中华烧伤杂志
中華燒傷雜誌
중화소상잡지
Chinese Journal of Burns
2015年
5期
372-377
,共6页
王洋%张亮平%雷睿%沈毅忱%沈辉%吴智南%徐靖宏
王洋%張亮平%雷睿%瀋毅忱%瀋輝%吳智南%徐靖宏
왕양%장량평%뢰예%침의침%침휘%오지남%서정굉
瘢痕%成纤维细胞%转化生长因子β1%Smad 4蛋白质%结缔组织生长因子%可溶性受体
瘢痕%成纖維細胞%轉化生長因子β1%Smad 4蛋白質%結締組織生長因子%可溶性受體
반흔%성섬유세포%전화생장인자β1%Smad 4단백질%결체조직생장인자%가용성수체
Cicatrix%Fibroblasts%Transforming growth factor beta 1%Smad 4 protein%Connective tissue growth factor%Soluble receptor
目的 探讨阻断TGF-β/Smads通路的双位点对人皮肤Fb生成瘢痕相关蛋白的影响.方法 将携带可溶性TGF-β受体Ⅱ(sTβRⅡ)、突变型Smad 4——Smad 4△M4基因的2种慢病毒载体按最适感染复数(MOI)50分别转染人皮肤Fb细胞株人包皮Fb 1(HFF-1)细胞,蛋白质印迹法测定2种转染细胞的sTβRⅡ、Smad 4△M4蛋白表达情况,并与未转染细胞进行对比.采用随机数字表法将HFF-1细胞分为6组,每组6皿,每皿1×104个.共转染组,将2种病毒按MOI=50、1:1混合共转染细胞后用5 ng/mL TGF-β1刺激72 h;sTβRⅡ组,慢病毒sTβRⅡ取MOI =50转染细胞,余处理同前;Smad 4△M4组,慢病毒Smad 4△M4取MOI=50转染细胞,余处理同前;阴性病毒对照组,空载体病毒转染细胞,余处理同前;阳性对照组,仅用5 ng/mL TGF-β1刺激72 h;空白对照组,常规培养细胞,不作任何其他处理.刺激结束后,采用蛋白质印迹法、实时荧光定量RT-PCR法分别检测各组细胞纤维连接蛋白的蛋白和mRNA表达,分别采用ELISA法和Sircol法检测各组细胞培养上清液中结缔组织生长因子(CTGF)及总胶原蛋白表达量.对数据行单因素方差分析及SNK-q检验.结果 (1)慢病毒sTβRⅡ转染的HFF-1细胞sTβRⅡ蛋白表达明显高于未转染的HFF-1细胞,慢病毒Smad 4△M4转染的HFF-1细胞Smad 4△M4蛋白表达亦明显高于未转染的HFF-1细胞.经验证,2种慢病毒转染的细胞均能有效表达相应的目的蛋白.(2)6组细胞纤维连接蛋白的蛋白及mRNA表达量总体均差异明显(F值分别为53.536和24.365,P值均小于0.001).阳性对照组细胞纤维连接蛋白的蛋白与mRNA表达量分别为1.60±0.18、1.99±0.40,与阴性病毒对照组的1.60±0.15、1.94±0.28相近(q值分别为0.091和0.419,P值均大于0.05),但高于其余4组(q值为5.245～ 18.228,P值均小于0.05);共转染组细胞纤维连接蛋白的蛋白与mRNA表达量分别为0.60±0.05、0.70±0.11,明显低于sTβRⅡ组的0.89±0.13、1.24±0.17和Smad4△M4组的0.91±0.14、1.28±0.19(q值为3.964 ～4.294,P值均小于0.05).(3)6组细胞培养上清液中CTGF及总胶原蛋白表达量总体均差异明显(F值分别为107.680和38.347,P值均小于0.001).阳性对照组细胞培养上清液中CTGF及总胶原蛋白表达量与阴性病毒对照组相近(q值分别为1.106和0.491,P值均大于0.05),但明显高于其余4组(q值为6.414 ～ 26.420,P值均小于0.05);共转染组细胞培养上清液中CTGF及总胶原蛋白表达量均明显低于sTβRⅡ组和Smad 4△M4组(q值为3.424～7.143,P值均小于0.05).结论 在皮肤Fb中,阻断TGF-β/Smads通路双位点在抑制TGF-β1导致的瘢痕相关蛋白上调方面优于阻断单个位点.
