本文關(guān)鍵詞：創(chuàng)傷后瘢痕形成機制及治療的研究，由筆耕文化傳播整理發(fā)布。

        研究背景病理性瘢痕是創(chuàng)傷愈合后最常見的并發(fā)癥之一,一直是外科修復(fù)領(lǐng)域研究的難點與熱點。病理性瘢痕不但可以影響患者的容貌,而且產(chǎn)生的攣縮可導(dǎo)致不同程度的功能障礙,為該類患者后期治療帶來沉重經(jīng)濟和心理負擔(dān)。其形成機制及防治一直是醫(yī)學(xué)界研究的熱點。目前其形成機制仍不清楚,尚無療效確切的治療方法。多年來,眾多學(xué)者從不同角度探討增生性瘢痕的形成、消退機理,期望從中找到治療瘢痕的突破口。目前較一致的看法是：①增生性瘢痕以細胞過度增殖和細胞外基質(zhì)過度沉積為特征,其中成纖維細胞是主要的效應(yīng)細胞；②增生性瘢痕在生物學(xué)層面主要表現(xiàn)為膠原代謝紊亂；③TGF-β1/smad信號通路與成纖維細胞的增殖、分化、遷徙、凋亡及膠原代謝等多種生理、病理過程密切相關(guān)。成纖維細胞作為創(chuàng)傷愈合過程中的主要效應(yīng)細胞,常常表現(xiàn)為過度增殖和功能活躍,從而引起一系列級聯(lián)反應(yīng),造成創(chuàng)面修復(fù)失控,導(dǎo)致細胞外間質(zhì)的過多沉積和創(chuàng)傷的過度愈合,形成病理性瘢痕。由此可見,成纖維細胞的功能異常在病理性瘢痕過度增生中扮演著重要的角色。Lekic等認為它是創(chuàng)傷修復(fù)的工程師、建筑者和管理員。自Convey等于1959年培養(yǎng)瘢痕成纖維細胞獲得成功以來,開始了體外研究瘢痕的新階段,為從細胞水平上認識瘢痕異常增生的機理提供了有力的證據(jù)和資料。近年來,病理性瘢痕的基礎(chǔ)研究不斷深入,成纖維細胞與瘢痕的關(guān)系以及瘢痕增生和侵襲性生長機制已成為醫(yī)學(xué)研究的熱點。但是,瘢痕成纖維細胞的原代培養(yǎng)對于初學(xué)者仍較為困難,失敗率較高。因此,建立高效的培養(yǎng)方法及對瘢痕成纖維細胞進行鑒定及活力測定顯得尤為重要。目前普遍的觀點認為,增生性瘢痕的形成在生物學(xué)層面主要表現(xiàn)為膠原代謝紊亂。較多的研究表明在瘢痕增生過程中,膠原蛋白的大量合成和Ⅰ、Ⅲ型膠原纖維比例發(fā)生變化是形成增生性瘢痕的重要機制。膠原代謝紊亂與MMPs、 TIMPs間的動態(tài)平衡以及TGF-p/smad信號通路的異常具有密切的關(guān)系。但是,目前關(guān)于膠原在增生性瘢痕組織中的變化及其調(diào)控機制仍不清晰,需進一步澄清。MMPs(?)(?)TIMPs(?)司的動態(tài)平衡是影響膠原代謝的重要因素。但是在創(chuàng)傷后增生性瘢痕組織中,膠原酶的變化說法不一。Ghahary等報道在體外培養(yǎng)的人增生性瘢痕成纖維細胞間質(zhì)膠原酶nRNA表達水平明顯低于正常皮膚成纖維細胞,而兩者的金屬蛋白酶組織抑制因子表達無顯著差異。國內(nèi)趙燁德等研究證實MMP-3mRNA在HS組FB中表達水平低于正常組,而且降解DNP-多肽(膠原酶底物)能力也明顯降低；同時將MMP-3基因轉(zhuǎn)入HS成纖維細胞后,提高了MMP-3表達水平,促進了ECM降解,得出了類似的結(jié)果。同樣,在對皮膚異常瘢痕的研究中發(fā)現(xiàn),異常瘢痕TIMP-1的表達明顯高于正常組織。在肝和腎的纖維化病變中也發(fā)現(xiàn)TIMP-1表達增加,可通過抑制MMPS的活性阻止過量的膠原降解。另外也存在較多與上述結(jié)果相反的報道。探索瘢痕組織內(nèi)MMPs(?)(?)TIMPs的變化規(guī)律及其影響因素,可能為瘢痕形成機制提供線索。SMADs介導(dǎo)的轉(zhuǎn)化生長因子信號通路在增生性瘢痕膠原代謝中發(fā)揮著重要的作用。SMADs是信號從受體到核內(nèi)的細胞內(nèi)主要轉(zhuǎn)導(dǎo)分子,也是目前瘢痕增生研究最多、最為重要的信號分子。根據(jù)不同分型,對成纖維細胞具有雙向調(diào)控作用。目前大量研究已經(jīng)證實,其中Smad2/3是具有促進作用的因子,而SAMD7是抑制性的中間作用分子。但是,由于分子內(nèi)部各種信號通路的相互作用,對于Smad2/3和SAMD7的變化規(guī)律及其影響因素仍是研究的重點。雖然大量的研究工作針對HS的發(fā)病機制和治療,并且取得了豐碩的成果,但是對于增生性瘢痕的治療目前仍缺乏有效的方案。外科手術(shù)切除是增生性瘢痕最常用的治療。盡管各方面的改進,如：“z”或“w”成形術(shù)局部減張,切除瘢痕的纖維部分,保留外層皮膚。但是,手術(shù)仍可能因愈合失控或再次創(chuàng)傷而導(dǎo)致瘢痕過量形成。本組因功能障礙及影響美觀,均采用手術(shù)的方法進行治療。手術(shù)標本用作本實驗。大量的研究表明,燈盞花素可抑制纖維化病變進展,燈盞花素是否能抑制病理性瘢痕成纖維細胞的增生,尚未見文獻報道。也有研究認為細胞凋亡與HS的形成、消退過程密切相關(guān),通過調(diào)控成纖維細胞凋亡有望成為有效防治HS的新亮點。本實驗通過建立高效的瘢痕成纖維細胞原代培養(yǎng)方法,觀察其生長規(guī)律,并對其進行生物學(xué)鑒定,首次探討燈盞花素對瘢痕成纖維細胞增殖和凋亡的影響,希望尋找到有效治療增生性瘢痕的藥物。同時對瘢痕組織真皮層內(nèi)影響膠原纖維代謝相關(guān)分子進行研究,挖掘其中重要分子的變化規(guī)律；為增生性瘢痕機制的闡明及治療提供思路。研究目的1、尋求一種高效的瘢痕成纖維細胞體外原代培養(yǎng)方法。2、了解瘢痕成纖維細胞生長過程中的形態(tài)學(xué)變化和鑒定方法。3、了解病理性瘢痕組織形態(tài)學(xué)、分子生物學(xué)變化規(guī)律,更加深入地了解增生性瘢痕的形成機制。4、從細胞學(xué)方面初步探討燈盞花素對瘢痕成纖維細胞生長增殖及凋亡的影響,為尋找有效的天然藥物提供理論依據(jù)。