中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2011年
38期
2714-2718
,共5页
刘振中%姜笃银%蔡景龙%宗宪磊%张基勋%单菲%王文婷%王魏
劉振中%薑篤銀%蔡景龍%宗憲磊%張基勛%單菲%王文婷%王魏
류진중%강독은%채경룡%종헌뢰%장기훈%단비%왕문정%왕위
瘢痕疙瘩%成纤维细胞%肽库%转化生长因子β1%细胞增殖
瘢痕疙瘩%成纖維細胞%肽庫%轉化生長因子β1%細胞增殖
반흔흘탑%성섬유세포%태고%전화생장인자β1%세포증식
Keloid%Fibroblasts%Peptide libra.%Transforming growth factor-beta 1%Cellproliferation
目的 从噬菌体随机12肽库中筛选获得转化生长因子(TGF)β1噬菌体模拟肽,评估其抑制瘢痕疙瘩成纤维细胞增殖的作用.方法 以人TGF-β1单克隆抗体为靶,生物淘选噬菌体模拟肽.噻唑蓝(MTT)比色法定量测定活细胞的数量.Annexin V-FITC/PI凋亡检测试剂盒和流式细胞仪检测对成纤维细胞的凋亡作用.免疫荧光测定检测模拟肽与成纤维细胞的亲和力.实时定量PCR分析方法检测成纤维细胞核因子κB(NF-κB),结缔组织生长因子(CTGF)的表达水平.结果 共获得10种噬菌体模拟肽,具有与TGF-β1、TGF-β2、TGF-β受体Ⅱ(TβRⅡ)、TGF-β诱导因子、NF-κB或细胞分裂原素活化蛋白激酶(MAPK)相似的序列.MTT比色测定结果显示4种(第7~10组)噬菌体模拟肽组能够抑制瘢痕疙瘩成纤维细胞增殖(P<0.05).免疫荧光测定显示是噬菌体上的模拟肽,而不是噬菌体本身,能够与瘢痕疙瘩成纤维细胞相结合;凋亡实验显示这4种噬菌体模拟肽能够轻度促使瘢痕疙瘩成纤维细胞发生轻度的晚期凋亡;实时定量PCR的结果显示这4种噬菌体模拟肽NF-κB的表达降低,表达量分别是阴性对照组的0.28、0.26、0.46、0.30倍,4种噬菌体模拟肽CTGF的表达降低,表达量分别是阴性对照组的0.26、0.60、0.34、0.17倍.结论 噬菌体模拟肽可能是通过调节NF-κB及CTGF的表达,调节瘢痕疙瘩成纤维细胞的增殖.
目的 從噬菌體隨機12肽庫中篩選穫得轉化生長因子(TGF)β1噬菌體模擬肽,評估其抑製瘢痕疙瘩成纖維細胞增殖的作用.方法 以人TGF-β1單剋隆抗體為靶,生物淘選噬菌體模擬肽.噻唑藍(MTT)比色法定量測定活細胞的數量.Annexin V-FITC/PI凋亡檢測試劑盒和流式細胞儀檢測對成纖維細胞的凋亡作用.免疫熒光測定檢測模擬肽與成纖維細胞的親和力.實時定量PCR分析方法檢測成纖維細胞覈因子κB(NF-κB),結締組織生長因子(CTGF)的錶達水平.結果 共穫得10種噬菌體模擬肽,具有與TGF-β1、TGF-β2、TGF-β受體Ⅱ(TβRⅡ)、TGF-β誘導因子、NF-κB或細胞分裂原素活化蛋白激酶(MAPK)相似的序列.MTT比色測定結果顯示4種(第7~10組)噬菌體模擬肽組能夠抑製瘢痕疙瘩成纖維細胞增殖(P<0.05).免疫熒光測定顯示是噬菌體上的模擬肽,而不是噬菌體本身,能夠與瘢痕疙瘩成纖維細胞相結閤;凋亡實驗顯示這4種噬菌體模擬肽能夠輕度促使瘢痕疙瘩成纖維細胞髮生輕度的晚期凋亡;實時定量PCR的結果顯示這4種噬菌體模擬肽NF-κB的錶達降低,錶達量分彆是陰性對照組的0.28、0.26、0.46、0.30倍,4種噬菌體模擬肽CTGF的錶達降低,錶達量分彆是陰性對照組的0.26、0.60、0.34、0.17倍.結論 噬菌體模擬肽可能是通過調節NF-κB及CTGF的錶達,調節瘢痕疙瘩成纖維細胞的增殖.
