不同阶段增生性瘢痕skp2，E2F1的表达及意义
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摘要
目的:通过对不同时期增生期瘢痕成纤维细胞的细胞周期相关基因skp2,E2F1及蛋白的表达变化与同时期成纤维细胞所处的细胞周期的相关性进行探讨,为基因干扰治疗用于增生性瘢痕的防治提供前期研究基础。方法:手术切取三个月,六个月,一年及两年的增生性瘢痕及正常皮肤各8例。用实时荧光定量PCR(RT-PCR)法检测各时期增生性瘢痕及正常皮肤中skp2,E2F1的mRNA表达;蛋白质免疫印迹(Western blotting)法检测标本中二者蛋白表达水平;用流式细胞术检测各时期增生性瘢痕中成纤维细胞的细胞周期分布情况。结果:(1)随着HS的发展,增生性瘢痕的成纤维细胞中skp2的mRNA及蛋白表达强度由强变弱,其表达趋势基本一致。3个月组、6个月组分别与1年组、2年组及正常组比较差异有统计学意义(P＜0.05),前两者之间差异有统计学意义(P＜0.05),后三著之间差异无统计学意义(P＞0.05)。E2F1的mRNA及蛋白表达在增生性瘢痕早期也是明显高于晚期,而且两者的表达趋势也基本一致。3个月组、6个月组分别与1年组、2年组、正常组比较差异有统计学意义(P＜0.05);3个月组与6个月组比较差异无统计学意义(P＞0.05);1年组、2年组、正常组之间比较差异也无统计学意义(P＞0.05)。(2)流式细胞仪检测结果:3个月、6个月增生性瘢痕成纤维细胞主要分布在S、G2/M期;而1年组、2年组增生性瘢痕及正常组的成纤维细胞多处于G0/G1期。G0/G1、S期比例:3个月组和6个月组比较无差异(P＞0.05);3个月、6个月组分别与1年、2年及正常组有差异(P＜0.05);1年组与2年组及正常组(P＜05);2年组与正常组无差异(P＞0.05)。G2\M期比例:3个月组高于6个月组(P＜0.05);3个月、6个月组分别高与1年、2年和正常组(P＜0.05);1年、2年和正常组无差异(P＞0.05)。结论:(1)增生性瘢痕早期存在细胞周期调控因子skp2,E2F1的表达异常,可能与增生性瘢痕早期成纤维细胞异常增殖有重要关系。(2)不同时期增生性瘢FB中skp2,E2F1mRNA与蛋白的表达趋势基本一致,并能较好的调控成纤维细胞细胞周期运行。(3)skp2,E2F1是调控增生性瘢痕的处于增生期的重要调控因子,在增生性瘢痕发生发展的早期阶段对这对调控基因进行干扰治疗,有可能会控制增生性瘢痕的发生和发展。
Objective: To study the relationship that the expression of skp2, E2F1mRNA and protein and cell cycle of fibroblasts at the different stages of hypertrophic scars, in order to provide an initial basis research about gene interference for future prevention and treatment of hypertrophic scars. Methods: The samples of hypertrophic scars(n=8 in each group) were collected respectively from hypertrophic scars of 3-month, 6-month, 1-year,, 2-year and normal skin. The expressions of skp2, E2F1 mRNA and protein were detected by RT-PCR and Wetern Blot.. And at the same time, the flow cytometer was used to detect the fibroblasts cell life cycle. Results: (1) With development of HS. the expression mRNA and protein of skp2 and E2F1 in fibroblasts of hypertrophic scar experiences from high to low level,, and that of in 3 months group and 6 months group was higher respectively than that of 1years, 2 years and normal group (P <0.05) , and that of in 1 year group, 2 years group and normal group were no difference (P>0.05); The expression intensity mRNA and protein of skp2 in 3 months group was higher than that of 6 months (P <0.05), The expression intensity mRNA and protein of E2F1 in 3 months group and 6 months group was no difference (P>0.05). (2) Results of flow cytometry: Most FB were in S and G2/M stage (cycle) in 3 months,6months group; And most FB were in G0/G1 stage (cycle) in 1 year , 2 years group and normal group. the rate of FB in S and G2/M stage (cycle) were no difference in 3 month and 6 month groups(P>0.05), and that in 3 months group and 6 months group were different that of 1years, 2 years and normal group(P <0.05 ), lyear group and 2years, normal group were difference(P <0.05), 2years and normal group was no difference(P>0.05). The rate of FB in G2/M: 3 months group and 6 months group was no difference(P>0.05); 3 months group and 6 months group was lower respectively than that of 1years, 2 years and normal group(P <0.05); 1year group was lower than 2years and normal groups . 2years and normal group was no difference(P0.05). Conclusion: (1) The abnormal expression of cell cycle regulatory gene skp2 and E2F1 are Closely correlation with the development of hypertrophic scar. (2) The mRNA and protein expressions of skp2 and E2F1 in different stages of hypertrophic scar are basically consistent, And the distribution of fibroblastic in cell life cycle match to the function of skp2 and E2F1. (3) The high percentage of fibroblastic cells at S and G2/M in esrly stage of hypertrophic scar suggest that an interventing in early stage be an important target to the genesis and development of hypertrophic scar.