研究方法1、標本收集、收集標本均來自來自廣東省第二人民醫(yī)院和廣州市紅十字會醫(yī)院2011-06～2011-12創(chuàng)傷性瘢痕手術(shù)患者標本24例,年齡18～56歲。標本的獲取均征得患者的知情同意,并簽訂知情同意書。標本入選和排除標準：A、經(jīng)臨床醫(yī)生鑒定為瘢痕組織,并初步進行分類(增生性、瘢痕疙瘩)；B、排除垂體、腎上腺疾病、傳染病、皮膚病及免疫性疾病患者,排除局部感染、潰瘍患者；C、手術(shù)前未接受針對瘢痕的任何治療。標本的獲取均征得病人的同意,簽知情同意書。2、標本的處理、分組組織切取后冷凍干燥法保存,均于4小時內(nèi)進行實驗。標本分三部分處理：一部分用于細胞原代培養(yǎng),另一部分用10%福爾馬林甲醛溶液固定,用于形態(tài)學(xué)鑒定(如免疫組化等)；第三部分置于液氮中凍存?zhèn)溆?提取蛋白和RNA用于后續(xù)試驗)。選取同期因其他手術(shù)切除的正常皮膚組織3例作為正常對照組進行研究。3、組織微粒法進行瘢痕成纖維細胞原代培養(yǎng),總結(jié)和完善瘢痕成纖維培養(yǎng)的方法,并對建立的細胞系進行命名和記錄相關(guān)資料；觀察瘢痕成纖維細胞生長規(guī)律和生長特點,MTT法繪制細胞生長曲線圖；根據(jù)細胞形態(tài)以及α-SMA抗體的細胞免疫組化對瘢痕成纖維細胞進行鑒定。4、對增生性瘢痕組織進行HE染色和masson三色染色觀察其形態(tài)學(xué)特征,比較正常組織和瘢痕組織的差異性。5、CollagenⅠ, CollagenⅢ的免疫組織化學(xué)染色,分析其在瘢痕組織內(nèi)分布特點和表達量。6、應(yīng)用RT-qPCR技術(shù)檢測病理性瘢痕組織真皮內(nèi)Collagen Ⅰ, CollagenⅢ, MMP-1, TIMP-1, SMAD3, SMAD7基因表達情況,并與正常組織進行比較,分析其差異性；應(yīng)用Western Blot技術(shù)檢測MMP-1、SMAD3、TIMP-1蛋白在瘢痕組織內(nèi)的表達。7、使用MTT法檢測不同濃度組瘢痕成纖維細胞增殖情況,繪制抑制率曲線圖,并進行統(tǒng)計學(xué)分析各組差異性；使用流式細胞儀Annexin V/PI雙染色法檢測不同濃度燈盞花素對增生性瘢痕成纖維細胞凋亡的影響；半定量RT-PCR檢測細胞Collagen Ⅰ和Collagen ⅢmRNA表達。研究結(jié)果1、瘢痕成纖維細胞形態(tài)學(xué)觀察和鑒定(1)病理性瘢痕組織微粒在接種后大約2-7天,少量細胞從組織周圍放射狀或者旋渦狀爬出(圖1-1a、b)。原代成纖維細胞呈梭形或不規(guī)則三角形,胞質(zhì)向外伸出多個長短不同的突起,多呈放射狀、編織狀或漩渦狀排列,有時細胞排列紊亂,有明顯的交叉重疊現(xiàn)象并呈團塊狀。細胞免疫組化α-SMA染色顯示細胞核呈類圓形或長橢圓。(2)病理性瘢痕成纖維細胞傳代培養(yǎng)時,成纖維細胞剛接種尚未貼壁時呈球形(圖1-1c),貼壁后細胞逐漸伸展,呈梭形、長條形、多角形或不規(guī)則形(圖1-1d)。2-3d即呈匯合狀態(tài)(圖1-1e)。細胞密度低時細胞之間排列疏松,有較大細胞間隙(圖1-1e)。細胞密度高時,細胞相互平行排列或呈放射狀和漩渦狀排列(圖1-1f)。隨著細胞代齡的增高,細胞逐步出現(xiàn)老化現(xiàn)象,鏡下觀察可見細胞體積變大、形狀扁平,過度伸展,伸展末端細長有分支,生長緩慢。(3)生長曲線顯示：瘢痕成纖維細胞接種后經(jīng)過短暫的潛伏期,開始增長的趨勢,于6-8天生長進入對數(shù)期,10天以后進入停滯期(圖1-3)。(4) α-SMA細胞免疫組化染色,出現(xiàn)較多陽性染色細胞的結(jié)果。結(jié)合其分離時的組織來源,以及其長梭形、柵欄狀、漩渦狀生長的特點,證實其為成纖維細胞。2、瘢痕組織HE染色觀察結(jié)果：(1)正常皮膚成纖維細胞和微血管數(shù)目較少(圖2-3a)。(2)瘢痕組織內(nèi)可見成纖維細胞和微血管增多,微血管呈縮窄傾向(圖2-3e)。真皮內(nèi)見編織狀排列的大片致密嗜伊紅染同質(zhì)性粗大膠原纖維,排列紊亂,無一定的方向,膠原纖維相互交叉呈漩渦狀或結(jié)節(jié)狀分布,結(jié)節(jié)中的膠原纖維較細,各結(jié)節(jié)之間由粗大的膠原纖維束分隔,粗大的膠原纖維束間可見分布有數(shù)量不等的成纖維細胞和小血管。(3)瘢痕組織(圖2-3b、d)表皮厚度較正常表皮(圖2-3a)明顯增厚。3、Masson三色染色觀察發(fā)現(xiàn)：瘢痕組織真皮層大量膠原纖維,粗細不一,顯示真皮乳頭層及附近血管增生,膠原纖維排列紊亂(圖2-4a)；真皮網(wǎng)狀層膠原纖維排列致密,近表皮處紊亂,近皮下組織處排列規(guī)則,較多血管分布,部分膠原纖維交叉形成膠原結(jié)節(jié),以近乳頭層處明顯(圖2-4b)。4、免疫組化結(jié)果(圖)顯示：瘢痕組織Ⅰ型膠原抗體在真皮層的細胞間質(zhì)中,主要分布于真皮網(wǎng)狀層,較為粗大,交織成結(jié)節(jié)狀,周圍散在分布成纖維細胞和血管壁。Ⅲ型膠原散在分布于真皮層的細胞間質(zhì)中,以真皮乳頭層分布較多,膠原束較細,排列疏松,走行方向不一,互相交織。血管壁上均有兩種纖維的分布。5、RT-qPCR結(jié)果顯示：瘢痕真皮組織和正常皮膚真皮組織內(nèi)均可檢測到collagen Ⅰ、collagen Ⅲ、MMP-1、TIMP-1、SMAD3、SMAD7的基因表達。與正常組織相比,瘢痕組織內(nèi)collagen Ⅰ (t=5.85, P<0.01)、collagenⅢ(t=4.515, P<0.05)、MMP-1(t=-13.22,P<0.01)、TIMP-1(t=2.52,P<0.05),smad7(t=-2.503,P<0.05)基因表達差異具有顯著性；由圖可見collagen Ⅰ、collagenⅢ、TIMP-1表達較正常組織升高,而MMP-1表達較正常組織降低；Smad3(t=-0.339,P>0.05)基因表達未見明顯差異。