中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2013年
6期
397-400
,共4页
杨晓红%曹毅%解凡%李园园%翁鹤%罗宏宾
楊曉紅%曹毅%解凡%李園園%翁鶴%囉宏賓
양효홍%조의%해범%리완완%옹학%라굉빈
神经纤毛蛋白质1%角蛋白细胞%血管内皮生长因子类%糖尿病足
神經纖毛蛋白質1%角蛋白細胞%血管內皮生長因子類%糖尿病足
신경섬모단백질1%각단백세포%혈관내피생장인자류%당뇨병족
Neuropilin-1%Keratinocytes%Vascular endothelial growth factors%Diabetic foot
目的 探讨神经纤毛蛋白-1(neuropilin-1,NRP-1)和细胞外调节蛋白激酶(ERK)在血管内皮生长因子(vascular endothelial growth factor,VEGF)促HaCaT细胞增殖中的可能机制.方法 培养HaCaT细胞,转染NRP-1质粒EX-O0008-M02,采用逆转录(RT)-PCR法检测转染后NRP-1 mRNA表达,蛋白免疫印迹法检测NRP-1蛋白质表达.将部分培养的HaCaT细胞分为脂质体对照组、对照质粒组以及目的质粒组分别予以脂质体、对照质粒pReceiver-M02及目的质粒EX-O0008-M02转染,各组分别加入PBS、MEK1/2抑制剂(U0126)、VEGF或U0126联合VEGF进行处理后,噻唑蓝(MTT)法检测HaCaT细胞增殖的改变,蛋白免疫印迹法观察ERK1/2的磷酸化情况以及增殖细胞核抗原(PCNA)的表达情况.采用One-way ANOVA检验组间差异,LSD法进行多重比较和校正.结果 RT-PCR和蛋白免疫印迹实验结果提示EX-O0008-M02质粒转染可有效促进HaCaT细胞NRP-1 mRNA及其蛋白质的表达.与脂质体对照组(A570值0.63±0.07)及对照质粒组(A570值0.62±0.13)比较,目的质粒组HaCaT细胞增殖活性(A570值0.88±0.14)明显增强,F=8.755,P＜0.05.与对照质粒VEGF刺激组(A570值0.88±0.10)比较,目的质粒VEGF刺激组HaCaT细胞的增殖活性(A570值1.14±0.18)亦明显增强,F=4.591,P＜0.05.与目的质粒VEGF刺激组比较,U0126预处理可以明显抑制VEGF对HaCaT细胞的促增殖作用(A570值0.50±0.13,F=42.106,P＜0.01).蛋白免疫印迹结果提示与对照质粒转染细胞比较,目的质粒转染后VEGF对HaCaT细胞ERK1/2的促磷酸化及促PCNA表达得到明显提高,然而此作用可以被U0126有效抑制.结论 ERK1/2信号通路的激活在NRP-1蛋白介导的VEGF促HaCaT细胞增殖作用中可能发挥关键性作用.
目的 探討神經纖毛蛋白-1(neuropilin-1,NRP-1)和細胞外調節蛋白激酶(ERK)在血管內皮生長因子(vascular endothelial growth factor,VEGF)促HaCaT細胞增殖中的可能機製.方法 培養HaCaT細胞,轉染NRP-1質粒EX-O0008-M02,採用逆轉錄(RT)-PCR法檢測轉染後NRP-1 mRNA錶達,蛋白免疫印跡法檢測NRP-1蛋白質錶達.將部分培養的HaCaT細胞分為脂質體對照組、對照質粒組以及目的質粒組分彆予以脂質體、對照質粒pReceiver-M02及目的質粒EX-O0008-M02轉染,各組分彆加入PBS、MEK1/2抑製劑(U0126)、VEGF或U0126聯閤VEGF進行處理後,噻唑藍(MTT)法檢測HaCaT細胞增殖的改變,蛋白免疫印跡法觀察ERK1/2的燐痠化情況以及增殖細胞覈抗原(PCNA)的錶達情況.採用One-way ANOVA檢驗組間差異,LSD法進行多重比較和校正.結果 RT-PCR和蛋白免疫印跡實驗結果提示EX-O0008-M02質粒轉染可有效促進HaCaT細胞NRP-1 mRNA及其蛋白質的錶達.與脂質體對照組(A570值0.63±0.07)及對照質粒組(A570值0.62±0.13)比較,目的質粒組HaCaT細胞增殖活性(A570值0.88±0.14)明顯增彊,F=8.755,P＜0.05.與對照質粒VEGF刺激組(A570值0.88±0.10)比較,目的質粒VEGF刺激組HaCaT細胞的增殖活性(A570值1.14±0.18)亦明顯增彊,F=4.591,P＜0.05.與目的質粒VEGF刺激組比較,U0126預處理可以明顯抑製VEGF對HaCaT細胞的促增殖作用(A570值0.50±0.13,F=42.106,P＜0.01).蛋白免疫印跡結果提示與對照質粒轉染細胞比較,目的質粒轉染後VEGF對HaCaT細胞ERK1/2的促燐痠化及促PCNA錶達得到明顯提高,然而此作用可以被U0126有效抑製.結論 ERK1/2信號通路的激活在NRP-1蛋白介導的VEGF促HaCaT細胞增殖作用中可能髮揮關鍵性作用.
