A型肉毒毒素引起人增生性瘢痕成纤维细胞的凋亡A型肉毒毒素引起人增生性瘢痕成纤维细胞的凋亡

doi:10.3969/j.issn.2095-4344.2013.24.009

摘要/Abstract

摘要：

背景：A型肉毒毒素临床上可以治疗增生性瘢痕，体外细胞培养研究发现可以抑制增生性瘢痕成纤维细胞的增殖。
目的：观察A型肉毒毒素对人增生性瘢痕成纤维细胞增殖的抑制作用，以及对人增生性瘢痕成纤维细胞凋亡的影响。
方法：通过消化法分离培养出人增生性瘢痕成纤维细胞，分别用不同浓度的A型肉毒毒素对细胞的生长增殖过程进行干预，通过MTT染色，于酶联免疫检测仪570 nm测定吸光度来研究细胞生长增殖情况，计算抑制率。通过Hoechst33342及PI染色检测人增生性瘢痕成纤维细胞凋亡情况，并计算凋亡率。
结果与结论：人增生性瘢痕成纤维细胞生长过程中呈梭形，细胞生长旺盛，细胞融合成单层，细胞排列成高度一致性。经A型肉毒毒素处理后，细胞增殖速度明显减慢，细胞数量减少，细胞排列方向散乱。MTT染色后吸光度减弱，随A型肉毒毒素浓度增加，吸光度明显减弱，与对照细胞比较，差异有显著性意义(P < 0.05)，半数抑制率出现在0.4 IU/L。在荧光显微镜下，人增生性瘢痕成纤维细胞经Hoechst33342和PI染色后细胞核呈现蓝色，细胞核为光滑的圆形或椭圆形外观。A型肉毒毒素干预后细胞核致密浓缩，染色不均匀，折光性增强，核膜皱缩，部分细胞核碎裂，出现碎块，有凋亡小体出现。随着A型肉毒毒素浓度的增加细胞凋亡率逐渐增高，与对照细胞比较差异有显著性意义(P < 0.05)，半数凋亡率在0.4 IU/L。说明A型肉毒毒素可以抑制人增生性瘢痕成纤维细胞的增殖，其主要通过引起细胞凋亡的途径来抑制成纤维细胞的增殖。

关键词: 组织构建, 皮肤组织构建, A型肉毒毒素, 增生性瘢痕, 成纤维细胞, 细胞核, 细胞培养, 细胞凋亡, 增殖, 皮肤, 瘢痕, 烧伤

Abstract:

BACKGROUND: Botulinum toxin type A can be used to treat human hypertrophic scar clinically and inhibit the proliferation of human hypertrophic scar fibroblasts in vitro.
OBJECTIVE: To investigate the effect of botulinum toxin type A on the proliferation and apoptosis of human hypertrophic scar fibroblasts.
METHODS: Human hypertrophic scar fibroblasts were isolated and cultured using digestion method. The cells were treated with botulinum type A at different concentrations. After 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide dyeing, the cell solutions were detected with enzyme-linked immunosorbent detector at 570 nm. The inhibitory rate was calculated. The cultured cells were stained with hoechst 33342 and PI. The apoptotic cells were observed under fluorescence microscopy and the apoptotic rate was calculated.
RESULTS AND CONCLUSION: The hypertrophic scar fibroblasts represented the spindle-like character with uniform composition and strong proliferation. After treated with botulinum toxin type A, the cells grew slowly, the cell amount reduced and the arrangement of the cells was scattered. The absorbance after 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide dyeing decreased. There was a statistically significant difference compared with the control group (P < 0.05). The half inhibitory rate was 0.4 IU/L. After hoechst 33342 and PI dyeing, the nuclei became blue and showed a smooth round or oval appearance. After treated with botulinum toxin type A, the nuclei were condensed with uneven dyeing. Some of the nuclei were fragmented and apoptotic bodies appeared. The apoptotic rate was increased with increasing concentrations of botulinum toxin type A. There was a statistically significant difference between the control group and the treated group (P < 0.05). The half apoptotic rate was 0.4 IU/L. These findings indicate that botulinum toxin type A can cause apoptosis of the human hypertrophic scar fibroblasts, thus inhibiting the proliferation of the cells.

