21 21 2017 07 28 Chinese Journal of Tissue Engineering Research July 28, 2017 Vol.21, No.21 1α ( 423000). 1α [J]. 2017 21(21):3281-3286. DOI:10.3969/j.issn.2095-4344.2017.21.001 ORCID: 0000-0002-2701-9583() 1α 1981 2010 423000 (5 7 d)(1.0 1.5 mm/d) 1 1 1 1 :R318 :A :2095-4344 (2017)21-03281-06 2017-04-26 1α 2 000 µstrain 1α 1α 1α GYY4137 YC-1 1 α (81301651)(N2015-006) Hydrogen sulfide promotes the osteogenesis of bone marrow mesenchymal cells through hypoxia-inducible factor-1 alpha under tensile stress Jiang Xiao-wen, Zhang Yi, Fan Xiao-sheng, Chen Yan-ze (Department of Stomatology, the First People s Hospital of Chenzhou & Institute of Translation Medicine, University of China South, Chenzhou 423000, Hunan Province, China) Abstract BACKGROUND: Hydrogen sulfide signaling has been proved to promote distraction osteogenesis; however, the underlying mechanism remains unclear. OBJECTIVE: To evaluate the relationship between hydrogen sulfide and hypoxia-inducible factor-1α (HIF-1α) Jiang Xiao-wen, Associate chief physician, Department of Stomatology, the First People s Hospital of Chenzhou & Institute of Translation Medicine, University of China South, Chenzhou 423000, Hunan Province, China Corresponding author: Jiang Xiao-wen, Department of Stomatology, the First People s Hospital of Chenzhou & Institute of Translation Medicine, University of China South, Chenzhou 423000, Hunan Province, China ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 3281
. 1α during the osteogenesis of rat bone marrow mesenchymal cells under tensile stress. METHODS: 2 000 µ strain was loaded on the in vitro cultured rat bone marrow mesenchymal cells by a four-point bending apparatus, and hydrogen sulfide donor or HIF-1α inhibitor was adopted in the tensile unit. Subsequently, the levels of osteogenic markers were detected. RESULTS AND CONCLUSION: Exogenous hydrogen sulfide signaling could promote the osteogenesis of rat bone marrow mesenchymal cells under tensile stress. However, this promotion was obviously eliminated when the endogenous HIF-1α expression was inhibited. These results show that hydrogen sulfide signaling system promotes the osteogenesis of rat bone marrow mesenchymal cells under tensile stress probably through HIF-1α. Subject headings: Tissue Engineering; Hydrogen Sulfide; Hypoxia-Inducible Factor 1, alpha Subunit Funding: the National Natural Science Foundation of China, No. 81301651; the Key Project of the First People s Hospital of Chenzhou, No. N2015-006 Cite this article: Jiang XW, Zhang Y, Fan XS, Chen YZ. Hydrogen sulfide promotes the osteogenesis of bone marrow mesenchymal cells through hypoxia-inducible factor-1 alpha under tensile stress. Zhongguo Zuzhi Gongcheng Yanjiu. 2017;21(21):3281-3286. 