蛋白质芯片在增生性瘢痕研究中的应用
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摘要
背景：病理性瘢痕在临床上可分为增生性瘢痕和瘢痕疙瘩两种类型。二者均由胶原纤维和细胞以及新生血管构成，其特征是大量的成纤维细胞增生和过度的胶原沉积。成纤维细胞是产生胶原的主要细胞，与病理性瘢痕的形成有重要关系。随着现代细胞生物学和分子生物学的迅猛发展，有人利用基因芯片技术对瘢痕组织与正常皮肤进行研究，已经证实了基因表达差异的存在。这些研究结果为本课题提供了有力的基因学依据。蛋白质芯片指纹图谱(SELDI)技术将蛋白质芯片与飞行质谱相结合，使它既具有芯片的高通量、高效率和重复性好的特点，又具有飞行质谱的高灵敏度检测的功能，可直接对血清及组织粗样品中的各种蛋白质进行检测，其理论和技术的发展为各种疾病的研究提供了新的方法。
目的：对体外培养的增生性瘢痕及正常皮肤组织中的成纤维细胞，利用赛弗吉的SELDI-TOF-MS(表面加强激光解吸电离飞行时间质谱)技术，分别建立增生性瘢痕成纤维细胞与正常皮肤组织成纤维细胞的蛋白质指纹图谱，找寻两者的蛋白质表达差异，建立两者的蛋白质指纹差异图谱，为增生性瘢痕形成机制的研究提供新的思路。
方法：选择增殖期的增生性瘢痕18例，部分瘢痕个体的正常皮肤组织6例。标本全部手术切取，立即行细胞培养。经3～4次传代后，将成纤维细胞裂解，裂解液上样于蛋白质芯片，用SELDI-TOF-MS芯片阅读仪读取数据，建立增生性瘢痕及正常皮肤组织成纤维细胞的蛋白质指纹图谱。进一步用生物信息学方法分析比较所得数据，选择有意义的蛋白质位点，建立一个敏感可靠的的生物信息学模型。
结果：共检测出394个波峰，其中m／z值在2～5KD之间的有143个；5～10KD之间的71个；10～20KD之间的47个；20～30KD之间的18个；30～40KD之间的34个；40～50KD之间的17个。主要的峰值集中在2～10KD之间。经过Ciphergen Biomarker Wizard软件处理，得出m／z位于2805Da、2910Da、
Background: Pathological scars may be divided into two kinds: hypertrophic scars and keloids. They are all made up of collagen fibers, cells and new-born blood vessels with the characteristic of fibroblasts' hyperplasia and collagen' s over-deposit. Whereas the collagen is secreted mainly by fibroblasts, which, it is concerned , has a close relationship with the formation of pathological scars. Along with the rapid development of cellular and molecular biology in recent years, the comparison of gene profiling of normal skin with hypertrophic scars has identified some gene segment responsible for the formation of scar. Such are important evidences for our study. SELDI is a new technology which developed after gene chip. SELDI combines protein chip technology with Mass Spectrometry . It has some merits such as high quantum, high efficiency, good repeatability of chip , and high sensitivity of Mass also. The development of knowledge and technology in proteomics has provided a powerful method in studying diseases.
Objective: To screen and evaluate protein biomarkers for the fibroblastic cells of hypertrophic scar and normal skin. By Ciphergen's SELDI-TOF-MS , all different protein spots were detected from the protein chips. From the point set, some special spots were picked out as the part of desired protein fingerprint.. Combining with bioinformatics, a specific patterns was established successfully to discriminate hypertrophic scar from normal skin.
Methods: The fibroblastie cells from eighteen cases of hypertrophic scar and six cases of normal skin were cultured individually in vitro.
