中华糖尿病杂志
中華糖尿病雜誌
중화당뇨병잡지
CHINES JOURNAL OF DLABETES MELLITUS
2012年
2期
115-119
,共5页
薛声能%雷娟%杨川%林刁珠%朱平%徐明彤%严励
薛聲能%雷娟%楊川%林刁珠%硃平%徐明彤%嚴勵
설성능%뢰연%양천%림조주%주평%서명동%엄려
基质金属蛋白酶9%组织金属蛋白酶抑制剂1%糖尿病足%大鼠
基質金屬蛋白酶9%組織金屬蛋白酶抑製劑1%糖尿病足%大鼠
기질금속단백매9%조직금속단백매억제제1%당뇨병족%대서
Matrix metalloproteinase 9%Tissue inhibitors of metalloproteinase 1%Diabetic foot%Rats
目的 观察外源性组织金属蛋白酶抑制剂-1( TIMP-1)对基质金属蛋白酶-9(MMP-9)高表达大鼠皮肤成纤维细胞生物学行为的影响,为寻找治疗糖尿病足的新方法提供实验依据.方法 应用SD大鼠皮肤成纤维细胞株CRL-1213用高糖(22 mmol/L)+高同型半胱氨酸(100 μmol/L)联合培养建立体外MMP-9高表达皮肤成纤维细胞模型,用外源性TIMP-1(100 μg/L)抑制MMP-9的活性,采用实时PCR法和ELISA法检测MMP-9基因及蛋白表达量,用明胶酶谱法检测MMP-9蛋白活性,采用流式细胞术检测细胞增殖功能、CCK-8检测细胞活力、ELISA法检测细胞胶原(羟脯氨酸)分泌能力、划痕实验评价细胞横向迁移能力、Transwell法评价细胞纵向迁移能力.采用单因素方差分析进行统计学分析.结果 TIMP-1干预组大鼠皮肤成纤维细胞MMP-9 mRNA和蛋白的表达量与MMP-9高表达组比较差异无统计学意义(P＞0.05),但MMP-9蛋白活性受到明显抑制(1.47 ±0.13vs 0.43 ±0.13,t=12.495,P＜0.01).与对照组比较,MMP-9高表达组大鼠皮肤成纤维细胞S期的比例[ (9.31 ±0.24)％ vs(6.54 ±0.29)％]、增殖指数[(13.8±0.5)％vs(11.3±0.6)％]、细胞活力(1.76 ±0.13 vs 1.35 ±0.12)、胶原(羟脯氨酸)分泌量[(1126±63) vs (353 ±61) μg/L]、6h横向迁移率[(38.7±2.6)％vs(21.3±2.1)％]和纵向迁移细胞数(91 ±4 vs 71 ±4)均明显减少(t=16.433、7.328、5.113、19.847、11.529、8.124,均P＜0.05)；TIMP-1干预后,大鼠皮肤成纤维细胞S期的比例[(6.54±0.29)％vs(7.75±0.27)％]、增殖指数[(11.3±0.6)％vs(12.5±0.5)％]、细胞活力(1.35 ±0.12 vs 1.64 ±0.14)、胶原(羟脯氨酸)分泌量[(353 ±61) vs (829 ±59)μg/L]、6h横向迁移率[(21.3±2.1)％vs(31.5±2.7)％]和纵向迁移细胞数(71 ±4 vs 85 ±4)与MMP-9高表达组比较得到明显改善[t =6.881、3.292、3.615、12.590、6.644、6.015,均P＜0.05].结论 MMP-9高表达大鼠皮肤成纤维细胞的生物学行为受到抑制,外源性TIMP-1能够减缓这种抑制作用.
