1、 毕业论文(设计)题目 0 姜黄素介导 NSC-34 细胞抗氧化应激能力的调节专业 药学目 录0文00 10文00 200 30文 41 00000 41.1 00000 41.2 细胞00000 41.3 CCK-8 00000细胞0力 51.4 00细胞0 MDA0 GSH 00 51.5 Real-time PCR 00细胞0 GST0 GCLC 的000化 71.6 0计000 72 00 82.1 细胞000 82.2 姜黄素0000细胞0 MDA0 GSH 00的00 92.3 姜黄素调节细胞0 GST0 GCLC 的000化 113 0论 130论 1400文0 1600 190
2、0 241摘 要目的0 00 NSC-34 细胞00氧化应激细胞00 hSOD1 G93A 000 ALS 细胞0000000抗氧化0姜黄素0细胞抗氧化应激0的00000000000000化000的000000的00000000 CCK-8 00000000000的 H2O2 0姜黄素00细胞0细胞0力的0000000计00000姜黄素00000 H2O2 0000 hSOD1 G93A000 NSC-34 细胞0000 (MDA)00000 (GSH)的000化00 real-time PCR 00细胞0 GSH 0000 GST0 GCLC 的000化0000 CCK-8 00000000
3、00 H2O2 0姜黄素0细胞0力的00000000000 10 M H2O2 0 10 M 姜黄素00000000000000H2O2(10 M)00的00 hSOD1 G93A 000 NSC-34 细胞0000000000000姜黄素 (10 M)0000细胞0 MDA 0000000氧化应激0000000 H2O2 00 24 h 0000细胞0 GSH 00000姜黄素 (10 M)000000000细胞0 GSH 的000000 real-time PCR 0000姜黄素0000细胞0 GST 0 GCLC mRNA 的0000 (P 0.01)00论0 姜黄素0能00000细胞抗
4、氧化0000的调节000 ALS 细胞00抗氧化应激的能力00000 姜黄素0000000化0000氧化应激0000000002AbstractObjective: The effect of curcumin on the antioxidant stress in the cells was established by using NSC-34 cells to establish a model of oxidative stress injury. It provides a solid experimental basis for the development of drugs
5、for prevention and treatment of amyotrophic lateral sclerosis syndrome.Methods: CCK-8 Colorimetric Method were used to detect the effects of H2O2 and Curcumin-treated Cell on cell viability. Spectrophotometer were used to detect the changes of MDA and GSH in hSOD1G93A mutant transfected NSC-34 cells
6、 (SOD1G93A NSC-34) treated with H2O2 after pre-treated with curcumin. And the changes of GST and GCLC in the cells were detected by real-time PCR. Results: CCK-8 results were compared with different concentrations of H2O2 and curcumin on the cell viability, we selected the final concentration of 10
7、M H2O2 and 10 M curcumin as a follow-up study conditions. Compared with the transfected hSOD1 G93A mutant NSC-34 cells treated with H2O2 (10 M), spectrophotometry showed that the content of MDA in the cells pretreated with curcumin (10 M) was significantly decreased and the oxidative stress was sign
8、ificantly improved. After incubation with H2O2 for 24 h, the concentration of GSH in the cells was significantly decreased, and the concentration of GSH was increased by co-incubation with curcumin (10 M). In addition, real-time PCR results showed that the expression of GST and GCLC mRNA was increas
9、ed by curcumin pretreatment (P 0.01).Conclusion: Curcumin may enhance the ability of anti-oxidative stress in ALS cell models by enhancing the regulation of cell antioxidant production.Key words: Curcumin; Amyotrophic lateral sclerosis; Oxidative stress; MDA; GSH3前 言0 0 0 0 0 0 化 0 (Amyotrophic late
