【摘要】：第一章MAPK信號通路介導活化素A、B對成纖維細胞的調(diào)控研究背景和目的組織修復、創(chuàng)傷愈合是當今醫(yī)學治療領域面臨的難題。從臨床醫(yī)學定義來看,創(chuàng)傷愈合是指機體遭受外力作用,皮膚等器官出現(xiàn)離斷或缺損后的修復過程,涉及到組織的再生、肉芽組織增生、瘢痕組織形成等一系列復雜的過程,表現(xiàn)出各種過程的協(xié)同作用。在人體器官創(chuàng)傷修復中,皮膚創(chuàng)傷愈合是最基本的模型,也是當今科學研究的熱點。皮膚是人體最大的器官,主要承擔著保護身體、排汗、感覺冷熱和壓力的功能。人和高等動物的皮膚由表皮、真皮、皮下組織三層組成。表皮是皮膚最外面的一層,真皮來源于中胚葉,由纖維、基質(zhì)、細胞構成,皮下組織來源于中胚葉,在真皮的下部,由疏松結締組織和脂肪小葉組成,其下緊臨肌膜[3]。由于其與外界環(huán)境直接接觸,所以非常容易損傷,隨著慢性病所致的皮膚損傷的增加,皮膚創(chuàng)傷愈合是目前臨床急需解決的問題之一[4]。皮膚創(chuàng)傷愈合是一個復雜的生物學過程,它啟動一系列復雜的生物學事件,主要包括炎癥、組織新生及組織重構。機體在受損刺激后,原來相對靜止的各種細胞被激活,表型發(fā)生變化。其中表皮干細胞、角質(zhì)形成細胞、成纖維細胞以及在創(chuàng)傷周圍殘存的上皮及附屬結構細胞在創(chuàng)面愈合過程中發(fā)揮了重要作用[6]。成纖維細胞是皮膚真皮和疏松結締組織的主要細胞。皮膚創(chuàng)傷愈合的早期其分泌前膠原、纖連蛋白和膠原酶等細胞外基質(zhì)成分,與新生毛細血管、炎癥細胞等共同形成肉芽組織,通過肉芽組織填補傷口組織缺損,為表皮細胞的覆蓋創(chuàng)造條件,另一方面其可遷移到傷口進行增殖,促進傷口愈合。在傷口愈合過程中成纖維細胞主要來源于真皮乳頭層的局部成纖維細胞和未分化的間充質(zhì)細胞,以及血管周圍的成纖維細胞和周細胞。一方面是由成纖維細胞通過分裂增殖而來,另一方面,更多的是由鄰近的間充質(zhì)細胞、纖維細胞和毛細血管周細胞等演變或游走至傷處[9]。在創(chuàng)傷修復的后期,成纖維細胞通過分泌膠原酶參與修復后組織的改建。其在以膠原的過度合成與沉積形成的纖維結締組織為特征的瘢痕組織形成中起了重要的作用。目前一致認為成纖維細胞是創(chuàng)傷愈合及瘢痕形成中的關鍵細胞。創(chuàng)傷愈合過程中細胞的增殖、遷移和分化,其各個步驟均有細胞生長因子的參與和調(diào)控,主要有表皮生長因子(EGF)、成纖維細胞生長因子(bFGF)、轉(zhuǎn)化生長因子p(TGF-β)、血小板源性生長因子(PDGF)等�；罨�(Activin)屬生長轉(zhuǎn)化因子β(trans-forming growth factor beta, TGF-β)超家族成員,具有廣泛的生物學活性,通過其信號轉(zhuǎn)導通路調(diào)控生殖和胚胎發(fā)育過程、調(diào)節(jié)紅細胞分化、參與病理炎癥過程、誘導細胞凋亡、促進損傷后的修復過程,并參與器官纖維化的形成等。因此,以Activin為靶向分子的研究在皮膚創(chuàng)傷修復中意義重大。課題組前期研究發(fā)現(xiàn),MAPK上游MEKK1基因敲除小鼠存在出生眼瞼閉合障礙,和表皮細胞遷移異常有關。進一步研究顯示：JNK-MAPK以及RhoA信號通路協(xié)同介導Activin B參與皮膚創(chuàng)傷愈合,調(diào)控角質(zhì)形成細胞的細胞骨架重組進而影響細胞遷移。絲裂原激活的蛋白激酶(mitogen-activation protein kinase, MAPK)是由一組級聯(lián)活化的絲／蘇氨酸蛋白激酶組成,介導細胞生物學應答的重要信號系統(tǒng),廣泛存在于哺乳動物中,在細胞遷移,分化,增殖等生物學功能中都發(fā)揮了重要作用。鑒于成纖維細胞在皮膚創(chuàng)傷愈合過程中的重要作用,我們推鋇MAPK信號通路在Activin介導的成纖維細胞中可能發(fā)揮一定的作用。為了探索MAPK信號通路是否介導成纖維細胞參與創(chuàng)傷修復,我們設計首先給予成纖維細胞Activin刺激,觀察其激動蛋白的變化,進而通過特異性阻斷或者特異性激活上述信號通路,以探究該信號通路是否參與Activin介導的成纖維細胞肌動蛋白的變化,從而揭示Activin調(diào)控成纖維細胞發(fā)揮作用的信號機制。本實驗設計在細胞水平觀察MAPK介導Activin A、Activin B對成纖維細胞的調(diào)控,為后續(xù)探討MAPK信號通路在皮膚創(chuàng)傷愈合中介導Activin A、Activin B調(diào)控成纖維細胞的作用奠定基礎。旨在給臨床治療皮膚創(chuàng)傷及瘢痕提供新的理論依據(jù)。研究方法1.分離培養(yǎng)C57BL/6乳鼠皮膚成纖維細胞；2.第4-6代成纖維細胞分成Activin A組、Activin B組、PBS組,Phallotoxins染色觀察30min, 1h,2h,6h,12h成纖維細胞骨架變化,Ativin A應用20ng/ml, Activin B應用10ng/ml;3.第4-6代成纖維細胞分成Activin A 10min、30min、120min組,Activin B 10min、30min、120min組,PBS組,提取細胞總蛋白,Western blot檢測JNK, ERK, p38的磷酸化活性；4.第4-6成纖維細胞分別用JNK特異性抑制劑SP600125 (5μmol)、p38特異性抑制劑SB202190 (5μmol)、ERK特異性抑制劑SL327 (5μmol)處理細胞30min后,給予Activin A、Activin B刺激,應用Western Blot檢測Activin A、Activin B誘導的成纖維細胞p-JNK, p-ERK和p-p38的表達,利用Phallotoxins染色觀察Activin A、Activin B誘導的細纖維細胞骨架蛋白的變化。5.第4-6代成纖維細胞分成Activin A組、Activin B組、PBS組,細胞劃痕實驗觀察24h,48h,72h成纖維細胞遷移。