小干扰RNA抑制CyclinD1表达诱导瘢痕疙瘩成纤维细胞凋亡的研究
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摘要
一、研究背景及目的
增生性瘢痕(Hypertrophic scar,HS)与瘢痕疙瘩(Keloid,K)是常见的病理性瘢痕,一般认为它们是由创伤引起,以成纤维细胞异常增殖,胶原等大量细胞外基质(extracellular matrix,ECM)的过度产生和沉积为特征的皮肤纤维增生性疾病,其发病率较高,尤其是瘢痕疙瘩,具有过度生长、超过原伤口界限、并侵犯临近组织、始终不退化和单纯手术切除后易复发等特点。瘢痕疙瘩不仅可继发畸形和功能障碍,影响美观,而且常伴有明显的瘙痒和疼痛症状,无论在功能上还是外观上都给患者身心健康带来极大的危害。病理性瘢痕的发病机制目前尚未完全清楚,虽然人们对其病因及治疗作了大量的研究,但至今仍未获得有效的治疗手段。
对于病理性瘢痕病因的研究,至今国内外许多学者从组织学、病理学、生理学、免疫学和遗传学等众多方面开展了一些列的研究,提出了诸如胶原合成与降解失调、细胞因子促进、成纤维细胞凋亡障碍、免疫反应和遗传学等许多学说,取得了不少成果,虽仍无法阐明病理性瘢痕的真正原因,但其中一点已经明确,即无论是何种体内或体外因素的影响,最终结果都是造成病理性瘢痕成纤维细胞过度增殖,胶原过量沉积。这是所有病理性瘢痕包括瘢痕疙瘩的病理特点。正是由于成纤维细胞的异常增殖才造成病理性瘢痕临床上一系列表现。由此可见,只要能在创伤愈合过程中,无论应用何种手段,早期抑制病理性瘢痕成纤维细胞的增殖,或者促进增殖细胞凋亡,减少胶原的过量沉积,就可能达到治疗病理性瘢痕的目的。因此研究病理性瘢痕成纤维细胞增殖凋亡的调节机制尤其是明确早期细胞异常增贿关键调控因子是本研究前提,而细胞的增殖分化调节又与细胞周期密切相关,细胞周期循环中的调控核心决定了细胞成长的最终去向。
细胞周期的概念的建立和近几年细胞周期的研究取得的突破性进展,为现代研究细胞的增殖、分化和凋亡奠定了基础。调控细胞周期的核心包括周期蛋白(Cyclin)、依赖周期蛋白激酶(CDK)以及依赖周期蛋白激酶抑制因子(CKI)。这三者有机地结合,使细胞分裂得以正常的进行。细胞周期调控核心的研究成果给人们探索细胞异常增殖分化机理带来了新的理论基础。研究表明,体内细胞的分裂是受时间、环境等条件影响的,正常细胞的分裂是相互制约、密切协调从而构成正常有序的细胞群,但如果细胞一旦不受控制、不受约束地无限制分裂下去,便可能造成病理改变,找到细胞周期过程中的CDK、Cyclin、CKI,也就找到如何控制分裂的异常细胞继续分裂的方法。而其中Cyclin对维持整个正常细胞周期起决定性作用。
在细胞周期中G1期的启动是细胞周期的关键步骤,因而调控G1/S期的Cyclin尤其重要。CyclinD1是细胞周期中主要的正态调控因子之一,由于它主要调节细胞周期G1期启动进入S期,它是细胞周期早期调控关键因子,起到加速细胞周期进程的作用,其过量表达就会引起细胞增殖的异常,因此,各种原因引起的CyclinD1高表达是造成细胞生长增殖过快的主要原因之一。在对众多的肿瘤的研究发现,肿瘤的发生、发展与G1/S调控点成分的异常密切相关。而瘢痕疙瘩所具有的独特生长特性以及临床表现,被众多学者视为一种创伤愈合后形成的良性肿瘤,成纤维细胞是瘢痕疙瘩形成的功能性细胞,其增殖与凋亡的失衡是导致瘢痕疙瘩不断增生而且难已退化的细胞学基础。瘢痕的形成可能是由于多种因素作用于细胞周期调控因子,引起CyclinD1合成增加,造成细胞周期进程加速,从而造成功能细胞的增殖与凋亡紊乱。为此,我们收集了增生性瘢痕和瘢痕疙瘩典型标本,观察成纤维细胞内CyclinD1的表达情况,尤其是瘢痕疙瘩周边部位和中央部位的表达差异对研究瘢痕疙瘩临床表现和指导治疗有重要意义。
目前,大量对病理性瘢痕的研究主要在明确病因方面,而治疗还局限在单纯的药物注射和病灶手术切除,无法从根本上彻底治愈,治疗后常复发,尤其瘢痕疙瘩手术后增生更明显。临床上迫切需要一种可以在创伤愈合瘢痕形成早期抑制瘢痕成纤维细胞增殖的方法。细胞周期调控细胞增殖凋亡的机制为我们在瘢痕成纤维细胞分裂增殖层面抑制细胞增殖提供了一条新的思路。随着基因工程技术的出现和迅猛发展,基因治疗将成为今后治疗病理性瘢痕的理想方法。已有学者开始尝试应用反义核酸技术,以序列特异性方式抑制促瘢痕形成因子基因表达,或者是向增生性瘢痕的成纤维细胞内转入抑制基因,以增加胶原的降解,但是效果均不理想。RNA干扰技术(RNA interfering,RNAi)的出现,进一步为基因治疗病理性瘢痕提供了广阔的空间。RNAi是指将外源双链RNA(double-strandedRNA)导入细胞后引起与其序列同源的特异基因mRNA,降解的现象,属于转录后基因沉默(postt ranscriptional gene silencing,PTGS)的一种形式。最早由Fire等在进行线虫基因功能研究中所提出,后被证实RNA干扰现象是自然产生的保护基因组的机制,普遍存在于动植物体中。经过大量研究学者提出一个在哺乳动物细胞内RNAi作用的模式:外源或内源产生的dsRNA,进入细胞后在核酸酶(Dicer)作用下被切割成3′端有2个碱基突出的21～23nt长度的小分子干扰RNA(small interfering RNAs,siRNAs),这些siRNA与RNAi特异性酶结合,形成RNA诱导沉默复合体(RNA-induced silencing complex,RISC),而后RISC在siRNA反义链的指导下与siRNA同源靶RNA相结合,并在近中点位置将其切割,达到抑制靶基因表达的作用。切割后产物经过聚合酶链式反应再次生成新的dsRNA继续作用于靶mRNA,最终完全降解靶mRNA。因此少量dsRNA就可以完成显著的干扰效应。但是,实验发现大于30bp的dsRNA导入后会启动细胞内的病毒防御机制,细胞内的干扰素合成增加,同时激活蛋白激酶K(PKR)和2′5′-寡腺苷酸合成酶(2′5′-AS)。活化的PKR磷酸化翻译启始因子eIF2α使其活性降低致使细胞内全部蛋白合成关闭。经激活的2′5′-AS合成2′5′-寡腺苷酸则可活化一种核糖核酸酶L(RNase L)降解非特异性mRNA。结果是非特异性的和全面的抑制基因表达,最终导致细胞凋亡。该因素在一短时间内限制了RNAi的发展。随着siRNA可以以各种方式得到而不必通过dsRNA并在体内和体外应用,使siRNA可以高特异性和高效率的阻止或抑制靶基因的表达。此后RNAi在医学方面探索和应用逐步成为研究的热点。
成功地应用RNAi技术敲除疾病相关基因表明该技术开启了新的治疗途径大门。RNAi目前已经在广泛的领域包括医学领域里取得了令人瞩目的进展,已经广泛应用于医疗各中疾病尤其是肿瘤相关方面的治疗。虽然有整形外科学者开始应用RNAi技术特异性抑制瘢痕形成的相关因子基因的表达,但是应用体外合成siRNA对瘢痕疙瘩成纤维细胞CyclinD1表达抑制的研究国内外尚未见报道。由此我们设想,如果病理性瘢痕成纤维细胞中CyclinD1存在着高表达,并因此而造成了细胞早期的异常增殖,那么应用RNAi技术阻止或抑制CyclinD1基因的表达,减少CyclinD1蛋白合成,从而抑制成纤维细胞的细胞周期早期增殖,就有可能达到治疗瘢痕增生的目的。因此,我们针对人CyclinD1基因设计并合成相应siRNA分子,转染瘢痕疙瘩成纤维细胞下调CyclinD1mRNA,抑制CyclinD1的有效合成,同时应用多种方法观测成纤维细胞异常增殖的抑制效果,为病理性瘢痕的RNAi治疗研究提供初步的理论依据,也希望能为临床进一步开展病理性瘢痕RNAi治疗提供新的思路。
二、材料和方法
1.研究对象
标本均取自南方医院整形外科手术患者。增生性瘢痕标本77例,其中男56例,女21例,年龄4岁～45岁,瘢痕增生时间40d～5a;瘢痕疙瘩标本10例,其中男6例,女4例,年龄18岁～46岁。24例正常皮肤(Normal skin,NS)均取自上述患者供皮区,为手术取皮修剪后残余皮片。患者没有长时间外用各种防治瘢痕药物史,不伴有肿瘤及其它严重疾病。取材前向患者说明取材目的,并征得患者同意。瘢痕疙瘩标本病变部位分别为耳垂、三角肌区及前胸。直视下区分各标本中央部和周边部,瘢痕组织中央较平且色淡区为中央部,周围明显隆起呈暗红色区域为周边部,均经临床及病理诊断证实。正常皮肤均取自上述患者供皮区,为手术取皮修剪后残余皮片。增生性瘢痕标本按瘢痕增生时间分为5组:1、1月～3月;2、4月～6月;3、7月～9月;4、10月～12月;5、12月以上。瘢痕疙瘩标本按中间2/3和周围1/3部分分为中央部和周边部2组。以NS组为对照。以PBS代替一抗作为阴性对照。
2.方法
(1)免疫组化染色检测CyclinD1
应用免疫组织化学SP法检测CyclinD1在增生性瘢痕不同病理时期和瘢痕疙瘩不同部位成纤维细胞中的表达差异。
(2) siRNA-cyclinD1分子的设计与合成
应用ambion公司在线软件siRNA target finder设计siRNA-cyclinD1分子,其正义链为5'-CAAACAGAUCAUCCGCAAAtt,反义链为5'-UUUGCGGAUGAUCUGUUUGtt。siRNA-cyclinD1分子干扰Cyclin D1基因的第664～684位核苷酸,靶序列为AACAAACAGATCATCCGCAAA。采用化学法分别合成siRNA-cyclinD1分子的正义链和反义链,经过变性退火处理后得到双链siRNA-cyclinD1分子。
(3)实验分组及转染siRNA-cyclinD1分子。
