摘要
增生性瘢痕(hypertrophic scar)是皮肤的纤维增殖紊乱疾病,通常发生于皮肤严重创伤和烧伤后。增生性瘢痕不仅影响患者的外观,而且会严重影响患者的心理健康和功能,使患者的生活质量显著下降,给患者家庭、社会带来了沉重的经济负担。
增生性瘢痕的主要特点是细胞外基质的过度沉积。由于绝大部分的实验动物比如大鼠、兔子、小鼠和猪不能形成增生性瘢痕,因此增生性瘢痕的研究一直都比较困难。
以前的研究表明,相比正常皮肤的成纤维细胞,增生性瘢痕组织来源的成纤维细胞分泌更多细胞外基质蛋白(Ⅰ型和Ⅲ胶原等)、蛋白多糖(versican和biglycan等)以及生长因子(TGF-β和胰岛素样生长因子等),但是表达较低的重塑酶,包括胶原酶和其它的基质金属蛋白酶以及小蛋白多糖decorin等。因此成纤维细胞在增生性瘢痕的形成中起着非常关键的作用。
细胞自身分泌的细胞因子对细胞自身的功能有着重要的作用,影响细胞自身的增值、分化、迁移以及其它的多种功能。因此,在本实验中,我们选择增生性瘢痕组织来源的成纤维细胞和正常皮肤来源的成纤维细胞的培养上清为研究对象,找出两种上清的差异蛋白,以期找到与皮肤瘢痕增生密切相关的蛋白,为探讨瘢痕的形成机制以及治疗和预防瘢痕打下初步的基础。
方法:
1.正常皮肤组织和增生性瘢痕组织的成纤维细胞的分离培养:
正常皮肤组织来源于西南医院泌尿外科包皮组织,增生性瘢痕组织来源于西南医院烧伤研究所烧伤患者。正常成纤维细胞来自本院泌尿外科,增生性瘢痕成纤维细胞来自我所烧伤后一年内患者。分离与培养方法如下:将组织块放入无菌培养皿内,PBS冲洗三遍,无菌剪刀除去表皮部分,剪碎皮下组织;将剪碎后的组织放入25mL锥形培养瓶内,加入0.5%胰酶10mL,室温,轻微振荡2h;加入10mL 10%小牛血清DMEM终止消化,将悬液通过无菌滤网过滤后弃去组织碎片;离心,400g/min×10min,弃上清,加入10mL 10%小牛血清DMEM洗三遍,再次离心;弃上清后,重悬细胞于10mL10%小牛血清DMEM中,移至75cm2培养瓶中,37℃,5% CO2培养箱内进行培养,24h后更换培养基,同时除去未贴壁细胞。待细胞完全长满后,即可进行传代培养。实验时选用第6代左右的成纤维细胞。
2.两种细胞培养上清的收集和分离:
用无血清培养基培养两种细胞六天后,收集培养上清,3000rpm,离心10min,收集上清,然后加入4倍体积–20℃预冷的丙酮,–20℃过夜后,12000rpm,离心30min,弃上清,待丙酮充分挥发后,将蛋白溶于裂解缓冲液,采用Bradford方法进行蛋白定量,然后用10%的SDS-PAGE将上清蛋白分离。
3.蛋白胶内酶解:
将含有目的带白的条带切下,胶条片断脱色,冲洗,在二硫苏糖醇还原以及碘乙酰胺烷化后,蛋白在37°C用胰蛋白酶消化过夜。用30%乙腈、0.3%三氟乙酸(TFA)、以及100%乙腈将得到的胰蛋白酶肽从胶条片断中提取出来。
4.质谱鉴定和分析:
采用HPLC-CHIP-MS/MS鉴定酶解肽段,质谱仪参数:分离器电压设置在40.0 V;Cap Exit为250.2 V;Oct1 DC为12.0 V;Oct2 DC为4.62 V;Trap Drive为85.0;Oct RF为216.3 Vpp;Lens 1设置为–5 V,Lens 2为–60 V。采用Spectrum Mill(Rev A.03.02.060; Agilent)搜索引擎软件进行数据分析。根据使用说明书,以胰酶为蛋白酶,在默认的条件下获得原始数据,然后在swiss-prot数据库中搜索针对人的蛋白数据库,允许有两个不完全裂解位点,且包括在搜索中氧化甲硫氨酸和N-末端谷胺酰胺转化成5-氧-2-吡咯烷羧酸的变量修正。如果前向-逆转得分大于2,列1-列2得分大于2,得分阈值大于7.67,得分百分比峰强度大于70%,我们认为肽段是有效的。只有当蛋白具有两个或两个以上的有效肽段、总得分大于25的时候,进行报告。
5. GO分析:
通过GO分析,找出上清中的分泌型蛋白以及细胞外基质蛋白,并对这些蛋白进行初步的功能分析。然后查询相关的文献和数据库,找出与增生性瘢痕形成相关的蛋白。
6.回复性验证:
通过Western Blotting回复性验证前胶原C内肽酶增强子1在两种上清中的表达情况。通过免疫组化检测其在正常皮肤组织和增生性瘢痕中的表达情况。
结果:
1.通过HPLC-CHIP-MS/MS鉴定,正常皮肤成纤维细胞培养上清共鉴定出82个蛋白,瘢痕组织来源的鉴定出79个蛋白。经过GO注释,正常皮肤来源的成纤维细胞的上清内有43个蛋白被注释为胞外蛋白,瘢痕来源的有37个。通过比较发现两种细胞共同分泌的蛋白有25个,瘢痕组织成纤维细胞特异性分泌的有12个,正常皮肤的有18个。
2.通过GO注释,我们对两种细胞分泌蛋白的功能进行了初步分析。从图2可知,两种成纤维细胞分泌蛋白主要具有结合功能,分别为41个和33个蛋白,占各自总分泌蛋白的95.3%和89.2%。正常成纤维细胞分泌蛋白分别有9个(20.9%)具有信号转导和结构分子功能,瘢痕组织的分别为5个(13.5%)和10个(27%).两种细胞分别有8个(18.6%)和9个(24.3%)蛋白具有催化活性。正常皮肤的成纤维细胞分别有1个蛋白具有转运和抗氧化活性,而瘢痕组织的成纤维细胞有6个具有酶调节活性。
3.通过生物信息学分析和查询相关的数据库以及参考文献,我们认为Vasorin蛋白和前胶原C内肽酶增强子1(Procollagen C-endopeptidase enhancer 1)与瘢痕的形成有关。