목적 탐토신경섬모단백-1(neuropilin-1,NRP-1)화세포외조절단백격매(ERK)재혈관내피생장인자(vascular endothelial growth factor,VEGF)촉HaCaT세포증식중적가능궤제.방법 배양HaCaT세포,전염NRP-1질립EX-O0008-M02,채용역전록(RT)-PCR법검측전염후NRP-1 mRNA표체,단백면역인적법검측NRP-1단백질표체.장부분배양적HaCaT세포분위지질체대조조、대조질립조이급목적질립조분별여이지질체、대조질립pReceiver-M02급목적질립EX-O0008-M02전염,각조분별가입PBS、MEK1/2억제제(U0126)、VEGF혹U0126연합VEGF진행처리후,새서람(MTT)법검측HaCaT세포증식적개변,단백면역인적법관찰ERK1/2적린산화정황이급증식세포핵항원(PCNA)적표체정황.채용One-way ANOVA검험조간차이,LSD법진행다중비교화교정.결과 RT-PCR화단백면역인적실험결과제시EX-O0008-M02질립전염가유효촉진HaCaT세포NRP-1 mRNA급기단백질적표체.여지질체대조조(A570치0.63±0.07)급대조질립조(A570치0.62±0.13)비교,목적질립조HaCaT세포증식활성(A570치0.88±0.14)명현증강,F=8.755,P＜0.05.여대조질립VEGF자격조(A570치0.88±0.10)비교,목적질립VEGF자격조HaCaT세포적증식활성(A570치1.14±0.18)역명현증강,F=4.591,P＜0.05.여목적질립VEGF자격조비교,U0126예처리가이명현억제VEGF대HaCaT세포적촉증식작용(A570치0.50±0.13,F=42.106,P＜0.01).단백면역인적결과제시여대조질립전염세포비교,목적질립전염후VEGF대HaCaT세포ERK1/2적촉린산화급촉PCNA표체득도명현제고,연이차작용가이피U0126유효억제.결론 ERK1/2신호통로적격활재NRP-1단백개도적VEGF촉HaCaT세포증식작용중가능발휘관건성작용.
Objective To explore the possible role of neuropilin-1 (NRP-1) and extracellular regulated protein kinases (ERK) in the mechanisms underlying the promotive effect of vascular endothelial growth factor (VEGF) on the proliferation of HaCaT cells.Methods HaCaT cells were cultured in vitro and transfected with a NRP-1 expression plasmid EX-O0008-M02.Reverse transcription (RT)-PCR and Western blot were performed to detect the mRNA and protein expression of NRP-1 in HaCaT cells respectively before and after the transfection.Some HaCaT cells were divided into three groups to be transfected with liposome (liposome control group),control plasmid pReceiver-M02 (plasmid control group),and objective plasmid EX-O0008-M02 (objective plasmid group),respectively,and each of the three groups was classified into several subgroups to be treated with phosphate buffer solution (PBS),U0126 (MEK1/2 inhibitor) and VEGF alone or in combination.After additional culture,methyl thiazolyl tetrazolium (MTT) assay was performed to determine the proliferative activity of HaCaT cells,Western blot to quantify the expression of total and phosphorylated ERK1/2 as well as proliferating cell nuclear antigen (PCNA) protein in HaCaT cells.Intergroup differences were assessed by one-way analysis of variance (ANOVA),and multiple comparisons and correction were done by using the least significant difference (LSD) test.Results RT-PCR and Western blot analysis confirmed that the transfection with NRP-1 effectively promoted the mRNA and protein expression of NRP-1 in HaCaT cells.A significant increase was observed in cellular proliferative activity (absorbence value at 570 nm) in the objective plasmid group compared with the liposome control group and plasmid control group (0.88 ± 0.14 vs.0.63 ± 0.07 and 0.62 ± 0.13,F =8.755,P ＜ 0.05),also in the VEGF-stimulated objective plasmid group compared with the VEGF-stimulated plasmid control group (1.14 ± 0.18 vs.0.88 ± 0.10,F =4.591,P ＜ 0.05).The U0126 pretreatment markedly suppressed the VEGF-induced proliferation of A375 cells in the objective plasmid group (0.50 ± 0.13 vs.1.14 ± 0.18,F =42.106,P ＜ 0.01).As Western blot analysis suggested,the objective plasmid significantly enhanced the VEGF-induced increase in ERK1/2 phosphorylation degree and PCNA expression intensity in HaCaT cells compared with the control plasmid,but the enhancing effect of objective plasmid was effectively inhibited by U0126.Conclusion The activation of ERK1/2 signaling pathway may play a key role in the NRP-1 protein-mediated promotive effect of VEGF on the proliferation of HaCaT cells.
