丙戊酸致骨肉瘤细胞辐射增敏作用及其分子机制研究

发布时间：2018-03-23 11:14

本文选题：丙戊酸　切入点：骨肉瘤细胞　出处：《山东大学》2017年硕士论文

【摘要】：目的骨肉瘤(osteosarcoma)主要好发于20岁以下青少年与儿童,是一种恶性骨肿瘤,临床上主要用放疗来治疗,但由于骨肉瘤对辐射有一定抵抗性,因此效果并不理想。为增强辐射对肿瘤细胞的杀伤作用,寻找有效的辐射增敏剂成为该领域研究的热点。在临床上,丙戊酸(ValproicAcid,VPA)是一种治疗癫痫病和其他痉挛性疾病的常见药物,也是最具代表性的一类组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitors,HDACis)。我们课题组及其他研究小组的实验结果均证明,VPA能够增强乳腺癌细胞、结肠癌细胞和肺癌细胞等多种肿瘤细胞对辐射的敏感性。我们课题组研究还揭示在治疗癫痫病的安全血药浓度(0.3-0.8 mM)下的VPA对乳腺肿瘤细胞辐射增敏作用与DNA损伤修复功能障碍有关,主要是破坏修复DNA双链断裂(DNAdouble strand breaks,DNA DSBs)的同源重组(homologous recombination,HR)分子机制。但是,HDACis对骨肉瘤细胞辐射增敏作用目前还不十分清楚。为此,本课题将探讨安全剂量(0.5 mM)与安全临界剂量(1.0mM)下的VPA对骨肉瘤U20S细胞辐射敏感性的作用,以及VPA辐射增敏作用是否与干扰HR和非同源末端连接(non-homologous end-joining,NHEJ)两种修复机制有关,目的在于为VPA用于骨肉瘤临床放射治疗提供重要的理论指导和实验依据。方法1.细胞克隆形成实验观察VPA对骨肉瘤细胞U20S辐射敏感性的影响单独VPA组是指给予0.5 mM的VPA作用于U20S细胞24 h;单独电离辐射(inozing radiation,IR)组是指用0、2 Gy、4 Gy及6 Gy辐射剂量处理细胞,联合组是先给予U20S细胞0.5 mM VPA作用24 h再分别给予0、2 Gy、4 Gy及6Gy辐射剂量处理,每组设三个平行样品。处理结束后继续培养14天,统计各组细胞克隆形成率。2.彗星实验检测VPA对IR所致的U20S细胞DSBs损伤的影响单独VPA组是指VPA作用U20S细胞24 h,单独IR组是用4 Gy辐射剂量处理细胞;联合组是先给予U20S细胞0.5 mM VPA作用24 h再进行4 Gy辐射剂量处理。收集各组细胞并制成单细胞悬液,进行彗星实验,观察细胞拖尾情况以及统计分析各组细胞的尾距变化。3.DNA DSBs标志物γH2AX与53BP1焦点形成,以及BRCA1焦点形成情况分析用0.5mMVPA、8Gy或VPA与IR联合分别处理U20S细胞,辐射后6h及24 h,应用免疫荧光技术观察各组细胞核内γH2AX焦点、53BP1焦点以及BRCA]焦点形成情况,并统计各组含不同蛋白焦点的细胞百分率。4.流式细胞仪检测VPA对细胞NHEJ修复效率的影响以及免疫印记技术检测参与NHEJ机制的关键修复蛋白的表达首先将I-SceI核酸酶转染到稳定表达非同源底物EJ5-GFP的U20S细胞系,造成DNA DSBs损伤,同时用0.5 mM VPA或1.0 mM VPA分别处理细胞,收集处理后的细胞并制成单细胞悬液,利用流式细胞术测定VPA对细胞NHEJ修复效率的影响。收集各处理组细胞蛋白裂解液用免疫印记技术检测NHEJ修复通路中主要蛋白KU70,KU80及DNA-PKcs蛋白表达水平。5.荧光原位杂交(FISH)技术检测VPA对基因组稳定性的影响单独VPA组的细胞给予0.5 mM的VPA并作用24 h;单独IR组细胞给予2 Gy辐射剂量处理,联合组的细胞先给予0.5 mMVPA作用24 h再进行2 Gy处理。辐射后24 h时,加入秋水仙碱使细胞停止于分裂期,收集分裂期细胞,按照常规FISH方法检测染色体变异情况。6.流式细胞仪检测VPA对细胞周期的影响用0.5 mM和1.0 mM的VPA分别处理细胞后,收集各组细胞,按照常规方法用流式细胞仪检测各组细胞的细胞周期变化。结果1.VPA能够增加IR引起的骨肉瘤细胞内DNA DSBs损伤的进一步蓄积彗星实验结果显示,与对照组相比,单独VPA组,细胞DNA拖尾长度有轻微增加,单独IR组DNA尾距则有明显增加,VPA与IR联合处理组,细胞拖尾长度为四组中最长(P0.05)。与单独IR组相比,在辐射后30min和120min,VPA与IR联合处理组的细胞彗星拖尾长度分别显著增加14.02%(P0.05)和16.49%(P0.05)。免疫荧光结果提示,与对照组相比,0.5 mM和1.0 mM VPA处理组含γH2AX焦点细胞的比率分别增加了 1.2倍和1.3倍。辐射后6h结果表明,与单独IR处理组相比,VPA与IR联合组含有γH2AX焦点阳性细胞比率分别增加了 1.6倍和2.1倍,差异有统计学意义(P0.05)。与对照组相比,0.5mM和1.0mMVPA处理组含有53BP1焦点的阳性细胞比率分别增加1.9倍和2.7倍(P0.05)。与单独IR组相比,VPA与IR联合组含53BP1焦点阳性细胞比率分别增加2.1倍和3.2倍(P0.05)。2.VPA增加骨肉瘤细胞对辐射的敏感性与正常组比较,单独VPA组的细胞存活率明显下降;与单独IR组相比,0.5 mM VPA与IR联合组在2 Gy、4 Gy及6 Gy各剂量下的细胞存活率均明显降低(P0.05),降低的比例分别为23.3%,32.4%及43.6%。3.安全和临界剂量VPA能够抑制细胞HR修复通路辐射后24 h时,0.5 mM或1.0 mM VPA与IR联合组含有H2AX焦点细胞的阳性率显著地高于单独IR组的2.24倍和3.43倍(P0.05),0.5 mM或1.0 mM VPA与IR联合组含有53BP1焦点细胞的阳性率也显著地高于单独IR组的1.27倍和1.62倍(P0.05)。辐射后6 h,VPA与IR联合组含有BRCA1焦点的细胞阳性百分比明显低于单纯IR组,降低的比例分别为26.5%(P0.05);辐射后24h,VPA与IR联合组含有BRCA1焦点的细胞阳性百分比也低于单纯IR组,为15.3%(P0.05)。克隆形成实验结果提示,0.5 mM VPA或者1.0 mM VPA与10μM ABT888联合作用使细胞克隆形成率均显著低于其他各组(P0.05)。4.安全和临界剂量VPA能够抑制细胞NHEJ修复通路与对照组相比,单独0.5 mM或1.0 mM VPA处理组I-SceI诱导的NHEJ重组效率分别降低至63.8%(P0.01)或61%(P0.01),两处理组之间无明显差异(P0.05)。KU80蛋白表达水平在单独VPA组和VPA与IR联合处理组表现为显著下降,但DNA-PKcs和KU70蛋白表达水平在各组细胞中无明显变化。5.安全剂量VPA对骨肉瘤细胞的细胞周期无明显影响单独0.5 mM或1.0 mM VPA处理组在G1、S与G2/M各期的细胞比率与对照组没有显著差异。6.安全剂量VPA能够增加骨肉瘤细胞基因组不稳定性对于无VPA处理组,辐射处理前后细胞的染色单体与染色质断裂百分率无统计学差异(P0.05),但是辐射状异常染色体结构形成的百分率由1.59%增加到7.34%(P0.05)。与单独VPA处理组相比,VPA与IR联合组的细胞染色单体断裂的百分率由4.57%增加到17.24%(P0.05)、染色质断裂的百分率由18.27%增至到43.1%(P0.05)以及辐射状异常染色体结构形成的百分率也表现出相同的变化趋势。结论1.应答DNA损伤反应时,安全剂量和安全临界剂量下的VPA都可进一步增加IR所致的DNADSBs损伤在骨肉瘤细胞内的蓄积。2.安全剂量下的VPA能够抑制骨肉瘤细胞生长并且增加细胞对辐射的敏感性。3.安全剂量下的VPA增加骨肉瘤细胞对辐射的敏感性可能是通过干扰BRCA1介导的HR和KU80介导的NHEJ分子机制所致。4.安全剂量下的VPA能够增加骨肉瘤细胞基因组不稳定性。