0 Introduction (distraction osteogenesis DO) [1] [1] [2-10] [11] (H 2 S) L [12] [13-14] [15] H 2 S3 H 2 S β 3 L H 2 S [16] H 2 S H 2 S H 2 S [17-19] (hypoxia-inducible factors HIFs) [20] H 2 S [21-22] H 2 S (bone marrow mesenchymal cell BMMC) 1α 1α H 2 S 1Materials and methods 1.1 1.2 2015 9 2016 2 1.3 2 SD250 350 g SYXK( ) 2010-0006 1.4 1.4.1 Percoll( Gibco ) 5% PBS 3 10% LG-DMEM( Gibco ) 2 d 70% 80% 0.25% 3 1.4.2 1.5 10 5 /cm 2 [1] 24 h 3282 P.O. Box 10002, Shenyang 110180
. 1α 60 min( ( ) 0.5 Hz 1 mm 2 000 µ strain0.2% 4 8 1 2 1α 1 α (YC-1) 80 µmol/l 3 H 2 S GYY41374 mmol/l 4 1α /H 2 S 3 YC-180 µmol/l 2 h 1.4.3 24 h PBS 1.4.4 H 2 S (10 ml 1 cm 0.8 cm ) 1 mol/l HCl 0.5 mlh 2 S 1 mol/l NaOH 0.5 ml H 2 S37 4 h H 2 S [17] 1.4.5 2 h PBS 3 ml 2 ml PBS 90 µl PBS 3 ( )450 nm U/mg 1.4.6 450 nm 2 h 1 ml 10 µl 40 µl 37 30 min 50 µl 37 30 min 37 15 min 450 nm 1.4.7 (PINP) ( ) 450 nm 2 h 1 ml 5 µl 45 µl PINP 30 min 150 µl 30 min 30 min 450 nm PINP 1.4.8 Real time-pcr 2 h Trizol(Gibco) RNA; (TaKaRa) BioRad 1 000 TM cdna 1 cdna SYBR Green RT-PCR GAPDH PCR 5 -CTT CGT CAG CGT CCT ATC AGT TC-3 5 -CAG CGT CAA CAC CAT CAT TCT G-3 2 (runt-related transcription factor 2 Runx2) mrna 5 -TAT GAC TCT ACC CAC GGC AAG T-3 5 -ATA CTC AGC ACC AGC ATC ACC-3 PCR95 30 s 95 5 s 57 30 s 40 Runx2 GAPDH 1.4.9 Western Blot 1α BCA ( Gibco ) 20 µg SDS-PAGE PVDF (1α1 500 Abcam ) (lgg 1 500 )37 1 h 1.5 H 2 S (PINP) Runx2 1α 1.6 SPSS 13.0 x _ ±s LSD P < 0.05 2Results 2.1 4 2 h 4 (P > 0.05 1) 2.2 H 2 S 4 2 h H 2 S 1α/ H 2 SH 2 S 2(P < 0.05 1) 2.3 H 2 S 1α/H 2 S (P < 0.05 1) H 2 S 2.4 H 2 S 3 (P < 0.05 1) 2.5 PINP 1α /H 2 S (P < 0.05 1) H 2 S PINP 2.6 Runx2 mrna H 2 S Runx2 mrna 4 (P < 0.05 1) ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 3283
. 1α 1 H 2S Table 1 Levels of osteogenic markers and hydrogen sulfide at the end of tensile stress loading (x _ ±s n=8) 1α H 2S 1α / H 2S (10 6 ) 6.22±0.80 5.67±0.75 6.21±0.66 6.38±0.61 H 2S(U/mg) 17.84±1.48 15.70±3.95 35.45±3.29 a 35.43±3.44 a (U/mg) 194.12±15.40 183.69±22.98 241.35±26.81 a 209.06±27.18 a (U) 1.48±0.16 1.17±0.17 1.91±0.25 a 1.28±0.18 PINP(µg/L) 8.00±0.26 2.23±0.35 4.81±0.57 a 3.16±0.37 Runx2 mrna 10.97±1.29 9.23±1.21 15.06±2.65 a 11.13±0.81 1α a P < 0.05 PINP A B C D M r 120 000 1α β-actin 43 000 1 1α Figure 1 Protein expression of hypoxia-inducible factor-1α in rat bone marrow mesenchymal cells at the end of tensile stress loading A D 1α H 2S 1α /H 2S B D 1α 2.7 1α Western blot4 hh 2 S 1α H 2 S 1α 1α1α /H 2 S ( 1) 3Discussion H 2 S 3 H 2 S [23-25] H 2 S H 2 S [17-18] GYY4137 H 2 S H 2 S [19] [26] [20] H 2 S H 2 S H 2 S Runx2 PINP Runx2 PINPH 2 S H 2 S H 2 S H 2 S H 2 S [27-28] α 3 α (1α 2α 3α) 1α H 2 S [29] Liu [21] H 2 S Bir [22] H 2 S [30] H 2 S [31] H 2 S 1α 1α mrna 1α [32-51] Wan [52] 3284 P.O. Box 10002, Shenyang 110180
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