MAPK信号通路介导活化素A、B对成纤维细胞的调控

发布时间：2018-08-26 16:42

【摘要】：第一章MAPK信号通路介导活化素A、B对成纤维细胞的调控研究背景和目的组织修复、创伤愈合是当今医学治疗领域面临的难题。从临床医学定义来看,创伤愈合是指机体遭受外力作用,皮肤等器官出现离断或缺损后的修复过程,涉及到组织的再生、肉芽组织增生、瘢痕组织形成等一系列复杂的过程,表现出各种过程的协同作用。在人体器官创伤修复中,皮肤创伤愈合是最基本的模型,也是当今科学研究的热点。皮肤是人体最大的器官,主要承担着保护身体、排汗、感觉冷热和压力的功能。人和高等动物的皮肤由表皮、真皮、皮下组织三层组成。表皮是皮肤最外面的一层,真皮来源于中胚叶,由纤维、基质、细胞构成,皮下组织来源于中胚叶,在真皮的下部,由疏松结缔组织和脂肪小叶组成,其下紧临肌膜[3]。由于其与外界环境直接接触,所以非常容易损伤,随着慢性病所致的皮肤损伤的增加,皮肤创伤愈合是目前临床急需解决的问题之一[4]。皮肤创伤愈合是一个复杂的生物学过程,它启动一系列复杂的生物学事件,主要包括炎症、组织新生及组织重构。机体在受损刺激后,原来相对静止的各种细胞被激活,表型发生变化。其中表皮干细胞、角质形成细胞、成纤维细胞以及在创伤周围残存的上皮及附属结构细胞在创面愈合过程中发挥了重要作用[6]。成纤维细胞是皮肤真皮和疏松结缔组织的主要细胞。皮肤创伤愈合的早期其分泌前胶原、纤连蛋白和胶原酶等细胞外基质成分,与新生毛细血管、炎症细胞等共同形成肉芽组织,通过肉芽组织填补伤口组织缺损,为表皮细胞的覆盖创造条件,另一方面其可迁移到伤口进行增殖,促进伤口愈合。在伤口愈合过程中成纤维细胞主要来源于真皮乳头层的局部成纤维细胞和未分化的间充质细胞,以及血管周围的成纤维细胞和周细胞。一方面是由成纤维细胞通过分裂增殖而来,另一方面,更多的是由邻近的间充质细胞、纤维细胞和毛细血管周细胞等演变或游走至伤处[9]。在创伤修复的后期,成纤维细胞通过分泌胶原酶参与修复后组织的改建。其在以胶原的过度合成与沉积形成的纤维结缔组织为特征的瘢痕组织形成中起了重要的作用。目前一致认为成纤维细胞是创伤愈合及瘢痕形成中的关键细胞。创伤愈合过程中细胞的增殖、迁移和分化,其各个步骤均有细胞生长因子的参与和调控,主要有表皮生长因子(EGF)、成纤维细胞生长因子(bFGF)、转化生长因子p(TGF-β)、血小板源性生长因子(PDGF)等。活化素(Activin)属生长转化因子β(trans-forming growth factor beta, TGF-β)超家族成员,具有广泛的生物学活性,通过其信号转导通路调控生殖和胚胎发育过程、调节红细胞分化、参与病理炎症过程、诱导细胞凋亡、促进损伤后的修复过程,并参与器官纤维化的形成等。因此,以Activin为靶向分子的研究在皮肤创伤修复中意义重大。课题组前期研究发现,MAPK上游MEKK1基因敲除小鼠存在出生眼睑闭合障碍,和表皮细胞迁移异常有关。进一步研究显示：JNK-MAPK以及RhoA信号通路协同介导Activin B参与皮肤创伤愈合,调控角质形成细胞的细胞骨架重组进而影响细胞迁移。丝裂原激活的蛋白激酶(mitogen-activation protein kinase, MAPK)是由一组级联活化的丝／苏氨酸蛋白激酶组成,介导细胞生物学应答的重要信号系统,广泛存在于哺乳动物中,在细胞迁移,分化,增殖等生物学功能中都发挥了重要作用。鉴于成纤维细胞在皮肤创伤愈合过程中的重要作用,我们推钡MAPK信号通路在Activin介导的成纤维细胞中可能发挥一定的作用。为了探索MAPK信号通路是否介导成纤维细胞参与创伤修复,我们设计首先给予成纤维细胞Activin刺激,观察其激动蛋白的变化,进而通过特异性阻断或者特异性激活上述信号通路,以探究该信号通路是否参与Activin介导的成纤维细胞肌动蛋白的变化,从而揭示Activin调控成纤维细胞发挥作用的信号机制。