Objective: To study the relationship that the expression of skp2, E2F1mRNA and protein and cell cycle of fibroblasts at the different stages of hypertrophic scars, in order to provide an initial basis research about gene interference for future prevention and treatment of hypertrophic scars. Methods: The samples of hypertrophic scars(n=8 in each group) were collected respectively from hypertrophic scars of 3-month, 6-month, 1-year,, 2-year and normal skin. The expressions of skp2, E2F1 mRNA and protein were detected by RT-PCR and Wetern Blot.. And at the same time, the flow cytometer was used to detect the fibroblasts cell life cycle. Results: (1) With development of HS. the expression mRNA and protein of skp2 and E2F1 in fibroblasts of hypertrophic scar experiences from high to low level,, and that of in 3 months group and 6 months group was higher respectively than that of 1years, 2 years and normal group (P <0.05) , and that of in 1 year group, 2 years group and normal group were no difference (P>0.05); The expression intensity mRNA and protein of skp2 in 3 months group was higher than that of 6 months (P <0.05), The expression intensity mRNA and protein of E2F1 in 3 months group and 6 months group was no difference (P>0.05). (2) Results of flow cytometry: Most FB were in S and G2/M stage (cycle) in 3 months,6months group; And most FB were in G0/G1 stage (cycle) in 1 year , 2 years group and normal group. the rate of FB in S and G2/M stage (cycle) were no difference in 3 month and 6 month groups(P>0.05), and that in 3 months group and 6 months group were different that of 1years, 2 years and normal group(P <0.05 ), lyear group and 2years, normal group were difference(P <0.05), 2years and normal group was no difference(P>0.05). The rate of FB in G2/M: 3 months group and 6 months group was no difference(P>0.05); 3 months group and 6 months group was lower respectively than that of 1years, 2 years and normal group(P <0.05); 1year group was lower than 2years and normal groups . 2years and normal group was no difference(P0.05). Conclusion: (1) The abnormal expression of cell cycle regulatory gene skp2 and E2F1 are Closely correlation with the development of hypertrophic scar. (2) The mRNA and protein expressions of skp2 and E2F1 in different stages of hypertrophic scar are basically consistent, And the distribution of fibroblastic in cell life cycle match to the function of skp2 and E2F1. (3) The high percentage of fibroblastic cells at S and G2/M in esrly stage of hypertrophic scar suggest that an interventing in early stage be an important target to the genesis and development of hypertrophic scar.
引文
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4 任丽红,郝立君,段国新,等.五倍子、蜈蚣对瘢痕成纤维细胞增殖及胶原合成的影响.实用美容整形外科杂志,2003,14(6):324-327.
5 刘丽忠,李国辉.甲基莲心碱在体外对瘢痕FB生长的影响.中草药,2002,33(11):1018-1020.
6 沈丹培,夏隆庆,鞠强,等.五倍子对体外培养的人皮肤瘢痕组织F6生长的影响.中华皮肤科杂志,2002,35(4):299-300.
7 吕远东,罗少军,刘嘉琦,等.己基可可碱对成纤维细胞的生物学影响。实用美容整形外科杂志,2002,13(2):71-73.
8 汤苏阳,徐获荣,王 强,等.苦参碱对人增生性瘢痕成纤维细胞细胞周期和DNA含量的影响.中华医学美学美容杂志,2002,8(1):30-32.
9 解伟光,姜会庆,李汉保.雷公藤提取物抑制增生性瘢痕成纤维细胞的实验研究.中华烧伤杂志2002,1 8(1):32-33.
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2 Hertwell LH,Kastan MB.Cell cycle control and cancer.Science,1994,266(5192):1821-1828.
3 Kastner A,Moyse E,Bauer S,et al.Unusal yegulation of cyclinD1 and cyclin-dependent kinases cdk2 and cdk4 during in vivo mitotic stimulation of olfact-ory neuron progenitors in adult mouse.J Neurochem,2000,74(6):2343-2349.
