ISSN 1007-7626 CN 11-3870 / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology 2011 8 27 8 755 ~ 760 ω-3 fat-1 1 2 1 * 1 1 1 2 1 266021 2 266109 ω-6pufas / ω-3pufas ω- 3PUFAs ω-6pufas ω-3pufas ω-3 Caenorhabditis elegans ω-3 fat-l cdna pegfpc1-fat-1 RT- PCR pegfpc1-fat-1 ω-6pufas / ω-3pufas MTT P < 0 05 fat-1 ω-6pufas ω-6pufas ω-3pufas 3T3 ω-3 Q812 Overexpression of ω-3 Fatty Acid Dehydrogenase Gene Protects Mouse Embryonic Fibrocytes from ω-6 PUFAs-induced Apoptosis 1 LI Fang-Fang 2 GE Yin-Lin 1 * XUE Mei-Lan 1 ZHANG Jin-Yu 1 LI Quan 1 SHAN Hu 2 1 Department of Biochemistry and Molecular Biology Medical School of Qingdao University Qingdao 2 School of Animal Science and Veterinary Medicine Qingdao Agricultural University Qingdao 266021 China 266109 China Abstract ω-3 polyunsaturated fatty acids ω-3pufas are important for the normal function of mammals However it is difficult to gain ω-3pufas in human body and there is no ω-3pufas dehydrogenase in vivo to catalyze ω-6pufas into ω-3pufas But the expressed product of fat-1 gene from Caenorhabditis elegan is ω-3 PUFAs dehydrogenase Hence to express fat-1 gene in mouse embryonic fibrocyte is the aim of this study First the eukaryotic expression vector pegfpc1-fat-1 containing fat-1 gene cdna was constructed and transfected into mouse embryonic fibrocyte Then the expression level was detected by RT-PCR and Laser Scanning Confocal Microscope the change of cellular ω-6pufas / ω-3pufas ratio was examined by gas chromatography the inhibiting rate of cells proliferation was observed by MTT method and cell apoptosis was evaluated by flow cytometry Results showed that the cells proliferation rate was higher and cells apoptosis magnitude was lower than those in the control cells In conclusion fat-1 gene could significantly decrease the ratio of cellular ω-6pufas / ω-3pufas and inhibit apoptosis of the 3T3 cell It shows strong cell-protective effects on mouse embryonic fibrocyte 2011-03-15 2011-06-13 No 30671079 No 20061065003 * Tel 0532-82991209 E-mail geyinlin@ 126 com Received March 15 2011 Accepted June 13 2011 Supported by National Natural Science Foundation of China No 30671079 and Foundation from Ministry of Education No 20061065003 * Corresponding author Tel 0532-82991209 E-mail geyinlin@ 126 com
756 27 Key words apoptosis ω-3 polyunsaturated fatty acids dehydrogenase gene expression embryo fibroblast cell PUFAs 6PUFAs 20 4ω-6PUFAs Sigma 1 2 pegfpc1-fat-1 fat-1 cdna WBGene00001393 PUFAs ω-6pufas ω-3pufas 1 2 ω-6pufas 5'-GGGGTACCGCCACCATGGTCGCTCAT TCCT-3' KpnⅠ 5'-CGCGGATCCTCCCTTGGCCTTTGCCTT- 3 4 ω-3pufas 3' BamHⅠ pcaggs-fat-1 PCR fat-1 5 6 cdna PCR ω-6pufas ω-3pufas 4 1 ~ 6 KpnⅠ / BamHⅠ 1 10 1 ~ 30 1 ω- pegfpc1 3PUFAs TOP10 Kanamycin 7 8 ω-6pufas ω-3pufas TaKaRa - 70 ω-3 ω-3pufas 1 3 9 10 DMEM 5 10 5 Caenorhabditis