目的：对体外培养的增生性瘢痕及正常皮肤组织中的成纤维细胞，利用赛弗吉的SELDI-TOF-MS(表面加强激光解吸电离飞行时间质谱)技术，分别建立增生性瘢痕成纤维细胞与正常皮肤组织成纤维细胞的蛋白质指纹图谱，找寻两者的蛋白质表达差异，建立两者的蛋白质指纹差异图谱，为增生性瘢痕形成机制的研究提供新的思路。
方法：选择增殖期的增生性瘢痕18例，部分瘢痕个体的正常皮肤组织6例。标本全部手术切取，立即行细胞培养。经3～4次传代后，将成纤维细胞裂解，裂解液上样于蛋白质芯片，用SELDI-TOF-MS芯片阅读仪读取数据，建立增生性瘢痕及正常皮肤组织成纤维细胞的蛋白质指纹图谱。进一步用生物信息学方法分析比较所得数据，选择有意义的蛋白质位点，建立一个敏感可靠的的生物信息学模型。
结果：共检测出394个波峰，其中m／z值在2～5KD之间的有143个；5～10KD之间的71个；10～20KD之间的47个；20～30KD之间的18个；30～40KD之间的34个；40～50KD之间的17个。主要的峰值集中在2～10KD之间。经过Ciphergen Biomarker Wizard软件处理，得出m／z位于2805Da、2910Da、
Background: Pathological scars may be divided into two kinds: hypertrophic scars and keloids. They are all made up of collagen fibers, cells and new-born blood vessels with the characteristic of fibroblasts' hyperplasia and collagen' s over-deposit. Whereas the collagen is secreted mainly by fibroblasts, which, it is concerned , has a close relationship with the formation of pathological scars. Along with the rapid development of cellular and molecular biology in recent years, the comparison of gene profiling of normal skin with hypertrophic scars has identified some gene segment responsible for the formation of scar. Such are important evidences for our study. SELDI is a new technology which developed after gene chip. SELDI combines protein chip technology with Mass Spectrometry . It has some merits such as high quantum, high efficiency, good repeatability of chip , and high sensitivity of Mass also. The development of knowledge and technology in proteomics has provided a powerful method in studying diseases.
Objective: To screen and evaluate protein biomarkers for the fibroblastic cells of hypertrophic scar and normal skin. By Ciphergen's SELDI-TOF-MS , all different protein spots were detected from the protein chips. From the point set, some special spots were picked out as the part of desired protein fingerprint.. Combining with bioinformatics, a specific patterns was established successfully to discriminate hypertrophic scar from normal skin.
Methods: The fibroblastie cells from eighteen cases of hypertrophic scar and six cases of normal skin were cultured individually in vitro.
引文
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8. HarropAR, Ghahary A, Scott P, et al. Regulation If collagen synthesis and m RNA expression in normal and hypertrophic scar fibroblasts in vitro by interferon-γ. J Surg Res,1995,58(5):471.
9. Robson MC, Barnetts RA. Prevention and treatment of post-burn scars and contracture. World J Surg, 1992,16:87-96.
10. David W, Friedman, Charles.et al. Regulation of collagen gene expression in keloids and hypirtrophic scar. J Surg. Res, 1993:55:214-222.
11. Reed M J, Vernon RB, Abrass IB, et al. TGFβ1 induces the expression of type Ⅰ collagen and SPARC, and enhances contraction of collagen gels, by fibroblasts from young and aged donors. J Cell Physiol, 1994,158:169-179.
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13. Garner WL, Karmiol S, Rodriguez JZ. et al. Phenotypic differences in cytokine responsiveness of hypertrophic scar versus normal dermal fibroblasts. J Invest Dermatol, 1993, 101:875.
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18. Pierce GF, Berg JV, Rudloph R, et al. Platelet derived growth factor-b and transforming growth factors-beta1 selectively. Modulate glucose amino-glycans, collagen, and myofibroblasts in excisonal wounds. Am J Pathol 1991, 138:629-646.
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2．蔡景龙 张宗学[M]现代瘢痕治疗学．北京：人民卫生出版社．104-105．
3．国外医学，创伤与外科基本问题分册，1998，19(1)：23．
4．杨松林，何清廉，林子豪，等．瘢痕成纤维细胞三维培养的实验研究．中华整形烧伤外科杂志，1996，12(1)：2．
5．刘德伍，李国辉，曹勇，等．粉防己碱对人皮肤成纤维细胞生长的抑制作用观察．中国中药杂志，1998，23(1)：55．
6．杨松林，何清廉，林子豪，等．γ干扰素对瘢痕成纤维细胞的生物学作用．中华整形烧伤外科杂志，1998，14(2)：107．
7. Koonin AJ. The aetiology of keloids: a review of the literature and a new hypothesis. South Afr Med J, 1964, 38:913-918.
8. HarropAR, Ghahary A, Scott P, et al. Regulation If collagen synthesis and m RNA expression in normal and hypertrophic scar fibroblasts in vitro by interferon-γ. J Surg Res,1995,58(5):471.
9. Robson MC, Barnetts RA. Prevention and treatment of post-burn scars and contracture. World J Surg, 1992,16:87-96.
10. David W, Friedman, Charles.et al. Regulation of collagen gene expression in keloids and hypirtrophic scar. J Surg. Res, 1993:55:214-222.
