P311在皮肤创面再上皮化和肾纤维化中的作用及机制研究

发布时间：2018-05-05 23:33

本文选题：P311 + 表皮干细胞　；参考：《第三军医大学》2016年博士论文

【摘要】：第一部分:P311在皮肤创面再上皮化中的作用及机制研究背景:创面修复是一个动态持续的过程,需要机体多种类型细胞、细胞外基质及一系列细胞因子和生长因子的共同参与。难愈创面目前已成为一个重要的临床难题,其原因除了传统的烧伤、创伤及外科手术外,一些慢性疾病如静脉曲张、糖尿病等也可引起创面难以愈合。难愈创面常常可导致终生残疾,给病人、家庭和社会造成巨大的经济社会负担。据统计,目前患有慢性难愈创面的病人数量正在全球范围内逐渐增加。鉴于此,深入研究创面愈合的相关机制,发展新的创面治疗方法,提高创面愈合质量已成为该领域研究的热点、重点和难点问题之一。哺乳动物皮肤创面愈合一般分为四个阶段:凝血期、炎症期、新生组织形成和组织重塑。新生组织形成起始于新生表皮迁移覆盖受损真皮。表皮干细胞主要分布于毛囊隆突部位,表皮细胞基底层和皮脂腺组织中,在保持皮肤表皮更新、促进创面愈合过程中起着至关重要的作用。当创面形成后,表皮干细胞活化增殖产生更多短暂扩充细胞,进一步增殖、分化迁移至创面发挥修复作用。表皮干细胞目前已成为临床上治疗慢性难愈创面的候选细胞。P311基因定位于人5号染色体和小鼠18号染色体,首次在小鼠胚胎大脑发现,并且在成年小鼠小脑、海马和嗅球中保持较高水平表达。P311蛋白是一个分子量为8-k Da的胞内蛋白,包含68个氨基酸,目前不属于任何已知的蛋白家族。P311蛋白N末端包含PEST结构域(富含脯氨酸、谷氨酸、丝氨酸和苏氨酸),在人、小鼠和鸡中高度保守。PEST结构域在目的蛋白通过泛素/蛋白酶体通路降解及蛋白与蛋白之间相互作用和活化过程中发挥重要作用。目前研究发现P311在神经和肺泡再生、血压稳态维持、神经胶质细胞瘤侵袭和肌成纤维细胞活化等方面发挥重要作用。我们前期研究发现,P311在早期增生性瘢痕肌成纤维细胞和创面肉芽组织活化的成纤维细胞中表达增高,可以通过促进TGF-β1表达在增生性瘢痕的形成过程中发挥一定作用。但目前P311在皮肤创面再上皮化过程是否发挥一定作用尚不清楚。在此基础上,我们对P311在皮肤创面再上皮化过程中的作用进行了研究,主要涉及以下三个方面:1)P311在皮肤创面新生表皮和正常皮肤表皮中的表达和分布;2)P311在表皮干细胞迁移中的作用;3)P311促进表皮干细胞迁移的分子机制。结果:1、P311在人和小鼠皮肤创面新生表皮中表达增高。我们收集了5例人皮肤创面组织标本和同体正常皮肤对照,同时构建了小鼠全层皮肤缺损模型,检测样本中P311基因表达。免疫组织化学技术检测发现P311主要表达于人和小鼠皮肤创面新生表皮细胞胞浆中,而正常皮肤表皮细胞中无P311表达。2、P311可以促进表皮干细胞迁移。利用Ⅳ型胶原快速黏附法分离了健康青年男性包皮中表皮干细胞,应用细胞免疫荧光和流式细胞技术检测了分离细胞中表皮干细胞分子标记物表达。细胞免疫荧光发现分离细胞中角蛋白K19和β1整合素高表达,并且二者于培养细胞中共定位表达。流式细胞技术检测发现培养细胞高表达α6整合素,而阴性标志物CD71表达阴性。我们进一步利用细胞体外创伤模型检测了P311腺病毒载体转染表皮干细胞后对细胞迁移功能的影响。结果发现P311增强表达后可以促进表皮干细胞迁移。我们分离了P311-/-和P311+/+新生小鼠皮肤表皮干细胞并应用流式细胞技术进行分子鉴定,结果发现培养细胞高表达α6整合素,而阴性标志物CD71表达阴性。进一步利用细胞体外创伤模型检测了P311敲除后对表皮干细胞迁移功能的影响,结果发现P311敲除后可以表皮干细胞迁移明显下降。我们构建了小鼠全层皮肤缺损模型检测P311敲除后对皮肤创面再上皮化的影响。结果发现小鼠全层皮肤缺损模型可以显著抑制小鼠皮肤创面收缩,P311+/+小鼠皮肤创面再上皮化速度明显快于P311-/-小鼠,P311敲除后小鼠皮肤创面再上皮化能力减弱。3、Rho GTPases在P311促进表皮干细胞迁移中的作用研究。为了探索P311促进表皮干细胞迁移的作用机制,我们应用Pulldown技术检测了Rho GTPases Rho A、Rac1和Cdc42活性。结果发现:P311增强表达后Rho A、Rac1和Cdc42活性明显增高,同时P311敲除后Rho A、Rac1和Cdc42活性下降。我们应用细胞体外创伤模型检测了Rho A、Rac1和Cdc42特异性抑制剂对P311促进表皮干细胞的影响。结果发现Rho A和Rac1抑制剂可以显著抑制P311对表皮干细胞迁移能力的增强作用,而Cdc42抑制剂无明显影响。综上所述,我们的研究首次发现P311可能通过增强Rho A和Rac1活性促进表皮干细胞迁移和创面再上皮化。本研究拓展了P311在创面愈合中的认识,可能为今后创面愈合的研究和治疗提供新的线索。第二部分:P311在肾纤维化中的作用及机制研究背景:肾纤维化是肾脏组织在多种损伤因素的刺激下自我修复过程失调的结果,临床上,有很大部分比例的肾纤维化病人最终将发展为肾功能衰竭,需要终生进行血液透析或肾脏移植治疗维持生命。大量流行病学研究表明,肾功能衰竭病人的患病率在世界范围内正逐渐增加。肾纤维化疾病现在已发展成为全球范围内的公共健康问题,给患者个人、家庭及社会造成了巨大的社会经济负担。目前,临床上针对肾纤维化疾病尚无十分有效的治疗方法。鉴于此,深入研究肾纤维化形成的病理机制,寻找肾纤维化形成的相关特异性基因是目前该领域研究的热点、重点和难点问题之一。肾脏纤维化是多种慢性肾脏疾病中不可逆的、进行性动态发展的病理过程,以肾脏小球及小管间质中大量的细胞外基质的合成和积聚为主要特征,该过程需要肾脏多种类型细胞的参与。基质产生细胞的活化被认为是肾纤维化病理过程中的关键,而基质产生细胞的来源主要包括成纤维细胞,肾小管上皮细胞、血管平滑肌细胞和巨噬细胞。肾小管上皮细胞是肾纤维化晚期阶段不可逆发展过程中最主要的效应细胞,在多种促纤维化因子(尤其是TGF-β1)的作用下,肾小管上皮细胞经历上皮细胞间质转型(epithelial-mesenchymal transition,EMT)过程,细胞表型发生改变,转分化成为肌成纤维细胞。TGF-β1是肾小管上皮细胞EMT过程中关键的调控分子,可以介导多种慢性肾脏疾病的病理发展过程,可以诱导肾小管上皮细胞表达波形蛋白(vimentin,一种成纤维细胞标志)和α肌动蛋白(α-smooth muscle actin,α-SMA,一种肌成纤维细胞细胞标志)。P311最初在小鼠胚胎大脑中发现,在成年小鼠小脑、海马和嗅球中保持较高水平表达。P311基因定位于人5号染色体、小鼠18号染色体,编码分子量约8-k Da胞内蛋白。P311蛋白包含68个氨基酸,尚不属于任何已知蛋白质家族。