研究結果：1. Phallotoxins染色：Activin A刺激成纖維細胞30min后,F-actin由之前少量集中于細胞膜附近變成大量積聚分布于整個細胞漿中,2h最明顯,6h可見稍微減弱,12h回到刺激前水平；Activin B刺激成纖維細胞30min后, F-actin從細胞周邊大量積聚到整個細胞漿中,2h達到高峰,6h大量積聚的actin,6h后開始減弱,12h回到刺激前水平。給予ERK特異性抑制劑SL327后,Activin A、Activin B誘導的成纖維細胞F-actin聚集現(xiàn)象減弱,但是沒有完全抑制,給予JNK特異性抑制劑SP600125后,Activin A、Activin B誘導的成纖維細胞F-actin聚集現(xiàn)象完全被抑制,p38特異性抑制劑SB202190處理后,Activin A、Activin B誘導的成纖維細胞F-actin聚集沒有變化。2. Western blot結果：Activin A、Activin B刺激成纖維細胞后p-JNK, p-ERK表達較PBS組均增高,p-p38無明顯變化。p-JNK、p-ERK在Activin A、 Activin B均在刺激后10min高表達,之后降低,120min降到刺激前水平。給予JNK特異性抑制劑和ERK特異性抑制劑后,Activin A、Activin B誘導的JNK和ERK磷酸化活化被抑制；給予p38特異性抑制劑后,p38磷酸化活性沒有變化。提示JNK、ERK/MAPK介導Activin A、Activin B對成纖維細胞骨架變化的調(diào)控,p38不參與Activin A、Activin B對成纖維細胞骨架變化的調(diào)控。3.細胞劃痕結果：Activin A組、Activin B組、PBS組成纖維細胞遷移無差異。研究結論JNK、ERK/MAPK介導Activin A、Activin B調(diào)控成纖維細胞骨架變化,而p38通路未發(fā)現(xiàn)參與Activin A、ActivinB調(diào)控成纖維細胞骨架。第二章表皮特異性Cdc42、Rac 1基因敲除小鼠的鑒定及相關質(zhì)粒構建研究背景和目的Rho家族是小鳥苷酸三磷酸酶,又稱小G蛋白。Rho家族的主要成員有Rho(RhoA, RhoB, RhoC)、Rac (Rac1, Rac2). Cdc42。Rho家族蛋白是一種重要的細胞內(nèi)信號分子,通過多種信號通路調(diào)節(jié)細胞的功能。Xunwei Wu等人用表皮特異性Cdc42、Rac1敲除轉(zhuǎn)基因小鼠發(fā)現(xiàn)其毛發(fā)生長發(fā)育障礙和毛囊異常。毛囊是維持毛發(fā)生長的基礎,通過多種途徑影響皮膚創(chuàng)傷愈合[20]。毛囊中有毛囊干細胞,有學者認為毛囊干細胞是皮膚創(chuàng)傷愈合的種子細胞之一,同時皮膚創(chuàng)傷愈合中皮膚附屬器官如毛囊的再生,是衡量愈合質(zhì)量的重要標準。在細胞水平上,Rho-GTPase通過調(diào)控細胞粘附斑、應力纖維、絲狀偽足、板狀偽足進而調(diào)控細胞遷移、增殖、分化等多種生物學功能,影響皮膚創(chuàng)傷愈合[23]。這些都提示：Rho-GTPase在皮膚創(chuàng)傷愈合中發(fā)揮重要的調(diào)控。為研究Rho-GTPase在皮膚創(chuàng)傷愈合中的作用,我們設計構建表皮特異性Cdc42、Rac1基因敲除小鼠。繼而構建Rho蛋白家族中Cdc42的顯性負效突變體(Cdc42NA)和組成型活性突變體(Cdc42L61)的慢病毒,為后續(xù)進一步研究Cdc42蛋白在皮膚創(chuàng)傷愈合中的作用及其上下游通路提供實驗工具。研究方法1. Cre-loxP系統(tǒng)構建表皮特異性Rac1、Cdc42敲除轉(zhuǎn)基因小鼠并鑒定：引進KRT14-cre、Rac1-loxP、Cdc42-loxP, Rac1-loxP轉(zhuǎn)基因小鼠,FO代用Rac1loxp/ loxp/Cre-,Cdc42 loxp/loxp/Cre-與KRT14-Cre+雜交,得到F1代Rac1 loxp/+/Cre+,Cdc42 loxp/+/Cre+雜合子,F1代和Rac1loxp/loxp/Cre-,Cdc42 loxp/loxp/Cre-雜交,得到F2代Rac1loxp/loxp/Cre+, Cdc42 loxp/loxp/Cre+純合子小鼠。異丙醇、DNA提取試劑盒提取小鼠DNA,紫外分光光度計測量DNA濃度、純度,記錄數(shù)據(jù)。SPSS統(tǒng)計軟件兩獨立樣本t檢驗分析數(shù)據(jù),P0.05有統(tǒng)計學意義。根據(jù)敲除前后DNA序列設計PCR引物,PCR鑒定小鼠基因型。2.構建Rho家族重組質(zhì)粒Plenti6/v5-cdc42NA, Plenti6/v5-cdc42L61:利用限制性內(nèi)切酶及DNA連接酶構建質(zhì)粒Plenti6/v5-Cdc42NA, Plenti6/v5-Cdc42L61,重組質(zhì)粒送Invitrogen公司測序,大腸桿菌擴增質(zhì)粒,質(zhì)粒提取純化,質(zhì)粒酶切鑒定。制備慢病毒：ViraPower慢病毒包裝系統(tǒng)制備相應的慢病毒,熒光顯微鏡觀察293FT細胞熒光,檢測慢病毒制備效果。研究結果1.KRT14啟動的表皮特異性Cdc42敲除轉(zhuǎn)基因小鼠純合子出生24小時內(nèi)死亡,雜合子正常成活,表皮特異性Rac1敲除轉(zhuǎn)基因小時純合子毛發(fā)生長障礙,雜合子正常成活,與文獻報道一致。PCR鑒定結果顯示敲除成功,可見敲除帶。酒精異丙醇提取DNA純度高,濃度高,與試劑盒提取在濃度上差別無統(tǒng)計學意義,純度比較有統(tǒng)計學差異,異丙醇組純度更高。2.質(zhì)粒經(jīng)酶切,瓊脂糖凝膠電泳,提示構建正確�；驕y序與NCBI公布的序列一致。熒光顯微鏡下觀察制備慢病毒的293FT細胞有綠色熒光蛋白表達且表達效率高達90%,提示慢病毒制備成功。研究結論1.表皮特異性Rac1、Cdc42敲除轉(zhuǎn)基因小鼠構建成功,PCR鑒定方法穩(wěn)定。酒精異丙醇提取DNA方法簡便易行,效果好,具有很高的實用價值。2.成功構建了Rho家族重組質(zhì)粒Plenti6/v5-Cdc42NA, Plenti6/v5-Cdc42L61,制備了Rho蛋白家族中Cdc42的顯性負效突變體(Cdc42NA)和組成型活性突變體(Cdc42L61)的慢病毒。
