羧甲基壳多糖对瘢痕成纤维细胞增殖和胶原合成的影响
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摘要
背景：瘢痕疙瘩是皮肤损伤后引发的胶原异常积聚所致的过度瘢痕化，与增生性瘢痕不同表现为过度生长，并超过原伤口界限，侵犯邻近组织，呈瘤样增生，造成功能障碍，是皮肤科等多个学科的难治性疾病。目前尚不能确定其确切的发病机理，也没有理想的治疗药物及方法，其主要问题是难以控制其较高的复发率，大量研究证明，病灶区成纤维细胞参与了该病的发生。目前国内研究的热点是如何抑制或控制这种成纤维细胞的增殖能力来达到治疗瘢痕疙瘩的目的。壳多糖及衍生物因具有良好的组织相容性，并抑制细菌、真菌生长、促进伤口愈合，已成为人工皮肤的理想选择材料，而其对瘢痕疙瘩作用的研究和应用还未见报道。目的：观察羧甲基壳多糖对体外培养的瘢痕成纤维细胞增殖和胶原合成的作用，探讨其治疗瘢痕疙瘩的机理，指导临床实践。方法：取新鲜瘢痕疙瘩组织，体外培养瘢痕疙瘩成纤维细胞，应用不同浓度的羧甲基壳多糖作用于3～9代瘢痕成纤维细胞，同时设阴性(无血清培养液)及阳性(曲安萘德)对照组。分别采用四甲基偶氮唑盐(MTT)定量测定法及血球板计数法测定羧甲基壳多糖对瘢痕成纤维细胞增殖的影响，同时采用羟脯氨酸比色法及Ⅲ型前胶原放免试剂盒测定不同浓度羧甲基壳多糖作用于瘢痕成纤维细胞后其上清液中胶原及Ⅲ型前胶原含量，最后将羧甲基壳多糖制成药膜，观察瘢痕成纤维细胞在其中生长情况。结果：MTT结果提示：与阴性对照组比较，羧甲基壳多糖在较低浓度(10μg／ml，50μg／ml，100μg／ml)作用24小时，对瘢痕成纤维细胞增殖无明显抑制作用(P＞0.05)。当延长作用时间至48小时、72小时后，三种浓度羧甲基壳多糖对瘢痕成纤维细胞增殖有明显抑制作用(P＜0.05)。当羧甲基壳多糖浓度增至200μg／ml，在24小时、48小时、72小时观察均显示明显抑制增殖作用(P＜0.05)。上述抑制作用随羧甲基壳多糖浓度增高或作用时间的延长而加强。呈明显的剂量依赖关系和时间依赖关系。羧甲基壳多糖浓度为200μg／ml作用24h、48h、72h对瘢痕成纤维细胞增殖抑制作用与曲安萘德组相比无显著差异(P＞0.05)。血球板计数法显示了与MTT法相一致的结果。羟脯氨酸比色法结果提示与阴性对照组相比，务实验浓度的羧甲基壳多糖(10μg／ml，50μg／ml，100μg／ml，200μg／ml)对瘢痕成纤维细胞作用48小时后，
中文摘要
能够明显抑制该细胞胶原的合成与分泌。(P<0.01)，但这种抑制作用与阳性对照
组曲安蔡德组相比亦有显著性差异(P<0.01)。用放射免疫法测定上清液中m型前
胶原结果显示:梭甲基壳多糖在较低浓度时(10拼创饥1，50ILg/ml)对瘫痕成纤维细
胞fll型前胶原的合成与分泌无明显抑制作用(P>0.05)，在较高浓度对疲痕成纤维
细胞m前胶原的合成与分泌有明显抑制作用(P<0.05)，这种抑制作用与阳性对照
组无明显差异(P闭.05)。结论:梭甲基壳多糖对体外培养的瘫痕成纤维细胞增殖
有明显抑制作用，抑制作用随梭甲基壳多糖浓度的增高或作用时间的延长而加强，
呈明显的剂量依赖关系和时间依赖关系，狡甲基壳多糖能够明显抑制瘫痕成纤维
细胞胶原的合成与分泌，这种抑制作用也随浓度的增高而加强。在较高浓度时可
以抑制瘫痕成纤维细胞中m型胶原的合成与分泌。
Background: Keloids are benign tumours of the skin. They tend to occur in younger patients after different kinds of injuries, infection or they may develop spontaneously. In contrast to hypertrophic scars, keloids are not confined to the original wound, but grow into the corresponding healthy skin .The rarely recede within time. Most patients complain of itching and suffer from impairment of their quality of life. Only little is known about the pathogenesis of keloids, although they have been clinically well characterized for a long time. Successful treatment of keloids is difficult because of frequent recurrences. Fibroblasts are isolated form keloids tissue and maintained in cell culture continue to express an increased capacity to proliferate and to produce collagen. Nowadays, one of the focus problems of keloids is how to inhibit these actions of keloid fibroblast,then find effective ways to treat keloids. Chitosan and its derivatives exhibit myriad biological actions, such as antitimicrobial, biodegradability, biocompatibility and accelerated wound healing properties. So it becomes an optimal biological dressing of articial skin. But there are few reports on their fuction on keloid and KFB.