目前研究发现P311主要在神经和肺泡发育、神经胶质瘤细胞侵袭及迁移、血压稳态维持、肌成纤维细胞分化和运动中发挥重要作用。本课题组研究发现,P311基因在早期增生性瘢痕中高表达,可以通过调节TGF-β1和α-SMA表达促进成纤维细胞向肌成纤维细胞分化,在增生性瘢痕形成中发挥一定的促进作用。在此基础上,我们对P311在肾纤维化中的作用进行了研究,主要涉及以下三个方面:1)P311在肾纤维化组织和正常肾脏组织中的表达和分布;2)P311敲除后对肾纤维化的影响;3)P311促进肾脏纤维化的分子机制。结果：1、P311在人和小鼠肾纤维化组织表达增高。我们收集了22例人肾纤维化组织标本和2例人正常肾脏组织标本,同时构建了小鼠单侧输尿管结扎模型成功地诱导了小鼠肾脏组织纤维化,检测样本中P311基因表达。HE染色显示肾纤维化组织可见典型的肾小管扩张、萎缩塌陷和大量炎症细胞浸润,Masson染色可见肾脏间质大量胶原纤维沉积和积聚。免疫组织化学技术检测发现P311主要表达于人和小鼠肾纤维化组织部分肾小管上皮细胞胞浆中,而正常肾脏组织中无P311表达。2、P311可能通过诱导肾小管上皮细胞EMT促进肾脏组织纤维化。我们应用masson染色、Real-Time PCR和western bloting技术检测了P311+/+和P311-/-小鼠肾纤维化组织中胶原含量和波形蛋白的表达。Masson染色结果显示P311-/-小鼠肾纤维化组织中胶原含量明显低于P311+/+小鼠。Real-Time PCR结果显示P311-/-小鼠肾纤维化组织中Ⅰ胶原m RNA水平明显低于P311+/+小鼠。Western Bloting技术检测结果显示P311敲除后,小鼠肾纤维化组织中波形蛋白含量明显下降。α-SMA是组织纤维化过程中肌成纤维细胞活化的典型标志物,因此我们利用免疫组织化学、Real-Time PCR和western bloting检测了P311+/+和P311-/-小鼠肾纤维化组织中α-SMA表达。免疫组织化学结果显示α-SMA蛋白主要表达于肾纤维化组织部分肾小管上皮细胞胞浆中,并且于P311阳性的细胞共定位表达。Real-Time PCR和Western Bloting结果显示α-SMA m RNA和蛋白在P311-/-小鼠肾纤维化组织中表达明显低于P311+/+小鼠。3、P311敲除后抑制了肾纤维化组织中TGF-β/Smad信号通路。TGF-β1是肾纤维化过程中肾小管上皮细胞EMT最关键的细胞因子,可以被损伤肾小管上皮细胞和浸润的巨噬细胞大量分泌,因此我们利用免疫组织化学、Real-Time PCR和western bloting检测了P311+/+和P311-/-小鼠肾纤维化组织中TGF-β1表达和巨噬细胞数量。免疫组织化学结果显示TGF-β1蛋白主要表达于肾纤维化组织部分肾小管上皮细胞胞浆中,并且于P311和α-SMA阳性的细胞共定位表达。Real-Time PCR结果显示TGF-β1 m RNA表达水平在P311+/+和P311-/-小鼠肾纤维化组织中无显著性差异,western bloting结果显示TGF-β1蛋白在P311-/-小鼠肾纤维化组织中表达明显低于P311+/+小鼠。TGF-β/Smad信号在肾纤维化病理发展过程中其中是否重要的作用,因此我们进一步利用Real-Time PCR和western bloting技术检测TGF-β1相关受体和Smad蛋白的表达。Real-Time PCR结果显示与P311+/+小鼠相比,TGF-β1Ⅰ型受体和TGF-β1Ⅱ型结果:受体m RNA表达水平在P311-/-小鼠肾纤维化组织明显下降。并且p-Smad2,Smad2,p-Smad3,Smad3,Smad4和Smad7蛋白P311-/-小鼠肾纤维化组织中表达明显低于P311+/+小鼠。综上所述,我们的研究发现P311可能通过增强TGF-β/Smad信号通路促进肾小管上皮细胞EMT和肾脏纤维化。本研究拓展了P311在纤维化疾病中的认识,可能为今后肾纤维病理过程的研究和治疗提供新的线索。
[Abstract]:The first part: the role of P311 in the re epithelialization of skin wound and its mechanism research background: wound repair is a dynamic and continuous process, which requires the joint participation of various types of cells, extracellular matrix and a series of cytokines and growth factors. Some chronic diseases, such as varicose veins and diabetes, can also cause the wounds to be difficult to heal. Hard healing wounds often lead to life-long disability, resulting in a huge economic and social burden on patients, families and society. According to statistics, the number of patients with chronic refractory wounds is now increasing worldwide. In view of this, in-depth study of the related mechanisms of wound healing, developing new wound healing methods and improving the quality of wound healing have become one of the key and difficult issues in this field. The healing of skin wounds in mammals is generally divided into four stages: coagulation, inflammation, new tissue formation and tissue remodeling. New tissue is formed. Epidermal stem cells are mainly distributed in the protuberance of the hair follicle, the basal layer of the epidermis and the sebaceous gland, which play a vital role in