统计学意义(P0.05)。1Gy、2Gy组MicroRNA-128a表达明显增加,其增加程度具有统计学意义(P0.05)。 8.辐射后2h,2Gy组较其他组的ROS水平有所增加。辐射后12h,各辐射组ROS水平均较对照组有所增加。应用NAC处理后U87MG胶质瘤细胞后,再用8Gy X线处理细胞,发现X线对U87MG细胞的抑制率明显降低。结论 1.U87MG胶质瘤细胞具有辐射抵抗性和抗凋亡的特点。 2.X射线可以抑制U87MG胶质瘤细胞的生长,与剂量强度具有相关性,其抑制效应是通过细胞衰老实现的。 3.U87MG胶质瘤细胞接受辐射后可能存在着逃逸衰老现象,这可能是辐射抵抗性的产生机制之一。 4.ROS是X线辐射杀伤U87MG胶质瘤的重要分子之一,降低辐射时产生的ROS水平能增加U87MG胶质瘤细胞的辐射抵抗性。 5.Bmi-1可能参与U87MG胶质瘤辐射后逃逸衰老的过程并在其中具有重要地位。 6. U87MG胶质瘤辐射后Micro128a的表达与剂量强度相关,并有可能参与Bmi-1表达的调控。
[Abstract]:Purpose

MicroRNA - 128a plays an important role in the treatment of glioma , and it can reduce the recurrence rate of glioma . It can reduce the recurrence rate of glioma . It can reduce the recurrence rate of glioma . It can reduce the recurrence rate of glioma . To investigate the expression of Bmi - 1 by regulating the expression of MicroRNA - 128a , it is possible to alter the radiosensitivity of glioma cells . It is expected to provide references for scientific research on microRNAs - 128a and Bmi - 1 as a new target for radiosensitization of glioma . Materials and Methods

1 . Human Glioblastoma cell line U87MG was purchased from the cell bank of Chinese Academy of Sciences , cultured by conventional cell culture method , passaged , frozen and recovered .

2 . The U87MG glioma cells were cultured , and the experimental cells were radiated with the linear accelerator . The radiation - related parameters and the required radiation dose and division mode were reported to the experienced physical division and the radiation plan was designed by using the source - skin - distance irradiation technique , and the radiation plan was accurately dose - verified before the experiment .

3 . After administration of U87MG cells with different doses of X - rays , the apoptosis of U87MG cells and flow cytometry were detected by PI - annexin - V FITC - labeled U87MG cells .

4 . Different doses of X - ray intervention U87MG cells were administered . The senescence cells usually have a large volume and a high enzyme activity of p - galactosidase when expressed at pH 6.0 . The detection of cell senescence was performed by using p - galactosidase staining .

5 . After administration of U87MG cells with different doses of X - rays , the total protein was extracted from the cells to be detected . After the BCA method was used to determine the protein concentration , the Bmi - 1 protein expression was detected by Western blotting .

6 . After administration of U87MG cells with different doses of X - rays , RNA quality test and purity determination were carried out after the total RNA was purified . After the samples were qualified , the expression of Bmi - 1mRNA and MicroRNA - 128a was detected by real - time quantitative PCR .

7 . After administration of U87MG cells with different doses of X - rays , fluorescent probe DCFH - DA labeled cells were used to detect the fluorescence of 2 ' , 7 ' - dichlorofluorescein by flow cytometry , and the intracellular ROS level was assessed .

1 . From the growth curve of the cells , there was no significant difference in the number of cells in each group . There was no significant difference in the proliferation of X - ray after irradiation . The proliferation of the group was significantly higher than that in the control group at 24h , 1Gy and 2Gy after irradiation .

2 . From the result inhibition curve , it can be seen that 1Gy , 2Gy group exhibited obvious stimulation proliferative effect in 5 days after receiving radiation , and the result was statistically significant ( P0.05 ) .
The inhibitory rate of X - ray on U87MG glioma cells was significantly inhibited after irradiation on the 7th day after radiation . The inhibition rate increased with the time of radiation , and the inhibition rate was significantly correlated with the radiation dose . The average inhibitory rate was as follows : 20.35 % in 1Gy group , 20.53 % in 2Gy group , 48.8 % in 4Gy group , 68.51 % in 6Gy group and 83.15 % in 8Gy group .

3 . Apoptosis of U87MG glioma cells could not be induced by X - ray irradiation at 72 h after X - ray irradiation . It was suggested that X - ray could not induce apoptosis of U87MG glioma cells .

4 . After radiation of different doses , the morphological changes of the cells were observed under the phase contrast microscope . It was observed that the volume of aging cells increased significantly , the cell flattened degree increased , the nucleus increased , the staining was deep , and the granular inclusion bodies were observed in the nucleus . The proportion of 尾 - galactosidase staining was positively correlated with the radiation intensity , and the proportion of aging cells in each group was statistically significant compared with the control group ( P0.05 ) . This suggested that the inhibitory effect of X - ray on U87MG glioma cells was realized by promoting cell senescence .

The percentage of cells in G0 / G1 phase increased with the increase of radiation intensity .

6 . Bmi - 1 protein expression level and mRNA expression level of U87MG glioma cells were up - regulated in 6Gy and 8Gy group 72 hours after irradiation .

7 . The expression of MicroRNA - 128a in 8Gy group was significantly decreased at 72h after radiation ( P0.05 ) . The expression of MicroRNA - 128a in group 1Gy and 2Gy increased significantly ( P0.05 ) .

8 . After irradiation , the levels of ROS were increased in the 2Gy group than in the other groups . After treatment with NAC , the levels of ROS in each group increased . After treatment with NAC , the cells were treated with 8Gy X - ray , and the inhibitory rate of X - ray on U87MG cells was significantly decreased . Conclusion

1 . U87MG glioma cells have the characteristics of radiation resistance and anti - apoptosis .

2 . X - ray can inhibit the growth of U87MG glioma cells and has a correlation with dose intensity , and its inhibitory effect is realized by cell aging .

3.U87MG glioma cells can escape aging after receiving radiation , which may be one of the mechanisms of radiation resistance .

4 . ROS is one of the important molecules of X - ray radiation killing U87MG glioma , and the level of ROS produced during irradiation can increase the radiation resistance of U87MG glioma cells .

5 . Bmi - 1 may participate in the process of escaping aging after U87MG glioma radiation and has important status in it .

6 . The expression of Micro128a after U87MG glioma irradiation is related to dose intensity and is likely to be involved in the regulation of Bmi - 1 expression .
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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