[Abstract]:Objectiveosteosarcoma (osteosarcoma) mainly occurs in the following 20 years old adolescents and children, is a kind of malignant bone tumors, the major clinical treatment with radiotherapy, but because osteosarcoma has certain resistance to radiation, so the effect is not ideal. In order to enhance the radiation killing effect on tumor cells, to find effective radiation sensitizer this has become a hot research field. In clinic, valproic acid (ValproicAcid, VPA) is a common medicine for treating epilepsy and other spastic disease, is the most representative of a class of histone deacetylase inhibitors (histone deacetylase, inhibitors, HDACis). Our group and other research groups the experimental results show that VPA can enhance the sensitivity of some breast cancer cells, colon cancer cells and lung cancer cells to radiation. Our study also revealed that in the safety of blood drug treatment for epilepsy disease concentrated The degree of sensitization (0.3-0.8 mM) associated with the dysfunction of DNA damage repair under VPA radiation on breast cancer cells, the main damage is the repair of DNA double strand breaks (DNAdouble strand breaks DNA DSBs (homologous recombination) by homologous recombination, HR) molecular mechanism. However, HDACis on osteosarcoma cell radiation sensitizing effect is still not very clear. Therefore, this paper will explore the safe dose (0.5 mM) and safety critical dose (1.0mM) under the effect of VPA on radiation sensitivity of osteosarcoma U20S cells, and VPA radiation sensitizing effect and HR interference and non homologous end joining (non-homologous end-joining, NHEJ) on two kinds of repair mechanism, objective is to provide an important theoretical guidance and experimental basis for the treatment of VPA for osteosarcoma clinical radiotherapy. Methods 1. cell clone formation assay, VPA on osteosarcoma cell U20S radiation sensitivity effect of VPA alone group Refers to the role of VPA in U20S cells treated with 0.5 mM 24 h; alone (inozing radiation, IR radiation group) refers to the use of 0,2 Gy, 4 Gy and 6 Gy radiation dose treatment group is given combined with cells, U20S cells of 0.5 mM VPA 24 h and Gy 4 were treated with 0,2, Gy and 6Gy radiation the dose of treatment, each group had three parallel samples. After cultured for 14 days, calculated the clone formation effect of U20S cells on DSBs damage induced by IR rate.2. comet assay VPA alone group VPA VPA refers to the role of U20S cells in IR group was 24 h, alone with 4 Gy radiation dose treatment cell the combined group is first given; U20S 0.5 mM VPA 24 h cells and 4 Gy radiation dose. Cells were collected and made into single cell suspension, by comet assay, comet cell observation and statistical analysis of the situation of each cell from the tail changes of.3.DNA markers of DSBs gamma H2AX and 53BP1 focus The formation of BRCA1, and the formation of the focus analysis with 0.5mMVPA, 8Gy or VPA combined with IR were