maintaining the skin epidermal growth and promoting the healing process. The.P311 gene of epidermal stem cells has now become a candidate cell for the clinical treatment of chronic refractory wounds. The.P311 gene is located on the human chromosome 5 and chromosome 18. It was first found in the mouse embryonic brain and maintained a high level of.P31 in the small brain, hippocampus and olfactory bulb of adult mice. 1 protein is a molecular weight of 8-K Da intracellular protein containing 68 amino acids and does not belong to any known protein family.P311 protein N terminal containing the PEST domain (rich in proline, glutamate, serine and threonine). In human, mice and chickens, the highly conserved.PEST domain is reduced by the ubiquitin / proteasome pathway in the target protein. P311 plays an important role in the interaction and activation of protein and protein. The present study has found that the regeneration of nerve and alveolus, the maintenance of blood pressure homeostasis, the invasion of glioblastoma and the activation of myofibroblast are important. Our previous study found that P311 is in the early proliferative scar myofibroblasts and in the early stage. The expression of TGF- beta 1 can play a role in the formation of hypertrophic scar, but it is not clear whether P311 plays a role in the re epithelialization of skin wounds. On this basis, we have made a study of the re epithelialization of P311 in the skin wound. The study mainly involves three aspects: 1) expression and distribution of P311 in skin wound surface and normal skin; 2) the role of P311 in the migration of epidermal stem cells; 3) the molecular mechanism of P311 to promote the migration of epidermal stem cells. Results: 1, the expression of P311 in the skin wound surface of human and mouse skin is increased. 5 cases of human skin wound tissue and normal skin were compared, and the whole layer skin defect model of mice was constructed, and the expression of P311 gene was detected in the samples. The immunohistochemical staining showed that P311 was mainly expressed in the cytoplasm of newborn epidermis cells in human and mouse skin wounds, while no P311 expressed.2 in normal skin epidermal cells, P311 Epidermal stem cell migration was promoted. The epidermal stem cells in the male prepuce of healthy young men were separated by the rapid adhesion method of type IV collagen. Cell immunofluorescence and flow cytometry were used to detect the expression of molecular markers in the epidermal stem cells in the isolated cells. Cytokeratin K19 and beta 1 integrin were detected by cell immunofluorescence. And the two were expressed in the cultured cells. Flow cytometry showed that the cultured cells expressed high expression of alpha 6 integrin, but negative marker CD71 expression was negative. We further detected the effect of P311 adenovirus vector transfection on the cell migration