10、ral sclerosis, ALS)0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 的 0 0 0 0 0 0 0 0 0 0 0 0 0 0 的 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 45 0 65 0 0 0 0 0 0 0 0 化 0 0 0 ALS 00 0 0 0 0 目 0 ALS 0 0 0 0 0 能 0 0 的 0 0 0 0 0 0 0 0 0 0 0 0 的 0 00 0 1,20 0 0 0 0 0 0 0 0 0 0 0 的 0 0 0 0 00 0 0 0 0 0 0 ALS 0 0 0 0 0 0 0
11、 0 0 0 0 氧 化 应 激 0 0 0 0 0 0 0的 0 0 0 0 0 0 0 0 0 能 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 素 0 0 00 氧 化 0 0 调 节 0 0 导 0 的 氧 化 应 激 0 0 0 0 0 氧 化 应 激 0 0 导 0 0 0 0 0 00 0 0 (0 0 0 0 0 0 0 DNA 0 )的 氧 化 0 0 30 0 0 0 0 0 导 0 0 0 细 胞 0能 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 化 0 0 0 0 0 0 0 0 能 0 0 0 0 0 0 0 00 0 0
12、 0 0 0 的 0 0 0姜 黄 素 (curcumin)0 0 姜 0 姜 黄 0 0 0 姜 黄 (Curcumalonga L.)0 0 0 00 0 0 0 0 0 0 0 0 0 0 化 0 0 0 0 0 0 0 0 0 抗 0 0 抗 氧 化 0 抗 0 0 0 抗 00 0 抗 0 0 0 抗 0 0 抗 0 0 0 化 0 抗 0 0 0 0 0 化 0 0 0 0 0 的 0 0 0 0 0 00 0 0 0 0 0 0 应 0 40 0 0 0 0 姜 黄 素 0 0 氧 0 0 0 0 0 0 0 0 0 0 0 的0 0 能 力 0 0 0 细 胞 0 0 0 0
13、0 0 0 0 (glutathione, GSH)0 0 0 0 0 0 0H2O2 0 0 的 细 胞 0 0 0 氧 (reactive oxygens pecies, ROS)0 0 0 0 0 细 胞 0 氧 化0 0 0 0 0 0 0Dong H 0 0 的 0 0 0 0 0 0 15 M 姜黄素0000000细胞0000000000 (FALS)0000 (SALS)0000细胞00氧化应激00000能00导0的00000 500000姜黄素000 ALS 000000的0力00 0 0 0 0 0 0 姜 黄 素 0 细 胞 抗 氧 化 应 激 能 力 的 调 节 0 0
14、0 姜 黄 素 的 0 00 0 0 0 0 0 0 0 0 0 ALS 0 0 的 0 0 0 0 0 论 0 0 04正 文1 方法与材料1.1 00000姜黄素 Cayman Chemical 00H2O2 00 XX 00000000Cell Counting Kit-8 (CCK-8 000 ) 00 XX 0000000000氧化00000 MDA00000 00 XX 0000000000000氧化000 GSH00000 00 XX 000000000000 0000000000000DMEM 00000 HyCloneThermo Fisher Scientific 0000
15、0000 Multiskan MK3 00 Thermo 000氧化00000 HERAcell 150i 00 Thermo 00000000计0 V-5000 00 METASH 00real-time PCR(CFX96TM Optics 0 ) singapore 001.2 细胞00000NSC-34 细胞000000细胞00000 (1214 0 )00000000000的000000细胞000000000000000000000000000000000000000000 ALS 00的000000000000的000000000000000000000000000000 NSC-
16、34 细胞00000 10 %0000的 DMEM 00000 37 0 0 5 % CO2 的000000000 48 h 00000000000细胞000 80 % - 90 %0000000000000细胞000000 NSC-34 细胞0 2 106/0000 6 0000 37 0 0 5 % CO2 000000 24 h 0000 2 g pEGFP-G93A-SOD1 0000000000000 Lipofectamine2000 00000000000 37 0 0 5 % CO2 00000 6 h 000500000000 000 24 h 00000001.3 CCK-
17、8 00000细胞0力0000的 NSC-34 细胞0 10000 0 /0000 96 00000000 37 0 0 5 % CO2 00000 24 h 000000001.3.1 H2O2 的0000的000000000 5 M 0 10 M 0 20 M 0 40 M 的 H2O2 00细胞00 0 M H2O2 00000000设0 6 0000000 37 0 0 5 % CO2 00000 24 h000 CCK-8 00000细胞0力0000000000 H2O2 的000001.3.2 姜黄素的0000的000000000 5 M 0 10 M 0 20 M 0 40 M
