【作者】 孙志成；

【导师】 李世荣；

【作者基本信息】 第三军医大学 ， 外科学， 2011， 博士

【摘要】 1.研究背景及研究目的创面愈合的理想结果应该是不但恢复皮肤正常的功能和外观,而且没有生长增生性瘢痕。但是严重烧伤和创伤后部分个体经常发生增生性瘢痕,导致功能障碍和外观改变。增生性瘢痕富含有大量的成纤维细胞和细胞外基质(ECM)。尽管增生性瘢痕的形成机制目前还不十分清楚,但是研究表明增生性瘢痕的形成与成纤维细胞的增殖过度和凋亡抑制密切相关。近些年研究发现凋亡相关基因,如bcl-2,fas,smad3和p53等,参与了成纤维细胞的增殖与凋亡的调节。前期工作我们利用基因芯片技术研究发现在增生性瘢痕和正常皮肤之间有许多基因表达存在显著差异。在我们发现的97个差异基因中,SFRP2,一种与凋亡相关的基因,在增生性瘢痕组织中高表达。研究显示SFRP2在心肌修复,肿瘤发生过程中参与了细胞的增殖与凋亡的调控。SFRP2是一种分泌型的糖蛋白,被认为是wnt信号的抑制剂,影响细胞的增殖、凋亡和分化。经典Wnt /β-catenin信号通路的基本过程是:配体Wnt蛋白结合细胞表面受体frizzled(Fz)家族蛋白和LRP5、LRP6,经过一系列过程导致β-catenin在胞质内稳定地积累。转位到细胞核内的β-catenin与淋巴细胞增强因子/T细胞因子(LEF / TCF)结合,调节wnt通路靶基因的表达,如c-myc和cyclin D1等。下游的靶基因参与调解细胞的增殖、凋亡和分化等功能。SFRP2在增生性瘢痕形成过程中的作用目前尚不清楚。为此,本实验旨在研究SFRP2在增生性瘢痕组织中高表达的意义和相关wnt信号通路的作用机制。2.方法2.1组织标本和细胞培养成纤维细胞由人的正常皮肤组织和增生性瘢痕组织培养获得。组织标本来自第三军医大学西南医院整形科。患者知情同意,并报西南医院道德委员会批准。2.2稳定高表达SFRP2的增生性瘢痕成纤维细胞的建立从增生性瘢痕成纤维细胞中获得总RNA,利用RT-PCR技术获得SFRP2的cDNA,将目的基因克隆到真核表达载体pcDNA3.0中。然后,将pcDNA3.0/SFRP2稳定转染到增生性瘢痕成纤维细胞,筛选稳定高表达SFRP2的增生性瘢痕成纤维细胞。2.3稳定低表达SFRP2的增生性瘢痕成纤维细胞的建立针对SFRP2的基因序列设计三条shRNA,分别命名为shRNA1、shRNA2和shRNA3。将合成的shRNA序列克隆到真核表达载体pSilencer 2.1-U6 neo中。然后,将pSilencer 2.1-U6 neo/SFRP2 shRNA稳定转染到增生性瘢痕成纤维细胞,筛选稳定低表达SFRP2的增生性瘢痕成纤维细胞。2.4 SFRP2对增生性瘢痕成纤维细胞增殖及凋亡的影响使用构建好的稳定高表达和低表达SFRP2的增生性瘢痕成纤维细胞,利用生长曲线和MTT方法检测SFRP2对增生性瘢痕成纤维细胞细胞活性的影响,进而利用BrdU标记方法检测SFRP2对增生性瘢痕成纤维细胞增殖的影响;利用caspase3活性和Annexin-V FITC/PI分析的方法检测SFRP2对增生性瘢痕成纤维细胞凋亡的影响。2.5 SFRP2 shRNA对增生性瘢痕成纤维细胞功能的影响使用构建好的稳定低表达SFRP2的增生性瘢痕成纤维细胞,利用realtime PCR技术检测成纤维细胞Ⅰ、Ⅲ前胶原蛋白和肌动蛋白α-SMA的mRNA表达;利用western blot检测α-SMA的蛋白表达水平;利用FPCL体外细胞模型检测SFRP2对成纤维细胞体外收缩功能的影响。2.6 SFRP2对增生性瘢痕成纤维细胞经典wnt信号通路的调控首先,利用western blot技术检测增生性瘢痕成纤维细胞和正常皮肤成纤维细胞的经典wnt信号上游关键蛋白wnt1,wnt3a和wnt8b的差异表达。其次,利用筛选出来的wnt3a体外干预高表达和低表达SFRP2的增生性瘢痕成纤维细胞,利用caspase3活性和Annexin-V FITC/PI分析的方法检测增生性瘢痕成纤维细胞凋亡的变化。最后,利用western blot方法检测wnt3a干预的高表达和低表达SFRP2的增生性瘢痕成纤维细胞经典wnt信号通路下游关键蛋白β-catenin和c-myc的表达。3结果3.1从增生性瘢痕成纤维细胞中提取总RNA,利用RT-PCR技术和SFRP2的特异性引物获得SFRP2目的片段,成功构建pcDNA3.0/SFRP2载体,并稳定转染到增生性瘢痕成纤维细胞。与未转染的增生性瘢痕成纤维细胞对比,pcDNA3.0/SFRP2稳定转染的增生性瘢痕成纤维细胞SFRP2的表达水平显著升高(p<0.01)。3.2将设计好的3条SFRP2 shRNA成功地构建到pSilencer 2.1-U6 neo载体中。将pSilencer 2.1-U6 neo/SFRP2 shRNA稳定转染到增生性瘢痕成纤维细胞。与其它干扰组和正常增生性瘢痕成纤维细胞对比,shRNA2抑制增生性瘢痕成纤维细胞表达SFRP2的效果最显著(p<0.01)。3.3生长曲线结果显示增生性瘢痕成纤维细胞5天达到生长平台期,SFRP2对增生性瘢痕成纤维细胞的生长平台期时间没有显著影响。MTT结果显示正常皮肤成纤维细胞的细胞活性显著弱于增生性瘢痕成纤维细胞(p<0.01),而SFRP2的过表达和低表达对成纤维细胞MTT的结果没有显著影响。此外,利用BrdU标记法进一步验证,也得到了同样的结果。3.4 Caspase3结果显示SFRP2 shRNA可以显著地提高增生性瘢痕成纤维细胞的caspase3的活性(p<0.01)。而增生性瘢痕成纤维细胞的SFRP2表达上调后,与未作干预的增生性瘢痕成纤维细胞对比,caspase3的活性差异不显著(p>0.05)。正常皮肤成纤维细胞的caspase3活性显著地高于增生性瘢痕成纤维细胞(p<0.05)。