(圖2-7,表2-1)6、Western Blot顯示：這些樣品中瘢痕真皮組織和正常皮膚真皮組織內(nèi)均可檢測到MMP-1、TIMP-1、SMAD3蛋白表達,但是表達量均低于正常組織。(圖2-9)7、燈盞花素作用前后成纖維細胞形態(tài)未見明顯變化；燈盞花素對體外培養(yǎng)FB生長增殖具有明顯的抑制作用((F=3.309,P<0.01)(表3-1)。與對照組相比,(80、100、150)μmol·mL-1濃度(實驗)組明顯抑制瘢痕成纖維細胞生長(P<0.05)。結(jié)合抑制率曲線可知,燈盞花素對瘢痕成纖維細胞的抑制作用呈劑量依賴性(圖1)。IC50=123.5μmol·mL-1。8、流式細胞儀Annexin V/PI雙染色法檢測FB凋亡結(jié)果顯示：IC50處瘢痕成纖維細胞凋亡率為14.2050±0.95553,未加藥組瘢痕成纖維細胞凋亡率為4.0220±0.31412,兩者相比差異具有統(tǒng)計學(xué)意義(t=-22.653,P<0.05)。：RT-PCR結(jié)果顯示隨著燈盞花素濃度的升高,對瘢痕成纖維細胞collagen Ⅰ和collagen Ⅲ mRNA產(chǎn)生明顯的影響,差異具有統(tǒng)計學(xué)意義。(如圖3-4,表3-1)結(jié)論1、本研究改良了瘢痕成纖維細胞培養(yǎng)方法,減少了培養(yǎng)失敗率,進一步補充了瘢痕成纖維細胞培養(yǎng)相關(guān)理論,為細胞培養(yǎng)提供一種高效方法。2、瘢痕成纖維細胞和正常成纖維細胞形態(tài)上無明顯的區(qū)別,可利用特異性標志α-SMA對其進行鑒別。3、增生性瘢痕組織中collagen Ⅰ和collagen Ⅲ明顯的增加,MMPs表達減少,以及TIMPs表達增高,與創(chuàng)面愈合纖維瘢痕形成關(guān)系密切。4、瘢痕增生過程中是多種信號通道、多因素相互作用的復(fù)雜過程。5、燈盞花素可通過抑制瘢痕成纖維細胞的增殖,并且誘導(dǎo)其發(fā)生凋亡,在瘢痕的治療方面存在較大的應(yīng)用前景,為尋找有效治療增生性瘢痕的藥物提供了新的思路。

    Background:Pathological scar, one of complications after skin trauma and post-operative, has a bad effect on the patient’s appearance and psychological. Its mechanism and prevention has been the focus of the medical profession.There is no clear conclusion on its mechanism, prevention and treatment.For many years, many scholars approach the problem from different perspectives on hyperplastic scar formation and regression,expect to find a breakthrough in the treatment of scar. A more consistent view are that:①hypertrophic scar is characterized by excessive cell proliferation and extracellular matrix, in which fibroblasts are the major effector cells;②The hypertrophic scar is mainly the result of collagen metabolism disorders at the biological level;③TGF-β1/smad signaling pathways are closely related with physiological and pathological procession of fibroblasts, such as proliferation, differentiation, migration, apoptosis and collagen metabolism and so on.Fibroblasts,as the major effector cells in the wound healing process, often shows excessive proliferation and active function, causing a cascade of reactions, out of control in wound repair, excessive deposition of extracellular matrix, the formation of pathological scar. Thus, the abnormal function of fibroblasts plays an important role in pathological scar hyperplasia. Lekic etal. describes fibroblasts as engineers, builders and administrators of wound repair.Since Convey etal. cultured hypertrophic scar fibroblasts in1959,it started a new stage of understanding of the mechanism of scar in vitro and the cellular level. In recent years, with the deepening basic research of pathological scar,relationship of fibroblasts and scar and mechanism of scar formation and invasive growth have become a hot topic