[Abstract]:Chapter 1 MAPK signaling pathway mediates the regulation of activin A and B on fibroblasts and tissue repair. Wound healing is a difficult problem in the field of medical treatment nowadays. Skin wound healing is the most basic model in wound healing of human organs, and it is also the hot spot of scientific research nowadays. Skin is the largest organ of the human body, mainly responsible for protecting the body, sweating, feeling cold and hot. The epidermis is the outermost layer of the skin. The dermis originates from the mesoderm and is composed of fibers, matrix and cells. The subcutaneous tissue originates from the mesoderm, and in the lower part of the dermis is composed of loose connective tissue and fat lobules, which are close to the myometrium [3]. Skin wound healing is a complex biological process that initiates a series of complex biological events, including inflammation, tissue regeneration and tissue formation. Tissue remodeling. After injury stimulation, the relatively static cells are activated and their phenotypes change. Epidermal stem cells, keratinocytes, fibroblasts, and the remaining epithelial and accessory structural cells around the wound play an important role in wound healing [6]. Fibroblasts are dermis and loose skin. The main cells of connective tissue. In the early stage of skin wound healing, they secrete extracellular matrix components such as procollagen, fibronectin and collagenase, and form granulation tissue together with new capillaries and inflammatory cells. The granulation tissue fills the defect of wound tissue and creates conditions for epidermal cells to cover the wound. On the other hand, it can migrate to the wound. During wound healing, fibroblasts are mainly derived from local fibroblasts and undifferentiated mesenchymal cells in the dermal papilla, fibroblasts and perivascular cells. Mesenchymal cells, fibroblasts and pericapillary cells evolve or migrate to the wound [9].In the late stage of wound healing, fibroblasts participate in tissue remodeling by secreting collagenase, which plays an important role in the formation of scar tissue characterized by excessive collagen synthesis and deposition of fibrous connective tissue. It was previously agreed that fibroblasts are the key cells in wound healing and scar formation. Cell proliferation, migration and differentiation in the process of wound healing involve and regulate cell growth factors, including epidermal growth factor (EGF), fibroblast growth factor (bFGF), transforming growth factor P (TGF - beta), platelet source. Activin is a member of the trans-forming growth factor beta (TGF-beta) superfamily and has a wide range of biological activities. Activin