此外,进一步利用Annexin-V FITC/PI分析的方法检测成纤维细胞的早期凋亡水平,也得到了相同的结果。3.5与增生性瘢痕成纤维细胞比较,体外FPCL模型结果显示SFRP2低表达的增生性瘢痕成纤维细胞与正常皮肤成纤维细胞的收缩能力显著降低(p<0.01)。Realtime PCR结果显示,SFRP2低表达的增生性瘢痕成纤维细胞和正常皮肤成纤维细胞α-SMA与Ⅰ、Ⅲ前胶原蛋白mRNA的水平显著低于增生性瘢痕成纤维细胞(p<0.01)。利用western blot检测α-SMA的蛋白水平,得到了同样的结果。3.6 Western blot结果显示wnt3a在增生性瘢痕成纤维细胞中的表达水平显著高于正常皮肤成纤维细胞(p<0.01),而wnt1和wnt8b差异不显著(p>0.05)。与未受wnt3a干预的增生性瘢痕成纤维细胞对比,wnt3a可以显著上调增生性瘢痕成纤维细胞caspase3的活性(p<0.01)。shRNA下调增生性瘢痕成纤维细胞的SFRP2表达后,wnt3a诱导成纤维细胞caspase3的活性显著提高(与各对照组相比,p<0.01)。SFRP2过表达后明显地抑制了wnt3a诱导成纤维细胞caspase3的活性(p<0.01)。采用Annexin-V FITC/PI分析的方法检测增生性瘢痕成纤维细胞的凋亡水平,得到了相近的结果。3.7在体外,用wnt3a分别刺激过表达和低表达SFRP2的增生性瘢痕成纤维细胞。与未受wnt3a干预的增生性瘢痕成纤维细胞对比,western blot结果显示wnt3a可以显著上调成纤维细胞胞浆和胞核β-catenin和全细胞的c-myc的表达水平(p<0.01)。shRNA下调SFRP2的表达后,wnt3a进一步地显著提高了成纤维细胞胞浆和胞核的β-catenin和全细胞的c-myc的表达(与各对照组相比,p<0.01)。SFRP2过表达后,wnt3a诱导成纤维细胞胞浆和胞核的β-catenin和全细胞的c-myc的表达水平显著地受到抑制(p<0.01)。4结论4.1成功获得了高表达和低表达SFRP2的增生性瘢痕成纤维细胞。4.2在体外,SFRP2 shRNA促进增生性瘢痕成纤维细胞的凋亡,对增生性瘢痕成纤维细胞的增殖没有显著作用。而SFRP2过表达对增生性瘢痕成纤维细胞的凋亡和增殖的影响均没有显著作用。提示SFRP2参与调控增生性瘢痕成纤维细胞的凋亡,可能是增生性瘢痕形成的机制之一。4.3在体外,SFRP2 shRNA抑制增生性瘢痕成纤维细胞Ⅰ、Ⅲ型胶原蛋白mRNA的表达,在基因和蛋白水平抑制α-SMA的表达,从而抑制体外FPCL模型收缩。提示SFRP2参与调控增生性瘢痕成纤维细胞ECM的形成,影响增生性瘢痕的增生与挛缩。4.4 wnt3a在增生性瘢痕成纤维细胞中的表达显著高于正常皮肤成纤维细胞。SFRP2抑制wnt3a诱导增生性瘢痕成纤维细胞的caspase3活性和凋亡率。进一步研究表明SFRP2能够显著地抑制增生性瘢痕成纤维细胞经典wnt信号通路下游关键蛋白β-catenin和c-myc的表达。提示SFRP2可能通过抑制wnt3a/β-catenin信号通路,调节靶基因c-myc的表达,从而影响增生性瘢痕成纤维细胞的凋亡。4.5本实验的结果初步表明SFRP2调控增生性瘢痕成纤维细胞的凋亡作用与wnt3a-β-catenin- Tcf/Lef-c-myc信号密切相关。SFRP2 shRNA可能是干预增生性瘢痕成纤维细胞凋亡新的策略。更多还原

【Abstract】 1. IntroductionThe ideal outcome of human wounding is functional and scarless healing. However, hypertrophic scar（HS） often occurs following cutaneous injury, such as burns and traumas which often result in contractures, severe functional and esthetic defects, and even permanent disability.HS is characterized by abnormal accumulation of a large number of fibroblasts and excessive deposition of extracellular matrix （ECM） components which result from abnormal fibroblast apoptosis and proliferation. Additionally, the differentiation from fibroblasts to myofibroblasts in HS plays an important role in the contracture of scars. Although the mechanism of HS remains unclear, it has been recognized that the inhibition of fibroblast apoptosis and proliferation promotion are key processes in the formation of HS.It is well known that wound healing is regulated by a delicate balance between cell proliferation and cell apoptosis. It was reported that a variety of genes including Bcl-2, Fas, smad3 