of medical research. However, the primary culture of scar fibroblasts is still a more difficult for beginners for a high failure rate.Therefore, the establishment of efficient cultivation methods is particularly necessary for scar fibroblasts.The prevailing view is that The hypertrophic scar is mainly the result of collagen metabolism disorders at the biological level. The excessive synthesis of collagen and collagen proportion change during the scar formation process is the important reason of hypertrophic scar.Collagen metabolism disorder has a close relationship with the dynamic balance of TIMPs and MMPs and TGF-beta signaling pathway. However,the changes of collagen and its regulation in hypertrophic scar is still unknown.The dynamic balance between MMPs and TIMPs is an important factor affecting the metabolism of collagen.But there are different opinions on changes of collagenase.Ghahary et al reported that mRNA expression of MMP-1in human scar fibroblast derived fibroblast collagenase was significantly lower than normal skin fibroblasts, while there was no significant difference in the mRNA expression of TIMP-1.Zhao etal. confirmed that MMP-3mRNA expression level in the HS group was lower than the normal group,which reached the same results.Likely, in the study of abnormal skin scars,the expression of TIMP-1was significantly higher than that of normal tissue. The same thing occurred in the liver and kidney fibrosis.In addition,many opposite conclusions were reported.it is possible to provide clues for the mechanism of scar formation by exploring the changes of MMPs and TIMPs in the scar tissue and its related factors.TGF-β1/smad signaling pathways plays an important role in the collagen metabolism of hypertrophic scar.SMADs is the main transduction molecules from the receptor to the cell nucleus.Different Smad have two-way regulation on fibroblast. Numerous studies have confirmed that Smad2/3is the promoting factor, while SAMD7being the inhibitory role.However, variation and its influencing factors of Smad2/3and SAMD7is also the focus of the study because of the interaction of various signaling pathways.Lots of work have been done for the pathogenesis and treatment of the HS and fruitful results have been achieved, but there still no effective or satisfactory treatment for HS so far. Surgical resection is the most common treatment for hypertrophic scar. Despite the improvements were done, Surgical resection may still be re-trauma healing and lead to excessive scar formation. This group have adopted surgical treatment due to the dysfunction and ugly appearance.The surgical specimens were used for this experiment. It