骨橋蛋白增強(qiáng)自發(fā)性高血壓大鼠血管外膜成纖維細(xì)胞的遷移活性

1、: Acta Physiologica Sinica, February 25, 2004, 56 (1): 21-24 21 , : , , , * , 200025 ( spontaneously hypertensive rats, SHR) PCR WKY ( 4.47, P<0.05) WKY (1863.23 (Wistar-Kyoto rats, WKY) mRNA , SHR 68.29, P<0.01) 38.60 , , , Transwell bFGF , SHR : 35.20 5.26 vs 22.2 3.27, P<0.05; bFGF: 30.2

2、3 4.54 (osteopontin, OPN) mRNA OPN (100 µmol/L 5.98 vs 26.61 3.84, P<0.05) SHR SHR OPN vs 19.20 43.91 vs 326.24 ( OPN SHR WKY, OPN : ; : Q463 ; ; ; ; ; Osteopontin enhances migratory ability of cultured aortic adventitial fibroblasts from spontaneously hypertensive rats SUN Ai-Jun, GAO Ping-

3、Jing, LIU Jian-Jun, JI Kai-Da, ZHU Ding-Liang* Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200032 Abstract: Migration of adventitial fibroblasts (AF) is involved in the neointimal formation which is one of the common pathological processes in sev

4、eral vascular diseases. The observation of whether the migratory response of AF from hypertensive animal is different from that of controls may provide an explanation of vascular remodeling in hypertension. We examined whether there is any difference between the migratory activity of AF derived from

5、 spontaneously hypertensive rat (SHR) and that from their normotensive counterpart Wistar-Kyoto rats (WKY). In addition, the role of osteopontin (OPN) in cell migration was also examined. Primary cultures of aortic adventitial fibroblasts were derived from SHR and age-matched WKY. Migration of fibro

6、blasts was determined with the Transwell method. The mRNA expression level of OPN was measured by a realtime quantitative PCR. When compared with WKY-derived cells, migration of adventitial fibroblasts from SHR exhibited an increased response when stimulated by 10% serum (cell number per field 35.20

7、 5.26 vs 22.2 3.27, P<0.05). Chemotaxis induced by 10 ng/ml bFGF showed a similar difference (cell number per field 30.23 4.54 vs 19.20 4.47, P<0.05). We also found that SHR-derived fibroblasts expressed a higher level of OPN mRNA than the cells from WKY (1863.23 43.91 vs 326.24 68.29, P<0.

8、01). To verify if OPN is associated with the enhanced migratory ability in AF from SHR, we designed the Received 2003-06-04 Accepted 2003-07-24 This work was supported by the National Natural Science foundation of China (M30070303, 30270544 ). Corresponding author. Tel: +86-21-64154883; E-mail : f97

9、075 22 Acta Physiologica Sinica, February 25, 2004, 56(1): 21-24 antisense oligonucleatide of OPN. The results showed that the antisense OPN oligonucleatide significantly inhibited AF migration (cell number per field 38.60 5.98 vs 26.61 3.84, P<0.05), while sense and mismatch OPN oligonucleotide

10、had no effect on cell migration. Therefore, the migration of adventitial fibroblasts appeared to be enhanced in cultures derived from SHR. OPN might be involved in the difference observed. Key words: aorta; adventitia; fibroblast; osteopontin; hypertension; cell migration; remodeling , 1 5´AACC

11、ACTGCCAGTCTCAT3´, AGACTGGCAGTGGTT3´, TATCAGTCTCGT3´ 1.2.3 10 µl, h PCR Trizol RNA 1 µg , 1 RNA WKY 5´ATG5´AACTACOPN mRNA (spontaneously hypertensive rats, SHR) , SHR 3 2 buffer, dNTP 2 mmol/L, : 37ºC 1 PCR OPN PCR RT-PCR (VSMC) SHR SHR WKY , 10 ng/L, MMLVase 4

12、00 U, 95ºC 5 min, PCR : 5´GCTTGGCTTATGGACTGAGGT-3´, 5´ACGAAGTTTCACAGCCATGAG-3´ 10 4 10 6 10 8 10 10 , 20 µl, 1.1 Trizol DMEM Gibco , bFGF SHR WKY , Transwell MoP. Corning SYBR Green PCR 0.5 µl, 1 mmol/L, Taq Green 2000 1.2.4 buffer, dNTP 2.5 mmol/L, MgCl 2 25 1.25