转染实验分2组:无关siRNA转染组,siRNA-cyclinD1转染组。设立未处理细胞为对照组。每组实验重复3次。
(4) RT-PCR法检测CyclinD1基因表达
成纤维细胞经siRNA-cyclinD1转染24h、48h、72h后分别提取细胞总RNA,然后进行逆转录反应,检测CyclinD1基因的mRNA相对表达量的改变。
(5)双标记流式细胞术分析细胞凋亡
成纤维细胞经siRNA-cyclinD1转染24h、48h、72h后,分别收集10~6个细胞进行流式细胞术检测,经Cellquest软件分析4个区域(UL,UR,LL,LR)内的细胞百分数。UR和LR区域的细胞百分数代表了凋亡细胞百分比。
(6)细胞凋亡DNA片段检测
成纤维细胞转染24h、48h、72h后,分别收集siRNA-cyclinD1转染组(实验组)和无关siRNA转染组(对照组)细胞各1×10~5个,提取DNA后进行1.2%琼脂糖凝胶电泳。
(7) MTT法检测成纤维细胞活性
siRNA-cyclinD1转染成纤维细胞24h,48h,72h后,MTT法检测成纤维细胞活性,以转染无关siRNA的细胞为对照组。计算各个时间点细胞增殖抑制率,计算公式为:细胞增殖抑制率=(1—转染siRNA-cyclinD1后的A570值/对照组细胞A570值)×100%。各时间点实验重复3次。
(8)数据处理
数据分析采用SPSS12.0统计软件,进行非参数检验——多组独立样本秩和检验(Kruskal—Wallis H检验)、单因素方差分析和以及LSD法进行组间多重比较。
三、结果
(1)增生性瘢痕不同时期成纤维细胞CyclinD1的表达
在增生性瘢痕早期,CyclinD1阳性表达的成纤维细胞较多,中晚期阳性表达的成纤维细胞明显减少,部分标本成纤维细胞中CyclinD1表达阴性。表达随增生性瘢痕的发展明显呈现由强至弱的变化。统计学处理显示:不同时期增生瘢痕和正常皮肤成纤维细胞中CyclinD1的表达差别有统计学意义(X~2=68.53,P＜0.001)。两两比较结果:第四和第五组差异无统计学意义,其他各组两两比较均差异有统计学意义。
(2)瘢痕疙瘩不同部位成纤维细胞CyclinD1的表达
瘢痕疙瘩周边部,CyclinD1阳性表达高,而在中央部,阳性表达明显减少。统计学处理显示:边缘部与NS组比较、边缘部与中央部比较以及中央部与NS组比较,CyclinD1蛋白表达均有显著性差异(P＜0.001)。
(3)转染后成纤维细胞形态的变化:
转染特异性小干扰性RNA的24h后部分成纤维细胞从梭形转变为圆形或椭圆形,并且随着时间增加,圆形或椭圆形细胞所占的比例逐渐增加,而未处理组和转染空脂质体组72h后细胞形态基本相同。
(4)转染siRNA-cyclinD1后对CyclinD1基因mRNA水平的影响
成纤维细胞转染50nMsiRNA-cyclinD1后,成纤维细胞的CyclinD1基因相对表达量在24h～72h内逐渐减少。
(5) siRNA-cyclinD1转染后对成纤维细胞周期的影响
成纤维细胞转染特异性siRNA-cyclinD1分子24h、48h、72h后,重复3次实验并采用SPSS12.0软件对结果进行单因素方差分析显示:(1)siRNA-cyclinD1转染影响各组细胞的G0/G1期(F=22.18,P＜0.001);(2)siRNA-cyclinD1转染影响各组细胞的S期(F=29.88,P＜0.001);(3)siRNA-cyclinD1转染不影响各组细胞的G2/M期(F=2.77,P=0.11)。组间两两比较结果显示:G0/G1期细胞平均百分率分别为(60.38±1.77)%、(64.94±1.62)%、(65.18±2.20)%,显著高于无关siRNA转染72h后的对照组细胞(54.45±1.77)%(P值分别＜0.01,＜0.001,＜0.001);S期细胞平均百分率分别为(18.15±1.28)%、(17.28±0.89)%、(11.54±1.56)%,显著低于对照组(22.17±1.68)%(P值分别为0.019,0.007,0.001);G2/M期细胞平均百分率分别为(21.47±3.04)%、(17.78±2.27)%、(23.28±1.83)%,与对照组(22.17±1.68)%无显著差别(P值分别为0.73,0.09,1.0)。
(6) siRNA-cyclinD1转染成纤维细胞后诱导细胞凋亡情况
特异性siRNA-cyclinD1分子转染成纤维细胞24h、48h、72h后,重复3次实验得到的细胞周期结果并进行单因素方差分析,结果:siRNA-cyclinD1转染影响各组细胞的凋亡率(F=458.77,P＜0.001)。组间多重比较(LSD法)结果为:siRNA-cyclinD1转染成纤维细胞24h、48h、72h后,细胞平均凋亡率分别为(7.82±0.45)%、(16.07±1.58)%、(18.49±0.82)%,均显著高于对照组(0.68±0.12)%,差异均有统计学意义(P值均＜0.001)。
(7) siRNA-cyclinD1转染后细胞DNA片段化
成纤维细胞转染特异性siRNA-cyclinD1后,24h内未见DNA小片段,48h～72h内出现大小介于100～400bp之间的DNA小片段;而转染无关siRNA的对照组细胞72h内未出现DNA小片段。
(8) siRNA-cyclinD1转染后成纤维细胞后的细胞增殖情况
成纤维细胞转染siRNA-cyclinD1后的24h、48h、72h,应用MTT法测定细胞生长情况(A570),重复3次实验并采用SPSS12.0软件进行单因素方差分析,结果为:siRNA-cyclinD1转染24、48、72h各个时间点内均影响各组细胞增殖(P值均＜0.001)。同一时间点内进行组间多重比较(LSD法),结果为:(1)与无关siRNA转染组相比,siRNA-cyclinD1转染组细胞生长受到抑制,差异有统计学意义(P＜0.001);(2)与未处理组相比,siRNA-cyclinD1转染组细胞生长受到抑制,差异有统计学意义(P＜0.001);(3)与未处理组相比,无关siRNA转染组细胞生长差异无统计学意义(P=0.854)。根据公式:细胞增殖抑制率=(1—转染后的A570/未处理细胞A570)×100%,计算siRNA转染后的细胞生长抑制率,分别为18%、37%和50%,与无关siRNA转染组相比呈逐步上升趋势。
四、结论
1.CyclinD1在病理性瘢痕的形成中起着重要作用,通过缩短G1期进程导致成纤维细胞异常增殖而促进了病理性瘢痕的形成。
2.CyclinD1在增生性瘢痕早期成纤维细胞中的高表达与增生性瘢痕早期增生旺盛密切相关。
3.CyclinD1在瘢痕疙瘩周边部成纤维细胞中的高表达,不仅促进了瘢痕疙瘩的形成,并可能是瘢痕疙瘩侵犯局部正常组织,浸润性生长的重要原因之一。
4.siRNA-cyclinD1分子对靶基因CyclinD1的抑制效果明显,靶基因mRNA量明显降低,从而使CyclinD1蛋白表达量下降,成纤维细胞细胞从G1期进入S期受到明显抑制而停留于G1期,随时间推移细胞发生凋亡,成纤维细胞数目减少。
5.本研究证实siRNA-cyclinD1分子能够干扰CyclinD1基因表达,进而抑制瘢痕疙瘩早期异常增殖。
Ⅰ.Background and aim
Hypertrophic scar(HS) and keloid(K) are common pathological scars.Usually believed to be caused with wounds,they are proliferative diseases of dermatofibroma characterized in abnormal proliferation of fibroblast,overproduction and deposit of such extracellular matrix(ECM) as collagen with high incidence.Keloid,in particular,has such characteristics as producing too much,proceeding beyond the boundary of wounds to encroach on tissues nearby,never degenerating and being easy to recur after pure surgery.It not only can cause dysmorphia and functional disturbance,but also has influence on the outlook.What is more,it often goes with evident pruritus and pains.Therefore,it brings great harzards to the physical and mental health of the patient both in functions and outlook.However,we have not found out completely the pathogensy of this pathologican scar,or effective methods for its treatment,though a great deal of research has done on its cause.
