CAJ | 학술논문

目的探讨二氯化钴(CoCl2)化学缺氧对瘢痕Fb整合素连接激酶(ILK)表达的影响,以及其对细胞增殖的影响. 方法体外培养7例患者增生性瘢痕Fb,取第5～6代细胞进行实验.7例患者的Fb各取6瓶,分别加入含6种终浓度为0、50、100、150、200、250 μmol/L CoCl2的DMEM培养液培养24 h,采用实时荧光定量PCR法检测ILK mRNA表达.选择最适CoCl2浓度(100 μmol/L)进行缺氧刺激,观察ILK蛋白于CoCl2作用0、1、2、4、12、24 h的表达.将细胞分为正常对照组、阴性对照组、ILK小干扰RNA(siRNA)组,分别将con-siRNA及ILK siRNA转染入后2组细胞,对照组仅以培养液培养,24 h后弃培养液,置于含6种浓度CoCl2的培养液中培养24 h.各组各浓度4个复孔.采用3,3′-[1-(苯氨酰基)-3,4-四氮唑]-二(4-甲氧基-6-硝基)苯磺酸钠(XTT)法检测各组细胞增殖水平.对数据进行单因素方差分析以及重复测量方差分析,多重比较采用LSD法. 结果 100 μmol/L CoCl2作用24 h时ILK mRNA表达最高,与其他浓度CoCl2作用的Fb相比差异有统计学意义(F=50.958,P＜0.001).100 μmol/L CoCl2作用1h时Fb ILK蛋白表达量(0.243±0.009)较0 h(0.387±0.017)降低,2 h(0.361±0.010)开始增高,4h(0.584±0.028)、12 h(0.730±0.029)、24 h(0.785±0.031)ILK蛋白表达强度逐渐增强.其中1、4、12、24 h的ILK蛋白表达量与0h相比差异具有统计学意义(P值均小于0.05).XTT结果显示,正常对照组在100 μmol/L CoCl2作用时细胞增殖水平最高(F=488.026,P＜0.001),从150 μmol/L起细胞增殖水平开始下降,250 μmol/L时细胞增殖水平显著低于0 μmol/L时水平(P值均小于0.05).ILK siRNA组细胞增殖水平在各浓度CoCl2作用下无明显变化(F=2.542,P=0.056).ILK siRNA组的细胞增殖水平显著低于正常对照组及阴性对照组(F=2519.542,P＜0.001). 结论 ILK可能是Fb对缺氧应答的关键蛋白,轻度缺氧可以提高细胞ILK的表达,促进瘢痕Fb增殖；而重度缺氧可以降低ILK的表达,抑制细胞增殖. 目的探討二氯化鈷(CoCl2)化學缺氧對瘢痕Fb整閤素連接激酶(ILK)錶達的影響,以及其對細胞增殖的影響. 方法體外培養7例患者增生性瘢痕Fb,取第5～6代細胞進行實驗.7例患者的Fb各取6瓶,分彆加入含6種終濃度為0、50、100、150、200、250 μmol/L CoCl2的DMEM培養液培養24 h,採用實時熒光定量PCR法檢測ILK mRNA錶達.選擇最適CoCl2濃度(100 μmol/L)進行缺氧刺激,觀察ILK蛋白于CoCl2作用0、1、2、4、12、24 h的錶達.將細胞分為正常對照組、陰性對照組、ILK小榦擾RNA(siRNA)組,分彆將con-siRNA及ILK siRNA轉染入後2組細胞,對照組僅以培養液培養,24 h後棄培養液,置于含6種濃度CoCl2的培養液中培養24 h.各組各濃度4箇複孔.採用3,3′-[1-(苯氨酰基)-3,4-四氮唑]-二(4-甲氧基-6-硝基)苯磺痠鈉(XTT)法檢測各組細胞增殖水平.對數據進行單因素方差分析以及重複測量方差分析,多重比較採用LSD法. 結果 100 μmol/L CoCl2作用24 h時ILK mRNA錶達最高,與其他濃度CoCl2作用的Fb相比差異有統計學意義(F=50.958,P＜0.001).100 μmol/L CoCl2作用1h時Fb ILK蛋白錶達量(0.243±0.009)較0 h(0.387±0.017)降低,2 h(0.361±0.010)開始增高,4h(0.584±0.028)、12 h(0.730±0.029)、24 h(0.785±0.031)ILK蛋白錶達彊度逐漸增彊.其中1、4、12、24 h的ILK蛋白錶達量與0h相比差異具有統計學意義(P值均小于0.05).XTT結果顯示,正常對照組在100 μmol/L CoCl2作用時細胞增殖水平最高(F=488.026,P＜0.001),從150 μmol/L起細胞增殖水平開始下降,250 μmol/L時細胞增殖水平顯著低于0 μmol/L時水平(P值均小于0.05).ILK siRNA組細胞增殖水平在各濃度CoCl2作用下無明顯變化(F=2.542,P=0.056).ILK siRNA組的細胞增殖水平顯著低于正常對照組及陰性對照組(F=2519.542,P＜0.001). 結論 ILK可能是Fb對缺氧應答的關鍵蛋白,輕度缺氧可以提高細胞ILK的錶達,促進瘢痕Fb增殖；而重度缺氧可以降低ILK的錶達,抑製細胞增殖.
