【作者】 刁建升；

【导师】 郭树忠； 夏文森； 夏炜；

【作者基本信息】 第四军医大学 ， 外科学， 2013， 博士

【摘要】 瘢痕疙瘩是皮肤创伤后引发的以成纤维细胞异常增殖并分泌大量细胞外基质（extracellular matrix，ECM）为主要特征的一种皮肤纤维化疾病，治疗效果不佳，复发率较高，发病机制尚不清楚。近年来研究发现，组蛋白去乙酰化酶（histonedeacetylase，HDAC）活性增高与多种组织和器官纤维化的发生发展密切相关，使用HDAC抑制剂降低HDAC活性可抑制皮肤正常成纤维细胞ECM的合成。同时HDAC活性增高还是多种肿瘤发生的病理机制，使用HDAC抑制剂可以通过激活凋亡信号，使异常增殖的肿瘤细胞生长停滞，诱导其凋亡。所以我们也推测HDAC活性增加可能与瘢痕疙瘩成纤维细胞的增殖和分泌大量ECM密切相关。本课题将研究I型HDAC（HDAC1和HDAC2）在瘢痕疙瘩成纤维细胞中的表达情况，并使用HDAC抑制剂TSA进行体内和体外实验进行干预，以期阐明I型HDAC是如何参与瘢痕疙瘩形成的病理机制，为HDAC抑制剂类药物临床应用预防和治疗瘢痕疙瘩提供理论基础。实验一HDAC1和HDAC2在瘢痕疙瘩成纤维细胞中表达的实验研究目的：本实验旨在研究I型HDAC（HDAC1和HDAC2）在瘢痕疙瘩成纤维细胞中的表达情况，并与正常皮肤成纤维细胞相比较，以期为瘢痕疙瘩提供新的发病机制，及治疗的新切入点。方法：我们首先进行瘢痕疙瘩成纤维细胞与正常皮肤成纤维细胞原代培养。接着通过RT-PCR和western blot方法检测HDAC1和HDAC2在瘢痕疙瘩成纤维细胞中的表达情况，并与正常皮肤成纤维细胞相比较。结果：我们通过RT-PCR方法检测发现，瘢痕疙瘩成纤维细胞HDAC1和HDAC2mRNA的表达均比正常皮肤成纤维细胞明显增高。接着，用western blot方法检测同样证实瘢痕疙瘩成纤维细胞HDAC1和HDAC2蛋白的表达也均比正常皮肤成纤维细胞要高。结论：我们的研究证实HDAC1和HDAC2在瘢痕疙瘩成纤维细胞中的表达明显增高。HDAC1和HDAC2的活性增强，可以使原有的基因表达平衡状态被打破，从而导致细胞增殖、凋亡和纤维化等生物学特征的异常，这可能是瘢痕疙瘩成纤维细胞过度增殖并合成大量ECM的病理新机制。实验二HDAC抑制剂TSA诱导瘢痕疙瘩成纤维细胞凋亡的实验研究目的：HDAC抑制剂TSA可以引起多种恶性增殖的细胞生长停滞，促进其发生凋亡，本实验旨在研究TSA是否可以诱导瘢痕疙瘩成纤维细胞发生凋亡及TSA对细胞HDAC1和HDAC2蛋白表达的影响。方法：首先，我们向培养的瘢痕疙瘩成纤维细胞加入不同浓度的HDAC抑制剂TSA（200nmol/L、400nmol/L、600nmol/L和800nmol/L），运用MTT法检测TSA对细胞活力的影响。然后，通过Hoechst染色和流式细胞仪检测TSA诱导瘢痕疙瘩成纤维细胞发生凋亡的作用。最后，运用western blot方法检测TSA对瘢痕疙瘩成纤维细胞HDAC1和HDAC2蛋白表达的影响。结果：通过MTT法检测TSA对细胞增殖的影响，结果发现随着浓度和时间的变化，TSA抑制细胞的增殖存在量效性和时效性的特点。成纤维细胞对于200nmol/L TSA作用耐受性良好，在600nmol/L和800nmol/LTSA作用下，细胞活力明显下降。随后600nmol/L TSA单独作用瘢痕疙瘩成纤维细胞，通过Hoechst染色和流式细胞仪检测同样证实TSA可以诱导瘢痕疙瘩成纤维细胞发生凋亡。最后，运用western blot方法检测发现600nmol/LTSA处理瘢痕疙瘩成纤维细胞48h后，瘢痕疙瘩成纤维细胞HDAC1和HDAC2蛋白的表达明显受到了抑制。结论：我们的研究证实HDAC抑制剂TSA可以抑制瘢痕疙瘩成纤维细胞增殖，诱导其发生凋亡，这可能是通过抑制HDAC1和HDAC2的表达来实现的。实验三HDAC抑制剂TSA抑制瘢痕疙瘩成纤维细胞胶原合成的实验研究目的：本实验旨在研究HDAC抑制剂TSA是否可以抑制TGF-β促瘢痕疙瘩成纤维细胞胶原合成的作用。方法：我们分别通过RT-PCR和western blot方法检测600nmol/LTSA单独作用或者联合5ng/ml TGF-β1蛋白对瘢痕疙瘩成纤维细胞I型胶原（collagen I）mRNA和蛋白表达的影响。结果：通过RT-PCR检测发现，向培养的瘢痕疙瘩成纤维细胞加入5ng/ml TGF-β1，可以促进细胞collagen I mRNA合成，而同时加入600nmol/L TSA后，可以抑制TGF-β1促进细胞collagen I mRNA合成的作用。单独使用600nmol/L TSA后这种抑制作用会更加显著。Western blot方法检测同样证实TSA可以抑制细胞collagen I蛋白的合成。结论：我们的研究证实HDAC抑制剂TSA可以抑制瘢痕疙瘩成纤维细胞胶原合成，这就为这种已经在临床治疗多种肿瘤的HDAC抑制剂应用于瘢痕疙瘩临床预防和治疗提供实验基础。实验四HDAC抑制剂TSA抑制兔耳增生性瘢痕形成的实验研究目的：已证实HDAC抑制剂TSA能够有效抑制体外培养的皮肤成纤维细胞胶原合成而起到抗纤维化作用，本部分实验拟通过兔耳增生性瘢痕模型验证TSA是否能够在体内实验抑制瘢痕的形成。方法：建立兔耳增生性瘢痕模型。每个兔耳做4个直径为1cm，间距为2cm的圆形全层皮肤缺损创面。每只兔的左耳为TSA治疗组，分别在创面上皮化后创面局部注射TSA，每个创面使用0.02%TSA0.05ml。连续注射1周。右耳为对照组，分别在相同时间点局部注射相同剂量生理盐水0.05ml。术后第23d收集瘢痕标本，进行RT-PCR和western blot检测collagen I和纤维连接蛋白（fibronectin）mRNA和蛋白的表达情况。术后第45d收集瘢痕标本，行HE染色切片计算瘢痕增生指数（scarelevation index，SEI）。结果：TSA治疗组瘢痕比对照组厚度较薄，术后45d时间点其SEI在TSA治疗组明显下降（1.45±0.09vs2.07±0.10），差异有统计学意义。同时，RT-PCR和western blot检测也证实在术后第23d（TSA治疗1周后），TSA可以有效的抑制collagen I和fibronectin表达。结论：我们的研究证实HDAC抑制剂TSA可以在体通过抑制collagen I和fibronectin的合成，有效的抑制兔耳增生性瘢痕的形成。更多还原

【Abstract】 Keloid is a pathological wound healing response to cutaneous injury andcharacterized by fibroblastic proliferation and accumulation of extracellular matrix.Clinically, keloids have proven to be very resistant to treatment. Despite the relativelyhigh prevalence of keloids in the general population, the mechanisms underlying keloidformation are only partially understood. Recent studies indicate that histone deacetylase（HDAC）activity is also associated with the development and progression of somechronic diseases characterized by