elegans ω-3 24 h 70% 96 fat-1 ω-3 300 ng pegfpc1-fat-1 0 09 μl Xfect ω-6pufas Polymer 24 2 0 μg pegfpc1-fat-1 ω-3pufas fat-1 0 6 μl Xfect Polymer 6 6 μg ω- pegfpc1-fat-1 1 8 μl Xfect Polymer 6PUFAs ω-3pufas pegfpc1 Xfect Polymer 10 s 11 12 fat-1 10 min DNA-Polymer pegfpc1-fat-1 37 5% CO 2 4 h RT-PCR 48 h fat-l ω-6pufas ω-3pufas MTT 1 4 pegfpc1 EGFP 488 nm RT- 1 PCR RNAiso Plus RNA 1 1 fat-1 cdna pcaggs-fat- 1 13 Kit 42 30 min cdna PrimeScript RT-PCR Kit β-actin 5'-CCAAGGCCAACCGCGAGA AGATGAC-3' 5'-AGGGTACATGGTGGTG CCGCCAGAC-3' 490 bp Fat-1 cdna ATG 630 bp TaKaRa Alexa Fluor 488 annexin V and PI for Flow Cytometry kit Invitrogen Xfect Polymer Clontech 75% RNase-free RNA PrimeScript RT-PCR Gibco 5'-GGTTCCCAGTGTACACTCTGTTCGGTTTCTGTGAT DNA Omega 18 2ω- G-3' fat-1 1209 bp 5'-
8 ω-3 fat-1 757 CGCGGATCCTCCCTTGGCCTTTGC CTT-3' 579 bp 94 3 min 94 30 s 55 30 s 72 1 min 30 1 5 48 h 0 01% BHT 0 5 mol / L KOH 1 ml 80 2 h 14% BF3 1 ml 80 1 h Fig 1 The KpnⅠ / BamHⅠ digestion of pegfpc1-fat-1 0 2 ml The Kpn Ⅰ / BamH Ⅰ digested plasmid pegfpc1 and fat-1 PUFAs gene were ligated by T4 DNA ligase at 4 for 16 hours Then 1 6 MTT ligation mixture was transformed into TOP10 competent cells 1 3 A The recombinant plasmid pegfpc1-fat-1 extracted from the 60 μmol / L 18 2 ω-6pufas Linoleic acid positive clone was identified by KpnⅠ / BamHⅠ digestion M 1 kb DNA ladder 1 2 Kpn I / BamHI digestion of pegfpc1-20 4 ω-6pufas arachidonic acid B fat-1 C pegfpc1 60 μmol / L 18 2 20 4ω-6PUFAs D pegfpc1-fat-1 60 μmol / L 18 2 20 4ω-6PUFAs 24 ~ 30 48 h MTT 5 mg / ml 20 μl 4 h 150 μl fat-1 DMSO 10 min 490 nm SPSS 1 7 48 h PBS 1 1 AnnexinV Fig 2 3 1 10 6 / ml 100 μl Alexa FluorR 488 annexinv 5 μl 100 μg / ml PI 1 μl 15 min 400 μl 1 annexin- 2 2 1 pegfpc1-fat-1 PCR KpnⅠ / BamHⅠ Fig 1 Fig 2 RT-PCR identification of fat-1 transcript levels DNAMAN GenBank After transfection 24 hours the total cellular RNA was fat-1 cdna extracted by RNAiso Plus and chloroform then the total TaKaRa RNA was reverse transcript into cdna and the following SJQ1031 RT-PCR was carried out at 94 3 min 94 30 s 2 2 Fat-1 55 30 s 72 1 min 30 cycles M 100 bp plus 48 h RT-PCR DNA ladder 1 RT-PCR products using pegfpc1 as pegfpc1-fat-1 fat-1 pegfpc1 fat-1 488 nm pegfpc1-fat-1 pegfpc1 pegfp-fat-1 template 2 3 RT-PCR products using pegfpc1-fat-1 as template
758 27 2 3 ω-6pufas / ω-3pufas 24 h 48 h 72 h ω-6pufas / ω-3pufas 14 24 h 48 h 72 h fat-1 ω- 6PUFAs ω-3pufas 48 h ω-6 PUFAs / ω-3 PUFAs 4 76 1 8 96 1 72 h ω-6 PUFAs / ω-3 PUFAs 4 84 16 99 1 Fig 4 Cell proliferation assessment by MTT method P < 0 05 72 h Mouse embryonic fibroblasts were transfected by pegfpc1 Table 1 fat-1 or pegfpc1-fat-1 with 60 μmol / L 18 2 ω-6 PUFAs ω-6pufas linoleic acid and 60 μmol / L 20 4 ω-6 PUFAs ω-3pufas ω-6pufas / arachidonic acid in the culture medium After