本文選題：丙戊酸　切入點：骨肉瘤細胞　出處：《山東大學》2017年碩士論文

【摘要】：目的骨肉瘤(osteosarcoma)主要好發(fā)于20歲以下青少年與兒童,是一種惡性骨腫瘤,臨床上主要用放療來治療,但由于骨肉瘤對輻射有一定抵抗性,因此效果并不理想。為增強輻射對腫瘤細胞的殺傷作用,尋找有效的輻射增敏劑成為該領(lǐng)域研究的熱點。在臨床上,丙戊酸(ValproicAcid,VPA)是一種治療癲癇病和其他痙攣性疾病的常見藥物,也是最具代表性的一類組蛋白去乙�；敢种苿�(histone deacetylase inhibitors,HDACis)。我們課題組及其他研究小組的實驗結(jié)果均證明,VPA能夠增強乳腺癌細胞、結(jié)腸癌細胞和肺癌細胞等多種腫瘤細胞對輻射的敏感性。我們課題組研究還揭示在治療癲癇病的安全血藥濃度(0.3-0.8 mM)下的VPA對乳腺腫瘤細胞輻射增敏作用與DNA損傷修復功能障礙有關(guān),主要是破壞修復DNA雙鏈斷裂(DNAdouble strand breaks,DNA DSBs)的同源重組(homologous recombination,HR)分子機制。但是,HDACis對骨肉瘤細胞輻射增敏作用目前還不十分清楚。為此,本課題將探討安全劑量(0.5 mM)與安全臨界劑量(1.0mM)下的VPA對骨肉瘤U20S細胞輻射敏感性的作用,以及VPA輻射增敏作用是否與干擾HR和非同源末端連接(non-homologous end-joining,NHEJ)兩種修復機制有關(guān),目的在于為VPA用于骨肉瘤臨床放射治療提供重要的理論指導和實驗依據(jù)。方法1.細胞克隆形成實驗觀察VPA對骨肉瘤細胞U20S輻射敏感性的影響單獨VPA組是指給予0.5 mM的VPA作用于U20S細胞24 h;單獨電離輻射(inozing radiation,IR)組是指用0、2 Gy、4 Gy及6 Gy輻射劑量處理細胞,聯(lián)合組是先給予U20S細胞0.5 mM VPA作用24 h再分別給予0、2 Gy、4 Gy及6Gy輻射劑量處理,每組設(shè)三個平行樣品。處理結(jié)束后繼續(xù)培養(yǎng)14天,統(tǒng)計各組細胞克隆形成率。2.彗星實驗檢測VPA對IR所致的U20S細胞DSBs損傷的影響單獨VPA組是指VPA作用U20S細胞24 h,單獨IR組是用4 Gy輻射劑量處理細胞;聯(lián)合組是先給予U20S細胞0.5 mM VPA作用24 h再進行4 Gy輻射劑量處理。收集各組細胞并制成單細胞懸液,進行彗星實驗,觀察細胞拖尾情況以及統(tǒng)計分析各組細胞的尾距變化。3.DNA DSBs標志物γH2AX與53BP1焦點形成,以及BRCA1焦點形成情況分析用0.5mMVPA、8Gy或VPA與IR聯(lián)合分別處理U20S細胞,輻射后6h及24 h,應用免疫熒光技術(shù)觀察各組細胞核內(nèi)γH2AX焦點、53BP1焦點以及BRCA]焦點形成情況,并統(tǒng)計各組含不同蛋白焦點的細胞百分率。4.流式細胞儀檢測VPA對細胞NHEJ修復效率的影響以及免疫印記技術(shù)檢測參與NHEJ機制的關(guān)鍵修復蛋白的表達首先將I-SceI核酸酶轉(zhuǎn)染到穩(wěn)定表達非同源底物EJ5-GFP的U20S細胞系,造成DNA DSBs損傷,同時用0.5 mM VPA或1.0 mM VPA分別處理細胞,收集處理后的細胞并制成單細胞懸液,利用流式細胞術(shù)測定VPA對細胞NHEJ修復效率的影響。收集各處理組細胞蛋白裂解液用免疫印記技術(shù)檢測NHEJ修復通路中主要蛋白KU70,KU80及DNA-PKcs蛋白表達水平。5.熒光原位雜交(FISH)技術(shù)檢測VPA對基因組穩(wěn)定性的影響單獨VPA組的細胞給予0.5 mM的VPA并作用24 h;單獨IR組細胞給予2 Gy輻射劑量處理,聯(lián)合組的細胞先給予0.5 mMVPA作用24 h再進行2 Gy處理。輻射后24 h時,加入秋水仙堿使細胞停止于分裂期,收集分裂期細胞,按照常規(guī)FISH方法檢測染色體變異情況。6.流式細胞儀檢測VPA對細胞周期的影響用0.5 mM和1.0 mM的VPA分別處理細胞后,收集各組細胞,按照常規(guī)方法用流式細胞儀檢測各組細胞的細胞周期變化。結(jié)果1.VPA能夠增加IR引起的骨肉瘤細胞內(nèi)DNA DSBs損傷的進一步蓄積彗星實驗結(jié)果顯示,與對照組相比,單獨VPA組,細胞DNA拖尾長度有輕微增加,單獨IR組DNA尾距則有明顯增加,VPA與IR聯(lián)合處理組,細胞拖尾長度為四組中最長(P0.05)。