4 李永林,陈璧.瘫痕增生的生物学基础研究进展中华整形烧伤外科1998,14(2):457-459
5 付小兵,程飚.进一步重视病理性瘢痕发生机制的研究.中国修复重建外科杂志,2005,19(1):1-5.
6 Wu Ct,Morris JR.Genes,Genetics,and Epigenetics:A Correspondence.Science,Aug 2001;293(5532):1103-5.
7 JF Liu,YM Zhang,CX Yi,et al.The expression and interaction of cyclin D1and p16 in fibroblasts of pathologic scars.Zhonghua Zheng Xing Wai Ke Za Zhi,Jul 2004;20(4):265-7.
8 R Wang,A Ghahary,Q Shen,et al.Hypertrophic scar tissues and fibroblasts produce more transforming growth factor-betal mRNA and protein than normal skin and cells.Wound Repair Regen,Mar 2000;8(2):128-37.
9 Gupat S,Kalra A.Efficacy and safety of intralesional 5-fluorouracil in treatment of keloids.Dermatology,2002,204(2):130 -132.
10 陈天新,武凤莲,王冬艳,等.5-FU对增生性瘢痕中cyclinD1、cdk4及p53基因表达的影响.中国美容整形外科杂志,2006,17(2):343-346.
11 查锡良,主编,医学分子生物学.2005年第1版.北京,人民卫生出版社.
12 Schulman BA,Carrano AC,Jeffrey PD,et al.Insights into SCF ubiquin ligases from the structure of the skp1-skp2 complex.Natur,2000,408(16):381-386.
13 Ganoth D,Bornstein G,Ko TK,et al.The cell-cycle regulatory protein CKstis required for SCF(Skp2)-mediated ubiquitiny lation of P27.Nat Cell Biol,2001,3(3):321-324.
14 Yokoi S,Yasui K,Saito OF,et al.A novel taraet gene,Skp2,within the 5P13amplicon that is frequently detected in small cell lung cancers.Am J Pathoc,2002,161(1):207-216.
15 吴文艺,朱世泽.Skp2和p27kip1蛋白在病理性瘢痕组织中的表达和意义.中华整形外科杂志,2005,21(6):448-451.
16 Nakayamak,Nagahamah,Minamishimaya,et al.Skp2-mediated degradation of p27 regulates progression into mitosis.DcvCcll,2004,6(5);661-672
17 张怀军,邢新.E2F1蛋白在病理性瘢痕组织中的表达.实用美容外科杂志,2003,14(4):169-171.
18 何正宏,白云凯.E2F-1与肾脏疾病的研究进展.医学综述.2008,15(2):2255-2258.
19 Harper JW,Adami GR,Wei N,et al.The p21 Cdk- interacting protein Ciplis a potent inhibitor of G1 cyclin-dependent kinases.Cell,1993;75(4):805-16.
20 El-Deiry,Tokino T,Velculescu VE.WAF1,a potential mediator of p53 tumor suppression.Cell,1993;75(4):817-825. Danielsen CC,Wiggers H,Andersen HR.Increased amounts of collagenase and gelatinase in porcine myocardium folling ischemia and reperfusion.J Mol cell Cardiol 1998;30(4):1431-7.
2 Cheng W,Saing S,Zhou H,et al.Ultrasound assessment of scald scars in Asian children receiving piessure gament therapy.J Pediatr surg,2001;36(3):466-9.
3 王芊,曹德君.丹参对培养的人FB生长和胶原形成的影响.中国临床药学杂志,2002,11(1):46-48.
4 任丽红,郝立君,段国新,等.五倍子、蜈蚣对瘢痕成纤维细胞增殖及胶原合成的影响.实用美容整形外科杂志,2003,14(6):324-327.
5 刘丽忠,李国辉.甲基莲心碱在体外对瘢痕FB生长的影响.中草药,2002,33(11):1018-1020.
6 沈丹培,夏隆庆,鞠强,等.五倍子对体外培养的人皮肤瘢痕组织F6生长的影响.中华皮肤科杂志,2002,35(4):299-300.
7 吕远东,罗少军,刘嘉琦,等.己基可可碱对成纤维细胞的生物学影响。实用美容整形外科杂志,2002,13(2):71-73.
8 汤苏阳,徐获荣,王 强,等.苦参碱对人增生性瘢痕成纤维细胞细胞周期和DNA含量的影响.中华医学美学美容杂志,2002,8(1):30-32.
9 解伟光,姜会庆,李汉保.雷公藤提取物抑制增生性瘢痕成纤维细胞的实验研究.中华烧伤杂志2002,1 8(1):32-33.
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