ω-3pufas transfection 48 hours MTT method was used to test the 2 4 cell viability Ctrl cells without any treatment LA MTT 60 Linoleic acid AA arachidonic acid V empty vector μmol / L 18 2 ω-6 PUFAs linoleic acid 20 transfection Fat-1 pegfpc1-fat1 transfection 4 ω-6 PUFAs arachidonic acid 48 h pegfpc1-fat-1 490 nm 0 982 ± 0 10 ω-6 PUFAs pegfpc1 490 nm 0 795 ± ω-3 PUFAs 3T3 0 17 OD = x 珋 ± s fat-1 P < 2 5 0 01 fat-1 ω-6pufas Table 1 4 8 h AnnexinV -Alex Fluor4 8 8 / PI Gas chromatography analysis of cellular PUFAs after transfection 72 hours x 珔 ± s n = 3 Number Peak retention time min Long chain fatty acids pegfpc1 pegfpc1-fat-1 1 11 892 14 0 2 72 ± 0 19 2 47 ± 0 13 2 14 125 15 0 0 30 ± 0 11 0 30 ± 0 02 3 16 550 16 0 24 25 ± 0 40 23 5 ± 0 42 4 17 089 16 1ω9 2 00 ± 1 18 2 21 ± 1 04 5 17 233 16 1ω7 3 26 ± 1 65 3 90 ± 1 70 6 17 531 16 1ω5 0 23 ± 0 05 0 27 ± 0 02 7 19 038 17 0 0 48 ± 0 13 0 49 ± 0 07 8 21 596 18 0 18 65 ± 0 29 17 92 ± 1 48 9 22 145 18 1ω9 21 30 ± 1 70 23 89 ± 0 02 10 22 317 18 1ω7 6 00 ± 0 51 6 39 ± 0 53 11 23 340 18 2ω6 5 58 ± 0 76 4 26 ± 1 17 12 24 155 18 3ω3 0 35 ± 0 06 0 39 ± 0 09 12 27 522 20 1ω9 0 42 ± 0 01 0 57 ± 0 07 13 27 796 20 1ω7 0 29 ± 0 07 0 32 ± 0 02 14 29 206 20 2ω6 0 50 ± 0 21 0 52 ± 0 09 15 30 185 20 3ω6 1 47 ± 0 13 1 31 ± 0 09 16 31 117 20 4ω6 10 01 ± 2 82 9 32 ± 2 40 17 33 983 20 5ω3EPA 0 39 ± 0 10 0 90 ± 0 12 18 48 514 22 6ω3DHA 1 77 ± 1 14 1 89 ± 0 53 Total ω-6 PUFAs / ω-3 PUFAs 6 99 4 84 Mouse embryonic fibroblasts were transfected by pegfpc1 and pegfpc1-fat-1 with 10 μmol / L Linoleic acid and 10 μmol / L Arachidonic acid After transfection 24 48 72 hours total cellular fatty acids were extracted and gas chromatography was used to examine the PUFAs composition
8 ω-3 fat-1 759 Fig 3 Fluorescence photos of mouse embryonic fibroblasts after transfection 48 hours 200 After transfection 48 hours the expressed protein level was detected by Laser Scanning Confocal Microscope at the 488 nm exciting light A Normal mouse embryonic fibroblasts without transfection B and D Mouse embryonic fibroblastss transfected by pegfpc1 C and E Mouse embryonic fibroblasts transfected by pegfpc1-fat-1 Fig 5 Representative of flow cytometry analysis of cell apoptosis After transfection 48 hours the mouse embryonic fibroblastsce were harvested and stained by the dyestuff PI and annexinv then the stained cells were analyzed by flow cytometry to measure fluorescence intensity of live cells apoptosis cells and the dead cells A Normal cells B Cells transfected by pegfpc1 C Cells transfected by pegfpc1-fat-1 Alexa FluorR 488-/ PI- Alexa FluorR 488 + / PI + Alexa FluorR 488 + / PI- Fig 5 DNA pegfpc1 13 9 ± 4 6% pegfpc1-fat-1 8 1 ± ω-3pufas 1 8% OD = xs P 珋 < 0 05 3 PUFAs 15 Fat-1 ω- 6PUFAs ω-3pufas ω-3pufas
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