18、姜黄素00细胞00 0 M姜黄素00000000设0 6 0000000 37 0 0 5 % CO2 00000 24 h000 CCK-8 00000细胞0力0000000000姜黄素的00000CCK-8 0000(1) 00000细胞0000000000000000 37 0 00的的 100 l0000000000000(2) 0000000000000 24 h0(3) 00000000 10 l CCK-8 0000000000000的0000000000000(4) 00000 37 0 0 5 % CO2 000000 4 h 000000(5) 00000000 450 n
19、m 0的000000 (OD 0 )0计0细胞0000细胞的000 =0000的 OD 0 00 OD 0 (000000 CCK-800细胞 )00000的 OD 0000 SD 0000细胞的0000 T/C %000 T 000细胞的 OD 00 C 000细胞的 OD 00细胞000 %=(00细胞的 OD 0 /00细胞的 OD 0 )100 %00 T/C = 50%0的00001.4 00细胞0 MDA0 GSH 00SOD1G93A 00细胞000000000000000000 10 M H2O2 00 24 h 0000000 10 M 姜黄素00000 4 h000000
20、10 M H2O2 000 24 h00000000细胞000细胞00000 MDA0 GSH 00000000060000000000000计0000000000的0000000计0细胞0 MDA0 GSH 000化0000000 100 200 1 MDA 00000000000000000000000000000000000000000000000000 95 0 00 40 min 0000000000000000计000 532 nm 00000 1 cm 00000000调0000000000MDA 00 (nM/mgprot)=(00 OD 0 -00 OD 0 )(00 OD
21、0 -00 OD 0 )00000 (10 nM/ml)00000000 (mgprot/ml)GSH 0000000000000000 0.5 ml 0000 2 ml 000 35004000 rpm/min 00 10 min0000000000应00 2 GSH 000应000 000 000000 (ml) 1.020 M/l GSH 00 (ml) 1.0000 (ml) 1.0000 (ml) 1.25 1.25 1.25000 (ml) 0.25 0.25 0.25000 (ml) 0.05 0.05 0.050000000000000000 5 min,000000计000
22、420 nm 00000 000 000 00010nM/ml 000 0.10000 (ml) 0.10000 (ml) 0.1 0.1000 I(ml) 4 4 4 4000 II(ml) 47000 1 cm 00000000调000000的0000 0GSH 00 (mgGSH/gprot)=(00 OD 0 -00 OD 0 )(00 OD 0 -00 OD 0 )00000 (2010-3 mM/l)000000000 00000000 (gprot/l)01.5 Real-time PCR 00细胞0 GST0 GCLC 的000化0000000细胞000细胞00000 Triz
23、ol 000000000细胞0RNA 000 Promega 0000应00000000应000000000000000000 PCR 000GST 00000000 GGATGGGGACTTCGTCTTGG00000TCAGGAGGTACGGGCTGTC0GCLC 00000000 GGGGTGACGAGGTGGAGTA00000GTTGGGGTTTGTCCTCTCCC0-actin 00000000 TTTCCAGCCTTCCTTCTTGGGTATG00000ATAGAGGTCTTTACGGATGTCAACG0PCR 0应000 95 0 000 10 min0 95 0 00 15 se
24、c0 58 0 00 1 min0 72 000 1 min000 40 0000000000 20 00 Ct 00001.6 0计00000000000 000 (mean S.E.M.)000应0 GraphPad Prism 5 0计000000素0000 (One-way ANOVA)000 Newman-Keuls 00000000 P 0.05 000000计000082 结果2.1 细胞0000000 CCK-8 000000000000 (0 - 40 M) H2O2 0000000 (0 - 40 M) 姜黄素0 SOD1G93A 00 NSC-34 细胞000的00000
25、000000000000002.1.1 H2O2 0000的0000 1 00000000000 5 M H2O2 0 SOD1G93A 00 NSC-34 细胞00000000 30 40 5 00000 10 M0 20 M0 40 M H2O2 00细胞0000000000的 58 %0 37 %0 26 %00000计000 (P 0.01)0000000 10 M H2O2 0激细胞 24 h 0000000000 1 H2O2 0导 SOD1G93A 00 NSC-34 细胞00的0000000000 S.E.M0 n = 40 * P 0.01(000000 )Fig1 Effe