regulates reproductive and embryonic development, regulates red blood cell differentiation, participates in pathological inflammation, induces apoptosis, and promotes injury. Previous studies have shown that MEKK1 knockout mice upstream of MAPK are associated with dysfunction of eyelid closure at birth and abnormal migration of epidermal cells. Mitogen-activated protein kinase (MAPK) is a cascade of serine/threonine protein kinases that mediate cellular biological responses. It is widely distributed in mammals and plays an important role in cell migration, differentiation, proliferation and other biological functions. In view of the important role of fibroblasts in skin wound healing, we suggest that barium MAPK signaling pathway may play a role in Activin-mediated fibroblasts. Whether the pathway mediates fibroblasts to participate in wound healing, we designed to first stimulate fibroblasts with Activin to observe the changes of its agonist protein, and then specifically block or activate the above signaling pathways to explore whether this signaling pathway is involved in Activin-mediated changes in fibroblasts actin, thereby This study was designed to observe the regulation of Activin A and Activin B on fibroblasts at the cellular level, and lay a foundation for further study on the role of MAPK signaling pathway in the regulation of Activin A and Activin B on fibroblasts during skin wound healing. Methods 1. Isolation and culture of C57BL/6 neonatal rat skin fibroblasts; 2. The 4th-6th generation fibroblasts were divided into Activin A group, Activin B group, PBS group and Phallotoxins staining to observe the changes of fibroblast skeleton in 30 minutes, 1 hour, 2 hours, 6 hours and 12 hours, Ativin A 20ng/ml, Activin B 10ng/ml; Progeny fibroblasts were divided into Activin A 10 min, 30 min, 120 min group, Activin B 10 min, 30 min, 120 min group, PBS group, extracting total protein, Western blot detection of JNK, ERK, p38 phosphorylation activity; 4. 4 - 6 fibroblasts were treated with JNK specific inhibitor SP600125 (5 micromol), p38 specific inhibitor SB202190 (5 micromol), ERK specific inhibitor respectively. Activin A and Activin B were used to stimulate the cells treated with SL327 for 30 minutes. The expression of p-JNK, p-ERK and p-p38 in fibroblasts induced by Activin A and Activin B was detected by Western Blot. The changes of cytoskeleton protein induced by Activin A and Activin B were observed by Phallotoxins staining. In A group, Activin B group, PBS group, cell scratch test observed 24 hours, 48 hours, 72 hours fibroblasts migration. Results: 1. Phallotoxins staining: Activin A stimulated fibroblasts 30 minutes later, F-actin from a small amount of concentration in the vicinity