Scholars at home and abroad have conducted some series of research on the causes of pathological scars from the point of view of histology,pathology, physiology,immunology and genetics,and great achievements have been made.The real causes of the disease still remain unclear.There is one point,however,that lots of experts have come into conformity,that is,the factors in vivo and vitro all contribute to the too much proliferation of fibroblast of the pathological scars,and the over deposit of collagan,which is typical of all pathological scars,including keloid.It is the abnormal proliferation of fibroblast that brings about the series of manifestations of the scars clinically.Thus,phologican scars may be treated if proliferation of their fibroblast can be inhibited with whatever means during the process of wounds healing,or something is done to enhance the apoptosis of reduplicated cells to reduce the over-deposit of collogan.It is therefore the precondition of this research to find out the mechanism to regulate the apoptosis of fibroblast proliferation of pathological scars,particularly the key factors regulating abnormal proliferation of cells at early stage,as the regulation of proliferation and differentiation is closely related with cell cycles,and the regulatory core in cyclnes determines the final terminal of cell growth.
That the establishment of the concept of cell cycle and the breakthrough in the research on it in recent years have laid down a foundation for the study of cell proliferation,differentiation and apoptosis in modern times.The discovery of Cyclin, CDK and CDI has certainly revealed the mechanism of such movement.The organic combination of the three brings about normal division of cells,becoming the core controlling cell cycle.The outcome:from the research on the core regulating cell cycle provides a new theoratical base for people to explore the mechanism of the abnormal cell proliferation and differentiation.Studies show that division of cell in vivo is affected by such factors as time and environment.Dissociation of normal cells is restricted by one another that coordinates closely to construct normal cell groups in order.Division would cause pathological change if it proceeds continuously without control or constraint.Therefore,if we find the CDK,Cyclin and CKI in the process of cell cycle,we would get the the way to control continuity of abnormal cell division.
Priming at G1 is a critical step in the cell cycle.It is particularly important, therefore,to regulate cyclins at G1/S.As one of normal major regulating factors, CyclinD1 mainly regulates cyclin at G1 to enter Phase S.It is the key factor for regulation at early stage to accelerate cell cycle.Its over expression,however,will bring about abnormal cell proliferation.Therefore,high expression of cyclinD1 due to various causes is one of the main reasons that makes cells grow too fast.Research on many a tumors has found that the occurrence and development of tumors are closely related to abnormality of the composition of G1/S at the regulating point.The unique growth characteristics and clinical manifestation of keloid shows have been taken by many scholars as a kind of innocent tumor developing after a wound heals.Fibroblasts are functional cells for keloid to develop,and their disequilibrium inin proliferation and apoptosis is the cytological base for keloids to grow continuously and be hard to become vestigial.There was research showing that cyclinD1 wass in a state of high expression in fibroblasts.This implies that keloids may be caused by the regulating factors working on cell cycles,for they increase synthesis of cyclinD1 and speed up cell cycles to cause confusion of functional cells in proliferation and apoptosis.Therefore,we have collected typical samples of hyperplastic scars and keloids to observe how cyclinD1 was expressed in fibroblasts.The differences in expression at the edge of keloids and in the center are of particular importance in the study of the clinical manifestation of keloids and in guidance in the treatment.
Presently a lot of research on pathological scars focus mainly on the cause,and treatment is limited to injection of medicine and surgical removal of focus of infection,which cannot thoroughly cure the diease,which may appear again later, particularly the scar fibroblast proliferation.There is an urgent need clinically for one method to inhibit the proliferation after the heeling of wounds but before the formation of scars.The mechanism to regulate the apoptosis of proliferation at cyclin provides a new way for us to inhibit proliferation at the level when scar fibroblasts break.With the emergence and rapid development of genetic engineering,gene therapy will become an ideal way to treat pathological scars.Some scholars have tried employing antisensenecleic acid technology to inhibit the gene expression of factors bringing about the formation of scars with sequence specificness,or transferring inhibiting genes into the fibroblasts of hyperplastic scars to build up collagenous degradation.This,however,does not seem to work well.The appearance of RNA interferring(RNAi) further provides vast space for treating genetically pathological scars.RNAi refers the phenomenon of degradation of the specific gene mRNA brought about by introducing exogenous double-stranded RNA.They are the congenerous in sequence,a sort of post-transcriptional gene silencing(PTGS),first put forward by Fire in I the research on the gene functions of edlworms,and then was confirmed that RNA interferring was the natural mechanism protecting gene groups, found commonly inside animals and plants.After a great deal of research,some scholars proposed a mode for RNAi to work in mammalian cells:exogenous or endoneous dsRNA was cut into small interferring RNAs(siRNA) with two basic group protrutions at 3' under the effect of Dicer after entering cells.The siRNA is combined with the specific enzyme of RNAi to develop RNA-induced silencing complex(RISC) which is further combined with RNA the exogenous target of siRNA under the instruction of siRNA antisense strand.They are cut at the near center to inhibit the expression of target genes.Through polymerase chain reaction the cut materials will develop into new dsRNA to work continuouly on mRNA until finally degrading target mRNA completely.Therefore,a small amount of dsRNA can complete the effect of remarkable interference.However,experiments have found that dsRNA introduced would initiate the virus protective mechanism in cells when it was larger than 30bp,which increased the synthesis of interferon in cells and activate simultaneouly the protein kinase K(PKR) and 2'5' adenyl acid synzyme(-AS).The activated PKR phosphorylation translates the initial factors eIF2αto decrease its activity so that the synthesis of protein in cells is closed completely.The activated 2'3'(-AS) is can activate a ribonuclease(RNase L) to degrade the non-specific mRNA,resulting in the non-specific and entire inhibition the expression of genes that causes apoptosis.This factor limits the development of RNAi in a short time.Now when it is easy to get it withouth dsRNA and employ it in and out of body,siRNA can specifically and effectively stop or inhibit the expression of target genes.RNAi will certainly become a hot point of research in medical field.
Surprising development has been made in the research on RNAi in extensive fields including medicine and it has been applied in widely in treating various diseases,particularly tumors.Although some scholars in plastics have-started to inhibit the expression of genes related to the formation of scars with the specifiness of RNAi technology,but there was no report about Apoptosis of human keloid fibroblast induced by small interfering RNA-mediated cyclinD1 gene silencing.We thus suppose that if the high expression of cyclinD1 in the fibroblasts of pathological scars causes the abnormal cell proliferation at early stage,we may knock out its synthesis of mRNA with RNAi technology to cut down the synthesis of CyclinD1 so as to inhibit the proliferation of fibroblasts at the early state of cell cycle.This may help us treat scar proliferation,which will provide new theoractical evidence for further research on the treatment of pathological scars.
Ⅱ.Materials and methods
1.Subjects
The Samples came from patients eceiving plastic surgery in Nanfang Hospital.These included 77 cases of hyperplastic scar,with 56 males and 21 females aged between 4 to 45 years old and the periods of scar hyperplasy ranging 40d～5a; there were 10 samples of keloid,among which 6 were males and 4 females aged between 18～46 years old.There were 24 cases of normal skin from the patients above.These were skin residues left after the surgery.The patients did not have a history of taking medication to prevent scars for long,nor did they suffer from tumors or other severe diseases.The patients were told about the purpose before the sampling and they agreed to it.The diseased parts with keloid sampled included the ear lobe, the deltoid region and the protothorax.The lower orthophoria part was divided into central and ambitus areas,both confirmed clinically and with pathologic diagnosis.The normal skins,taken from the skin-supply areas of the patients above, were residuces left after the surgery.The samples of hyperplastic scars were divided into five groups according to the time hyperplasy occurred:1.Those occurred at Months 1 to 3;2.those at Months 4 to 6;3.those at Months 7 to 9;4.those at month 10 to 12 and 5.those appeared 12 months after the surgery.Samples of keloid were divided into two groups:the central group with 2/3 of the center and the edge group with 1/3 of the edge.Those in the NS group were taken as the control group,and PBS as negative the control group.