treated U20S cells after radiation 6h and 24 h, each nucleus gamma H2AX immunofluorescence was applied to observe the focus, focus and focus of the formation of 53BP1 BRCA], and was analyzed with different focal cell percentage of.4. protein by flow cytometry to study the effect of VPA on cellular NHEJ repair efficiency and expression of key repair protein immunoblotting technology in NHEJ detection mechanism of the first U20S cell line was transfected into I-SceI nuclease stable expression of non homologous EJ5-GFP substrate, DNA caused DSBs damage at 0.5 mM VPA or 1 mM were treated with VPA cells, collecting treated cells and made into single cell suspension. By measuring the effects of VPA on cellular NHEJ repair efficiency by flow cytometry. From each treatment group cell lysates by immunoblotting with NHEJ detection technology The main protein KU70 repair pathway, the expression of KU80 and DNA-PKcs protein level of.5. fluorescence in situ hybridization (FISH) technique to detect the effect of VPA on genome stability alone VPA group were treated with 0.5 mM VPA and 24 h; group IR were treated with 2 separate Gy radiation treatment, the combination group received 0.5 mMVPA 24 cells h and 2 Gy. 24 h after radiation, adding colchicine stops cells in mitosis, mitotic cells were collected, according to the conventional FISH method for detection of chromosome variation of.6. flow cytometry cell respectively to study the effect of VPA on the cell cycle of 0.5 mM and 1 mM after VPA cells were collected. The change of cell cycle was detected by flow cytometry using conventional method. According to the results of 1.VPA to further accumulation of osteosarcoma cells increased IR induced DNA DSBs damage comet experiment results show that, with the control group Compared with VPA alone group, DNA cell tail length was increased slightly, IR alone group DNA tailmoment were significantly increased, the combined treatment group VPA and IR, the length of comet cell into four groups in the longest (P0.05). Compared with IR group, the radiation of 30min and 120min, VPA and IR combined treatment group the cell tail lengths were significantly increased (P0.05 14.02%) and 16.49% (P0.05). Immunofluorescence results show that, compared with the control group, the ratio of 0.5 mM and 1 mM VPA treatment group containing gamma H2AX focus cells were increased by 1.2 times and 1.3 times. The results show that 6h radiation, compared with IR alone group VPA, combined with IR group containing gamma H2AX focus positive cell rate were increased by 1.6 times and 2.1 times, the difference was statistically significant (P0.05). Compared with the control group, the positive cell ratio of 0.5mM and 1.0mMVPA treatment group containing 53BP1 focus was increased by 1.9 times and 2.7 times (P0.05) compared with IR group. VPA, combined with IR group containing 53BP1 focus positive cell rate was increased by 2.1 times and 3.2 times (P0.05).2.VPA increased compared with the normal group the sensitivity of osteosarcoma cells to radiation alone, the survival rate of group VPA decreased significantly; compared with IR group, 0.5 mM VPA combined with IR group at 2 Gy, 4 Gy and 6 