新制癌菌素诱导小鼠海马神经元细胞DNA损伤的作用研究
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摘要
为了探索化学诱导物新制癌菌素(NCS)对小鼠海马神经元细胞株HT22 DNA损伤的影响,研究以9Gy X射线辐照为阳性对照组,通过CCK8试剂盒法检测绘制NCS作用下HT22细胞生长曲线以明确NCS处理细胞的最佳浓度,经Hoechst染色和TUNEL染色观察该剂量NCS对细胞DNA损伤及凋亡的影响,用免疫荧光染色结合Western blot分析HT22细胞核中DNA损伤反应酶PARP1的表达变化.结果显示,NCS作用的终质量浓度为1 000 ng/m L时,从48 h起对HT22细胞增殖表现出显著的抑制作用;Hoechst染色可见NCS作用下HT22细胞DNA损伤加剧,TUNEL染色提示NCS能够诱导细胞凋亡;免疫荧光染色和Western-blot实验显示NCS能够促进细胞核内PARP1的表达.说明化学诱导剂NCS能够引起HT22细胞DNA损伤及凋亡以及上调DNA损伤反应酶PARP1的表达,可以用于模拟大剂量射线对细胞的辐射损伤效应.
To explore the effect of chemical inducer neocarzinostatin( NCS) on the DNA damage of mouse hippocampal neuron cell line HT22,9 Gy X-ray irradiation was used as a positive control group.The growth curve of HT22 cells after NCS treatment were detected by CCK8 kit method to verify the optimal concentration of NCS in treating cells in this study.Hoechst staining and TUNEL staining were used to investigate the effects of NCS at optimal concentration on the cellular DNA damage and apoptosis. Immunofluorescence staining and Western blot were used to analyze the expression changes of PARP1-DNA damage reaction enzyme in HT22 cell nuclei. The results showed that when the concentration of NCS was 1 000 ng/m L,the proliferation of HT22 cells were significantly inhibited from 48 h; Hoechst staining showed that DNA damage of HT22 cells were exacerbated by NCS.TUNEL staining suggested that NCS can induce cellular apoptosis.Immunofluorescence staining and Western blot showed that NCS can promote the expression of PARP1 in the nucleus.The results indicated that chemical inducer NCS can induce DNA damage and apoptosis in HT22 cells and up-regulate the expression of PARP1. It can be used to simulate the radiation damage effect of high dose radiation on cells.
To explore the effect of chemical inducer neocarzinostatin( NCS) on the DNA damage of mouse hippocampal neuron cell line HT22,9 Gy X-ray irradiation was used as a positive control group.The growth curve of HT22 cells after NCS treatment were detected by CCK8 kit method to verify the optimal concentration of NCS in treating cells in this study.Hoechst staining and TUNEL staining were used to investigate the effects of NCS at optimal concentration on the cellular DNA damage and apoptosis. Immunofluorescence staining and Western blot were used to analyze the expression changes of PARP1-DNA damage reaction enzyme in HT22 cell nuclei. The results showed that when the concentration of NCS was 1 000 ng/m L,the proliferation of HT22 cells were significantly inhibited from 48 h; Hoechst staining showed that DNA damage of HT22 cells were exacerbated by NCS.TUNEL staining suggested that NCS can induce cellular apoptosis.Immunofluorescence staining and Western blot showed that NCS can promote the expression of PARP1 in the nucleus.The results indicated that chemical inducer NCS can induce DNA damage and apoptosis in HT22 cells and up-regulate the expression of PARP1. It can be used to simulate the radiation damage effect of high dose radiation on cells.
引文
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[3]REZVANI M,BIRDS D A,HODGES H,et al.Modification of radiation myelopathy by the transplantation of neural stem cells in the rat[J].Radiat Res,2001,156(4):408-412.
[4]DOROVSKIKH G N,KOZHEDUB S A,GORLINA ALU,et al.Radiodiagnosis of cervical spine lesions[J].Vestn Rentgenol Radiol,2012,5-6(3):12-9.
[5]张晶,魏丽,孙万良,等.大鼠放射性脊髓损伤后神经元数量变化规律[J].现代生物学进展,2013(7):1 219-1 222.ZHANG J,WEI L,SUN W L,et al.Regulation of number of neurons following radiation-induced spinal cord injury in rats[J].Progress in Modern Biomedicine,2013,7:1 219-1 222.
[6]OKADA S,OKEDA R.Pathology of radiation myelopathy[J].Neuropathology,2001,21(4):247-265.
[7]WALD-ALTMAN S,PICHINUK E,KAKHLON O,et al.A differential autophagy-dependent response to DNA double-strand breaks in bone marrow mesenchymal stem cells from sporadic ALS patients[J].Dis Model Mech,2017,10(5):645-654.
[8]BERNADETTE H,GUILHEM L,ELISABETH A,et al.Reinvestigation of the proteolytic activity of neocarzinostatin[J].J Bacteriol,2000,182(7):1 812-1 818.
[9]ALBERT J M,CAO C,KIM K W.Inhibition of poly(ADP-ribose)polymerase enhances cell death and improves tumor growth delay in irradiated lung cancer models[J].Clin Cancer Res,2007,13:3 033-3 042.
[10]SINHA S,GHILDIYAL R,MEHTA V S,et al.ATMNFk B axis-driven TIGAR regulates sensitivity of glioma cells to radiomimetics in the presence of TNFα[J].Cell Death Dis,2013,4(5):e615.
[11]KUROMIZU K,TSUNASAWA S,MAEDA H,et al.Reexamination of the primary structure of an antitumor protein,neocarzinostatin[J].Arch Biochem Biophys,1986,246(1):199-205.
[12]NAPIER M A,HOLMQUIST B,STRYDOM D J,et al.Neocarzinostatin:spectral characterization and separation of a non-protein chromophore[J].Biochem Biophys Res Commun,1979,89(2):635-642.
[13]KAPPEN L S,NAPIER M A,GOLDBERG I H,et al.Roles of chromophore and apo-protein in neocarzinostatin action[J].Proc Natl Acad Sci U S A,1980,77(4):1 970-1 974.
[14]杨茂裕,顾觉奋.双烯二炔类抗癌药——新制癌菌素的研究进展[J].抗感染药学,2014,11(2):89-92.YANG M Y,GU J Q.Advances in researches on enediyne:Neocarzinostatin[J].Anti Infect Pharm,2014,11(2):89-92.
[15]FENG X,KOH D W.Roles of poly(ADP-ribose)glycohydrolase in DNA damage and apoptosis[J].Int Rev Cell Mol Biol,2013,304:227-281.
[16]KIM B K,IM J Y,HAN G,et al.p300 cooperates with c-Jun and PARP1 at the p300 binding site to activate Rho B transcription in NSC126188-mediated apoptosis[J].Biochim Biophys Acta,2014,1839(5):364-373.
[17]MC CORMICK A,SWAISLAND H.In vitro assessment of the roles of drug transports in the disposition and drug-drug interaction potential of olaparib[J].Xenobiotica,2016,47(10):1-47.