2.Methods
(1) Detecting cyclinD 1 with immunohistochemistry
Detect with SP method of immunohistochemistry the expression difference of CyclinD1 in different pathological phase of hyperplastic scar and fibroblast in different area of keloid.
(2) siRNA design and synthesis for CyclinD1
With siRNA target finder,the softwear online by Ambion,we designed s ome siRNA-cyclinD1 molecues whose positive-sense strand being 5'-CAAACA GAUCAUCCGCAAAtt and antisense strand 5'-UUUGCGGAUGAUCUGUUUGt t.The siRNA-cyclinD1 molecues interfered nucleotides of Cyclin D1 genes pos itioning at No.664-684,and the target serial was AACAAACAGATCATCCG CAAA.Then with chemical methods,we had the positive-sense and anti-sense s-trands of siRNA-cyclinD1 molecues synthesized respectively(entrusted to Sh anghai Jikai Genes Co.).They became double-stranded siRNA-cyclinD1 mole cues after being treated with denaturalized anneal.
(3) laboratory groups and transfected siRNA-cyclinD1 molecues
There were two groups for the transfection laboratory:blank control group in which no transfection was related to siRNA,and siRNA group spectific to Cyclin in which transfected Smad2 specific to siRNA.Tests were repeated three times in each group.
(4) Detecting CyclinD1 gene expression with RT-PCR
Gross RNA of cells were taken when the fibroblasts were transfected with siRNA-cyclinD1 for 24,48 and 72 hours and reverse transcription reaction was conducted for the observation of the change of the relative expression amount in mRNA in CyclinD1.
(5) Analyzing apoptosis with double tagged flow cytometry
10~6 cells after transfected siRNA-cyclinD1 molecues were collected to be detected with flow cytometry and the percent of cells in four areas(UL,UR,LL and LR) were analyzed with Cellquest softwear.The percent of cells in UR and LR standed for the percentage of apoptosed cells.
(6) Detecting DNA fragment of apoptosis
10~6 cells were collected respectively from the transfection siRNA-CyclinD1 group(the experimental group) and the siRNA group without transfection(the control group),and 10μl of DNA were colbcted from each for 1.2%agarose gel electophoresis.
(7) Detecting the activity of fiborblast with MTT
The activity of fibroblasts was detected with MTT when siRNA-cyclinD1 was transfected into fibroblasts for 24,48 and 72 hours,with the cells in siRNA without transfection in the control group.The rate of cell proliferation inhibited at different time point was worked out.The formulas was the rate of cell proliferation inhibited= (1—A570 value after transfected siPNA-cyclinD1/A750 value of cells in the control group)×100%.The tests were repeated three times at each time point.
(8) Data processing
Analysis on the data was done with SPSS12.0 for Kruskal-Wallis H test),and comparisons among groups were made for mono-factor variance with LSD method.
Ⅲ.Results
(1) The expression of CyclinD1 in fibroblast at different stage of hyperplastic scar
At the early stage of hyperplastic scar,more fibroblasts showed positive expression with CyclinD1,while fewer fibroblasts showed positive expression at middle and later stages,and in some samples the CyclinD1 had negative expression in the fibroblasts.The expression turned from being strong to weak with the development of the scars.Statistical analysis showed great difference in the expression of CyclinD1 protein between Group 1 and Group NS;between Group 2 and Group NS,and between Groups 1 and 5(X~2=68.53,p＜0.001);while comparisons in pairs in other groups showed no statistic significance.(P＞0.05)
(2) The expression of CyclinD1 in fibroblast at different parts of keloid
At the ambitus of a keloid,CyclinD1 showed high positive expression,while at the center,positive expression dropped greatly.Statistical treatment showed marked difference in the expression of Cyclin D1 protein between the ambitus and Group NS (P＜0.001),between the ambitus and the center(P＜0.001),and between the center and Group NS(P＜0.001).
(3) The change in form for fibroblast after being transfected
When siRNAwas transfected for 24 hours,some fibroblasts turned from fusiform into spherical or oval shape,and with time proceeding,the percentage of round or oval cells increased gradually.The cells in the untreated group and the hollow transfected bangosome group remained basically in the same shapes.
(4) The influence of transfected siRNA-cyclinD1 on the level of mRNA the CyclinD gene
When the fibroblasts were transfected with 50nM siRNA-cyclinD1,the relative expression amount of the CyclinD gene in the fibroblasts gradually became less within 24～72 hours.
(5) The influence oftransfected siRNA-cyclinD1 on the cycle of fibroblasts
When the fibroblasts were transfetected with specific siRNA-cyclinD1 molecules for 24,48 and 72 hours,the experiment was repeated for three times before the results were analyzed with SPSS12.0 in mon-factor variance,showing that transfection of siRNA-cyclinD1(1) had influence on cells in the groups at G0/G1 (F=22.18,P＜0.001);(2) affected the cells at Phase S(F=29.88,P＜0.001); and(3) did not have any influence on the cells at Phase G2/M(F=2.77,p＜0.001).Comaprisons made between pairs of groups showed that the average percentage of cells at Phase G0/G1 were(60.38±1.77)%,(64.94±1.62)%and (65.18±2.20)%respectively,much higher than those in the control group without siRNA after 72 hours(54.45±1.77%)(P＜0.01,＜0.001,＜0.001);the average percentages of cells at Phase S were(18.15±1.28)%,(17.28±0.89)%and (11.54±1.56)%,much lower than that(22.17±1.68)%(P values being 0.019, 0.007 and 0.001 respectively) in the control group;and the average percentages at Phase G2/M were(21.47±3.04)%,(17.78±2.27)%and(23.28±1.83)% respectively,showing no difference with that(22.17±1.68)%(P values being 0.73, 0.09,1.0 respectively).
(6) Apoptosis induced after siRNA-cyclinD1 was transfected into fibroblasts
When specific siRNA-cyclinD1 molecues were transfected into fibroblasts for 24,48 and 72 hours,and the experiment was repeated for three times before the results of cyclines were analyzed in mono-factor vairance with LSD method among groups that showed that transfection with siRNA-cyclinD1 had influence on the apoptosis rate(F=458.77,P＜0.01).Comparisons between pairs of groups indicated that when siRNA-cyclinD1 was transfected into fibroblasts for 24,48 and 72 hours,the average apoptosis rates were(7.82±0.45)),(16.07±1.58)%and (18.49±0.82)%respectively,much higher than that(0.68±0.12)%in the control group,the differences were all of statistic significance(the P values being＜0.001).
(7) Gragmentation of DNA when siRNA-cyclinD1 was transfected
When the fibroblasts were transfected with siRNA-cyclinD1,no fragmentswere found in 24 hours,but in 48～72 hours fragments as large as 100～400bp appeared; No fragments were found in the cells in the control group in 72 hours.
(8) The proliferation of fibroblasts after siRNA-cyclinD1 was transfected
24,48 and 72 hours after siRNA-cyclinD1 were transfected into fibroblasts,the growth of the cells were determined with MTT rules(A570).The experiment was repeated three times and analysis was made in multi-factor variance with SPSS12.0 with the results showing that transfection of siRNA-cyclinD1 had influence on the proliferation in each group(p＜0.001).Comparisons among groups with LSD method at the same time point made showed that(1) cell growth in the siRNA-cyclinD1 transfected group was inhibited in contrast with that in the group without siRNA transfection,the difference being statistically significant(p＜0.001); (2) cell growth in the siRNA-cyclinD1 transfected group was inhibited in contrast with that in the group without any treatment,the difference being statistically significant(p＜0.001);and(3) the cell growth in the group without siRNA transfection indicated no statistical significance(p=0.854).The cell proliferation inhibiting rates after siRNA was transfected was worked out in accordance with the formular:cell growth inhibiting rate=(1—transfected A570/untreated cells A570)×100%,and we got 18%,37%and 50%,tending to increase gradually,with the difference showing statistically significance(p＜0.001) compared with that in the group with siRNA transfection.
Ⅳ.Conclusion
1.CyclinD1 played an important role in the formation of pathological scars.Shortening proceeding of Phase G1 resulted in abnormal proliferation of fibroblasts that encouraged the formation of pathological scars.
2.The high expression of CyclinD1 in fibroblasts at early stage of hyperplastic scars was closely related to the prosperous hyperplasy of scars at early stage.
3.The high expression of CyclinD1 in fibroblasts at the edge of keloid not only promoted the their formation,but also probably one of the reasons for keloid to offent normal tissues locally and grow infiltratively.
4.siRNA-cyclinD1 molecues could effectively inhibit CyclinD1,the target genes, and the amount of mRNA dropped greatly to reduce the expression of CyclinD1.The cell cycline of fibroblasts was restricted when it moved from G1 to Phase S and had to stop at G1,and cell tended to apoptose with time going and cell proliferation was inhibited.
5.This research confirms that siRNA-cyclinD1 can interfere the cell expression of CyclinD1 and then inhibit the abnormal hyperplasy of keloid at early stage.