與單獨IR組相比,在輻射后30min和120min,VPA與IR聯(lián)合處理組的細胞彗星拖尾長度分別顯著增加14.02%(P0.05)和16.49%(P0.05)。免疫熒光結(jié)果提示,與對照組相比,0.5 mM和1.0 mM VPA處理組含γH2AX焦點細胞的比率分別增加了 1.2倍和1.3倍。輻射后6h結(jié)果表明,與單獨IR處理組相比,VPA與IR聯(lián)合組含有γH2AX焦點陽性細胞比率分別增加了 1.6倍和2.1倍,差異有統(tǒng)計學意義(P0.05)。與對照組相比,0.5mM和1.0mMVPA處理組含有53BP1焦點的陽性細胞比率分別增加1.9倍和2.7倍(P0.05)。與單獨IR組相比,VPA與IR聯(lián)合組含53BP1焦點陽性細胞比率分別增加2.1倍和3.2倍(P0.05)。2.VPA增加骨肉瘤細胞對輻射的敏感性與正常組比較,單獨VPA組的細胞存活率明顯下降;與單獨IR組相比,0.5 mM VPA與IR聯(lián)合組在2 Gy、4 Gy及6 Gy各劑量下的細胞存活率均明顯降低(P0.05),降低的比例分別為23.3%,32.4%及43.6%。3.安全和臨界劑量VPA能夠抑制細胞HR修復通路輻射后24 h時,0.5 mM或1.0 mM VPA與IR聯(lián)合組含有H2AX焦點細胞的陽性率顯著地高于單獨IR組的2.24倍和3.43倍(P0.05),0.5 mM或1.0 mM VPA與IR聯(lián)合組含有53BP1焦點細胞的陽性率也顯著地高于單獨IR組的1.27倍和1.62倍(P0.05)。輻射后6 h,VPA與IR聯(lián)合組含有BRCA1焦點的細胞陽性百分比明顯低于單純IR組,降低的比例分別為26.5%(P0.05);輻射后24h,VPA與IR聯(lián)合組含有BRCA1焦點的細胞陽性百分比也低于單純IR組,為15.3%(P0.05)�？寺⌒纬蓪嶒灲Y(jié)果提示,0.5 mM VPA或者1.0 mM VPA與10μM ABT888聯(lián)合作用使細胞克隆形成率均顯著低于其他各組(P0.05)。4.安全和臨界劑量VPA能夠抑制細胞NHEJ修復通路與對照組相比,單獨0.5 mM或1.0 mM VPA處理組I-SceI誘導的NHEJ重組效率分別降低至63.8%(P0.01)或61%(P0.01),兩處理組之間無明顯差異(P0.05)。KU80蛋白表達水平在單獨VPA組和VPA與IR聯(lián)合處理組表現(xiàn)為顯著下降,但DNA-PKcs和KU70蛋白表達水平在各組細胞中無明顯變化。5.安全劑量VPA對骨肉瘤細胞的細胞周期無明顯影響單獨0.5 mM或1.0 mM VPA處理組在G1、S與G2/M各期的細胞比率與對照組沒有顯著差異。6.安全劑量VPA能夠增加骨肉瘤細胞基因組不穩(wěn)定性對于無VPA處理組,輻射處理前后細胞的染色單體與染色質(zhì)斷裂百分率無統(tǒng)計學差異(P0.05),但是輻射狀異常染色體結(jié)構(gòu)形成的百分率由1.59%增加到7.34%(P0.05)。與單獨VPA處理組相比,VPA與IR聯(lián)合組的細胞染色單體斷裂的百分率由4.57%增加到17.24%(P0.05)、染色質(zhì)斷裂的百分率由18.27%增至到43.1%(P0.05)以及輻射狀異常染色體結(jié)構(gòu)形成的百分率也表現(xiàn)出相同的變化趨勢。結(jié)論1.應答DNA損傷反應時,安全劑量和安全臨界劑量下的VPA都可進一步增加IR所致的DNADSBs損傷在骨肉瘤細胞內(nèi)的蓄積。2.安全劑量下的VPA能夠抑制骨肉瘤細胞生長并且增加細胞對輻射的敏感性。3.安全劑量下的VPA增加骨肉瘤細胞對輻射的敏感性可能是通過干擾BRCA1介導的HR和KU80介導的NHEJ分子機制所致。4.安全劑量下的VPA能夠增加骨肉瘤細胞基因組不穩(wěn)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.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R738

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9 朱U，

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