was reported that breviscapine can inhibit fibrosis progression,but it has not been reported whether it can inhibit proliferation of pathological scar fibroblast.Other studies suggest that apoptosis is closely related to the formation of HS.And the regulation of fibroblast apoptosis is expected to become a new bright spot of the effective prevention and treatment of HS.In this study,we observe the growth variation and biological identification in the establishment of efficient primary culture of scar fibroblasts, and firstly explore the impact of Breviscapine on proliferation and apoptosis of scar fibroblast, hope to find effective drugs for the treatment of hypertrophic scars. The metabolism of collagen fibers in the dermis layer of scar tissue related molecules, and tap one of the important molecular variation; to provide ideas for the clarification and treatment of mechanism of hypertrophic scar. Objective:1. Searching for an efficient method of scar fibroblasts culture in vitro.2.Detecting the morphology, molecular biology of pathological scar,to understand formation mechanism of hyperplastic scar in depth.3.To study the effects of Breviscapine on proliferation and apoptosis of cultured scar fibroblasts. It may be developed as a new drug for the treatment of pathological.Methods:1. Specimen collectionThe24Traumatic scar specimens, age18to56years, was from the Second People’s Hospital of Guangdong Province and Guangzhou Red Cross Hospital from2011.06to2011.12. Access to specimens with the patient’s informed consent, and signed informed consent. Inclusion and exclusion criteria of specimen:1. The clinician identification of scar tissue, and a preliminary classification (hypertrophic, atrophic, keloid);2. Without the pituitary, adrenal diseases, infectious diseases, skin diseases and autoimmune diseases, local infection, ulcer;3. Receiving nothing treatment for the scar. Obtaining of specimens had the patient’s informed consent, and signed the Informed Consent Form.2. Specimen handling, groupingSpecimen was saved with freeze-drying method,and was used for primary culture within4hours. The specimens were divided into three parts:1. Cells primary culture;2. Fixed in10%formaldehyde solution for morphological identification (eg, immunohistochemistry, etc.);3. Frozen in liquid nitrogen alternate (extraction of protein and RNA). three normal skin used as normal controls3.Primary culture of scar fibroblasts:Observing the scar fibroblasts growth regulation and the growth characteristics; Drawing cell growth curves by MTT assay; identifition of scar fibroblasts. 