4.通过WB,对前胶原C内肽酶增强子1进行了回复性验证,证实了前胶原C内肽酶增强子1瘢痕组织来源的成纤维细胞培养上清中高表达,而在正常皮肤培养上清中不表达。免疫组化结果表明其在正常皮肤组织上皮高表达而在瘢痕组织上皮中低表达。
结论:
通过收集培养上清和HPLC-CHIP-MS/MS鉴定,我们建立了瘢痕成纤维细胞和正常皮肤成纤维细胞的分泌蛋白谱,并对其功能进行了初步的分析。通过对比增生性瘢痕组织和正常皮肤组织来源的成纤维细胞的培养上清,我们认为增生性瘢痕组织来源的成纤维细胞表达的Vasorin蛋白、前胶原C内肽酶增强子1与瘢痕组织的形成有密切的关系,为进一步阐明增生性瘢痕组织的发生机制和临床干预打下了初步的基础。
Hypertrophic scar is a skin fibroproliferative disorder, which usually happen after serious skin trauma and extensive burn. Hypertrophic scar not only deforms the appearance of the patients, but also severely affects the psychological health and body functions. Hypertrophic scar characterizes excessive deposition of extracellular matrix (ECM).
Because of most of the experimental animal such as rat, mouse, rabbit and pig cannot form hypertrophic scars, it’s very difficult to study hypertrophic scar directly.
Previous study demonstrated that, compared with fibroblasts from normal skin, fibroblasts from hypertrophic scar secreted more ECMs(typeⅠandⅢcollagen), versican, biglycan and other growth factors(such as TGF-βand insulin-like growth factor) and less remodeling enzymes, including collagenase and other matrix metalloproteinases, and small proteoglycan decorin, which plays a very important role in preventing scar formation. So fibroblasts play a key role in scar formation.
Self-secreted cytokines are very important to cells themselves, which can affect the proliferation, differentiation, migration and other physiological functions. So in this experiment, we chose culture supernatant of fibroblasts derived from both hypertrophic scar and normal skin tissues as our study object. We established the secreted protein profiles of both cells and found out that protein vasorin and procollagen C endopeptidase enhancer 1 were highly related with scar formation.
Methods:
1. Isolation of fibroblast from both normal skin and hypertrophic scar tissues: the normal skin and scar tissues were obtained from Southwest Hospital, and enzyme digestion method was used to isolate the fibroblasts.
2. Collection and separation of the supernatant proteins:
After six day culture with serum-free media, the culture supernatant was collected. Ice acetone was used to precipitate the proteins in the supernatant. After centrifugation at 12000rpm about 30min, the supernatant was discard. The obtained proteins were dissolved in lysis buffer and 10% SDS-PAGE was performed to separate the protein.