and p53, were associated with the regulation of fibroblast apoptosis and proliferation. Previously, we screened genes that were differentially expressed in HS and normal skin of burn patients using cDNA microarray and then identified 97 differentially expressed genes. Among these genes, only one anti-apoptotic gene named secreted frizzled-related protein 2 （SFRP2） was conspicuously up-regulated in HS tissue.SFRP2 is a secreted glycoprotein and it has been considered a modulator of Wnt signaling and involved in apoptosis, proliferation and differentiation. Kobayashi et al. confirmed that SFRP2 induced the cardiomyoblast apoptosis and favored collagen deposition and fibrosis. Further, up-regulation of SFRP2 in retinas of patients with retinitis pigmentosa suggested an anti-apoptotic role of SFRP2 in some degree. The role of SFRP2 in the HS formation is not clarified yet. In the present study we inspected the role and mechanim of SFRP2 in HS formation.2. Materials and methods2. 1 Patient specimens and primary cell cultureHSFBs were established as primary cell lines either from normal skin or from HS tissue obtained from patients whom were recovering from severe burn. Individuals knew and signed informed consents. This study was also approved by the Ethnic Committee of Southwest Hospital of the Third Military Medical University （Chongqing, China）.2.2 upregulating of SFRP2 by pcDNA3.0/SFRP2Obtaining cDNA of SFRP2 from HSFBs,and then establishing pcDNA3.0/SFRP2. HSFBs were transfected with pcDNA3.0/SFRP2 plasmids. The transfection efficiency was confirmed by immunoflurescence density of SFRP2. The expression levels of SFRP2 were measured by western blot.2.3 Silencing of SFRP2 by shRNAThree pairs of shRNAs targeting different regions of the human SFRP2 transcript （accession number in GenBank: NM₀03013） and one control shRNA were designed and synthesized. shRNA fragments were annealed and cloned into the pSilencer 2.1-U6 neo plasmid （Ambion, USA）. HSFBs were transfected with SFRP2 shRNA plasmids. The transfection efficiency was confirmed by immunoflurescence density of SFRP2. The expression levels of SFRP2 were measured by western blot at the second days after the transfection. After the examination, shRNA2 was selected as the most efficient sequence to block the expression of SFRP2.2.4 Cell proliferation assayUsing Cellular growth curve, MTT and BrdU Proliferation assay methods to evaluate the proliferation of HSFBs.2.5 Cell apoptosis