35 2 Vol 35 No 2 2016 6 Journal of South-Central University for Nationalities Nat Sci Edition Jun 2016 ASICs TNF-α 1 1 2 3 4 * 1 430074 2 437100 3 430200 4 437100 ASICs TNFα PCR ph 7 3 ph 5 5 ASICs TNF-α TNF-α ASICs TNF-α ASICs 1 PcTx1 TNF-α ASICs 3 APETX2 TNF-α ASICS 1 TNF-α PCR R392 9 A 1672-4321 2016 02-0051-05 Promoted TNF-α Expression of by ASICs under Acidic Tumor Microenvironment Hu Xin 1 Yi Pin 1 Ming Yin 2 Tong Shenglan 3 Gong Huihong 4 1 College of PharmacySouth-Central University for NationalitiesWuhan 430074China 2 Xianning City Spa Red Flag Road Middle School of Hubei ProvinceWuhan 437100China 3 Clinical Laboratory the First People's Hospital of Jiangxia District in WuhanWuhan 430200China 4 College of Biomedical EngineeringHubei University of Science and TechnologyXianning 437100China Abstract To investigate the relationship between the TNF-α expression and ASICs in under the tumor microenvironmentreal-time PCR was performed to determine the TNF-α expression changes in mice under normal physiological ph 7 3 acidic ph 5 5 and inhibitor-added conditionrespectively The results indicated that the expression of TNF-α significantly elevated in tumor under normal physiological condition as well as acidic condition In contrastthe expression was significantly inhibited by the addition of ASICs' nonspecific inhibitors amiloridediclofenacor ASICs subunit 1 inhibitor PcTx-1 Howeverpretreatment with ibuprofen or ASIC subunits 3 inhibitors APETX2 had no effect So the ASICs subunit 1 of was likely responsible for promoting the expression of TNF- α under the acidic tumor microenvironment Keywords TNF-α real-time PCR ASICs inhibitor 2015-11-26 * 1979- E-mail ghh7322@ 126 com 1977- E-mail huxin5540@ 126 com 81201610 2015ZY008 JYX14013
52 35 1 1 1 TAM 2 3 Percoll GE Hyclone cdna RPMI-1640 TNF-α 4 TNF-α 17 Thermo PCR kd stnf-α 26 kd tmtnfα 5 TNF-α NO ROS T Green Takara SW-CJ-2FD TNF-α CO 2 BPN-150CW Thermo IMS-50 6 7 GTR10-1 T TNF-α PCR 7500 8 9 10 11 1 2 Blab /c 18 ~ 20 g 6 ~ 8 ph 5 8 ~ 7 41 3 ph 7 2 ~ 7 4 5 mm 0 133 g 5mL 100 μm IL-22-20 500 mm 0 08 LAK g 5 ml TNF- 200 μm - 20 100 mm α IFN-γ TGF-β IL-10 IL-12 0 1g DMSO 200 μm 12 ASICsH + H + 4 26 ml 500 nm ENaC /DEG 10 nm APETX2 5 μg APETX2 ASICs 548 μl 2 mm 13 BMM T ASICs 2 1 ASICs TNF-α ml H22 3 ~ 4 Blab /c Blab /c TNF-α 6 Blab /C 1 10 6 H22 PCR 200 TNF-α ph 5 5 1000 r /min 10 min TNF-α ASICs 1 RNA DNA Marker RealTime PCR Master Mix SYBR ABI H22-20 PcTx1 10 μg PcTx1 100 nm 2 Blab /c 5 10 6 /0 2 75% 5 min EP 5 ml TNF-α 10 min
2 ASICs TNF-α 53 5 ml 10% FCS-RIPM 1640 25 3 min 200 2 4 1000 r /min 10min 2 ml 10% FCS-1640 p < 0 05 Percoll 2 ml PBS 2 ml 50% 60% 70% 100% Percoll 2900 r /min 30 min 2 80% MDSC 2 2 RNA ASICs 1 10 7 2000 g 5 min RNA cdna 2 3 30 mg PCR 7 2 μl 400 μl BufferR-I 1 ml 1 0 μl cdna 0 8 μl Master Mix 10 μl 8 ~ 10 1 5 PCR ml 150 μl BufferR- PCR II 15 ~ 30 s 12000 g 4 5 min Ct PCR 1 1 5 ml EP 250 μl Delta-delta Ct 2 ml EP mrna = 2 n n = 1 6000 Ct - Ct g 4 2 min EP Ct - Ct 2 ml EP 500 ml Buffer W1A 12000 g 3 min EP TNF-α 1 2 2 ml EP 700 μl BufferW2 12000 g 3 min 1 2 ml EP 12000 g 3 min 1 1 5 ml EP 70 μl 12000 g 4 3 min RNA 2 3 PCR Genebank TNF-α mrna NM-013693 2 TNF-α 700 bp 5'- ACCCTCACACTCAGATCATCTTCTC-3' 5'- 1 CAGATTGACCTCAGCGCTGAGTTG-3' β-actin 300bp 5'-TCACCCACACTGTGCCCA Fig 1 TCTACGA-3' 5'-CATCGGAACCGCTCGTTGC CAATAG-3' 96 200 μl PCR 94 3 min 94 30 s 57 4 30 s 72 50 s 35 72 3 min GraphPad 3 3 1 ASICs TNF-α ph 7 3 ph 5 5 16 h * p < 0 05 ** p < 0 01 *** p < 0 001 N7 3 normalph 7 3 T5 5 tumorph 5 5 ph TNF-α TNF-α expression in of various group under different ph or with the addition of amiloride 1 2 ph 7 3 TNF-α
54 35 * p < 0 05 ** p < 0 01 *** p < 0 001 N7 3 normalph7 3 T7 3 normalph7 3 T5 5 tumor ph5 5 2 3 PH PcTx1 APETX2 ph TNF-α TNF-α Fig 2 TNF-α expression in of various group under different ph or with the addition of diclofenac and ibuprofen Fig 2 TNF-α expression in of various group under different ph or with the addition of PcTx1 and APETX2 TNF-α TNF-α TNF-α p < 0 01p < 0 001 TNF-α ASICs TNF-α TNF-α TNF-α 3 2 ASICs 1 3 PcTx-1 RNA cdna PCR 3 1 TNF-α 3 PcTx1 TNF-α p < 0 01 APETX2 ASICs TNF-α TNF-α ASICs 1 TNF-α TNF-α ph ASIC1 ASIC3 ASIC2 ph TNF-α ASIC2 PcTx1 APETX2 ASIC1 ASIC2 PCR TNF-α 4 * p < 0 05 ** p < 0 01 *** p < 0 001 N7 3 normal ph7 3 T7 3 normal ph7 3 T5 5 tumor ph5 5 tmtnf-α NO IL-10 TGF-β APETX2 TNF-α CD4 + T CD8 + T MDSC ph ph 5 5pH 7 3 16 h ASICs PcTx1 APETX2 H + ASICs ph ASICs T DC BMM TNF-α TNF-α TNF-α ph TNF-α MDSC
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