Objective: To investigate the effects of CM-CH on keloid fibroblasts proliferation and collagen synthesis.
Methods: Keloid fibroblasts were isolated from fresh keloid tissue and cultured. The KFB(from the 3rd to 9th passages)were treated with various concentrations of CM-CH. Triamcinolone acetonide was used as appositive control, cell culture solution was used as a negative control. The effect of CM-CH on proliferation was examined by MTT and cell count technique. Human type III pre-collagen was determined with radio-immunoassa measurement and hydroxy proline (HP), was evaluated by colorimetric analysis. After CM-CH affected the KFB. The infuence of KFB on CM-CH film was observed.
Results: The MTT results showed that CM-CH at lower concentration (10μg/ml, 50μg/ml, 10μg/ml) after treating 24h, did not inhibited KFB proliferation significantly (p>0.05). After treating 48h and 72h, all of the three concentrations showed inhibition effects significantly (p<0.05). At higher concentration (200μg/ml) the significant inhibition effect on KFB proliferation was observed at any detected time (p<0.05). This
inhibition of CM-CH on KFB was not only the concentration but experiment time as well. The inhibition effect of CM-Ch on KFB is similar to that of triamcinolone acetonid (p>0.05) at 200μg/ml concentrations after 24h, 48h, 72h. The result of cell count technique is similar to MTT. The result of colorimetric analysis of hydroxy proline (HP) indicated that CM-CH experiment concentrations after 48h on KFB could markedly decrease the synthesis of collagen (p<0.01). But the inhibition effect of the synthesis of collagen was significantly different than that of triamcinolone acetonid (p<0.01). The result of human typelll pre-collagen determined with radio-immunoassa measurement showed that CM-CH at lower concentration (10μg/ml, 50μg/ml) did not decrease the content of III pre-collagen (p>0.05). The content of III pre-collagen was at lower level when CM-CH at high concentrations. (100μg/ml, 200μg/ml)(p<0.05) and showed no significant difference than that of triamcinolone acetonid.
Conclusion: CM-CH can inhibit the proliferation of KFB. The inhibition of CM-CH on KFB was not only the concentration but experiment time as well. CM-CH can inhibit the synthesis of collagen of KFB.in vitro by a concentration-depended manner, indicating that CM-CH may be effective in treatment of keloids.