of the cell membrane into a large number of accumulation and distribution in the whole cytoplasm, 2 hours the most obvious, 6 hours a slight weakening, 12 hours back to. Activin B stimulated fibroblasts to accumulate a large amount of F-actin from the periphery of the cells to the whole cytoplasm 30 minutes later, peaked at 2 hours, accumulated at 6 hours, began to weaken after 6 hours, and returned to the pre-stimulation level 12 hours later. Activin A and Activin B induced fibroblast F-actin aggregation was completely inhibited after JNK specific inhibitor SP600125 was given. Activin A and Activin B induced fibroblast F-actin aggregation did not change after treatment with p38 specific inhibitor SB202190. 2. Western blot results: Activin A and Activin B pricking The expression of p-JNK, p-ERK increased after stimulation of fibroblasts, but the expression of p-JNK, p-ERK remained unchanged. The expression of p-JNK, p-ERK in Activin A and Activin B increased 10 minutes after stimulation, then decreased to pre-stimulation level at 120 minutes. Activin A and Activin B-induced JNK and ERK phosphorylation was inhibited after JNK specific inhibitors and ERK specific inhibitors were given. It is suggested that JNK, ERK / MAPK mediate the regulation of Activin A and Activin B on the changes of fibroblast cytoskeleton, while p38 does not participate in the regulation of Activin A and Activin B on the changes of fibroblast cytoskeleton. 3. Cell scratch results: Activin A, Activin B, PBS constitute fibroblast migration. Conclusion JNK, ERK / MAPK mediate the cytoskeleton changes of Activin A and Activin B, but p38 pathway is not involved in the cytoskeleton changes of Activin A and Activin B. Chapter 2 Identification of epidermis-specific Cdc42, Rac 1 knockout mice and construction of related plasmids. The background and purpose of this study is that the Raho family is guanosine triglyceride. The main members of the Rho family are Rho (RhoA, RhoB, RhoC), Rac (Rac1, Rac2). Cdc42. Rho family proteins are important intracellular signaling molecules that regulate cell function through a variety of signaling pathways. Xunwei Wu et al. used epidermal-specific Cdc42, Rac1 knockout transgenic mice to detect hair growth and development disorders. Hair follicles are the basis of maintaining hair growth and affecting skin wound healing through various ways [20].There are hair follicle stem cells in hair follicles, some scholars believe that hair follicle stem cells are one of the seed cells of skin wound healing, and the regeneration of skin accessory organs such as hair follicles in skin wound healing is an important criterion to evaluate the quality of healing. Quasi. At the cellular level, Rho-GTPase