增生性瘢痕(Hypertrophic scar,HS)与瘢痕疙瘩(Keloid,K)是常见的病理性瘢痕,一般认为它们是由创伤引起,以成纤维细胞异常增殖,胶原等大量细胞外基质(extracellular matrix,ECM)的过度产生和沉积为特征的皮肤纤维增生性疾病,其发病率较高,尤其是瘢痕疙瘩,具有过度生长、超过原伤口界限、并侵犯临近组织、始终不退化和单纯手术切除后易复发等特点。瘢痕疙瘩不仅可继发畸形和功能障碍,影响美观,而且常伴有明显的瘙痒和疼痛症状,无论在功能上还是外观上都给患者身心健康带来极大的危害。病理性瘢痕的发病机制目前尚未完全清楚,虽然人们对其病因及治疗作了大量的研究,但至今仍未获得有效的治疗手段。
对于病理性瘢痕病因的研究,至今国内外许多学者从组织学、病理学、生理学、免疫学和遗传学等众多方面开展了一些列的研究,提出了诸如胶原合成与降解失调、细胞因子促进、成纤维细胞凋亡障碍、免疫反应和遗传学等许多学说,取得了不少成果,虽仍无法阐明病理性瘢痕的真正原因,但其中一点已经明确,即无论是何种体内或体外因素的影响,最终结果都是造成病理性瘢痕成纤维细胞过度增殖,胶原过量沉积。这是所有病理性瘢痕包括瘢痕疙瘩的病理特点。正是由于成纤维细胞的异常增殖才造成病理性瘢痕临床上一系列表现。由此可见,只要能在创伤愈合过程中,无论应用何种手段,早期抑制病理性瘢痕成纤维细胞的增殖,或者促进增殖细胞凋亡,减少胶原的过量沉积,就可能达到治疗病理性瘢痕的目的。因此研究病理性瘢痕成纤维细胞增殖凋亡的调节机制尤其是明确早期细胞异常增贿关键调控因子是本研究前提,而细胞的增殖分化调节又与细胞周期密切相关,细胞周期循环中的调控核心决定了细胞成长的最终去向。
细胞周期的概念的建立和近几年细胞周期的研究取得的突破性进展,为现代研究细胞的增殖、分化和凋亡奠定了基础。调控细胞周期的核心包括周期蛋白(Cyclin)、依赖周期蛋白激酶(CDK)以及依赖周期蛋白激酶抑制因子(CKI)。这三者有机地结合,使细胞分裂得以正常的进行。细胞周期调控核心的研究成果给人们探索细胞异常增殖分化机理带来了新的理论基础。研究表明,体内细胞的分裂是受时间、环境等条件影响的,正常细胞的分裂是相互制约、密切协调从而构成正常有序的细胞群,但如果细胞一旦不受控制、不受约束地无限制分裂下去,便可能造成病理改变,找到细胞周期过程中的CDK、Cyclin、CKI,也就找到如何控制分裂的异常细胞继续分裂的方法。而其中Cyclin对维持整个正常细胞周期起决定性作用。
在细胞周期中G1期的启动是细胞周期的关键步骤,因而调控G1/S期的Cyclin尤其重要。CyclinD1是细胞周期中主要的正态调控因子之一,由于它主要调节细胞周期G1期启动进入S期,它是细胞周期早期调控关键因子,起到加速细胞周期进程的作用,其过量表达就会引起细胞增殖的异常,因此,各种原因引起的CyclinD1高表达是造成细胞生长增殖过快的主要原因之一。在对众多的肿瘤的研究发现,肿瘤的发生、发展与G1/S调控点成分的异常密切相关。而瘢痕疙瘩所具有的独特生长特性以及临床表现,被众多学者视为一种创伤愈合后形成的良性肿瘤,成纤维细胞是瘢痕疙瘩形成的功能性细胞,其增殖与凋亡的失衡是导致瘢痕疙瘩不断增生而且难已退化的细胞学基础。瘢痕的形成可能是由于多种因素作用于细胞周期调控因子,引起CyclinD1合成增加,造成细胞周期进程加速,从而造成功能细胞的增殖与凋亡紊乱。为此,我们收集了增生性瘢痕和瘢痕疙瘩典型标本,观察成纤维细胞内CyclinD1的表达情况,尤其是瘢痕疙瘩周边部位和中央部位的表达差异对研究瘢痕疙瘩临床表现和指导治疗有重要意义。
目前,大量对病理性瘢痕的研究主要在明确病因方面,而治疗还局限在单纯的药物注射和病灶手术切除,无法从根本上彻底治愈,治疗后常复发,尤其瘢痕疙瘩手术后增生更明显。临床上迫切需要一种可以在创伤愈合瘢痕形成早期抑制瘢痕成纤维细胞增殖的方法。细胞周期调控细胞增殖凋亡的机制为我们在瘢痕成纤维细胞分裂增殖层面抑制细胞增殖提供了一条新的思路。随着基因工程技术的出现和迅猛发展,基因治疗将成为今后治疗病理性瘢痕的理想方法。已有学者开始尝试应用反义核酸技术,以序列特异性方式抑制促瘢痕形成因子基因表达,或者是向增生性瘢痕的成纤维细胞内转入抑制基因,以增加胶原的降解,但是效果均不理想。RNA干扰技术(RNA interfering,RNAi)的出现,进一步为基因治疗病理性瘢痕提供了广阔的空间。RNAi是指将外源双链RNA(double-strandedRNA)导入细胞后引起与其序列同源的特异基因mRNA,降解的现象,属于转录后基因沉默(postt ranscriptional gene silencing,PTGS)的一种形式。最早由Fire等在进行线虫基因功能研究中所提出,后被证实RNA干扰现象是自然产生的保护基因组的机制,普遍存在于动植物体中。经过大量研究学者提出一个在哺乳动物细胞内RNAi作用的模式:外源或内源产生的dsRNA,进入细胞后在核酸酶(Dicer)作用下被切割成3′端有2个碱基突出的21～23nt长度的小分子干扰RNA(small interfering RNAs,siRNAs),这些siRNA与RNAi特异性酶结合,形成RNA诱导沉默复合体(RNA-induced silencing complex,RISC),而后RISC在siRNA反义链的指导下与siRNA同源靶RNA相结合,并在近中点位置将其切割,达到抑制靶基因表达的作用。切割后产物经过聚合酶链式反应再次生成新的dsRNA继续作用于靶mRNA,最终完全降解靶mRNA。因此少量dsRNA就可以完成显著的干扰效应。但是,实验发现大于30bp的dsRNA导入后会启动细胞内的病毒防御机制,细胞内的干扰素合成增加,同时激活蛋白激酶K(PKR)和2′5′-寡腺苷酸合成酶(2′5′-AS)。活化的PKR磷酸化翻译启始因子eIF2α使其活性降低致使细胞内全部蛋白合成关闭。经激活的2′5′-AS合成2′5′-寡腺苷酸则可活化一种核糖核酸酶L(RNase L)降解非特异性mRNA。结果是非特异性的和全面的抑制基因表达,最终导致细胞凋亡。该因素在一短时间内限制了RNAi的发展。随着siRNA可以以各种方式得到而不必通过dsRNA并在体内和体外应用,使siRNA可以高特异性和高效率的阻止或抑制靶基因的表达。此后RNAi在医学方面探索和应用逐步成为研究的热点。
成功地应用RNAi技术敲除疾病相关基因表明该技术开启了新的治疗途径大门。RNAi目前已经在广泛的领域包括医学领域里取得了令人瞩目的进展,已经广泛应用于医疗各中疾病尤其是肿瘤相关方面的治疗。虽然有整形外科学者开始应用RNAi技术特异性抑制瘢痕形成的相关因子基因的表达,但是应用体外合成siRNA对瘢痕疙瘩成纤维细胞CyclinD1表达抑制的研究国内外尚未见报道。由此我们设想,如果病理性瘢痕成纤维细胞中CyclinD1存在着高表达,并因此而造成了细胞早期的异常增殖,那么应用RNAi技术阻止或抑制CyclinD1基因的表达,减少CyclinD1蛋白合成,从而抑制成纤维细胞的细胞周期早期增殖,就有可能达到治疗瘢痕增生的目的。因此,我们针对人CyclinD1基因设计并合成相应siRNA分子,转染瘢痕疙瘩成纤维细胞下调CyclinD1mRNA,抑制CyclinD1的有效合成,同时应用多种方法观测成纤维细胞异常增殖的抑制效果,为病理性瘢痕的RNAi治疗研究提供初步的理论依据,也希望能为临床进一步开展病理性瘢痕RNAi治疗提供新的思路。
二、材料和方法
1.研究对象
标本均取自南方医院整形外科手术患者。增生性瘢痕标本77例,其中男56例,女21例,年龄4岁～45岁,瘢痕增生时间40d～5a;瘢痕疙瘩标本10例,其中男6例,女4例,年龄18岁～46岁。24例正常皮肤(Normal skin,NS)均取自上述患者供皮区,为手术取皮修剪后残余皮片。患者没有长时间外用各种防治瘢痕药物史,不伴有肿瘤及其它严重疾病。取材前向患者说明取材目的,并征得患者同意。瘢痕疙瘩标本病变部位分别为耳垂、三角肌区及前胸。直视下区分各标本中央部和周边部,瘢痕组织中央较平且色淡区为中央部,周围明显隆起呈暗红色区域为周边部,均经临床及病理诊断证实。正常皮肤均取自上述患者供皮区,为手术取皮修剪后残余皮片。增生性瘢痕标本按瘢痕增生时间分为5组:1、1月～3月;2、4月～6月;3、7月～9月;4、10月～12月;5、12月以上。瘢痕疙瘩标本按中间2/3和周围1/3部分分为中央部和周边部2组。以NS组为对照。以PBS代替一抗作为阴性对照。
2.方法
(1)免疫组化染色检测CyclinD1
应用免疫组织化学SP法检测CyclinD1在增生性瘢痕不同病理时期和瘢痕疙瘩不同部位成纤维细胞中的表达差异。
(2) siRNA-cyclinD1分子的设计与合成
应用ambion公司在线软件siRNA target finder设计siRNA-cyclinD1分子,其正义链为5'-CAAACAGAUCAUCCGCAAAtt,反义链为5'-UUUGCGGAUGAUCUGUUUGtt。siRNA-cyclinD1分子干扰Cyclin D1基因的第664～684位核苷酸,靶序列为AACAAACAGATCATCCGCAAA。采用化学法分别合成siRNA-cyclinD1分子的正义链和反义链,经过变性退火处理后得到双链siRNA-cyclinD1分子。
(3)实验分组及转染siRNA-cyclinD1分子。
转染实验分2组:无关siRNA转染组,siRNA-cyclinD1转染组。设立未处理细胞为对照组。每组实验重复3次。