Postgraduate: Chen Hairong (dermatology ) Directed by: Professor Wu Yanfang
中文摘要
能够明显抑制该细胞胶原的合成与分泌。(P<0.01)，但这种抑制作用与阳性对照
组曲安蔡德组相比亦有显著性差异(P<0.01)。用放射免疫法测定上清液中m型前
胶原结果显示:梭甲基壳多糖在较低浓度时(10拼创饥1，50ILg/ml)对瘫痕成纤维细
胞fll型前胶原的合成与分泌无明显抑制作用(P>0.05)，在较高浓度对疲痕成纤维
细胞m前胶原的合成与分泌有明显抑制作用(P<0.05)，这种抑制作用与阳性对照
组无明显差异(P闭.05)。结论:梭甲基壳多糖对体外培养的瘫痕成纤维细胞增殖
有明显抑制作用，抑制作用随梭甲基壳多糖浓度的增高或作用时间的延长而加强，
呈明显的剂量依赖关系和时间依赖关系，狡甲基壳多糖能够明显抑制瘫痕成纤维
细胞胶原的合成与分泌，这种抑制作用也随浓度的增高而加强。在较高浓度时可
以抑制瘫痕成纤维细胞中m型胶原的合成与分泌。
Background: Keloids are benign tumours of the skin. They tend to occur in younger patients after different kinds of injuries, infection or they may develop spontaneously. In contrast to hypertrophic scars, keloids are not confined to the original wound, but grow into the corresponding healthy skin .The rarely recede within time. Most patients complain of itching and suffer from impairment of their quality of life. Only little is known about the pathogenesis of keloids, although they have been clinically well characterized for a long time. Successful treatment of keloids is difficult because of frequent recurrences. Fibroblasts are isolated form keloids tissue and maintained in cell culture continue to express an increased capacity to proliferate and to produce collagen. Nowadays, one of the focus problems of keloids is how to inhibit these actions of keloid fibroblast,then find effective ways to treat keloids. Chitosan and its derivatives exhibit myriad biological actions, such as antitimicrobial, biodegradability, biocompatibility and accelerated wound healing properties. So it becomes an optimal biological dressing of articial skin. But there are few reports on their fuction on keloid and KFB.
Objective: To investigate the effects of CM-CH on keloid fibroblasts proliferation and collagen synthesis.
Methods: Keloid fibroblasts were isolated from fresh keloid tissue and cultured. The KFB(from the 3rd to 9th passages)were treated with various concentrations of CM-CH. Triamcinolone acetonide was used as appositive control, cell culture solution was used as a negative control. The effect of CM-CH on proliferation was examined by MTT and cell count technique. Human type III pre-collagen was determined with radio-immunoassa measurement and hydroxy proline (HP), was evaluated by colorimetric analysis. After CM-CH affected the KFB. The infuence of KFB on CM-CH film was observed.
Results: The MTT results showed that CM-CH at lower concentration (10μg/ml, 50μg/ml, 10μg/ml) after treating 24h, did not inhibited KFB proliferation significantly (p>0.05). After treating 48h and 72h, all of the three concentrations showed inhibition effects significantly (p<0.05). At higher concentration (200μg/ml) the significant inhibition effect on KFB proliferation was observed at any detected time (p<0.05). This
inhibition of CM-CH on KFB was not only the concentration but experiment time as well. The inhibition effect of CM-Ch on KFB is similar to that of triamcinolone acetonid (p>0.05) at 200μg/ml concentrations after 24h, 48h, 72h. The result of cell count technique is similar to MTT. The result of colorimetric analysis of hydroxy proline (HP) indicated that CM-CH experiment concentrations after 48h on KFB could markedly decrease the synthesis of collagen (p<0.01). But the inhibition effect of the synthesis of collagen was significantly different than that of triamcinolone acetonid (p<0.01). The result of human typelll pre-collagen determined with radio-immunoassa measurement showed that CM-CH at lower concentration (10μg/ml, 50μg/ml) did not decrease the content of III pre-collagen (p>0.05). The content of III pre-collagen was at lower level when CM-CH at high concentrations. (100μg/ml, 200μg/ml)(p<0.05) and showed no significant difference than that of triamcinolone acetonid.
Conclusion: CM-CH can inhibit the proliferation of KFB. The inhibition of CM-CH on KFB was not only the concentration but experiment time as well. CM-CH can inhibit the synthesis of collagen of KFB.in vitro by a concentration-depended manner, indicating that CM-CH may be effective in treatment of keloids.
Postgraduate: Chen Hairong (dermatology ) Directed by: Professor Wu Yanfang
引文
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2. Ehrlich HP, Desmouliere A, Diegelmann RF, et al. Morphological and immunochemical differences between keloid and hypertrophic scar. Am J Pathol, 1994, 14: 105.
3. Raghow R. The role of extracelluar matrix in postiflammatory wound healing and fibrosis. FASEBJ, 1994, 8:823.
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6．张文君．刘万顺．异透明质酸治疗皮肤溃疡临床观察．中国皮肤性病学杂志，1995，9：(4)：212．
7. Chen XG, Wang Z, Liuws, et al. The effect of carboxymethyl-Chitosan on proliferation and collagen secretion of normal and keloid skin fibroblasts. Biomaterials, 2002 Dec; 23(23)4609-4614.