목적 탐토이록화고(CoCl2)화학결양대반흔Fb정합소련접격매(ILK)표체적영향,이급기대세포증식적영향. 방법 체외배양7례환자증생성반흔Fb,취제5～6대세포진행실험.7례환자적Fb각취6병,분별가입함6충종농도위0、50、100、150、200、250 μmol/L CoCl2적DMEM배양액배양24 h,채용실시형광정량PCR법검측ILK mRNA표체.선택최괄CoCl2농도(100 μmol/L)진행결양자격,관찰ILK단백우CoCl2작용0、1、2、4、12、24 h적표체.장세포분위정상대조조、음성대조조、ILK소간우RNA(siRNA)조,분별장con-siRNA급ILK siRNA전염입후2조세포,대조조부이배양액배양,24 h후기배양액,치우함6충농도CoCl2적배양액중배양24 h.각조각농도4개복공.채용3,3′-[1-(분안선기)-3,4-사담서]-이(4-갑양기-6-초기)분광산납(XTT)법검측각조세포증식수평.대수거진행단인소방차분석이급중복측량방차분석,다중비교채용LSD법. 결과 100 μmol/L CoCl2작용24 h시ILK mRNA표체최고,여기타농도CoCl2작용적Fb상비차이유통계학의의(F=50.958,P＜0.001).100 μmol/L CoCl2작용1h시Fb ILK단백표체량(0.243±0.009)교0 h(0.387±0.017)강저,2 h(0.361±0.010)개시증고,4h(0.584±0.028)、12 h(0.730±0.029)、24 h(0.785±0.031)ILK단백표체강도축점증강.기중1、4、12、24 h적ILK단백표체량여0h상비차이구유통계학의의(P치균소우0.05).XTT결과현시,정상대조조재100 μmol/L CoCl2작용시세포증식수평최고(F=488.026,P＜0.001),종150 μmol/L기세포증식수평개시하강,250 μmol/L시세포증식수평현저저우0 μmol/L시수평(P치균소우0.05).ILK siRNA조세포증식수평재각농도CoCl2작용하무명현변화(F=2.542,P=0.056).ILK siRNA조적세포증식수평현저저우정상대조조급음성대조조(F=2519.542,P＜0.001). 결론 ILK가능시Fb대결양응답적관건단백,경도결양가이제고세포ILK적표체,촉진반흔Fb증식；이중도결양가이강저ILK적표체,억제세포증식.
Objective To explore the expression of integrin-linked kinase (ILK) in fibroblasts (Fbs) of scar induced by cobalt chloride (CoCl2) and its effect on cell proliferation.Methods The human hypertrophic scar Fbs of seven patients were isolated and cultured in vitro.Cells from the 5th to the 6th passages were used in the experiment.Six bottles of Fbs were obtained from each of the seven patients,and they were respectively cultured with DMEM nutrient solution containing CoCl2 in the concentration of 0,50,100,150,200,and 250 μmol/L for 24 h.The expression of ILK mRNA was determined with real-time fluorescence quantitative PCR.Fbs were stimulated by CoCl2 in the most suitable concentration (100 μmol/L) and the protein expression of ILK was determined 0,1,2,4,12,and 24 h after the stimulation.Then the Fbs were divided into control group (cultured with nutrient solution),negative control group (transfected with con-siRNA),and ILK siRNA group (transfected with ILK siRNA).They were cultured with nutrient solution containing CoCl2 in different concentrations 24 h after transfection,with 4 wells for each concentration in each group.The cell proliferation was detected by XTT assay.Data were processed with one-way analysis of variance (ANOVA) and ANOVA for repeated measurement,and LSD method was used in multiple comparisons.Results The expression level of ILK mRNA was highest in Fbs cultured with 100 μmol/L CoCl2 for 24 h,with significant difference compared with those of Fbs cultured with other concentrations ofCoCl2 (F =50.958,P ＜ 0.001).The expression of ILK protein in Fbs cultured with 100 μmol/L CoCl2 for 1 h (0.243 ±0.009) was lower than that cultured for 0 h (0.387 ±0.017),and it started to increase from 2 h (0.361 ±0.010),and exaggerated at 4 h (0.584±0.028),12 h (0.730 ±0.029),and 24 h (0.785 ± 0.031).The expression levels of ILK protein at 1,4,12,24 h were statistically different from that at 0 h (P values all below 0.05).XTT showed that cell proliferation level was highest in control group when cultured with 100 μmol/L CoCl2 (F =488.026,P ＜ 0.001),which decreased from 150 μmol/L.The cell proliferation level in control group cultured with 250 μmol/L CoCl2 was significantly lower than that with 0 μmol/L (P values all below 0.05).There was no significant change in cell proliferation in ILK siRNA group among different concentrations of CoCl2 (F =2.542,P =0.056).The cell proliferation level in ILK siRNA group was significantly lower than that in control group and negative control group (F =2519.542,P ＜0.001).Conclusions ILK may be a key protein in response of hypoxia in Fbs.The mild hypoxia can stimulate the expression of ILK and promote the proliferation of Fbs,while severe hypoxia can reduce the expression of ILK and inhibit cell proliferation.