assayUsing caspase3 activity and fluorescence-activated cell sorting （FACS） methods to detect the apoptosis of HSFBs.2.6 Cell function assayUsing Fibroblast populated collagen lattice （FPCL） model to evaluate the contractility of HSFbs. Further, real-time PCR and western blot were used to measure the mRNA and protein expression ofα-SMA in HSFb. In addition, mRNA levels of typesⅠandⅢprocollagen were assayed by quantitative real-time PCR.2.7 Wnt proteins assayUsing Western blot to measure expression of Wnt1, wnt3a and wat8b of HSFBs. And then apoptosis of HSFBs treated with wnt3a was measured by caspase3 activity and fluorescence-activated cell sorting （FACS）.β-catenin and c-myc was detected by western blot.3. Results3.1 upregulating of SFRP2 by pcDNA3.0/SFRP2pcDNA3.0/SFRP2 is the most efficient to upregulate the expression of SFRP2 of HSFBS,and obtain HSFBs transfected with pcDNA3.0/SFRP2（versus HSFBs,p<0.01）.3.2 Silencing of SFRP2 by shRNAshRNA2 is the most efficient interference sequence to suppress the expression of SFRP2 of HSFBs, and obtain HSFBs transfected with psilencer 2.1-U6./SFRP2（versus HSFBs and shRNA1, shRNA3,p<0.01）.3.3 Cell proliferation assayMTT and BrdU results suggested that Upregulating and downregulating SFRP2 in HSFBs had no significant effect on HSFB proliferation （p>0.05）.3.4 Cell apoptosis assayUpregulating SFRP2 in HSFBs had no significant effect on HSFBs apoptosis. But compared with normal HSFBs, SFRP2 shRNA promoted cell caspase3 activity （p<0.01）. The results of FACS were same.3.5 Cell function assayCompared with normal HSFBs, the suppression of SFRP2 expression decreasdα-SMA mRNA and protein levels and resulted in loss of contractile ability of HSFBs （p<0.01）. SFRP2 shRNA2 downregulated the mRNAs of typesⅠandⅢprocollagen of HSFBs （p<0.01）.3.6 wnt proteins assayControlled with NSFBs, the results of WB suggested expression of Wnt3a was upregulated in HSFBs （p<0.01）; The level of apoptosis of HSFBs treated with wnt3a was suppressed by SFRP2 and SFRP2 suppressed expression ofβ-catenin and c-myc of HSFBs （p<0.01）.4. Conclusion4.1 Obtaining the HSFBs of overexpressing and downexpressing SFRP2.4.2 In vitro, SFRP2 had no effect on proliferation of HSFBS.4.3 In vitro, SFRP2 shRNA promoted HSFB apoptosis, but upregulating SFRP2 didn’t.4.4 SFRP2 suppressed apoptosis of HSFBs by influencing wnt3a/β-catenin.β-catenin and C-myc may be key factors in this progress.4.5 SFRP2 shRNA may be a novel treatment strategy.更多还原