11. Reed M J, Vernon RB, Abrass IB, et al. TGFβ1 induces the expression of type Ⅰ collagen and SPARC, and enhances contraction of collagen gels, by fibroblasts from young and aged donors. J Cell Physiol, 1994,158:169-179.
12. Sappino AP, Schurch W, Gabbiani G. Differentitation repertoire of fibroblastic cells: expression of cytoskeletal proteins as marker of phenotypic modulations. Lab Invest, 1990:63:144.
13. Garner WL, Karmiol S, Rodriguez JZ. et al. Phenotypic differences in cytokine responsiveness of hypertrophic scar versus normal dermal fibroblasts. J Invest Dermatol, 1993, 101:875.
14. Robb EC, Waymack JP, Warden GD, el al. A new model for studying the development of human hypertrophic burn scar formation. J Burn Care Rehabil, 1987, 8:371-375.
15. Tan EM, Qin H, Kennedy SH, et al. Platelet-derived growth factors-AA and-BB regulate collagen and collagenase gene expression differentially in human fibroblasts, Biochem J, 1995,310:585-588.
16. Lin RY, Sullivan KM, Argenta PA, et al. Exogenous transforming growth factors-beta amplifies its own expression and induces scar formation in a model of human fetal skin repair. Ann Surg, 1995,222:146-154.
17. Sullivan KM. Lorenz HP, Meuli M, et al. A model of scarless hurt fetal wound repair is deficient in transforming growth factor beta. J Pediatr Surg, 1995, 30:198-202.
18. Pierce GF, Berg JV, Rudloph R, et al. Platelet derived growth factor-b and transforming growth factors-beta1 selectively. Modulate glucose amino-glycans, collagen, and myofibroblasts in excisonal wounds. Am J Pathol 1991, 138:629-646.
19. Shah M, Foreman DM, Ferguson MW. Neutralisation of TGF-beta1 and 1TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci, 1995, 108:985-1002.
20. 20. Ghahary A, You JS, Poul GS, et al. Immunolocalization of TGF-β1 in human hypertrophic scar and normal dermal tissues. Cytokin, 1995,7:184-190.
21. Scott PG, Dodd CM, Tredget EE, et al. Immunohistochemical localization of the proteoglycans decorin, biglyean and versican and transforming growth factor 6 in human post-burn hypertrophic and mature scars. Hispotopah, 1995,26:423-431.
22. Choi BM, Kwak HJ, Jun CD, et al. Control of scarring in adult wounds using antisense transforming growth factor-beta1 oligodeoxy nucleotides. Immunol Cell Biol, 1996,74(2): 144.
23. 23. Massague J, Hata A, Liu F. TGF-beta signaling through the Smad pathway [J]. Trends Cell Biol, 1997, 7:187-192.
24. Sporn MB, Roberts AB. A major advance in the use of growth factors to enhance wound healing [J]. J Clin Invest, 1993, 92(6):2565-2566.
25. Hieter P, Boguski M. Functional Genomics: it's all how you read it. Science. 1997. 278: 601-602.
26. Kallioniemi OP, Wagner U, Kononen J. et al. Tissue microarray technology for high throughput molecular profiling of cancer. Hum Mol Genet, 2001, 10: 657-662.
27. Wang CM , Hyakusoku H , Asano G. Genetic predispositions to keloid and hypertrophic scars. J Jpn Plast Reconstr Surg, 2001, 21: 241-246.
28. Bettinger DA, Yager DR, Diegelmann RF. et al. The effect of TG beta on keloid fibroblast proliferation and collagen synthesis. Plast Reconstr Surg, 1996. 98: 827-833.
29. Tan EM , Rouda S, Greenbaum SS, et al. Acidic and basic fibroblast growth factors down-regulate collagen gene expression in keloid fibroblasts. Am J Pathol, 1993, 142: 463-470.
30. Sayah DN, Soc C, Shaw WW , et al. Down regulation ofapoptosis-related genes in keloid tissues. J Surg Res, 1999, 87: 209-216.
31. Banks RE, Dunn MJ, Hochstrasser DF, et al. Proteomics: new perspectives, new biomedical opportunities [J]. Lancet, 2000, 356(9243): 1749-1756.
32. Wasinger VC, Cordwell S J, Cerpa-Poljak A, et al. Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium [J]. Electrophoresis, 1995, 16(7): 1090-1094.
33. Arrell DK, Neverova I, and Van Eyk JE. Cardiovascular proteomics: evolution and potential [J]. Circ Res, 2001, 88(8): 763-773.
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