目的 探討阻斷TGF-β/Smads通路的雙位點對人皮膚Fb生成瘢痕相關蛋白的影響.方法 將攜帶可溶性TGF-β受體Ⅱ(sTβRⅡ)、突變型Smad 4——Smad 4△M4基因的2種慢病毒載體按最適感染複數(MOI)50分彆轉染人皮膚Fb細胞株人包皮Fb 1(HFF-1)細胞,蛋白質印跡法測定2種轉染細胞的sTβRⅡ、Smad 4△M4蛋白錶達情況,併與未轉染細胞進行對比.採用隨機數字錶法將HFF-1細胞分為6組,每組6皿,每皿1×104箇.共轉染組,將2種病毒按MOI=50、1:1混閤共轉染細胞後用5 ng/mL TGF-β1刺激72 h;sTβRⅡ組,慢病毒sTβRⅡ取MOI =50轉染細胞,餘處理同前;Smad 4△M4組,慢病毒Smad 4△M4取MOI=50轉染細胞,餘處理同前;陰性病毒對照組,空載體病毒轉染細胞,餘處理同前;暘性對照組,僅用5 ng/mL TGF-β1刺激72 h;空白對照組,常規培養細胞,不作任何其他處理.刺激結束後,採用蛋白質印跡法、實時熒光定量RT-PCR法分彆檢測各組細胞纖維連接蛋白的蛋白和mRNA錶達,分彆採用ELISA法和Sircol法檢測各組細胞培養上清液中結締組織生長因子(CTGF)及總膠原蛋白錶達量.對數據行單因素方差分析及SNK-q檢驗.結果 (1)慢病毒sTβRⅡ轉染的HFF-1細胞sTβRⅡ蛋白錶達明顯高于未轉染的HFF-1細胞,慢病毒Smad 4△M4轉染的HFF-1細胞Smad 4△M4蛋白錶達亦明顯高于未轉染的HFF-1細胞.經驗證,2種慢病毒轉染的細胞均能有效錶達相應的目的蛋白.(2)6組細胞纖維連接蛋白的蛋白及mRNA錶達量總體均差異明顯(F值分彆為53.536和24.365,P值均小于0.001).暘性對照組細胞纖維連接蛋白的蛋白與mRNA錶達量分彆為1.60±0.18、1.99±0.40,與陰性病毒對照組的1.60±0.15、1.94±0.28相近(q值分彆為0.091和0.419,P值均大于0.05),但高于其餘4組(q值為5.245～ 18.228,P值均小于0.05);共轉染組細胞纖維連接蛋白的蛋白與mRNA錶達量分彆為0.60±0.05、0.70±0.11,明顯低于sTβRⅡ組的0.89±0.13、1.24±0.17和Smad4△M4組的0.91±0.14、1.28±0.19(q值為3.964 ～4.294,P值均小于0.05).(3)6組細胞培養上清液中CTGF及總膠原蛋白錶達量總體均差異明顯(F值分彆為107.680和38.347,P值均小于0.001).暘性對照組細胞培養上清液中CTGF及總膠原蛋白錶達量與陰性病毒對照組相近(q值分彆為1.106和0.491,P值均大于0.05),但明顯高于其餘4組(q值為6.414 ～ 26.420,P值均小于0.05);共轉染組細胞培養上清液中CTGF及總膠原蛋白錶達量均明顯低于sTβRⅡ組和Smad 4△M4組(q值為3.424～7.143,P值均小于0.05).結論 在皮膚Fb中,阻斷TGF-β/Smads通路雙位點在抑製TGF-β1導緻的瘢痕相關蛋白上調方麵優于阻斷單箇位點.
목적 탐토조단TGF-β/Smads통로적쌍위점대인피부Fb생성반흔상관단백적영향.방법 장휴대가용성TGF-β수체Ⅱ(sTβRⅡ)、돌변형Smad 4——Smad 4△M4기인적2충만병독재체안최괄감염복수(MOI)50분별전염인피부Fb세포주인포피Fb 1(HFF-1)세포,단백질인적법측정2충전염세포적sTβRⅡ、Smad 4△M4단백표체정황,병여미전염세포진행대비.채용수궤수자표법장HFF-1세포분위6조,매조6명,매명1×104개.공전염조,장2충병독안MOI=50、1:1혼합공전염세포후용5 ng/mL TGF-β1자격72 h;sTβRⅡ조,만병독sTβRⅡ취MOI =50전염세포,여처리동전;Smad 4△M4조,만병독Smad 4△M4취MOI=50전염세포,여처리동전;음성병독대조조,공재체병독전염세포,여처리동전;양성대조조,부용5 ng/mL TGF-β1자격72 h;공백대조조,상규배양세포,불작임하기타처리.자격결속후,채용단백질인적법、실시형광정량RT-PCR법분별검측각조세포섬유련접단백적단백화mRNA표체,분별채용ELISA법화Sircol법검측각조세포배양상청액중결체조직생장인자(CTGF)급총효원단백표체량.대수거행단인소방차분석급SNK-q검험.결과 (1)만병독sTβRⅡ전염적HFF-1세포sTβRⅡ단백표체명현고우미전염적HFF-1세포,만병독Smad 4△M4전염적HFF-1세포Smad 4△M4단백표체역명현고우미전염적HFF-1세포.경험증,2충만병독전염적세포균능유효표체상응적목적단백.(2)6조세포섬유련접단백적단백급mRNA표체량총체균차이명현(F치분별위53.536화24.365,P치균소우0.001).양성대조조세포섬유련접단백적단백여mRNA표체량분별위1.60±0.18、1.99±0.40,여음성병독대조조적1.60±0.15、1.94±0.28상근(q치분별위0.091화0.419,P치균대우0.05),단고우기여4조(q치위5.245～ 18.228,P치균소우0.05);공전염조세포섬유련접단백적단백여mRNA표체량분별위0.60±0.05、0.70±0.11,명현저우sTβRⅡ조적0.89±0.13、1.24±0.17화Smad4△M4조적0.91±0.14、1.28±0.19(q치위3.964 ～4.294,P치균소우0.05).(3)6조세포배양상청액중CTGF급총효원단백표체량총체균차이명현(F치분별위107.680화38.347,P치균소우0.001).양성대조조세포배양상청액중CTGF급총효원단백표체량여음성병독대조조상근(q치분별위1.106화0.491,P치균대우0.05),단명현고우기여4조(q치위6.414 ～ 26.420,P치균소우0.05);공전염조세포배양상청액중CTGF급총효원단백표체량균명현저우sTβRⅡ조화Smad 4△M4조(q치위3.424～7.143,P치균소우0.05).결론 재피부Fb중,조단TGF-β/Smads통로쌍위점재억제TGF-β1도치적반흔상관단백상조방면우우조단단개위점.