function after the transfection of the adenovirus vector to the epidermal stem cells. The results showed that the P311 enhancement was enhanced. The expression of epidermal stem cells could promote the migration of epidermal stem cells. We isolated the skin epidermal stem cells of P311-/- and P311+/+ newborn mice and used the flow cytometry for molecular identification. The results showed that the cultured cells expressed the alpha 6 integrin, but the negative marker CD71 was negative. Furthermore, the P311 knockout was used to detect the P311 knockout. The effect of epidermal stem cell migration was found to decrease the migration of epidermal stem cells after P311 knockout. We constructed a full-thickness skin defect model in mice to detect the effect of P311 knockout on the re epithelialization of the skin wounds. The results showed that the full layer skin defect model in mice could significantly inhibit the contraction of the skin wound of the mice, and the P311+/+ was small. The rate of re epithelialization of skin wound in rat skin was faster than that of P311-/- mice. The re epithelialization ability of the skin wound after P311 knockout decreased.3, and Rho GTPases in P311 promoted the migration of epidermal stem cells. In order to explore the mechanism of P311 to promote the migration of epidermal stem cells, we should use Pulldown technology to detect Rho GTPases Rho A. And Cdc42 activity. The results showed that the activity of Rho A, Rac1 and Cdc42 increased significantly after the enhanced expression of P311, and Rho A, Rac1 and Cdc42 activity decreased after the P311 knockout. Inhibiting the enhancement of P311's migration ability to epidermal stem cells, while Cdc42 inhibitors have no obvious effect. To sum up, our study first found that P311 may promote epidermal stem cell migration and wound re epithelialization by enhancing Rho A and Rac1 activity. This study expands the understanding of P311 in wound healing and may be the healing of wound healing in the future. Research and treatment provide new clues. The second part: the role of P311 in renal fibrosis and its mechanism research background: renal fibrosis is the result of the disorder of the self repair process of renal tissue under a variety of damage factors. In clinical, a large proportion of renal fibrosis patients will eventually develop to renal failure and need to be carried out for a lifetime. Hemodialysis or kidney transplantation to maintain life. A large number of epidemiological studies have shown that the prevalence of renal failure is increasing worldwide. Renal fibrosis has now developed into a global public health problem, causing huge social and economic burdens for individuals, families and societies. There is no effective treatment for renal fibrosis in bed. In view of this, it is one of the key and difficult problems in this field to study the pathological mechanism of