4.Observing the differences morphological characteristics between hypertrophic scar and normal tissue by HE staining and masson trichrome staining.5.Distribution characteristics and expression of Collagen Ⅰ and Collagen Ⅲ was analyzed in the scar tissue by immunohistochemical staining.6.Gene expression of Collagen Ⅰ, Collagen Ⅲ, MMP-1, TIMP-1, SMAD3, SMAD7within the dermis is detected by RT-qPCR and the difference is compared between HS and the normal tissue. Protein expression of MMP-1, of SMAD3and TIMP-1by Western Blot technology.7.Fibroblasts were cultured as an experimental model and the effect of breviscapine were studied. Cell proliferation was determined by MTT assay, and Apoptosis of cultured fibroblasts is induced by breviscapine at the IC50with Annexin Ⅴ/PI staining and flow-cytometry.Results:1. Morphology and identification of scar fibroblast(1) A small number of cells climb out from HS organization in the shape of radial or spiral between two and seven days.(Fig1-1a, b) Primary fibroblasts were in the shape of spindle-shaped or irregular triangle. Nuclei were round or oblong ina-SMA of Immunohistochemistry staining.(2) It is spherical when the fibroblast has not been adherent in HS fibroblast subculture (Fig1-1c), and then extended, showing spindle-shaped, long rectangular, polygonal or rules-shaped (Fig1-1d). Cell was in the state of the convergence at2～3d (Fig1-1e). It is arranged loosely and in larger cell gap in Low cell density (Fig1-1e) and arranged in parallel or radial and swirling arrangement in high cell density,(Fig1-1f).(3) Growth curve:After a short incubation period, Scar fibroblasts showed a gradually increasing trend growth in6to8days (Fig1-3) and into the stagnation of the number after10days.(4) The results of α-SMA of Immunohistochemical staining is shown in fig1-5. We can identify fibroblasts by positive staining cells and its morphology.2. HE staining and Masson staining:1. Fewer fibroblast and microvascular in normal skin (Fig2-3a).2. Increased fibroblast and microvascular can be seen in Scar tissue (Fig2-3e). Thick collagen in dermis is disorganized, the collagen fiber was cross-cutting,swirling, nodular, among which there are collagen fibers,vessels and fibroblast(Fig2-3b, d).3. Marked thickening of the epidermal Scar tissue is thicker than normal epidermis (Fig2-3a).3. Result of IHC:Collagen Ⅰ is mainly distributed in the reticular dermis. The distribution of Collagen Ⅲ is in the papillary dermis. Both of two fibers are found in the vessel wall.4. RT-qPCR results:collagen Ⅰ, collagen Ⅲ, MMP-1and TIMP-1, SMAD3, SMAD7gene expression in dermal tissue of the scar and normal skin can be detected. Compared with normal tissue, gene expression of collagenⅠ (t=5.85, P<0.01)、 collagenⅢ (t=4.515, P<0.05), MMP-1(t=-13.22, P<0.01), TIMP-1(t=2.52, P<0.05)、smad7(t=-2.503, P<0.05) were significant different in scar. We can see collagenⅠ、collagenⅢ、TIMP-1is increased and MMP-1is decreased. There was no significant difference in gene expression of Smad3(t=-0.339, P>0.05), and Smad7(p=0.0629>0.05).