3. In-gel digestion:
In-gel digestion was performed as follows: the SDS-PAGE gel was cut into gel slices, each of which contained a stained protein(s). The resulting slices were destained at 37°C in 50mM NH4HCO3 containing 50% ethanol. After the dye (Coomassie Brilliant Blue) was completely removed, the slices were washed with 25mM NH4HCO3 (pH 8.0) and dehydrated with acetonitrile (ACN). After being dried in a SpeedVac concentrator, the gel-bound proteins were reduced in 10mM DTT and then alkylated in 55 mM iodoacetamide (IAA) containing 6M guanidine hydrochloride. The gel pieces were then washed with 25 mM NH4HCO3, and again dehydrated with ACN and dried in a SpeedVac. The dry gel pieces were reswollen with 25μL of 25mMNH4HCO3 containing 0.5μg of trypsin and 0.1% n-octyl glucoside (W/V). Digestion was carried out at 37°C overnight. The peptides were extracted two times with 50μL of 5% formic acid and 50% acetonitrile by sonication for 15min. The combined extracts were evaporated to about 2μL in a SpeedVac and stored at -80°C.
4. MS identification: The parameters for the mass spectrometer: the skimmer voltage was set at 40.0 V; the Cap Exit at 250.2 V; the Oct1 DC at 12.0 V; the Oct2 DC at 5.62 V; the Trap Drive at 85.0; the Oct RF at 216.3 Vpp; Lens 1 was set at–5 V and Lens 2 at–60 V. Data were analyzed using Spectrum Mill proteomics software (Rev A.03.02.060; Agilent).Raw data were extracted under default conditions and searched against mammalian sequences in the swiss-prot database using trypsin as the protease, allowing 2 missed cleavages, and including variable modifications of oxidized methionine and N-terminal glutamine conversion to pyroglutamic acid in the search. Peptides were considered valid with a forward–reverse score >2 and a rank 1–rank 2 score >2, a score threshold >7.67, and percentage-scored peak intensity >70%. Only proteins with 2 or more validated peptides and a total score >25 were considered valid for reporting. To compare identified proteins between treatment groups, the number of spectra and the summed ion intensity of peptides for each protein (total ion intensity) were used as indicators of protein amounts. Because these are semiquantitative metrics, we only considered those proteins with at least 5 additional spectra and at least a 10-fold increase in total ion intensity sufficiently different for reporting. To evaluate signs of matrix degradation, gel slices were used to estimate the molecular weight of proteins using the molecular weight marker as well as the molecular weight of some of the protein constituents.
5. Analysis of the identified proteins: GO annotation was performed to select out the serected proteins and extracellular matrix proteins. Then proteins related to scar formation was picked out and analyezed.
6. Retrospective verification: Western blotting and immunohistochemistry was applied to verify the proteins that were related to scar formation.
Results:
1. 82 and 79 proteins secreted by fibroblast from normal skin and scar tissues were identified respectively. Among these proteins, 43 and 37 proteins were annotated by GO as extracellular region proteins respectively. Further analysis showed that 25 proteins were secreted by both cells, 18 proteins secreted by normal skin fibroblast and 12 secreted by hypertrophic scar fibroblast specifically.
2. We analyzed the function of these proteins by GO annotation. Most of the proteins secreted by both cells involve in binding function, account for 95.3% (41) and 89.2% (33) respectively. 9 proteins secreted by normal skin fibroblasts have signal transduction function and 9with structural molecule function, while proteins secreted by fibroblasts from scar tissues with such functions are 5and 10 respectively. 8 proteins form normal skin fibroblasts and 9 from the other cells have catalytic activity.
3. We found that protein vasorin and Procollagen C-endopeptidase enhancer 1 secreted by fibroblast derived from scar tissues are highly related to scar formation.
4. WB detection demonstrated that Procollagen C-endopeptidase enhancer 1 was highly expressed in culture supernatant of hypertrophic scar fibroblasts. Immunohistochemistry detection showed that Procollagen C-endopeptidase enhancer 1 was highly expressed in epidermis of normal skin.
Conclusion:
We established the secretory protein profiles of fibroblast from both normal skin and hypertrophic scar tissues. After comparison with that of firbroblasts from normal skin, we considered that protein vasorin and procollagen C-endopeptidase enhancer 1 were related to scar formation. Further WB detection confirmed our results. So vasorin and Procollagen C-endopeptidase enhancer 1 may be a new target for the treatment of scar.
引文
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