本实验设计在细胞水平观察MAPK介导Activin A、Activin B对成纤维细胞的调控,为后续探讨MAPK信号通路在皮肤创伤愈合中介导Activin A、Activin B调控成纤维细胞的作用奠定基础。旨在给临床治疗皮肤创伤及瘢痕提供新的理论依据。研究方法1.分离培养C57BL/6乳鼠皮肤成纤维细胞；2.第4-6代成纤维细胞分成Activin A组、Activin B组、PBS组,Phallotoxins染色观察30min, 1h,2h,6h,12h成纤维细胞骨架变化,Ativin A应用20ng/ml, Activin B应用10ng/ml;3.第4-6代成纤维细胞分成Activin A 10min、30min、120min组,Activin B 10min、30min、120min组,PBS组,提取细胞总蛋白,Western blot检测JNK, ERK, p38的磷酸化活性；4.第4-6成纤维细胞分别用JNK特异性抑制剂SP600125 (5μmol)、p38特异性抑制剂SB202190 (5μmol)、ERK特异性抑制剂SL327 (5μmol)处理细胞30min后,给予Activin A、Activin B刺激,应用Western Blot检测Activin A、Activin B诱导的成纤维细胞p-JNK, p-ERK和p-p38的表达,利用Phallotoxins染色观察Activin A、Activin B诱导的细纤维细胞骨架蛋白的变化。5.第4-6代成纤维细胞分成Activin A组、Activin B组、PBS组,细胞划痕实验观察24h,48h,72h成纤维细胞迁移。研究结果：1. Phallotoxins染色：Activin A刺激成纤维细胞30min后,F-actin由之前少量集中于细胞膜附近变成大量积聚分布于整个细胞浆中,2h最明显,6h可见稍微减弱,12h回到刺激前水平；Activin B刺激成纤维细胞30min后, F-actin从细胞周边大量积聚到整个细胞浆中,2h达到高峰,6h大量积聚的actin,6h后开始减弱,12h回到刺激前水平。给予ERK特异性抑制剂SL327后,Activin A、Activin B诱导的成纤维细胞F-actin聚集现象减弱,但是没有完全抑制,给予JNK特异性抑制剂SP600125后,Activin A、Activin B诱导的成纤维细胞F-actin聚集现象完全被抑制,p38特异性抑制剂SB202190处理后,Activin A、Activin B诱导的成纤维细胞F-actin聚集没有变化。2. Western blot结果：Activin A、Activin B刺激成纤维细胞后p-JNK, p-ERK表达较PBS组均增高,p-p38无明显变化。p-JNK、p-ERK在Activin A、 Activin B均在刺激后10min高表达,之后降低,120min降到刺激前水平。给予JNK特异性抑制剂和ERK特异性抑制剂后,Activin A、Activin B诱导的JNK和ERK磷酸化活化被抑制；给予p38特异性抑制剂后,p38磷酸化活性没有变化。提示JNK、ERK/MAPK介导Activin A、Activin B对成纤维细胞骨架变化的调控,p38不参与Activin A、Activin B对成纤维细胞骨架变化的调控。3.细胞划痕结果：Activin A组、Activin B组、PBS组成纤维细胞迁移无差异。研究结论JNK、ERK/MAPK介导Activin A、Activin B调控成纤维细胞骨架变化,而p38通路未发现参与Activin A、ActivinB调控成纤维细胞骨架。第二章表皮特异性Cdc42、Rac 1基因敲除小鼠的鉴定及相关质粒构建研究背景和目的Rho家族是小鸟苷酸三磷酸酶,又称小G蛋白。Rho家族的主要成员有Rho(RhoA, RhoB, RhoC)、Rac (Rac1, Rac2). Cdc42。