中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2012年
7期
1242-1244,封3
,共4页
涂意辉%薛华明%夏志道%马童%刘晓东%杜靖远
塗意輝%薛華明%夏誌道%馬童%劉曉東%杜靖遠
도의휘%설화명%하지도%마동%류효동%두정원
乳铁蛋白%软骨细胞%增殖%细胞外调节蛋白激酶
乳鐵蛋白%軟骨細胞%增殖%細胞外調節蛋白激酶
유철단백%연골세포%증식%세포외조절단백격매
Lactoferrin%Chondrocytes%Proliferation%Extracellular signal regulated kinase
目的 观察人乳铁蛋白(hLF)对人骨关节炎软骨细胞(HACs)增殖活力及细胞外调节蛋白激酶(ERK)基因表达的影响. 方法 噻唑蓝(MTT)比色法检测HACs的增殖力,并选择hLF最佳工作浓度；LIVE/DEAD染色检测HACs的活力；实时定量聚合酶链反应(Real-time PCR)和免疫细胞化学染色检测磷酸化细胞外调节蛋白激酶( p-ERK )/ERK的表达.结果 MTT检测显示hLF对HACs的增殖促进作用呈剂量-效应关系(P＜0.05),以200 mg/L为最佳药物浓度.LIVE/DEAD染色显示hLF可以显著促进HACs的活力,与空白对照组和无血清培养组比较,差异有统计学意义[(98.53±2.76)％比(89.12 ±6.98)％比(65.37 ±2.08)％,P＜0.05].Real-time PCR检测显示,hLF作用48 h后ERK mRNA相对表达量为(1.589±0.113),与对照组(1.024±0.026)比较差异有统计学意义(P＜0.05).IN Cell Analyzer 2000定量检测,hLF能够显著促进p-ERK/ERK蛋白的表达,较对照组明显增高,差异有统计学意义[(0.887±0.003)比(0.637±0.002),P＜0.05].结论 hLF可明显促进HACs增殖力和活力,其作用机制可能与上调增殖基因p-ERK/ERK表达有关.
目的 觀察人乳鐵蛋白(hLF)對人骨關節炎軟骨細胞(HACs)增殖活力及細胞外調節蛋白激酶(ERK)基因錶達的影響. 方法 噻唑藍(MTT)比色法檢測HACs的增殖力,併選擇hLF最佳工作濃度；LIVE/DEAD染色檢測HACs的活力；實時定量聚閤酶鏈反應(Real-time PCR)和免疫細胞化學染色檢測燐痠化細胞外調節蛋白激酶( p-ERK )/ERK的錶達.結果 MTT檢測顯示hLF對HACs的增殖促進作用呈劑量-效應關繫(P＜0.05),以200 mg/L為最佳藥物濃度.LIVE/DEAD染色顯示hLF可以顯著促進HACs的活力,與空白對照組和無血清培養組比較,差異有統計學意義[(98.53±2.76)％比(89.12 ±6.98)％比(65.37 ±2.08)％,P＜0.05].Real-time PCR檢測顯示,hLF作用48 h後ERK mRNA相對錶達量為(1.589±0.113),與對照組(1.024±0.026)比較差異有統計學意義(P＜0.05).IN Cell Analyzer 2000定量檢測,hLF能夠顯著促進p-ERK/ERK蛋白的錶達,較對照組明顯增高,差異有統計學意義[(0.887±0.003)比(0.637±0.002),P＜0.05].結論 hLF可明顯促進HACs增殖力和活力,其作用機製可能與上調增殖基因p-ERK/ERK錶達有關.
목적 관찰인유철단백(hLF)대인골관절염연골세포(HACs)증식활력급세포외조절단백격매(ERK)기인표체적영향. 방법 새서람(MTT)비색법검측HACs적증식력,병선택hLF최가공작농도；LIVE/DEAD염색검측HACs적활력；실시정량취합매련반응(Real-time PCR)화면역세포화학염색검측린산화세포외조절단백격매( p-ERK )/ERK적표체.결과 MTT검측현시hLF대HACs적증식촉진작용정제량-효응관계(P＜0.05),이200 mg/L위최가약물농도.LIVE/DEAD염색현시hLF가이현저촉진HACs적활력,여공백대조조화무혈청배양조비교,차이유통계학의의[(98.53±2.76)％비(89.12 ±6.98)％비(65.37 ±2.08)％,P＜0.05].Real-time PCR검측현시,hLF작용48 h후ERK mRNA상대표체량위(1.589±0.113),여대조조(1.024±0.026)비교차이유통계학의의(P＜0.05).IN Cell Analyzer 2000정량검측,hLF능구현저촉진p-ERK/ERK단백적표체,교대조조명현증고,차이유통계학의의[(0.887±0.003)비(0.637±0.002),P＜0.05].결론 hLF가명현촉진HACs증식력화활력,기작용궤제가능여상조증식기인p-ERK/ERK표체유관.
Objective To investigate the effect of human lactoferrin (hLF) on proliferation,viability and extracellular signal regulated kinase ( ERK ) expression in vitro using cultured human osteoarthritis chondrocytes (HACs).Methods The cell proliferation was assessed by methyl thiazol tetrazolium (MTT) assay and the optimal concentration of hLF was determined for the next experiment.The cell viability was assessed by LIVE/DEAD staining. The mRNA and protein expression of phosphorylated ERK (p-ERK) and ERK were determined by using quantitative real-time polymerase chain reaction (Real-time PCR) and immunocytochemistry respectively.Results The MTT assay revealed that hLF could promote proliferation of chondrocytes in a dose-dependent manner (P ＜0.05 ),and 200 mg/L was appropriately selected as the optimal concentration of hLF for further experiment.The LIVE/DEAD staining showed that hLF significantly increased viability of HACs compared with blank control group and serum-free group [(98.53 ± 2.76 ) ％ vs ( 89.12±6.98) ％ vs (65.37 ± 2.08 ) ％,P ＜ 0.05].qRT-PCR indicate that mRNA expression level of ERK was ( 1.589 ± 0.113 ) after exposure to hLF,which was obviously enhanced compared with control group (1.024 ±0.026) (P ＜0.05).The IN Cell Analyzer 2000 demonstrated that the protein expression of p-ERK/ERK was significantly upregulated in cultured HACs treated with hLF compared with control group (0.887 ± 0.003 vs 0.637 ± 0.002,P ＜ 0.05 ).Conclusion hLF can significantly promote the proliferation and viability of HACs,which may be associated with up-regulated p-ERK/ERK gene expression.