Key words: tissue construction, skin tissue construction, botulinum toxin type A, hypertrophic scar, fibroblasts, nuclei, cell culture, apoptosis, proliferation, skin, scar, burn

中图分类号:

R318

李卫华，高玉伟，李德水，邢玉玺. A型肉毒毒素引起人增生性瘢痕成纤维细胞的凋亡A型肉毒毒素引起人增生性瘢痕成纤维细胞的凋亡[J]. 中国组织工程研究, 2013, 17(24): 4429-4435.

Li Wei-hua, Gao Yu-wei, Li De-shui, Xing Yu-xi. Botulinum toxin type A induces apoptosis of human hypertrophic scar fibroblasts[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(24): 4429-4435.

图/表（结果） 3

参考文献

[1] Wu BJ, Wu JM, Lin ZH, et al. Zhonghua Shaoshang Zazhi. 2006;22(06):469-470.吴包金,吴建明,林子豪,等.增生性瘢痕退行性变过程中的细胞凋亡和相关基因表达[J].中华烧伤杂志,2006,22(06):469-470.http://www.cnki.com.cn/Article/CJFDTotal-ZHSA200606028.htm

[2] Wang L, Tai NZ, Fan ZH, et al. ZHonghua Zhengxing Waike Zazhi. 2009;4:284-287.王琳,邰宁正,范志宏, 等. A型肉毒毒素对兔耳增生性瘢痕组织中P物质、β1转移生长因子、α平滑肌肌动蛋白的影响[J].中华医学美学美容杂志,2009,6:410-413.http://www.cqvip.com/QK/71135X/201107/32577654.html

[3] Wang L, Tai NZ, Fu MG, et al. Zhonghua Zhengxing Waike Zazhi. 2009;25(3):222-223.王琳,邰宁正,傅敏刚,等.A型肉毒毒素注射治疗瘢痕疙瘩患者的顽固性痛痒[J].中华整形外科杂志,2009,25(3): 222-223.http://www.cqvip.com/QK/71135X/201107/30537397.html

[4] Zhao BC, Qian L. Hunana Yike Daxue Xuebao. 2000;25(1): 73-76.赵柏程,钱利.增生性瘢痕和瘢痕疙瘩组织中细胞凋亡与增殖的研究[J]．湖南医科大学学报,2000,25(1):73-76.http://www.cnki.com.cn/Article/CJFDTotal-HNYD200001027.htm

[5] Yu B, Chen ML, Liu WG, et al. Zhonghua Yixue Meixue Meirong Zazhi. 2008;14(2):95-101.于波,陈敏亮,刘文阁,等.A型肉毒毒素预防与治疗早期增生性瘢痕的临床研究[J].中华医学美学美容杂志,2008,14(2):95-101.http://med.wanfangdata.com.cn/News/Article/1/1826.aspx

[6] Gauqlitz GG, Bureik D,Dombrowski Y,et al. Botulinum toxin a for the treatment of keloids. Skin Pharmacol Physiol. 2012;25 (6):318-319.http://www.ncbi.nlm.nih.gov/pubmed/22948093

[7] Li WH, Li DS, Gao YW, et al. Zhonguo Zuzhi Gongcheng Yanjiu. 2012;16(20):3667-3670.李卫华,李德水,高玉伟,等.A型肉毒毒素可抑制人增生性瘢痕成纤维细胞增殖和胶原蛋白合成[J].中国组织工程研究,2012,16 (20): 3667-3670.http://www.cnki.com.cn/Article/CJFDTotal-XDKF201220022.htm