26、cts of H2O2 induced cell insult in SOD1G93A transfected NSC-34 cellEach bar represents the meanS.E.M. n=4, *P 0.01 compared with control2.1.2 姜黄素0000的0000 2 00000000000 5 M0 10 M0 20 M 姜黄素0 SOD1G93A 00 NSC-34 细胞000000000000计0000 40 M 姜黄素0细胞00000000的 72 %00000000细胞000000000000计000 (P 90.01)0000000 10
27、 M 00姜黄素的000000000000000姜黄素10 M 00000细胞0 MDA 00000000 GSH 000000 (P 0.01)(030 4)00000000 10 M 00姜黄素00000 2 姜黄素0导 SOD1G93A 00 NSC-34 细胞000的0000000000 S.E.M0 n = 4, * P 0.01(000000 )Fig2 Effects of curcumin induced cell insult in SOD1G93A transfected NSC-34 cellEach bar represents the meanS.E.M. n=4,
28、*P 0.01 compared with control2.2 姜黄素0000细胞0 MDA0 GSH 00的002.2.1 细胞0 MDA 00的0化00 3 000000000000000 10 M H2O2 00的 SOD1G93A 00NSC-34 细胞0 MDA 的0000000000000计000 (P 0.01)0 10 M 姜黄素000 4 h 00 10 M H2O2 0000 24 h 0000000 10 M H2O2 000细胞0 MDA 0000000氧化00000000000000计000 (P 0.01)0100 3 姜黄素0 H2O2 0导的 SOD1G93A
29、 00 NSC-34 细胞0 MDA 00的0000000000 S.E.M0 n = 4, * P 0.01(000000 )0 #P 0.01(0 H2O2 000 )Fig3 Effects of curcumin on H2O2 induced MDA production in SOD1G93A transfected NSC-34 cellEach bar represents the meanS.E.M. n=4, *P 0.01 compared with control; #P 0.01 compared with the H2O2 group2.2.2 细胞0 GSH 00
30、的0化00 4 00000000000 SOD1G93A 00 NSC-34 细胞0000 10 M H2O2 000细胞0 GSH 0000 (P 0.05)0000细胞00 10 M 姜黄素00000 4 h000000 10 M H2O2 0 24 h000000000000 10 M 姜黄素000细胞0000000000000细胞0 GSH 的00 (P 0.01)00 4 姜黄素0 H2O2 0导的 SOD1G93A 00 NSC-34 细胞0 GSH 00的0000000000 S.E.M0 n = 4, * P 0.05, * P 0.01(000000 )0 #P 0.01(0
31、 H2O2 000 )Fig4 Effects of curcumin on H2O2 induced GSH production in SOD1G93A transfected NSC-34 cellEach bar represents the meanS.E.M. n=4, *P 0.05, *P 0.01 compared with control; #P 0.01 compared with the H2O2 group112.3 姜黄素调节细胞0 GST0 GCLC 的000化00 5 000 Real-time PCR 00000 00000000 SOD1G93A 00NSC
32、-34 细胞0000 10 M H2O2 000细胞0 GST (A)0 GCLC (B)0000 (P 0.05)0000000姜黄素000000000000000细胞0 GST0 GCLC的00000000计000 (P 0.01)00 5. 姜黄素0 H2O2 0导的 SOD1G93A 00 NSC-34 细胞0 GST, GCLC mRNA 00的000000000000 S.E.M0 n = 4, * P 0.05, * P 0.01(000000 )0 P 0.01(0 H2O2 000 )0Fig5. Effects of curcumin on H2O2 induced GST
33、, GCLC mRNA expression in SOD1G93A transfected NSC-34 cell.Each bar represents the meansS.E.M. n=4, *P 0.05, *P 0.01 compared with control; #P 0.01 compared with the H2O2 group.123 讨论ALS 000000的0000000000000000能的000000的00 600000000000000的0000 700000000000000的0000000的0000000氧化应激000000000的000000000000
34、0激0000细胞0000000000000 80氧化应激0细胞0氧化000抗氧化000000000的00应激0000000000氧0000000000000000000000的抗氧化000能00000氧00细胞00000000 DNA 00000000000细胞000000导0细胞的00 900 ALS 000000000000000氧化的0000 DNA0000000000的00 1000000000 20 %的000 ALS(FALS)00000000氧化00化0 (superoxidedismutase 1, SOD1)000000 G93A 00000000000000的0000000