목적 종서균체수궤12태고중사선획득전화생장인자(TGF)β1서균체모의태,평고기억제반흔흘탑성섬유세포증식적작용.방법 이인TGF-β1단극륭항체위파,생물도선서균체모의태.새서람(MTT)비색법정량측정활세포적수량.Annexin V-FITC/PI조망검측시제합화류식세포의검측대성섬유세포적조망작용.면역형광측정검측모의태여성섬유세포적친화력.실시정량PCR분석방법검측성섬유세포핵인자κB(NF-κB),결체조직생장인자(CTGF)적표체수평.결과 공획득10충서균체모의태,구유여TGF-β1、TGF-β2、TGF-β수체Ⅱ(TβRⅡ)、TGF-β유도인자、NF-κB혹세포분렬원소활화단백격매(MAPK)상사적서렬.MTT비색측정결과현시4충(제7~10조)서균체모의태조능구억제반흔흘탑성섬유세포증식(P<0.05).면역형광측정현시시서균체상적모의태,이불시서균체본신,능구여반흔흘탑성섬유세포상결합;조망실험현시저4충서균체모의태능구경도촉사반흔흘탑성섬유세포발생경도적만기조망;실시정량PCR적결과현시저4충서균체모의태NF-κB적표체강저,표체량분별시음성대조조적0.28、0.26、0.46、0.30배,4충서균체모의태CTGF적표체강저,표체량분별시음성대조조적0.26、0.60、0.34、0.17배.결론 서균체모의태가능시통과조절NF-κB급CTGF적표체,조절반흔흘탑성섬유세포적증식.
Objective To isolate the transforming growth factor-beta 1 (TGF-β1) phage model peptides from phage 12-mer display peptide library to inhibit the proliferation of keloid fibroblasts.Methods The phage display 12-mer peptide library was screened for 4 rounds with monoclonal anti-human TGF-β1 as the target to yield the specific phage model peptides.The 3-( 4,5-dimethyhhiazol-2-yl )-2,5-diphenyltetrazolium bromide (MTT) assay was used for the quantitative determination of cellular proliferation.Apoptosis was detected by the Annexin V-FITC/PI apoptosis detection kit and the cells were analyzed with flow cytometry.lmmunofluorescent assay was employed to show the binding affinity of model peptides for keloid fibroblasts.Quantitative real-time polymerase chain reaction (PCR) was performed to detect the expressions of nuclear factor kappa B (NF-κB) and connective tissue growth factor (CTGF).Results Ten phage model peptides were obtained and they were similar to TGF-β1,TGF-β2,TGF-βreceptor Ⅱ (TβRⅡ),TGF-β-induced factor,NF-κB or mitogen-activated protein kinase (MAPK).The results of MTT showed that four phage model peptides ( No.7 - 10) could inhibit the proliferation of keloid fibroblasts ( P < 0.05 ).The results of apoptotic assessment showed that phage model peptides ( No.7 - 10)could slightly trigger the late apoptotic stage of keloid fibroblasts.The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts.The findings of quantitative real-time PCR analysis suggested that the relative expression of NF-κB decreased in phage model peptides groups (No.7 -10).The quantitative expression was 0.28,0.26,0.46 and 0.30respectively versus the negative control group.The relative expression of CTGF decreased in phage model peptides groups (No.7- 10).The quantitative expression was 0.26,0.60,0.34 and 0.17 respectively versus the negative control group.Conclusion Four phage model peptides ( No.7 - 10) isolated from phage display 12-mer peptide library can inhibit the proliferation of keloid fibroblasts via regulating the expressions of NF-κB and CTGF.