Objective To explore the effects of blocking two sites of TGF-β/Smads signaling on the formation of scar-related proteins in human skin fibroblasts.Methods Two lentivirus vectors encoding soluble TGF-β receptor Ⅱ (sTβR Ⅱ) and mutant Smad 4—Smad 4△M4 were respectively transfected into human skin fibroblast cell line human foreskin fibroblast 1 (HFF-1) cells with the optimum multiplicity of infection (MOI) of 50.The protein expressions of sTβR Ⅱ and Smad 4△M4 of the two types of transfected cells were determined by Western blotting so as to compare with those of the untransfected cells.The HFF-1 cells were divided into 6 groups as named below according to the random number table, with 6 dishes in each group, 1 × 104 cells per dish.Co-transfection group, transfected with the two previous lentivirus vectors,mixed with the ratio of 1:1 and MOI of 50, and then stimulated with 5 ng/mL TGF-β1 for 72 h;sTβR Ⅱ group, transfected with lenti-sTβR Ⅱ with MOI of 50, with the other treatment as above;Smad 4△M4 group,transfected with lenti-Smad 4△M4 with MOI of 50, with the other treatment as above;negative virus group, transfected with empty lentivirus vector, with the other treatment as above;positive control group, stimulated with 5 ng/mL TGF-β1 for 72 h;and blank control group, conventionally cultured without any other treatment.After stimulation, Western blotting and real-time fluorescent quantitative RT-PCR were respectively used to determine the protein and mRNA expressions of fibronectin in cells of each group.ELISA and Sircol collagen assay were respectively used to determine the protein expressions of connective tissue growth factor (CTGF) and total collagen in the cell culture supernate of each group.Data were processed with one-way analysis of variance and SNK-q test.Results (1) HFF-1 cells transfected with lenti-sTβR Ⅱ and HFF-1 cells transfected with lenti-Smad 4△M4 respectively expressed higher levels of sTβR Ⅱ protein and Smad 4△M4 protein compared with those of untransfected cells, confirming that HFF-1 cells transfected with the two lentivirus vectors can efficiently express the target proteins.(2) There were statistically significant differences in the protein and mRNA expressions of fibronectin in cells of the 6 groups (with F values respectively 53.536 and 24.365, P values below 0.001).The protein and mRNA expressions of fibronectin in cells of positive control group (respectively 1.60 ± 0.18 and 1.99 ± 0.40) were similar with those of negative virus group (respectively 1.60 ± 0.15 and 1.94 ± 0.28, with q values respectively 0.091 and 0.419, P values above 0.05) , and they were significantly higher than those of the rest 4 groups (with q values from 5.245 to 18.228, P values below 0.05).The protein and mRNA expressions of fibronectin in cells of cotransfection group (respectively 0.60 ± 0.05 and 0.70 ± 0.11) were significantly lower than those of sTβR Ⅱ group (respectively 0.89 ± 0.13 and 1.24 ± 0.17) and Smad 4△M4 group (respectively 0.91 ± 0.14 and 1.28 ± 0.19, with q values from 3.964 to 4.294, P values below 0.05).(3) There were statistically significant differences in the protein expressions of CTGF and total collagen in the cell culture supernate of the 6 groups (with F values respectively 107.680 and 38.347, P values below 0.001).The protein expressions of CTGF and total collagen in the cell culture supernate of positive control group were similar with those of negative virus group (with q values respectively 1.106 and 0.491, P values above 0.05) , and they were significantly higher than those of the rest 4 groups (with q values from 6.414 to 26.420, P values below 0.05).The protein expressions of CTGF and total collagen in the cell culture supernate of co-transfection group were significantly lower than those of sTβR Ⅱ group and Smad 4△M4 group (with q values from 3.424to 7.143, P values below 0.05).Conclusions In human skin fibroblasts, blockage of two sites of TGF-β/Smads signaling can reduce the expression of scar related proteins which are up-regulated by TGF-β1to a greater extent than that of blocking one single site.