renal fibrosis and to find the specific genes in the formation of renal fibrosis. Renal fibrosis is irreversible and progressive in many chronic renal diseases. The main characteristics of the pathological process of dynamic development are the synthesis and accumulation of a large number of extracellular matrix in the renal pellets and tubulointerstitium. This process requires the participation of various types of cells in the kidney. The activation of the matrix producing cells is considered to be the key to the pathological process of renal fibrosis, and the source of the matrix producing cells mainly includes the fibroblast fineness. Cell, renal tubular epithelial cells, vascular smooth muscle cells and macrophages. Renal tubular epithelial cells are the most important effector cells in the irreversible development of renal fibrosis stage. Under the action of a variety of fibrotic factors (especially TGF- beta 1), renal tubular epithelial cells are transformed by epithelial-mesenchymal Transi Tion, EMT) process, the cell phenotype changes, and transdifferentiation into myofibroblast.TGF- beta 1 is a key regulator in the EMT process of renal tubular epithelial cells, which can mediate the pathological process of a variety of chronic renal diseases, and can induce the expression of the renal tubular epithelial cells (vimentin, a fibroblast marker) and alpha muscle movement. Protein (alpha -smooth muscle actin, alpha -SMA, a myofibroblast cell marker).P311 was initially found in the mouse embryonic brain and maintained a high level of expression in the cerebellum, hippocampus and olfactory bulb in adult mice. The.P311 gene was located on chromosome 5, chromosome 18, and the encoding molecular weight of 8-K Da intracellular protein.P311 protein contains 68 ammonia. Basic acids are not yet known as any known protein families. Current studies have found that P311 plays an important role in nerve and alveolar development, glioma cell invasion and migration, homeostasis of blood pressure, myofibroblast differentiation and exercise. The study found that the high expression of P311 in early hypertrophic scars can be mediated by modulation. The expression of TGF- beta 1 and alpha -SMA promotes the differentiation of fibroblasts into myofibroblasts and plays a certain role in the formation of hypertrophic scars. On this basis, we have studied the role of P311 in renal fibrosis, mainly involved in the following three aspects: 1) the expression and differentiation of P311 in renal fibrosis and normal renal tissue. Cloth; 2) the effect of P311 knockout on renal fibrosis; 3) the molecular mechanism of P311 to promote renal fibrosis. Results: 1, the expression of P311 in human and mouse renal fibrosis tissues was increased. We collected 22 specimens of renal fibrosis and 2 normal renal tissue specimens, and established a mouse unilateral ureteral ligation model successfully