[8] Haubner F, Ohmann E, Müller-Vogt U, et al. Effects of botulinum toxin a on cytokine synthesis in a cell culture model of cutaneous scarring. Arch Facial Plast Surg. 2012;14(2):122-126.http://www.ncbi.nlm.nih.gov/pubmed/22006235

[9] Hevia O.Retrospective review of 500 patients treated with abobotulinumtoxin A.J Drugs Dermatol. 2010;9(9):1081-1084.http://www.ncbi.nlm.nih.gov/pubmed/20865838

[10] Levy NS, Lowenthal DT. Application of botulinum toxin to clincal therapy: advances and cautions. Am J Ther. 2012; 19(4): 281-286.http://www.ncbi.nlm.nih.gov/pubmed/22481607

[11] Dresslor D. Parmacology of botulinum toxin drugs. HNO.2012; 60(6):496-504.http://www.ncbi.nlm.nih.gov/pubmed/23054049

[12] Cable MM. On the front lines: What’s new in botox and facial fillers. Mo Med.2010;36:2121-2134.http://www.ncbi.nlm.nih.gov/pubmed/21319685

[13] Beer Kr. Combined treatment for skin rejuvenation and soft-tissue augmentation of the aging face. J Drugs Dermatol. 2011;10(2):125-132.http://www.ncbi.nlm.nih.gov/pubmed/21283916

[14] Goldman A, Wollina U. Facial rejuvenation for middle-aged women: a combined approach with minimally invasive procedures. Clin Interv Aging.2010;23(5):293-299.http://www.ncbi.nlm.nih.gov/pubmed/20924438

[15] Raspaldo H, Baspeyras M,Bellity P,et al.Upper- and mid-face anti-aging treatment and prevention using onabotulinumtoxin A: the 2010 multidisciplinary French consensus—part 1. J Cosmet Dermatol.2011;10(1):36-50.http://www.ncbi.nlm.nih.gov/pubmed/21332914

[16] Sepehr A,Chauhan N,AlexanderAJ,et al.Botulinum toxin type a for facial rejuvenation: treatment evolution and patient satisfaction.Aesthetic Plast Surg.2010;34(5):583-586.http://www.ncbi.nlm.nih.gov/pubmed/20383497

[17] Andrade NN, Deshpande GS. Use of botulinum toxin (botox) in the management of masseter muscle hypertrophy: a simplified technique. Plast Reconstr Surg.2011;128(1):24-26.http://www.ncbi.nlm.nih.gov/pubmed/21701301

[18] Pary A,Pary K.Masseteric hypertrophy: considerations regarding treatment planning decisions and introduction of a novel surgical technique. J Oral Maxillafac Surg.2011;69(3): 944-949.http://www.ncbi.nlm.nih.gov/pubmed/21195532

[19] Jaspers GW, Pijpe J, Jansma J.The use of botulinum toxin type A in cosmetic facial procedures. Int Oral Maxillofac Surg. 2011; 40(2):127-133.http://www.ncbi.nlm.nih.gov/pubmed/20965695

[20] Kim NH, Park RH, Park JB. Botulinum toxin type A for the treatment of hypertrphy of the masseter muscle. Plast Reconstr Surg.2010;125(6):1693-1705.http://www.ncbi.nlm.nih.gov/pubmed/22842446

[21] Lee BJ,Jeong JH,Wang SG,et al.Effect of botulinum toxin type a on a rat surgical wound model.Clin Exp Otorhinolaryngol. 2009; 2(1):20-27.http://www.ncbi.nlm.nih.gov/pubmed/19434287

[22] Venus MR.Use of botulinum toxin type A to prevent widening of facial scars.Plast Reconstr Surg. 2007;19(1):423-424.http://www.ncbi.nlm.nih.gov/pubmed/16651948

[23] Gassner HG,Brissett AE,Otley CC,et al.Botulinum toxin to improve facial wound healing:A prospective,blinded, placebo-controlled study. Mayo Clin Proc.2006;81(8): 1023-1028.http://www.ncbi.nlm.nih.gov/pubmed/16901024