目的 觀察外源性組織金屬蛋白酶抑製劑-1( TIMP-1)對基質金屬蛋白酶-9(MMP-9)高錶達大鼠皮膚成纖維細胞生物學行為的影響,為尋找治療糖尿病足的新方法提供實驗依據.方法 應用SD大鼠皮膚成纖維細胞株CRL-1213用高糖(22 mmol/L)+高同型半胱氨痠(100 μmol/L)聯閤培養建立體外MMP-9高錶達皮膚成纖維細胞模型,用外源性TIMP-1(100 μg/L)抑製MMP-9的活性,採用實時PCR法和ELISA法檢測MMP-9基因及蛋白錶達量,用明膠酶譜法檢測MMP-9蛋白活性,採用流式細胞術檢測細胞增殖功能、CCK-8檢測細胞活力、ELISA法檢測細胞膠原(羥脯氨痠)分泌能力、劃痕實驗評價細胞橫嚮遷移能力、Transwell法評價細胞縱嚮遷移能力.採用單因素方差分析進行統計學分析.結果 TIMP-1榦預組大鼠皮膚成纖維細胞MMP-9 mRNA和蛋白的錶達量與MMP-9高錶達組比較差異無統計學意義(P＞0.05),但MMP-9蛋白活性受到明顯抑製(1.47 ±0.13vs 0.43 ±0.13,t=12.495,P＜0.01).與對照組比較,MMP-9高錶達組大鼠皮膚成纖維細胞S期的比例[ (9.31 ±0.24)％ vs(6.54 ±0.29)％]、增殖指數[(13.8±0.5)％vs(11.3±0.6)％]、細胞活力(1.76 ±0.13 vs 1.35 ±0.12)、膠原(羥脯氨痠)分泌量[(1126±63) vs (353 ±61) μg/L]、6h橫嚮遷移率[(38.7±2.6)％vs(21.3±2.1)％]和縱嚮遷移細胞數(91 ±4 vs 71 ±4)均明顯減少(t=16.433、7.328、5.113、19.847、11.529、8.124,均P＜0.05)；TIMP-1榦預後,大鼠皮膚成纖維細胞S期的比例[(6.54±0.29)％vs(7.75±0.27)％]、增殖指數[(11.3±0.6)％vs(12.5±0.5)％]、細胞活力(1.35 ±0.12 vs 1.64 ±0.14)、膠原(羥脯氨痠)分泌量[(353 ±61) vs (829 ±59)μg/L]、6h橫嚮遷移率[(21.3±2.1)％vs(31.5±2.7)％]和縱嚮遷移細胞數(71 ±4 vs 85 ±4)與MMP-9高錶達組比較得到明顯改善[t =6.881、3.292、3.615、12.590、6.644、6.015,均P＜0.05].結論 MMP-9高錶達大鼠皮膚成纖維細胞的生物學行為受到抑製,外源性TIMP-1能夠減緩這種抑製作用.
목적 관찰외원성조직금속단백매억제제-1( TIMP-1)대기질금속단백매-9(MMP-9)고표체대서피부성섬유세포생물학행위적영향,위심조치료당뇨병족적신방법제공실험의거.방법 응용SD대서피부성섬유세포주CRL-1213용고당(22 mmol/L)+고동형반광안산(100 μmol/L)연합배양건입체외MMP-9고표체피부성섬유세포모형,용외원성TIMP-1(100 μg/L)억제MMP-9적활성,채용실시PCR법화ELISA법검측MMP-9기인급단백표체량,용명효매보법검측MMP-9단백활성,채용류식세포술검측세포증식공능、CCK-8검측세포활력、ELISA법검측세포효원(간포안산)분비능력、화흔실험평개세포횡향천이능력、Transwell법평개세포종향천이능력.채용단인소방차분석진행통계학분석.결과 TIMP-1간예조대서피부성섬유세포MMP-9 mRNA화단백적표체량여MMP-9고표체조비교차이무통계학의의(P＞0.05),단MMP-9단백활성수도명현억제(1.47 ±0.13vs 0.43 ±0.13,t=12.495,P＜0.01).여대조조비교,MMP-9고표체조대서피부성섬유세포S기적비례[ (9.31 ±0.24)％ vs(6.54 ±0.29)％]、증식지수[(13.8±0.5)％vs(11.3±0.6)％]、세포활력(1.76 ±0.13 vs 1.35 ±0.12)、효원(간포안산)분비량[(1126±63) vs (353 ±61) μg/L]、6h횡향천이솔[(38.7±2.6)％vs(21.3±2.1)％]화종향천이세포수(91 ±4 vs 71 ±4)균명현감소(t=16.433、7.328、5.113、19.847、11.529、8.124,균P＜0.05)；TIMP-1간예후,대서피부성섬유세포S기적비례[(6.54±0.29)％vs(7.75±0.27)％]、증식지수[(11.3±0.6)％vs(12.5±0.5)％]、세포활력(1.35 ±0.12 vs 1.64 ±0.14)、효원(간포안산)분비량[(353 ±61) vs (829 ±59)μg/L]、6h횡향천이솔[(21.3±2.1)％vs(31.5±2.7)％]화종향천이세포수(71 ±4 vs 85 ±4)여MMP-9고표체조비교득도명현개선[t =6.881、3.292、3.615、12.590、6.644、6.015,균P＜0.05].결론 MMP-9고표체대서피부성섬유세포적생물학행위수도억제,외원성TIMP-1능구감완저충억제작용.
