小鼠真皮成纤维细胞成脂分化对金黄色葡萄球菌感染的抵抗效应及机制研究

Counteractive effect of mouse dermal fibroblasts during their adipogenic differentiation against Staphylococcus aureus infection and its mechanisms

目的探讨小鼠真皮成纤维细胞(MdFB)成脂分化过程抗金黄色葡萄球菌感染的效应及机制。方法从新生C57BL/6小鼠提取MdFB,采用脂肪诱导分化培养基培养48 h诱导成脂分化,之后采用分化维持培养基继续培养。采用实时荧光定量逆转录PCR(RT-PCR)检测MdFB成脂分化0~6 d抗菌肽蛋白(CAMP)基因mRNA相对表达水平;采用Western印迹法检测MdFB成脂分化培养基上清液CAMP蛋白表达趋势。取12孔板每孔加入1 ml 1×10^(6) CFU MdFB,再加入1×10^(7) CFU灭活金黄色葡萄球菌悬液或磷酸盐缓冲液(对照)刺激,分别采用成脂分化培养基或普通培养基培养4 h,分为共同作用组、分化对照组、金葡菌刺激组、对照组,收集细胞,采用Western印迹及RT-PCR检测CAMP蛋白及mRNA表达。采用成脂分化5 d的MdFB培养基上清液培养金黄色葡萄球菌(5×10^(4) CFU/ml),在10~24 h内每2小时评估生长活性,以普通培养基培养的MdFB上清液(正常对照组)和不含细胞的培养基上清液(阴性对照组)作为对照。采用非配对t检验或方差分析比较组间差异。结果成脂分化后0、1、2、4、6 d时CAMP mRNA相对表达差异(1.14±0.74、68.04±12.72、683.12±38.06、1390.68±226.21、454.57±204.12)有统计学意义(F=50.08,P<0.001),1、2、4、6 d时均高于0 d时(t=9.09、31.03、10.63、3.85,均P<0.05),CAMP基因的表达呈先升后降趋势。培养基上清液中CAMP蛋白含量在第2~5天达到较高水平,之后下降。对照组、金葡菌刺激组、分化对照组、共同作用组MdFB CAMP蛋白(0.433±0.176、0.574±0.176、1.007±0.176、1.217±0.176)及mRNA相对表达(0.08±0.02、0.38±0.10、0.49±0.11、0.80±0.03)差异均有统计学意义(F=46.79、43.25,均P<0.05),共同作用组蛋白及mRNA相对表达均高于分化对照组、金葡菌刺激组、对照组(P<0.05)。成脂分化组细胞上清液培养金黄色葡萄球菌10 h时生长活性(0.053±0.015)均低于正常对照组、阴性对照组(0.109±0.015、0.106±0.015,t=11.30、13.26,均P<0.05),在培养10~24 h的余时间点成脂分化组生长活性均低于正常对照组及阴性对照组(均P<0.05)。结论MdFB在成脂分化过程中向胞外分泌抗菌肽蛋白,可抑制金黄色葡萄球菌的增殖。

Objective To investigate the counteractive effect of mouse dermal fibroblasts(MdFBs)during their adipogenic differentiation against Staphylococcus aureus infection,and to explore its mechanisms.Methods MdFBs were obtained from newborn C57BL/6 mice,and their adipogenic differentiation was induced by culture in an adipogenic medium for 48 hours.Real-time fluorescence-based quantitative PCR(RT-PCR)was performed to determine the mRNA expression of cathelicidin antimicrobial peptide(CAMP)on days 0-6 during the adipogenic differentiation of MdFBs,and Western blot analysis to determine the protein expression of CAMP in the culture supernatant of MdFBs during their adipogenic differentiation.MdFBs were divided into 4 groups:co-stimulation group stimulated by S.aureus suspensions and cultured in an adipogenic medium,adipogenic control group cultured in an adipogenic medium,S.aureus-stimulation group stimulated by S.aureus suspensions and cultured in a common medium,and control group stimulated by phosphate-buffered saline and cultured in a common medium;Western blot analysis and RT-PCR were conducted to determine the protein and mRNA expression of CAMP.S.aureus(5×104 CFU/ml)was cultured with the culture supernatant of MdFBs after 5-day adipogenic differentiation(adipogenic group),and the growth activity was evaluated every 2 hours during 10-24 hours after the start of co-culture;S.aureus cultured with the culture supernatant of MdFBs in a common medium served as the normal control group,and that cultured with cell-free culture supernatant served as the negative control group.Differences between groups were assessed using unpaired t-test or analysis of variance.Results Significant differences were observed in the relative mRNA expression of CAMP among different time points(days 0,1,2,4,and 6)during the adipogenic differentiation of MdFBs(1.14±0.74,68.04±12.72,683.12±38.06,1390.68±226.21,454.57±204.12,F=50.08,P<0.001);the CAMP mRNA expression was significantly higher on days 1,2,4,and 6 than on day 0(t=9.09,31.03,10.63,3.85,respectively,all P<0.05),and showed an initial rise and subsequent fall during days 0-6.The CAMP protein expression in the culture supernatant of MdFBs peaked on days 2-5 and subsequently decreased.Significant differences were observed in the mRNA and protein expression of CAMP among the control group,S.aureus-stimulation group,adipogenic control group and co-stimulation group(mRNA:0.08±0.02,0.38±0.10,0.49±0.11,0.80±0.03,respectively,F=43.25,P<0.05;protein:0.433±0.176,0.574±0.176,1.007±0.176,1.217±0.176,respectively,F=46.79,P<0.05),and the relative mRNA and protein expression of CAMP was significantly higher in the co-stimulation group than in the adipogenic control group,S.aureus-stimulation group and control group(all P<0.05).At 10 hours during culture,the growth activity of S.aureus was significantly lower in the adipogenic group(0.053±0.015)than in the normal control group and negative control group(0.109±0.015,0.106±0.015,t=11.30,13.26,respectively,both P<0.05);during 10-24 hours,the growth activity of S.aureus also showed a significant decrease in the adipogenic group compared with the normal control group and negative control group(all P<0.05).Conclusion MdFBs secreted CAMP during the adipogenic differentiation,and could inhibit the proliferation of S.aureus.