[24] Wang L, Tai NZ, Fan ZH. Zhonghua Zhengxing Waike Zazhi. 2009;25(1):50-53. 王琳,邰宁正,范志宏.肉毒毒素A对大鼠皮肤及创面组织中SP、CGRP、TGF-β1和α-SMA的影响[J].中华整形外科杂志, 2009, 25(1):50-53.http://www.cqvip.com/QK/96147A/200901/29450569.html

[25] Xiao Z, Zhang F, Lin W, et al. Effect of botulinum toxin type A on transforming growth factor beta 1 in fibroblasts derived from hypertrophic scar: a preliminary report. Aesthetic Plast Surg. 2010;34(4):424-427.http://www.ncbi.nlm.nih.gov/pubmed/19802513

[26] Penn JW, Grobbelaar AO, Rolfe KJ. The role of the TGF-β family in wound healing, burns and scarring: a review. Int J Burns Trauma. 2012;2(1):18-28.http://www.ncbi.nlm.nih.gov/pubmed/22928164

[27] Huang C, Akaishi S, Oqawa R. Mechanosignaling pathways in cutaneous scarring. Arch Dermatol Res.2012;304(8): 589-597.http://www.ncbi.nlm.nih.gov/pubmed/22886298

[28] Abe M, Yokoyama Y, Ishikawa O. A possible mechanism of basic fibroblast growth factor-promoted scarless wound healing: the induction of myofibroblast apoptosis. Eur J Dermatol. 2012;22(1):46-53.http://www.ncbi.nlm.nih.gov/pubmed/22370167

[29] Hounardoust D, Varkey M, Marcoux Y, et al. Reduced decorin, fibromodulin, and transforming growth factor-β3 in deep dermis leads to hypertrophic scarring. J Burn Care Res. 2012; 33(2):218-227.http://www.ncbi.nlm.nih.gov/pubmed/22079916

[30] Liu J, Wang Y, Pan Q, et al. Wnt/β-catenin pathway forms a negative feedback loop during TGF-β1 induced human normal skin fibroblast-to-myofibroblast transition. J Dermatol Sci. 2012;65(1):38-49.http://www.ncbi.nlm.nih.gov/pubmed/22041457

[31] Moon H , Yong H, Lee AR. Optimum scratch assay condition to evaluate connective tissue growth factor expression for anti-scar therapy. Arch Pharm Res. 2012;35(2):383-388.http://www.ncbi.nlm.nih.gov/pubmed/22370794

[32] Guo F, Cater DE, Leask A. Mechanical tension increases CCN2/CTGF expression and proliferation in gingival fibroblasts via a TGFβ-dependent mechanism. Plos One. 2011;6(5):e19756.http://www.ncbi.nlm.nih.gov/pubmed/21611193

[33] Tao L, Liu J, Li Z, et al. Role of the JAK-STAT pathway in proliferation and differentiation of human hypertrophic scar fibroblasts induced by connective tissue growth factor. Mol Med Report. 2010;3(6):941-945.http://www.ncbi.nlm.nih.gov/pubmed/21472337

[34] Zhibo X,Miaobo Z.Botulinum toxin type A affects cell cycle distribution of fibroblasts derived from hypertrophic scar. J Plast Reconstr Aesthetic Surg.2008;61(9):1128-1129.http://www.ncbi.nlm.nih.gov/pubmed/18555763

[35] Chen W, Fu XB, Zhou G, et al. Zhonghua Shiyong Waike Zazhi. 2004;21(9):1111-1113.陈伟,付小兵,周岗,等. 增生性瘢痕内3种成纤维细胞生长因子及其受体基因的表达[J]. 中华实验外科杂志,2004,21(9): 1111-1113.http://www.cnki.com.cn/Article/CJFDTotal-ZHSY200409040.htm