Objective To explore the effect of tissue inhibitor of metalloproteinase 1 (TIMP-1) on the biological behaviors of rat dermal fibroblast with high matrix metalloproteinase 9 ( MMP-9),in order to provide an experimental basis for looking for a new way to treat diabetic foot.Methods The cell model of skin fibroblast was established by using dermal fibroblast cell line CRL-1213 from SD rats with high expression of MMP-9 by high glucose (22.0 mmol/L) + high homocysteine (100 μmol/L) co-culture.Exogenous TIMP-1 was used to inhibite the activity of MMP-9.Realtime PCR and ELISA were used to detect the expression of MMP-9 mRNA and protein.Gelatin zymography was used to detect the activity of MMP-9.Flow cytometry was used to detect cell proliferation. CCK-8 was used to detect cell viability.ELISA assay was used to detect collagen (hydroxyproline) secretion. Scratch test was used to evaluate horizontal migration of cells.Transwell method was used to evaluate vertical migration of cells.ANOVA was used for data analysis.Results The expression difference of MMP-9 mRNA and protein between TIMP-1 group and high MMP-9 group had no statistically significant (P ＞ 0.05).But the activity of MMP-9 protein in TIMP-1 group was lower than that in high MMP-9 group ( 1.47 ± 0.13 vs 0.43 ± 0.13,t =12.495,P ≤ 0.01 ).Compared with control group,the proportion of S phase cells [ ( 9.31 ± 0.24 ) ％ vs ( 6.54 ± 0.29 ) ％ ],proliferation index [ ( 13.8 ± 0.5 ) ％ vs ( 11.3 ± 0.6) ％ ],cell viability ( 1.76 ± 0.13 vs 1.35 ± 0.12),collagen (hydroxyproline) secretion [ (1126± 63 ) vs (353 ± 61 ) μg/L],6 h horizontal migration rate [ (38.7 ±2.6)％ vs (21.3 ±2.1 )％ ] and the number of vertical migration cells (91 ±4 vs 71 ±4) in high MMP-9 group were decreased (t =16.433,7.328,5.113,19.847,11.529 and 8.124 repectively,all P ＜ 0.05).After interfered by TIMP-1,the proportion of S phase cells [ (6.54 ± 0.29 ) ％ vs (7.75 ±0.27)％],proliferation index [(11.3 ±0.6)％ vs (12.5 ±0.5)％],cell viability (1.35 ±0.12 vs 1.64 ± 0.14 ),collagen ( hydroxyproline ) secretion [ ( 353 ± 61 ) vs ( 829 ± 59 ) μg/L ],horizontal migration rate [ (21.3 ± 2.1 ) ％ vs (31.5 ± 2.7) ％ ] and the number of vertical migration cells (71 ± 4 vs 85 ± 4 ) were increased compared with those in high M MP-9 group( t =6.881,3.292,3.615,12.590,6.644and 6.015 respectively,all P ＜ 0.05).Conclusion The biological behaviors of skin fibroblasts with high expression of MMP-9 is inhibited.The inhibited effect can be decreased by exogenous TIMP-1.