induced little. .HE staining of P311 gene expression in the test samples showed that the renal tubule dilatation, atrophy and infiltration of numerous inflammatory cells were found in the renal fibrosis tissue. Masson staining showed the accumulation and accumulation of mass collagen fibers in the renal interstitium. Immunohistochemistry showed that P311 was mainly expressed in human and mouse kidney fibers. In the cytoplasm of the renal tubular epithelial cells of the vascular tissue, there is no P311 expression in normal renal tissue and.2, P311 may promote renal tissue fibrosis by inducing renal tubular epithelial cells EMT. We used Masson staining, Real-Time PCR and Western bloting technique to detect the collagen content and waveform in P311+/+ and P311- / - mice renal fibrosis tissues. The results of protein expression.Masson staining showed that collagen content in renal fibrosis tissues of P311-/- mice was significantly lower than that of.Real-Time PCR in P311+/+ mice. The level of collagen m RNA in the renal fibrosis tissues of P311-/- mice was significantly lower than that of P311+/+ mice.Western Bloting technique. The content of vimentin is obviously decreased. Alpha -SMA is a typical marker of myofibroblast activation during tissue fibrosis. Therefore, we used immunohistochemistry, Real-Time PCR and Western bloting to detect the expression of alpha -SMA in the renal fibrosis tissues of P311+/+ and P311-/- mice. The results of immunohistochemical staining showed that the alpha -SMA protein was mainly expressed in the expression of alpha -SMA protein. The expression of.Real-Time PCR and Western Bloting in P311 positive cells showed that the expression of alpha -SMA m RNA and protein in the renal fibrosis tissue of P311-/- mice was significantly lower than P311+/+ mice.3. .TGF- beta 1 is the most critical cytokine in renal tubular epithelial cell EMT during renal fibrosis, which can be secreted by damaged renal tubular epithelial cells and infiltrating macrophages. Therefore, we used immunohistochemistry, Real-Time PCR and Western bloting to detect the expression of TGF- beta 1 in the renal fibrosis tissues of P311+/+ and P311- / mice. The number of macrophages. Immunohistochemical results showed that TGF- beta 1 protein was mainly expressed in the cytoplasm of renal tubular epithelial cells in renal fibrosis tissue, and the co localization and expression of.Real-Time PCR in P311 and alpha -SMA positive cells showed that there was no significant difference in the expression of TGF- beta 1 m RNA in the renal fibrosis tissues of P311+/+ and P311-/- mice, w Estern bloting results showed that the expression of TGF- beta 1 protein in renal fibrosis of P311-/- mice was significantly lower than that of P311+/+.TGF- /Smad /Smad signal in kidney.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R641;R692

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