[36] Jiang ZY, Fu XB, Sheng ZY, et al. Zhonghua Waike Zazhi. 2006,44(07):496-498.姜笃银,付小兵,盛志勇.瘢痕组织愈合中成纤维细胞凋亡抗性及其临床意义[J].中华外科杂志,2006,44(07):496-498.http://www.cnki.com.cn/Article/CJFDTotal-ZHWK200607017.htm

引言

材料方法

设计：细胞体外培养，对比观察。

时间与地点：实验于2011年6月至2012年3月在武警后勤学院完成。

材料：

标本的采集：纳入10例标本取自住院患者手术时切除的增生性瘢痕，其中男7例，女3例，年龄6-42岁，平均32岁。瘢痕为烧伤或创伤后8个月-3年，瘢痕凸出于皮肤表面，发红、痒、痛症状明显，瘢痕增生活跃。

增生性瘢痕诊断标准：经病理组织学检查确诊，病损区域内胶原纤维在真皮大量沉积，增厚、排列不规则，或呈旋涡形，或缠绕成绳索状，在旋涡形或绳索状的胶原中常有黏蛋白的沉积^[7]。

纳入标准：①3个月内未使用过类固醇激素、青霉胺或抗肿瘤药物等，且未接受过放射线治疗。②纳入患者对实验知情同意，并签署知情同意书，实验过程符合医学伦理学标准。

排除标准：①结缔组织疾病或影响结缔组织代谢的疾病。②有心、肺、肝、肾等慢性疾病。

A型肉毒毒素可引起人增生性瘢痕成纤维细胞凋亡实验的主要试剂与仪器：

Main reagents and instruments:

实验方法：

人增生性瘢痕成纤维细胞的分离与培养：手术中获取的瘢痕组织标本置入含青霉素和链霉素各100 U/mL的DMEM培养基中，然后转移至实验室。将皮下脂肪组织彻底剪除，将瘢痕组织标本浸于25%DispaseII中，置于37 ℃条件下消化2 h，完整撕下表皮组织。瘢痕组织用PBS反复冲洗3遍后，用剪刀剪切成1.0-2.0 mm³的小组织块。加入50倍体积的1 g/L的粗制胶原酶，于37 ℃条件下消化4 h。边消化边振荡，随时在显微镜下观察消化结果。当成纤维细胞大部分从包裹的胶原纤维组织中游离出来后，加入含胎牛血清的DMEM培养基终止消化。混合液用100目不锈钢网过滤，去除大的组织块残渣。滤液离心后，去除上清液，细胞计数后每25 mL培养瓶接种2×10⁵个细胞，以后每隔3 d换液。待细胞融合后，应用胰酶消化后传代。实验所用细胞为体外培养的第二三代细胞。

MTT法检测人增生性瘢痕成纤维细胞增殖：收集对数生长期细胞，以4 000 /孔接种于96孔培养板，待细胞贴壁后，吸去培养基，保留空白孔和对照孔，其他实验孔按照浓度梯度0.1，0.2，0.3，0.4，0.5 IU/L分别加入含有A型肉毒毒素DMEM培养基。每个实验组设定6个复孔，37.0 ℃，体积分数5%CO₂培养48 h。每孔加入 20 µL MTT(5 g/L)，37.0 ℃继续培养6 h，取出96孔培养板，小心吸掉上清液，每孔加入100 µL DMSO，以溶解结晶，溶解20 min后，于酶联免疫检测仪上570 nm测定吸光度。绘制细胞增殖曲线。计算半数抑制率。