(4) RT-PCR法检测CyclinD1基因表达
成纤维细胞经siRNA-cyclinD1转染24h、48h、72h后分别提取细胞总RNA,然后进行逆转录反应,检测CyclinD1基因的mRNA相对表达量的改变。
(5)双标记流式细胞术分析细胞凋亡
成纤维细胞经siRNA-cyclinD1转染24h、48h、72h后,分别收集10~6个细胞进行流式细胞术检测,经Cellquest软件分析4个区域(UL,UR,LL,LR)内的细胞百分数。UR和LR区域的细胞百分数代表了凋亡细胞百分比。
(6)细胞凋亡DNA片段检测
成纤维细胞转染24h、48h、72h后,分别收集siRNA-cyclinD1转染组(实验组)和无关siRNA转染组(对照组)细胞各1×10~5个,提取DNA后进行1.2%琼脂糖凝胶电泳。
(7) MTT法检测成纤维细胞活性
siRNA-cyclinD1转染成纤维细胞24h,48h,72h后,MTT法检测成纤维细胞活性,以转染无关siRNA的细胞为对照组。计算各个时间点细胞增殖抑制率,计算公式为:细胞增殖抑制率=(1—转染siRNA-cyclinD1后的A570值/对照组细胞A570值)×100%。各时间点实验重复3次。
(8)数据处理
数据分析采用SPSS12.0统计软件,进行非参数检验——多组独立样本秩和检验(Kruskal—Wallis H检验)、单因素方差分析和以及LSD法进行组间多重比较。
三、结果
(1)增生性瘢痕不同时期成纤维细胞CyclinD1的表达
在增生性瘢痕早期,CyclinD1阳性表达的成纤维细胞较多,中晚期阳性表达的成纤维细胞明显减少,部分标本成纤维细胞中CyclinD1表达阴性。表达随增生性瘢痕的发展明显呈现由强至弱的变化。统计学处理显示:不同时期增生瘢痕和正常皮肤成纤维细胞中CyclinD1的表达差别有统计学意义(X~2=68.53,P＜0.001)。两两比较结果:第四和第五组差异无统计学意义,其他各组两两比较均差异有统计学意义。
(2)瘢痕疙瘩不同部位成纤维细胞CyclinD1的表达
瘢痕疙瘩周边部,CyclinD1阳性表达高,而在中央部,阳性表达明显减少。统计学处理显示:边缘部与NS组比较、边缘部与中央部比较以及中央部与NS组比较,CyclinD1蛋白表达均有显著性差异(P＜0.001)。
(3)转染后成纤维细胞形态的变化:
转染特异性小干扰性RNA的24h后部分成纤维细胞从梭形转变为圆形或椭圆形,并且随着时间增加,圆形或椭圆形细胞所占的比例逐渐增加,而未处理组和转染空脂质体组72h后细胞形态基本相同。
(4)转染siRNA-cyclinD1后对CyclinD1基因mRNA水平的影响
成纤维细胞转染50nMsiRNA-cyclinD1后,成纤维细胞的CyclinD1基因相对表达量在24h～72h内逐渐减少。
(5) siRNA-cyclinD1转染后对成纤维细胞周期的影响
成纤维细胞转染特异性siRNA-cyclinD1分子24h、48h、72h后,重复3次实验并采用SPSS12.0软件对结果进行单因素方差分析显示:(1)siRNA-cyclinD1转染影响各组细胞的G0/G1期(F=22.18,P＜0.001);(2)siRNA-cyclinD1转染影响各组细胞的S期(F=29.88,P＜0.001);(3)siRNA-cyclinD1转染不影响各组细胞的G2/M期(F=2.77,P=0.11)。组间两两比较结果显示:G0/G1期细胞平均百分率分别为(60.38±1.77)%、(64.94±1.62)%、(65.18±2.20)%,显著高于无关siRNA转染72h后的对照组细胞(54.45±1.77)%(P值分别＜0.01,＜0.001,＜0.001);S期细胞平均百分率分别为(18.15±1.28)%、(17.28±0.89)%、(11.54±1.56)%,显著低于对照组(22.17±1.68)%(P值分别为0.019,0.007,0.001);G2/M期细胞平均百分率分别为(21.47±3.04)%、(17.78±2.27)%、(23.28±1.83)%,与对照组(22.17±1.68)%无显著差别(P值分别为0.73,0.09,1.0)。
(6) siRNA-cyclinD1转染成纤维细胞后诱导细胞凋亡情况
特异性siRNA-cyclinD1分子转染成纤维细胞24h、48h、72h后,重复3次实验得到的细胞周期结果并进行单因素方差分析,结果:siRNA-cyclinD1转染影响各组细胞的凋亡率(F=458.77,P＜0.001)。组间多重比较(LSD法)结果为:siRNA-cyclinD1转染成纤维细胞24h、48h、72h后,细胞平均凋亡率分别为(7.82±0.45)%、(16.07±1.58)%、(18.49±0.82)%,均显著高于对照组(0.68±0.12)%,差异均有统计学意义(P值均＜0.001)。
(7) siRNA-cyclinD1转染后细胞DNA片段化
成纤维细胞转染特异性siRNA-cyclinD1后,24h内未见DNA小片段,48h～72h内出现大小介于100～400bp之间的DNA小片段;而转染无关siRNA的对照组细胞72h内未出现DNA小片段。
(8) siRNA-cyclinD1转染后成纤维细胞后的细胞增殖情况
成纤维细胞转染siRNA-cyclinD1后的24h、48h、72h,应用MTT法测定细胞生长情况(A570),重复3次实验并采用SPSS12.0软件进行单因素方差分析,结果为:siRNA-cyclinD1转染24、48、72h各个时间点内均影响各组细胞增殖(P值均＜0.001)。同一时间点内进行组间多重比较(LSD法),结果为:(1)与无关siRNA转染组相比,siRNA-cyclinD1转染组细胞生长受到抑制,差异有统计学意义(P＜0.001);(2)与未处理组相比,siRNA-cyclinD1转染组细胞生长受到抑制,差异有统计学意义(P＜0.001);(3)与未处理组相比,无关siRNA转染组细胞生长差异无统计学意义(P=0.854)。根据公式:细胞增殖抑制率=(1—转染后的A570/未处理细胞A570)×100%,计算siRNA转染后的细胞生长抑制率,分别为18%、37%和50%,与无关siRNA转染组相比呈逐步上升趋势。
四、结论
1.CyclinD1在病理性瘢痕的形成中起着重要作用,通过缩短G1期进程导致成纤维细胞异常增殖而促进了病理性瘢痕的形成。
2.CyclinD1在增生性瘢痕早期成纤维细胞中的高表达与增生性瘢痕早期增生旺盛密切相关。
3.CyclinD1在瘢痕疙瘩周边部成纤维细胞中的高表达,不仅促进了瘢痕疙瘩的形成,并可能是瘢痕疙瘩侵犯局部正常组织,浸润性生长的重要原因之一。
4.siRNA-cyclinD1分子对靶基因CyclinD1的抑制效果明显,靶基因mRNA量明显降低,从而使CyclinD1蛋白表达量下降,成纤维细胞细胞从G1期进入S期受到明显抑制而停留于G1期,随时间推移细胞发生凋亡,成纤维细胞数目减少。
5.本研究证实siRNA-cyclinD1分子能够干扰CyclinD1基因表达,进而抑制瘢痕疙瘩早期异常增殖。
Ⅰ.Background and aim
Hypertrophic scar(HS) and keloid(K) are common pathological scars.Usually believed to be caused with wounds,they are proliferative diseases of dermatofibroma characterized in abnormal proliferation of fibroblast,overproduction and deposit of such extracellular matrix(ECM) as collagen with high incidence.Keloid,in particular,has such characteristics as producing too much,proceeding beyond the boundary of wounds to encroach on tissues nearby,never degenerating and being easy to recur after pure surgery.It not only can cause dysmorphia and functional disturbance,but also has influence on the outlook.What is more,it often goes with evident pruritus and pains.Therefore,it brings great harzards to the physical and mental health of the patient both in functions and outlook.However,we have not found out completely the pathogensy of this pathologican scar,or effective methods for its treatment,though a great deal of research has done on its cause.