13、U, 0.25 µmol/L, SYBR 2 µl 92ºC 15 s, 58ºC Rotor-gene t , lecular Probes Marche 1.2 1.2.1 WKY , 1.2.2 DMEM , 15% , 30 s, 72ºC 2 min, 35 10 , DMEM 35 (1) 10% , Transwell 6h : , SHR ANOVA , P<0.05 , Transwell 12 ng/ml bFGF 1 , PBS 10 5 2.1 SHR 10% WKY , SHR WKY , 37 , 4% , ,

14、 5 (10 (2) OPN DMEM OPN, MWG , 20) : OPN Transwell 1. 10% S 10 ng/ml bFGF SHR AF WKY Fig.1. Number of migratory AF from WKY and SHR under 10% serum (S) and bFGF. *P<0.05 vs WKY group. : 23 WKY ml) SHR 1 2.2 SHR mRNA 326.24 2 SHR , 19.2 22.2 (P<0.05) 4.47 30.2 3.27 35.2 5.26, 38.62 5.98 33.45 3

15、.84 OPN (P<0.05) , 3 6.34 35.68 , , 5.31 30.45 100 µmol/L 10 µmol/ 3 bFGF (10 ng/ WKY 4.54, 3 , PN L 4.16 26.62 50 µmol/L OPN 1 µmol/L (P<0.05) WKY OPN 68.29, SHR mRNA 1863.23 2.4 Transwell µmol/L OPN, 10% 10% 4.51 +100 µmol/L 40.87 4.12 OPN OPN SHR +100 OPN, , 4.

16、15 35.43 , WKY 43.91, 5 , 10% , 10% +100 µmol/L OPN 42.37 4.03 (P<0.01) 43.86 , 100 µmol/L (P<0.05) 3 4 , 2. WKY SHR OPN mRNA WKY Fig. 2. OPN mRNA expression of adventitial fibroblasts (AF) from WKY and SHR. *P<0.01 vs WKY group. 2.3 OPN Transwell SHR 4. OPN SHR 10% +10 µmol/

17、L OPN, 10% , , 10% +100 µmol/L +1 Fig. 4. Effects of antisense, sense and mismatch OPN on migration of AF from SHR. AS, antisence group; S, sense group; M, mismatch group. *P<0.05 vs control. µmol/L OPN, 10% OPN, 10% +50 µmol/L OPN , , Hsieh PDGF 3. OPN SHR / , VSMC 5 , SHR Fig. 3.

18、 Effect of antisense OPN on migration of AF from SHR. Control, 10% serum (S); antisense (AS) 1100, 10% S+1100 µmol/L ASOPN. P<0.05 vs control. * WKY 20 SHR SHR, VSMC 3 24 Acta Physiologica Sinica, February 25, 2004, 56(1): 21-24 1991 2 , WKY, SHR , 4,5 AF 2 phenotype. J Hypertens Suppl 1997;

19、15(6):S3-S7. Zhu DL, Herembert T, Marche P. Increased proliferation of adventitial fibroblasts from spontaneously hypertensive rat aorta. J Hypertens 1991;9:1161-1168. 3 Hsieh CC, Lau Y. Migration of vascular smooth muscle cells is enhanced in cultures derived from spontaneously hypertensive rats. P

20、lfugers Arch 1998; 435:286-292. 4 Tao T ( ), Zhu DL, Gong LS. Collagen distribution and its quantitative variation in the aortic adventitial of SHR , SHR AF OPN AF WKY, OPN SHR-AF Ye OPN 150 8 spontaneously hypertensive rats. Chin J Hyperten ( ), 2001;2:50-52 (Chinese, English abstract). : (1) VSMC

21、2 , AF , , AF , OPN 9 6,7 , 5 Gong Y ( ), Zhu Dl, Xiao JC, Yang J. Histological OPN 6 observation of the small arterial adventitia in renal Hhlum in hypertensive patients. Chin J Cardiol ( ), 2001,9:1-3 (Chinese, English abstract). Yue TL, McKenna PJ, Ohlstein EH, Farach-Carson MC, Butler WT, Johans

22、on K, McDevitt P, Feuerstein GZ, Stadel JM. Osteopontin-stimulated vascular smooth muscle cell migration is mediated by beta 3 integrin. Exp Cell Res 1994; 214:459-464. DD-PCR , , (myofibroblast, MF) PCR SHR AF WKY, SHR AF OPN , , OPN , OPN SHR AF OPN SHR-AF OPN , , SHR AF 8 7 Li G, Chen YF, Kelpke

23、SS, Oparil S, Thompson JA. Estrogen attenuates integrin-beta(3)-dependent adventitial fibroblast migration after inhibition of osteopontin production in vascular smooth muscle cells. Circulation 2000;101:2949-2955. Ye QH, Qin LX, Forgues M, He P, Kim JW, Peng AC. Predicting hepatitis B virus-positive metastatic hepat