Hoechst33342及PI染色检测人增生性瘢痕成纤维细胞凋亡：将消毒好的盖玻片置于24孔板内，按4×10⁴/孔接种入细胞，贴壁后加药处理，按照浓度梯度0.1，0.2，0.3，0.4，0.5 IU/L分别加入含有A型肉毒毒素的培养基作用48 h。吸尽培养液，加入0.2 mL固定液，固定20 min。去固定液，PBS洗3遍，每次3 min，吸尽液体，加入 0.2 mL hoechst33342染色液，染色5 min，加入PI继续染色5 min，PBS洗3遍，每次3 min。盖上贴有细胞的盖玻片。无色指甲油固定，激发波长在350 nm，发射波长在460 nm。荧光显微镜下观察细胞核形态。2人双盲观察，计数200个以上细胞，计算凋亡率(%)，绘制凋亡曲线，计算半数凋亡率。

主要观察指标：MTT染色后吸光度、细胞增殖曲线、荧光显微镜下细胞凋亡形态及细胞凋亡曲线。

统计学分析：由第一、第二作者采用SPPS 15.0统计学软件，进行t 检验，P < 0.05表示差异有显著性意义。

讨论

A型肉毒毒素目前在整形外科主要用于去除抬头纹、鱼尾纹等动力性皱纹，其主要原理是麻痹面部表情肌^[9-20]。由于瘢痕的形成与皮肤张力有很大的关系，有学者根据这个原理在面部整形手术后，于切口周围注射A型肉毒毒素，以减少术后瘢痕的形成，临床上达到很好的效果^[21-23]。于波等^[2]应用A型肉毒毒素预防和治疗早期增生性瘢痕，可以使增生性瘢痕变软变平，瘢痕增生明显改善。王琳等^[2]应用A型肉毒毒素治疗瘢痕疙瘩患者，不仅可以明显减轻患者的顽固性痒痛症状，而且注射3次(6个月)后，瘢痕明显的萎缩扁平。A型肉毒毒素治疗增生性瘢痕和瘢痕疙瘩在临床上取得良好的治疗效果。

由于瘢痕增生主要是人皮肤成纤维细胞过度增殖以及产生胶原蛋白过量所致，所以A型肉毒毒素对瘢痕增生的治疗作用肯定与人增生性瘢痕成纤维细胞生物学特性发生改变有关。作者的实验发现A型肉毒毒素不仅可以抑制人增生性瘢痕成纤维细胞的增殖，而且可以减少增生性瘢痕成纤维细胞合成胶原蛋白的量^[7]。

实验通过细胞凋亡实验，发现A型肉毒毒素可以引起人增生性瘢痕成纤维细胞的凋亡。初步阐明了A型肉毒毒素抑制人增生性瘢痕成纤维细胞增殖的机制。另有研究发现A型肉毒毒素可以影响瘢痕组织中转化生长因子β的生成^[24-25]。转化生长因子β刺激成纤维细胞产生的胶原及其他基质成分的沉积，抑制胶原酶，阻断血纤维蛋白溶酶原抑制物，刺激成纤维细胞纤维化，促进瘢痕形成^[26-30]。A型肉毒毒素也可以减少增生性瘢痕成纤维细胞结缔组织生长因子的表达，而增生性瘢痕成纤维细胞结缔组织生长因子也是瘢痕增生的一个重要因素^[31-33]。A型肉毒毒素也会影响增生性瘢痕成纤维细胞的生长周期，使大部分细胞由功能活跃的增殖期转入静止期，合成能力减弱^[34]。以上的实验结果证明A型肉毒毒素对于人增生性瘢痕成纤维细胞具有抑制增殖的作用，其原理可能是多方面的，包括细胞凋亡、影响细胞分泌生长因子以及改变细胞的细胞生长周期等。

细胞凋亡是一种安全的细胞死亡方式，A型肉毒毒素可引起人增生性瘢痕成纤维细胞发生凋亡，说明A型肉毒毒素在临床上治疗增生性瘢痕是一种比较安全有效的治疗方法^[35-36]，作者的实验为临床上应用A型肉毒毒素治疗增生性瘢痕提供了更扎实的理论依据。今后的实验应该更深入的研究A型肉毒毒素对增生性瘢痕成纤维细胞其他生物活性方面的影响。