Scholars at home and abroad have conducted some series of research on the causes of pathological scars from the point of view of histology,pathology, physiology,immunology and genetics,and great achievements have been made.The real causes of the disease still remain unclear.There is one point,however,that lots of experts have come into conformity,that is,the factors in vivo and vitro all contribute to the too much proliferation of fibroblast of the pathological scars,and the over deposit of collagan,which is typical of all pathological scars,including keloid.It is the abnormal proliferation of fibroblast that brings about the series of manifestations of the scars clinically.Thus,phologican scars may be treated if proliferation of their fibroblast can be inhibited with whatever means during the process of wounds healing,or something is done to enhance the apoptosis of reduplicated cells to reduce the over-deposit of collogan.It is therefore the precondition of this research to find out the mechanism to regulate the apoptosis of fibroblast proliferation of pathological scars,particularly the key factors regulating abnormal proliferation of cells at early stage,as the regulation of proliferation and differentiation is closely related with cell cycles,and the regulatory core in cyclnes determines the final terminal of cell growth.
That the establishment of the concept of cell cycle and the breakthrough in the research on it in recent years have laid down a foundation for the study of cell proliferation,differentiation and apoptosis in modern times.The discovery of Cyclin, CDK and CDI has certainly revealed the mechanism of such movement.The organic combination of the three brings about normal division of cells,becoming the core controlling cell cycle.The outcome:from the research on the core regulating cell cycle provides a new theoratical base for people to explore the mechanism of the abnormal cell proliferation and differentiation.Studies show that division of cell in vivo is affected by such factors as time and environment.Dissociation of normal cells is restricted by one another that coordinates closely to construct normal cell groups in order.Division would cause pathological change if it proceeds continuously without control or constraint.Therefore,if we find the CDK,Cyclin and CKI in the process of cell cycle,we would get the the way to control continuity of abnormal cell division.
Priming at G1 is a critical step in the cell cycle.It is particularly important, therefore,to regulate cyclins at G1/S.As one of normal major regulating factors, CyclinD1 mainly regulates cyclin at G1 to enter Phase S.It is the key factor for regulation at early stage to accelerate cell cycle.Its over expression,however,will bring about abnormal cell proliferation.Therefore,high expression of cyclinD1 due to various causes is one of the main reasons that makes cells grow too fast.Research on many a tumors has found that the occurrence and development of tumors are closely related to abnormality of the composition of G1/S at the regulating point.The unique growth characteristics and clinical manifestation of keloid shows have been taken by many scholars as a kind of innocent tumor developing after a wound heals.Fibroblasts are functional cells for keloid to develop,and their disequilibrium inin proliferation and apoptosis is the cytological base for keloids to grow continuously and be hard to become vestigial.There was research showing that cyclinD1 wass in a state of high expression in fibroblasts.This implies that keloids may be caused by the regulating factors working on cell cycles,for they increase synthesis of cyclinD1 and speed up cell cycles to cause confusion of functional cells in proliferation and apoptosis.Therefore,we have collected typical samples of hyperplastic scars and keloids to observe how cyclinD1 was expressed in fibroblasts.The differences in expression at the edge of keloids and in the center are of particular importance in the study of the clinical manifestation of keloids and in guidance in the treatment.
Presently a lot of research on pathological scars focus mainly on the cause,and treatment is limited to injection of medicine and surgical removal of focus of infection,which cannot thoroughly cure the diease,which may appear again later, particularly the scar fibroblast proliferation.There is an urgent need clinically for one method to inhibit the proliferation after the heeling of wounds but before the formation of scars.The mechanism to regulate the apoptosis of proliferation at cyclin provides a new way for us to inhibit proliferation at the level when scar fibroblasts break.With the emergence and rapid development of genetic engineering,gene therapy will become an ideal way to treat pathological scars.Some scholars have tried employing antisensenecleic acid technology to inhibit the gene expression of factors bringing about the formation of scars with sequence specificness,or transferring inhibiting genes into the fibroblasts of hyperplastic scars to build up collagenous degradation.This,however,does not seem to work well.The appearance of RNA interferring(RNAi) further provides vast space for treating genetically pathological scars.RNAi refers the phenomenon of degradation of the specific gene mRNA brought about by introducing exogenous double-stranded RNA.They are the congenerous in sequence,a sort of post-transcriptional gene silencing(PTGS),first put forward by Fire in I the research on the gene functions of edlworms,and then was confirmed that RNA interferring was the natural mechanism protecting gene groups, found commonly inside animals and plants.After a great deal of research,some scholars proposed a mode for RNAi to work in mammalian cells:exogenous or endoneous dsRNA was cut into small interferring RNAs(siRNA) with two basic group protrutions at 3' under the effect of Dicer after entering cells.The siRNA is combined with the specific enzyme of RNAi to develop RNA-induced silencing complex(RISC) which is further combined with RNA the exogenous target of siRNA under the instruction of siRNA antisense strand.They are cut at the near center to inhibit the expression of target genes.Through polymerase chain reaction the cut materials will develop into new dsRNA to work continuouly on mRNA until finally degrading target mRNA completely.Therefore,a small amount of dsRNA can complete the effect of remarkable interference.However,experiments have found that dsRNA introduced would initiate the virus protective mechanism in cells when it was larger than 30bp,which increased the synthesis of interferon in cells and activate simultaneouly the protein kinase K(PKR) and 2'5' adenyl acid synzyme(-AS).The activated PKR phosphorylation translates the initial factors eIF2αto decrease its activity so that the synthesis of protein in cells is closed completely.The activated 2'3'(-AS) is can activate a ribonuclease(RNase L) to degrade the non-specific mRNA,resulting in the non-specific and entire inhibition the expression of genes that causes apoptosis.This factor limits the development of RNAi in a short time.Now when it is easy to get it withouth dsRNA and employ it in and out of body,siRNA can specifically and effectively stop or inhibit the expression of target genes.RNAi will certainly become a hot point of research in medical field.
Surprising development has been made in the research on RNAi in extensive fields including medicine and it has been applied in widely in treating various diseases,particularly tumors.Although some scholars in plastics have-started to inhibit the expression of genes related to the formation of scars with the specifiness of RNAi technology,but there was no report about Apoptosis of human keloid fibroblast induced by small interfering RNA-mediated cyclinD1 gene silencing.We thus suppose that if the high expression of cyclinD1 in the fibroblasts of pathological scars causes the abnormal cell proliferation at early stage,we may knock out its synthesis of mRNA with RNAi technology to cut down the synthesis of CyclinD1 so as to inhibit the proliferation of fibroblasts at the early state of cell cycle.This may help us treat scar proliferation,which will provide new theoractical evidence for further research on the treatment of pathological scars.
Ⅱ.Materials and methods
1.Subjects
The Samples came from patients eceiving plastic surgery in Nanfang Hospital.These included 77 cases of hyperplastic scar,with 56 males and 21 females aged between 4 to 45 years old and the periods of scar hyperplasy ranging 40d～5a; there were 10 samples of keloid,among which 6 were males and 4 females aged between 18～46 years old.There were 24 cases of normal skin from the patients above.These were skin residues left after the surgery.The patients did not have a history of taking medication to prevent scars for long,nor did they suffer from tumors or other severe diseases.The patients were told about the purpose before the sampling and they agreed to it.The diseased parts with keloid sampled included the ear lobe, the deltoid region and the protothorax.The lower orthophoria part was divided into central and ambitus areas,both confirmed clinically and with pathologic diagnosis.The normal skins,taken from the skin-supply areas of the patients above, were residuces left after the surgery.The samples of hyperplastic scars were divided into five groups according to the time hyperplasy occurred:1.Those occurred at Months 1 to 3;2.those at Months 4 to 6;3.those at Months 7 to 9;4.those at month 10 to 12 and 5.those appeared 12 months after the surgery.Samples of keloid were divided into two groups:the central group with 2/3 of the center and the edge group with 1/3 of the edge.Those in the NS group were taken as the control group,and PBS as negative the control group.