Rho家族蛋白是一种重要的细胞内信号分子,通过多种信号通路调节细胞的功能。Xunwei Wu等人用表皮特异性Cdc42、Rac1敲除转基因小鼠发现其毛发生长发育障碍和毛囊异常。毛囊是维持毛发生长的基础,通过多种途径影响皮肤创伤愈合[20]。毛囊中有毛囊干细胞,有学者认为毛囊干细胞是皮肤创伤愈合的种子细胞之一,同时皮肤创伤愈合中皮肤附属器官如毛囊的再生,是衡量愈合质量的重要标准。在细胞水平上,Rho-GTPase通过调控细胞粘附斑、应力纤维、丝状伪足、板状伪足进而调控细胞迁移、增殖、分化等多种生物学功能,影响皮肤创伤愈合[23]。这些都提示：Rho-GTPase在皮肤创伤愈合中发挥重要的调控。为研究Rho-GTPase在皮肤创伤愈合中的作用,我们设计构建表皮特异性Cdc42、Rac1基因敲除小鼠。继而构建Rho蛋白家族中Cdc42的显性负效突变体(Cdc42NA)和组成型活性突变体(Cdc42L61)的慢病毒,为后续进一步研究Cdc42蛋白在皮肤创伤愈合中的作用及其上下游通路提供实验工具。研究方法1. Cre-loxP系统构建表皮特异性Rac1、Cdc42敲除转基因小鼠并鉴定：引进KRT14-cre、Rac1-loxP、Cdc42-loxP, Rac1-loxP转基因小鼠,FO代用Rac1loxp/ loxp/Cre-,Cdc42 loxp/loxp/Cre-与KRT14-Cre+杂交,得到F1代Rac1 loxp/+/Cre+,Cdc42 loxp/+/Cre+杂合子,F1代和Rac1loxp/loxp/Cre-,Cdc42 loxp/loxp/Cre-杂交,得到F2代Rac1loxp/loxp/Cre+, Cdc42 loxp/loxp/Cre+纯合子小鼠。异丙醇、DNA提取试剂盒提取小鼠DNA,紫外分光光度计测量DNA浓度、纯度,记录数据。SPSS统计软件两独立样本t检验分析数据,P0.05有统计学意义。根据敲除前后DNA序列设计PCR引物,PCR鉴定小鼠基因型。2.构建Rho家族重组质粒Plenti6/v5-cdc42NA, Plenti6/v5-cdc42L61:利用限制性内切酶及DNA连接酶构建质粒Plenti6/v5-Cdc42NA, Plenti6/v5-Cdc42L61,重组质粒送Invitrogen公司测序,大肠杆菌扩增质粒,质粒提取纯化,质粒酶切鉴定。制备慢病毒：ViraPower慢病毒包装系统制备相应的慢病毒,荧光显微镜观察293FT细胞荧光,检测慢病毒制备效果。研究结果1.KRT14启动的表皮特异性Cdc42敲除转基因小鼠纯合子出生24小时内死亡,杂合子正常成活,表皮特异性Rac1敲除转基因小时纯合子毛发生长障碍,杂合子正常成活,与文献报道一致。PCR鉴定结果显示敲除成功,可见敲除带。酒精异丙醇提取DNA纯度高,浓度高,与试剂盒提取在浓度上差别无统计学意义,纯度比较有统计学差异,异丙醇组纯度更高。2.质粒经酶切,琼脂糖凝胶电泳,提示构建正确。基因测序与NCBI公布的序列一致。荧光显微镜下观察制备慢病毒的293FT细胞有绿色荧光蛋白表达且表达效率高达90%,提示慢病毒制备成功。研究结论1.表皮特异性Rac1、Cdc42敲除转基因小鼠构建成功,PCR鉴定方法稳定。酒精异丙醇提取DNA方法简便易行,效果好,具有很高的实用价值。2.成功构建了Rho家族重组质粒Plenti6/v5-Cdc42NA, Plenti6/v5-Cdc42L61,制备了Rho蛋白家族中Cdc42的显性负效突变体(Cdc42NA)和组成型活性突变体(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
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R641

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