fibrosis. And inhibition of HDAC activity of fibroblastsby a number of structurally divergent classes of HDAC inhibitors causes significantinhibition of extracellular matrix synthesis. Furthermore, HDAC inhibitors have been shown to induce arrest, differentiation, and/or apoptosis of proliferating cells. So wehypothesize that increased activity of HDAC may be associated with keloid fibroblastproliferation and accumulation of extracellular matrix. In this study, we will research ofclass I HDAC（HDAC1and HDAC2）expressions in keloid fibroblasts, and throughrespectively in vivo and in vitro experimental intervention with HDAC inhibitor TSA, toclarify how class I HDACs are involved in keloid fibroblasts proliferation and collagensecretion. Our study will open up the possibility of testing HDAC inhibitors for clinicalprevention and treatment of keloids.Experiment1The study of HDAC1and HDAC2expressions in keloid fibroblastsPurpose: To investigate HDAC1and HDAC2expressions in keloid fibroblasts andnormal fibroblasts. To provide new pathogenetic mechanisms for keloid formation andnew treatment point.Methods: We cultured keloid fibroblasts and normal skin fibroblasts. Then, we researchedthe expressions of HDAC1and HDAC2in keloid fibroblasts and normal fibroblasts byRT-PCR and western blot.Results: The increase of HDAC1and HDAC2mRNA expressions were observed inkeloid fibroblasts compared to normal fibroblasts. These results were also confirmed bywestern blot analysis. Western blot analysis showed that the expressions of HDAC1andHDAC2protein were markedly up-regulated in keloid fibroblasts compared to normalfibroblasts.Conclusion: Our study confirmed that expressions of HDAC1and HDAC2in thekeloid-derived fibroblasts were significantly higher. Enhanced HDAC1and HDAC2activity, which makes the original gene expression balance has been broken, leading toabnormal cell proliferation, apoptosis and fibrosis and other biological characteristics.HDAC1and HDAC2activity may be a new pathogenetic mechanism for keloidformation. Experiment2Histone deacetylase inhibitor TSA induces apoptosis of keloidfibroblastsPurpose: As TSA could induce growth arrest and apoptosis of many kind of cells, the aimof this study is to determine whether HDAC inhibitor trichostatin A（TSA）could induceapoptosis of proliferating keloid fibroblasts and inhibit the expressions of HDAC1andHDAC2.Methods: We first studied the effect of escalating doses（200nmol/L,400nmol/L,600nmol/L or800nmol/L）of TSA on keloid fibroblasts viability using MTT proliferationassays. Then, to further investigate the inhibitory effect of TSA on proliferation of keloidfibroblasts, we stained cells using Hoechst staining after600nmol/L TSA treatment, andflow cytometry analysis was performed as a second independent method. Furthermore,after treatment with600nmol/L TSA for48h, we researched the expressions of HDAC1and HDAC2protein expressions in keloid fibroblasts by western blot.Results: HDAC inhibitor TSA inhibited the growth of keloid fibroblasts indose-dependent and time-dependent manner monitored using MTT proliferation assays.Lower TSA doses of200nmol/L were well tolerated with preserved cell viability relativeto control. A statistically significant decrease in cell viability was observed at aconcentration of