(Fig2-7and Tab2-1).5. Western blot:MMP-1and TIMP-1, of SMAD3can be detected in dermal of the scar tissue samples and normal skin.But with a lower Protein expression than normal tissue.(Fig2-9).6. In vitro, the proliferation of fibroblasts is significantly inhibited by breviscapine in the manner of dose-dependent(F=3.309,P<0.01). Compared with the control group, proliferation of fibroblasts is significantly inhibited in the concentration of80,100,150μmol·ml-1(P<0.05). Apoptosis rate of fibroblasts induced by breviscapine at the IC50is significantly increased.7. The results of Flow cytometric Annexin V/PI double dyeing show apoptosis rate of experimental group is14.2050±0.95553in IC50, Apoptosis rate of control group is4.0220±0.31412, a statistically significant difference in both (t=22.653, P<0.05). The results of RT-PCR show that with increasing of concentration, the scar fibroblasts collagen Ⅰ and collagen Ⅲ mRNA are influenced obviously, there is a statistically significant difference,(fig3-4, tab3-1)Conclusion:1. Establishing scar fibroblast cell lines and naming them, and complementing primary culture theory of scar fibroblast.2. A better understanding of different fibroblasts between HS and normal skin is observed by observing the morphological changes during the growth prosess.3. Collagen Ⅰ and collagen Ⅲ in the hypertrophic scar tissue increased significantly. The formation of HS is closely related with the reduced expression of MMPs, and increased expression of TIMPs.4. The formation process of scar is a complex process of multiple signal paths, multi-factor interactions.5. Breviscapine can significantly inhibit the proliferation and induce the apoptosis of cultured scar fibroblasts. It may be developed as a new drug for the treatment of pathological scar.

        創(chuàng)傷后瘢痕形成機制及治療的研究

摘要3-10ABSTRACT10-16第一部分 增生性瘢痕成纖維細胞的原代培養(yǎng)與鑒定19-33    1 材料和方法19-24    2 結(jié)果24-28    3 討論28-30    4 結(jié)論30    參考文獻30-33第二部分 病理性瘢痕膠原代謝及其影響因素的研究33-54    1 材料和方法33-40    2 結(jié)果40-49    3 討論49-52    4 結(jié)論52    參考文獻52-54第三部分 燈盞花素對創(chuàng)傷性瘢痕成纖維細胞增殖、凋亡及mRNA表達的影響54-64    1 材料和方法54-57    2. 結(jié)果57-60    3 討論60-62    4 結(jié)論62    參考文獻62-64全文總結(jié)64-65綜述65-76    參考文獻72-76中英文縮略詞對照表76-77攻讀學(xué)位期間主要的研究成果77-78致謝78-80附件80

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