8．司徒镇强，吴正军主编．细胞培养．第一版．西安：世界图书出版公司，1996．68-69．
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11．鄂征主编．组织培养与分子生物学技术．第一版．北京：北京出版社，1995．438-439．
12. HuszarG, Maiocco J, Naftolin F. Monitoring of collagen and collagen fragments in chromatography of protein mixture. Anal Biochem, 1980, 105(1): 424-429.
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2. Ehrlich HP, Desmouliere A, Diegelmann RF, et al. Morphological and immunochemical differences between keloid and hypertrophic scar. Am J Pathol, 1994, 14: 105.
3. Raghow R. The role of extracelluar matrix in postiflammatory wound healing and fibrosis. FASEBJ, 1994, 8:823.
4. Bloom D, Heredity of keloids: Review of the literature and report of a family with multiple keloids in five generations. NT, State J Med, 1956, 56: 511.
5. Murray J C, Pollack SV, Pinnell SR. Keloids: A review. J Am Acad Dermatol, 1981, 4: 461.
6．张文君．刘万顺．异透明质酸治疗皮肤溃疡临床观察．中国皮肤性病学杂志，1995，9：(4)：212．
7. Chen XG, Wang Z, Liuws, et al. The effect of carboxymethyl-Chitosan on proliferation and collagen secretion of normal and keloid skin fibroblasts. Biomaterials, 2002 Dec; 23(23)4609-4614.
8．司徒镇强，吴正军主编．细胞培养．第一版．西安：世界图书出版公司，1996．68-69．
9．鄂征主编．组织培养与分子生物学技术．第一版．北京：北京出版社，1995．87-88．
10．司徒镇强，吴正军主编．细胞培养．第一版．西安：世界图书出版公司，1996．186-187．
11．鄂征主编．组织培养与分子生物学技术．第一版．北京：北京出版社，1995．438-439．
12. HuszarG, Maiocco J, Naftolin F. Monitoring of collagen and collagen fragments in chromatography of protein mixture. Anal Biochem, 1980, 105(1): 424-429.
13．章静波主编．细胞生物学实验方法与技术．第二版．北京医科大学中国协和医科大学联合出版社出版，1996，1-2．
14．蔡景龙，张宗学主编．现代瘢痕治疗学．第一版．北京：人民卫生出版社，1998．263．
15. Tredget EE, Nedelec B, Scott PG, et al. Hypertrophic scar, Keloids and contracture: the cellular and molecular basis for therapy. Surg Clin North Am, 1997, 77: 701-703.
16．蔡景龙，张宗学主编．现代瘢痕治疗学．第一版，北京：人民卫生出版社．1998．17-30．
17．王文革，钱云良，商庆新．细胞因子与瘢痕形成机制的研究进展．中华整形烧伤外科杂志，1997，13(2)：128．
18. Rockwell WB, Cohen IK, Ehrlich HP. Keloids and hypertrophic scar:a comprehensive review Plast Reconstr Sury, 1989, 84(5): 827.
19. Muller MJ, Herndon DN. The challenge of burns Lancet, 1994, 343(8891): 216.
20．丁焕文，何锡煌．碱性成纤维细胞生长因子研究进展．细胞生物学杂志，1996，18(1)：14．
21. Minami S, Okamto Y, Hamada K, et al. Veterinary practice with chitin and Chitosan. EXS, 1999, 87: 265-77Review.
22. Ishihara M, Nakanishi K, Onok, et al. Photocrosslinkable Chitosan as a dressing for wound occlusion and accelerator in healing process. Biomaterials, 2002, 23(3): 833-840.
23. Ueno H, Nakamura F, Murakami M, et al. Evaluation effects of chitosan for the extrcellular matrix production by fibroblasts an growth factors production by macrophages. Biomaterials, 2001, 22(15): 2125-2130.
24．陈瑞芬，董晓黎，张澄波等．壳多糖对创伤愈合作用的形态学观察．首都医学院学报，1994，15(3)：176-179．
25．鲁元刚，伍津津，朱堂友等．复合壳多糖人工皮肤修复家兔全层皮肤缺损的实验研究．中华烧伤杂志，2002，18(1)：19-22．
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