TSA（600nmol/L and800nmol/L）after incubation for12h, compared tocontrol. Hoechst staining and flow cytometry analysis showed significantly moreapoptotic cells were observed in600nmol/L TSA treated group compared to controls.Furthermore, treatment with TSA abrogated HDAC1and HDAC2expressionsdramatically. Western blot analysis showed that treatment with TSA for48h, theexpressions of HDAC1and HDAC2were markedly decreased in keloid fibroblasts.Conclusion: On the basis of our in vitro evidence we can state that TSA could inhibitproliferation and induce apoptosis of keloid fibroblasts with the decreased expressions ofHDAC1and HDAC2.Experiment3Histone deacetylase inhibitor TSA inhibits collagen synthesis of keloid fibroblastsPurpose: The aim of this study is to determine whether HDAC inhibitor TSA could causeabrogation of TGF-β induced collagen synthesis in keloid fibroblasts.Methods: We researched the expressions of collagen I in keloid fibroblasts treatment with600nmol/L TSA or/and5ng/ml TGF-β1by RT-PCR and western blot.Results: The addition of5ng/ml TGF-β1up-regulated collagen I mRNA expressioncompared to controls by RT-PCR analysis. But incubation with600nmol/L TSA, theincrease of collagen I mRNA expression induced by TGF-β1was reduced by50%compared to controls. Furthermore, treatment with TSA alone abrogated collagen I mRNAexpression dramatically. These results were confirmed by western blot analysis. Westernblot analysis showed that treatment with TSA, the expression of collagen I protein inducedby TGF-β1was markedly decreased.Conclusion: On the basis of our in vitro evidence we can state that TSA inhibition ofECM may be an appropriate therapeutic strategy for the management of keloid. Our studyopens up the possibility of testing HDAC inhibitor, an agent already in clinical trials forthe treatment of many tumors, for clinical keloid treatment use.Experiment4Histone deacetylase inhibitor TSA reduces hypertrophic scarring in arabbit ear modelPurpose: To test the ability of HDAC inhibitor TSA to reduce hypertrophic scar formationin a rabbit ear model, which had been confirmed to have potent anti-fibrogenic effects andsuppress collagen synthesis of skin fibroblasts.Methods: We had developed a reliable model in rabbit that resulted in hypertrophicscarring. Four1cm full-thickness circular wounds were made on each ear. After thewounds re-epithelialized,0.02%TSA was intradermally injected to the wounds in thetreatment group. Collagen I and fibronectin expressions were detected by RT-PCR andwestern blot at postoperative day（POD）23. Scar hypertrophy was quantified by measurement of the scar elevation index（SEI）at POD45.Results: Compared to the control group, the injection of TSA led to much morenormal-looking of scars in the rabbit ear. SEI at POD45was significantly decreased afterinjections of TSA versus untreated scars（1.45±0.09vs.2.07±0.10）. Further more, weconfirmed the decreased expression of collagen I and fibronectin at POD23（after beingtreated with TSA for one week）in the treated scars compared to the control by RT-PCRand western blot analysis.Conclusions: The introduction of TSA can result in the decreased formation ofhypertrophic scars in a rabbit ear model, which is corroborated by evidence of decreasedcollagen I and fibronectin synthesis.更多还原