different doses of Gy the cell survival rate were significantly lower (P0.05), reduced respectively 23.3%, 32.4% and 43.6%.3. safety and the critical dose of VPA can inhibit cell HR repair pathway after radiation of 24 h, the positive rate was 0.5 mM or 1 mM VPA combined with IR group H2AX cells contain significant focus 2.24 times higher than that of IR alone group and 3.43 times (P0.05), the positive rate was 0.5 mM or 1 mM VPA combined with IR group 53BP1 cells containing the focus was significantly higher than that of 1.27 times and 1.62 times separately in IR group (P0.05). 6 h after irradiation, the percentage of VPA positive cells and IR group containing BRCA1 focus the Was obviously lower than that in group IR, reduce the ratio of 26.5% (P0.05); 24h after radiation, positive cells percentage of VPA combined with IR group containing BRCA1 focus is lower than that of the pure IR group, 15.3% (P0.05). Cloning experiment results suggest that the combined effect of 0.5 mM VPA or 1 mM VPA and 10 M ABT888 the clone formation rate was significantly lower than that of the other groups (P0.05) and.4. safety critical dose of VPA could inhibit the cell NHEJ repair pathway compared with the control group, 0.5 separate mM or 1 mM VPA treatment group I-SceI NHEJ induced by recombinant efficiency were reduced to 63.8% (P0.01) or 61% (P0.01), no significant difference between the two treatments group (P0.05) the expression level of.KU80 protein in VPA alone group and VPA combined with IR in treatment group decreased significantly, but the DNA-PKcs and KU70 protein expression in the cells of each group had no obvious change in.5. dose VPA on osteosarcoma cell cycle of ignorance On 0.5 separate mM or 1 mM VPA treatment group in G1, S and G2/M phases of the cell rate is no significant difference between the two groups of.6. safe dose of VPA can increase the osteosarcoma cell genomic instability for VPA treatment group, radiation treatment before and after chromatid breaks and chromatin cell percentage (no significant difference P0.05), but the percentage of radiation form abnormal chromosome structure increased from 1.59% to 7.34% (P0.05). Compared with VPA alone group, the percentage of chromatid breaks of VPA combined with IR group of cells increased from 4.57% to 17.24% (P0.05), the percentage of chromatin fracture increased from 18.27% to 43.1% (P0.05) and the percentage of radiation form of abnormal chromosome structure also showed the same trend. The conclusion of the 1. response to DNA damage response, safety and security critical dose dose of VPA can be further increased DNADSBs damage induced by IR The wound in the osteosarcoma cells in the accumulation of.2. under the safe dosage of VPA could inhibit osteosarcoma cell growth and increase cell sensitivity to radiation dose.3. security under the VPA increased sensitivity of osteosarcoma cells to radiation may be mediated by interfering with BRCA1 HR and KU80 NHEJ mediated molecular mechanism induced by.4. under the safe dosage VPA can increase the osteosarcoma cell genomic instability.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738

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