regulates cell migration, proliferation, differentiation and other biological functions by regulating cell adhesion plaques, stress fibers, filiform pseudopodia and lamellar pseudopodia, thus affecting skin wound healing [23]. We designed and constructed epidermis-specific Cdc42, Rac1 knockout mice, and then constructed the dominant negative mutant (Cdc42NA) of Cdc42 in the Rho protein family and the lentiviruses of the constitutive active mutant (Cdc42L61) in order to further study the role of Cdc42 protein in skin wound healing and its upstream and downstream pathways. Methods 1. The Cre-loxP system was used to construct epidermis-specific Rac1, Cdc42 knockout transgenic mice and identified: introduced KRT14-cre, Rac1-loxP, Cdc42-loxP, Rac1-loxP transgenic mice, FO-substituted Rac1 loxp/loxp/Cre-, Cdc42-loxp/Cre-and KRT14-Cre+hybridized to obtain F1 generation Rac1-xp/+, Cdc42-loxp/Cre+, Cdc42-loxp/Cre+heterozygote, F1 and F1 generation Rac1-loxp/Cre. C1loxp/loxp/Cre-, Cdc42 loxp/loxp/Cre-hybridization, F2 generation Rac1loxp/loxp/Cre+, Cdc42 loxp/loxp/Cre+, Cdc42 loxp/loxp/Cre+ homozygote mice. Isopropanol, DNA extraction kit, UV spectrophotometer measurement of DNA concentration, purity, recording data. SPSS statistical software two independent samples t test analysis data, P 0.05 statistical significance. Construction of Rho family recombinant plasmids Plenti6/v5-cdc42L61, Plenti6/v5-cdc42L61: Construction of plasmids Plenti6/v5-Cdc42NA, Plenti6/v5-Cdc42L61 with restriction endonuclease and DNA ligase, Plenti6/v5-Cdc42L61, Plenti6/v5-Cdc42L61, and delivery of recombinant plasmids to Invitrogen for sequencing, E.coli amplification, Plasmid Extraction and purification. Preparation of lentiviruses: ViraPower lentivirus packaging system to prepare the corresponding lentiviruses, fluorescence microscopy 293FT cell fluorescence, detection of lentiviral preparation effect. Results 1. KRT14-initiated epidermis-specific Cdc42 knockout transgenic mice homozygote death within 24 hours of birth, heterozygote normal survival, epidermis-specific R Homozygote hair growth disorder occurred after AC1 knockout, and heterozygotes survived normally, which was consistent with the literature. PCR results showed that the knockout was successful and the knockout band was visible. High. 2. The plasmids were digested by enzyme and agarose gel electrophoresis, suggesting that the construction was correct. The gene sequencing was consistent with the sequence published by NCBI. The method of extracting DNA by alcohol isopropanol is simple, effective and has high practical value. 2. Successful construction
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R641

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