2.Methods
(1) Detecting cyclinD 1 with immunohistochemistry
Detect with SP method of immunohistochemistry the expression difference of CyclinD1 in different pathological phase of hyperplastic scar and fibroblast in different area of keloid.
(2) siRNA design and synthesis for CyclinD1
With siRNA target finder,the softwear online by Ambion,we designed s ome siRNA-cyclinD1 molecues whose positive-sense strand being 5'-CAAACA GAUCAUCCGCAAAtt and antisense strand 5'-UUUGCGGAUGAUCUGUUUGt t.The siRNA-cyclinD1 molecues interfered nucleotides of Cyclin D1 genes pos itioning at No.664-684,and the target serial was AACAAACAGATCATCCG CAAA.Then with chemical methods,we had the positive-sense and anti-sense s-trands of siRNA-cyclinD1 molecues synthesized respectively(entrusted to Sh anghai Jikai Genes Co.).They became double-stranded siRNA-cyclinD1 mole cues after being treated with denaturalized anneal.
(3) laboratory groups and transfected siRNA-cyclinD1 molecues
There were two groups for the transfection laboratory:blank control group in which no transfection was related to siRNA,and siRNA group spectific to Cyclin in which transfected Smad2 specific to siRNA.Tests were repeated three times in each group.
(4) Detecting CyclinD1 gene expression with RT-PCR
Gross RNA of cells were taken when the fibroblasts were transfected with siRNA-cyclinD1 for 24,48 and 72 hours and reverse transcription reaction was conducted for the observation of the change of the relative expression amount in mRNA in CyclinD1.
(5) Analyzing apoptosis with double tagged flow cytometry
10~6 cells after transfected siRNA-cyclinD1 molecues were collected to be detected with flow cytometry and the percent of cells in four areas(UL,UR,LL and LR) were analyzed with Cellquest softwear.The percent of cells in UR and LR standed for the percentage of apoptosed cells.
(6) Detecting DNA fragment of apoptosis
10~6 cells were collected respectively from the transfection siRNA-CyclinD1 group(the experimental group) and the siRNA group without transfection(the control group),and 10μl of DNA were colbcted from each for 1.2%agarose gel electophoresis.
(7) Detecting the activity of fiborblast with MTT
The activity of fibroblasts was detected with MTT when siRNA-cyclinD1 was transfected into fibroblasts for 24,48 and 72 hours,with the cells in siRNA without transfection in the control group.The rate of cell proliferation inhibited at different time point was worked out.The formulas was the rate of cell proliferation inhibited= (1—A570 value after transfected siPNA-cyclinD1/A750 value of cells in the control group)×100%.The tests were repeated three times at each time point.
(8) Data processing
Analysis on the data was done with SPSS12.0 for Kruskal-Wallis H test),and comparisons among groups were made for mono-factor variance with LSD method.
Ⅲ.Results
(1) The expression of CyclinD1 in fibroblast at different stage of hyperplastic scar
At the early stage of hyperplastic scar,more fibroblasts showed positive expression with CyclinD1,while fewer fibroblasts showed positive expression at middle and later stages,and in some samples the CyclinD1 had negative expression in the fibroblasts.The expression turned from being strong to weak with the development of the scars.Statistical analysis showed great difference in the expression of CyclinD1 protein between Group 1 and Group NS;between Group 2 and Group NS,and between Groups 1 and 5(X~2=68.53,p＜0.001);while comparisons in pairs in other groups showed no statistic significance.(P＞0.05)
(2) The expression of CyclinD1 in fibroblast at different parts of keloid
At the ambitus of a keloid,CyclinD1 showed high positive expression,while at the center,positive expression dropped greatly.Statistical treatment showed marked difference in the expression of Cyclin D1 protein between the ambitus and Group NS (P＜0.001),between the ambitus and the center(P＜0.001),and between the center and Group NS(P＜0.001).
(3) The change in form for fibroblast after being transfected
When siRNAwas transfected for 24 hours,some fibroblasts turned from fusiform into spherical or oval shape,and with time proceeding,the percentage of round or oval cells increased gradually.The cells in the untreated group and the hollow transfected bangosome group remained basically in the same shapes.
(4) The influence of transfected siRNA-cyclinD1 on the level of mRNA the CyclinD gene
When the fibroblasts were transfected with 50nM siRNA-cyclinD1,the relative expression amount of the CyclinD gene in the fibroblasts gradually became less within 24～72 hours.
(5) The influence oftransfected siRNA-cyclinD1 on the cycle of fibroblasts
When the fibroblasts were transfetected with specific siRNA-cyclinD1 molecules for 24,48 and 72 hours,the experiment was repeated for three times before the results were analyzed with SPSS12.0 in mon-factor variance,showing that transfection of siRNA-cyclinD1(1) had influence on cells in the groups at G0/G1 (F=22.18,P＜0.001);(2) affected the cells at Phase S(F=29.88,P＜0.001); and(3) did not have any influence on the cells at Phase G2/M(F=2.77,p＜0.001).Comaprisons made between pairs of groups showed that the average percentage of cells at Phase G0/G1 were(60.38±1.77)%,(64.94±1.62)%and (65.18±2.20)%respectively,much higher than those in the control group without siRNA after 72 hours(54.45±1.77%)(P＜0.01,＜0.001,＜0.001);the average percentages of cells at Phase S were(18.15±1.28)%,(17.28±0.89)%and (11.54±1.56)%,much lower than that(22.17±1.68)%(P values being 0.019, 0.007 and 0.001 respectively) in the control group;and the average percentages at Phase G2/M were(21.47±3.04)%,(17.78±2.27)%and(23.28±1.83)% respectively,showing no difference with that(22.17±1.68)%(P values being 0.73, 0.09,1.0 respectively).
(6) Apoptosis induced after siRNA-cyclinD1 was transfected into fibroblasts
When specific siRNA-cyclinD1 molecues were transfected into fibroblasts for 24,48 and 72 hours,and the experiment was repeated for three times before the results of cyclines were analyzed in mono-factor vairance with LSD method among groups that showed that transfection with siRNA-cyclinD1 had influence on the apoptosis rate(F=458.77,P＜0.01).Comparisons between pairs of groups indicated that when siRNA-cyclinD1 was transfected into fibroblasts for 24,48 and 72 hours,the average apoptosis rates were(7.82±0.45)),(16.07±1.58)%and (18.49±0.82)%respectively,much higher than that(0.68±0.12)%in the control group,the differences were all of statistic significance(the P values being＜0.001).
(7) Gragmentation of DNA when siRNA-cyclinD1 was transfected
When the fibroblasts were transfected with siRNA-cyclinD1,no fragmentswere found in 24 hours,but in 48～72 hours fragments as large as 100～400bp appeared; No fragments were found in the cells in the control group in 72 hours.
(8) The proliferation of fibroblasts after siRNA-cyclinD1 was transfected
24,48 and 72 hours after siRNA-cyclinD1 were transfected into fibroblasts,the growth of the cells were determined with MTT rules(A570).The experiment was repeated three times and analysis was made in multi-factor variance with SPSS12.0 with the results showing that transfection of siRNA-cyclinD1 had influence on the proliferation in each group(p＜0.001).Comparisons among groups with LSD method at the same time point made showed that(1) cell growth in the siRNA-cyclinD1 transfected group was inhibited in contrast with that in the group without siRNA transfection,the difference being statistically significant(p＜0.001); (2) cell growth in the siRNA-cyclinD1 transfected group was inhibited in contrast with that in the group without any treatment,the difference being statistically significant(p＜0.001);and(3) the cell growth in the group without siRNA transfection indicated no statistical significance(p=0.854).The cell proliferation inhibiting rates after siRNA was transfected was worked out in accordance with the formular:cell growth inhibiting rate=(1—transfected A570/untreated cells A570)×100%,and we got 18%,37%and 50%,tending to increase gradually,with the difference showing statistically significance(p＜0.001) compared with that in the group with siRNA transfection.
Ⅳ.Conclusion
1.CyclinD1 played an important role in the formation of pathological scars.Shortening proceeding of Phase G1 resulted in abnormal proliferation of fibroblasts that encouraged the formation of pathological scars.
2.The high expression of CyclinD1 in fibroblasts at early stage of hyperplastic scars was closely related to the prosperous hyperplasy of scars at early stage.
3.The high expression of CyclinD1 in fibroblasts at the edge of keloid not only promoted the their formation,but also probably one of the reasons for keloid to offent normal tissues locally and grow infiltratively.
4.siRNA-cyclinD1 molecues could effectively inhibit CyclinD1,the target genes, and the amount of mRNA dropped greatly to reduce the expression of CyclinD1.The cell cycline of fibroblasts was restricted when it moved from G1 to Phase S and had to stop at G1,and cell tended to apoptose with time going and cell proliferation was inhibited.
5.This research confirms that siRNA-cyclinD1 can interfere the cell expression of CyclinD1 and then inhibit the abnormal hyperplasy of keloid at early stage.
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