35、000的00 7000氧化应激0 ALS 的00000000000000000抗氧化000000的0力0H2O2 0000000000000的00氧000000000000氧化00的0导0 1100000 H2O2 00 SOD1G93A 00 NSC-34 细胞0 MDA 00000 GSH 000000 H2O2 0导0 SOD1G93A 00 NSC-34 细胞氧化00000抗氧化能力000MDA 000细胞0氧化应激00的000000000细胞00氧化000的0000000的0应细胞氧化00的00 12,130 GSH 00000000000能的0000000000000000氧000
36、000000000000 GSH 00000细胞0抗氧化000000 SOD 0 CAT 的00000的00000000细胞00氧化00000000化0000导00000的 GSH00000MDA 0000的 14000000 GSH 00000的0000000的抗氧化应激00000000 -0000000000 (-GCS)0 0000000000 (GCL)00000 S 000 (GSTs)000000氧化00 (GPx)000素0氧0 (haem oxygenases, Hos)00000 GCL 0000 GSH 00的00000的0化00(GCLC)00000调 GCL 00000
37、0细胞0 GSH 0000导 GSH 0000 15013GST 0000抗氧化0000000000的0000000化000应000000的0氧化0应00000抗氧化000Jaroonwitchawan T 00000氧化应激000000000细胞00的000000姜黄素0能00000000000氧 (reactive oxygens pecies, ROS)的00000000能0000的0000000 160000000姜黄素0000细胞0000000导的氧化应激000细胞00 (iNOS 0 COX-2)的000000000ALS 0000000 ROS 00000导的 COX-2 的00
38、 17,180000姜黄素000000导00细胞000氧的00000细胞0 GSH 00000调节细胞0氧化00000抗细胞000细胞0000 1900000000姜黄素0能00000000的000000000000000姜黄素000000000000000000000激00细胞0000导细胞00 20000000姜黄素00细胞的00000000000000000000000的姜黄素00的细胞0000000000的00000000000的00000000000 (10 M)00姜黄素000000000000000 10 M 姜黄素000 4 h 0细胞0 MDA 0000000 GSH 000
39、0000Samarghandian S 00000姜黄素0应激0000导的00000氧化应激00的0000000000000 MDA0 GSH0 SOD00氧化00 (CAT)的000000000应激00000 MDA 000000 GSH 0 SOD 的00000000000姜黄素00000000000000姜黄素0000应激0导的00的00000000氧化00 21,220000000000000000 10 M 姜黄素0004 h 00000000 H2O2 000细胞0的氧化00000000细胞0 MDA 000000细胞0 GSH 的000 real-time PCR 0000姜黄素
40、0000细胞0GST 0 GCLC mRNA 的00000000000姜黄素0000细胞0抗氧化应激00的000000000000000000000000 ALS 的0000000的0000014结 论000000000 H2O2 000导00 hSOD1G93A 000 NSC-34 细胞000氧化应激000000000 10 M H2O2 0导0细胞0 MDA 00000 GSH 的0力0000000 10 M 姜黄素000 4 h 0细胞0氧化应激00000000000000 H2O2 0细胞00的000000000 MDA 的000000细胞0GSH 的00000000 GST0 GC
41、LC 的00000姜黄素00细胞氧化应激00的00000000000000000000000015参考文献1 Chi A, Logroscino G, Traynor B J, et al. Global epidemiology of amyotrophic lateral sclerosis: a systematic review of the published literature.J. Neuroepidemiology, 2013, 41(2): 118.2 00 . 000素000 hSOD1-G93A 的 NSC-34 细胞的000000000 D. 000000 , 201
42、5: 15-18.3 Teresa C M, Cristiana V, Francesca B, et al. Oxidative stress and mitochondrial damage: importance in non-SOD1 ALSJ. Front Cell Neurosci, 2015, 9(5): 41-48.4 Jurenka J S. Anti-inflammatory properties of curcumin, a major constituent of Curcuma longa: a review of preclinical and clinical researchJ. Altern Med Rev, 2009, 14(2): 141-153.5 Dong H, Xu L, Wu L, et al. Curcumin abolishes mutant TDP-43 induced excitability in a motoneuron-like
