發(fā)布時間：2018-05-05 23:33

  本文選題：P311 + 表皮干細(xì)胞　； 參考：《第三軍醫(yī)大學(xué)》2016年博士論文

【摘要】：第一部分:P311在皮膚創(chuàng)面再上皮化中的作用及機(jī)制研究背景:創(chuàng)面修復(fù)是一個動態(tài)持續(xù)的過程,需要機(jī)體多種類型細(xì)胞、細(xì)胞外基質(zhì)及一系列細(xì)胞因子和生長因子的共同參與。難愈創(chuàng)面目前已成為一個重要的臨床難題,其原因除了傳統(tǒng)的燒傷、創(chuàng)傷及外科手術(shù)外,一些慢性疾病如靜脈曲張、糖尿病等也可引起創(chuàng)面難以愈合。難愈創(chuàng)面常�？蓪�(dǎo)致終生殘疾,給病人、家庭和社會造成巨大的經(jīng)濟(jì)社會負(fù)擔(dān)。據(jù)統(tǒng)計,目前患有慢性難愈創(chuàng)面的病人數(shù)量正在全球范圍內(nèi)逐漸增加。鑒于此,深入研究創(chuàng)面愈合的相關(guān)機(jī)制,發(fā)展新的創(chuàng)面治療方法,提高創(chuàng)面愈合質(zhì)量已成為該領(lǐng)域研究的熱點、重點和難點問題之一。哺乳動物皮膚創(chuàng)面愈合一般分為四個階段:凝血期、炎癥期、新生組織形成和組織重塑。新生組織形成起始于新生表皮遷移覆蓋受損真皮。表皮干細(xì)胞主要分布于毛囊隆突部位,表皮細(xì)胞基底層和皮脂腺組織中,在保持皮膚表皮更新、促進(jìn)創(chuàng)面愈合過程中起著至關(guān)重要的作用。當(dāng)創(chuàng)面形成后,表皮干細(xì)胞活化增殖產(chǎn)生更多短暫擴(kuò)充細(xì)胞,進(jìn)一步增殖、分化遷移至創(chuàng)面發(fā)揮修復(fù)作用。表皮干細(xì)胞目前已成為臨床上治療慢性難愈創(chuàng)面的候選細(xì)胞。P311基因定位于人5號染色體和小鼠18號染色體,首次在小鼠胚胎大腦發(fā)現(xiàn),并且在成年小鼠小腦、海馬和嗅球中保持較高水平表達(dá)。P311蛋白是一個分子量為8-k Da的胞內(nèi)蛋白,包含68個氨基酸,目前不屬于任何已知的蛋白家族。P311蛋白N末端包含PEST結(jié)構(gòu)域(富含脯氨酸、谷氨酸、絲氨酸和蘇氨酸),在人、小鼠和雞中高度保守。PEST結(jié)構(gòu)域在目的蛋白通過泛素/蛋白酶體通路降解及蛋白與蛋白之間相互作用和活化過程中發(fā)揮重要作用。目前研究發(fā)現(xiàn)P311在神經(jīng)和肺泡再生、血壓穩(wěn)態(tài)維持、神經(jīng)膠質(zhì)細(xì)胞瘤侵襲和肌成纖維細(xì)胞活化等方面發(fā)揮重要作用。我們前期研究發(fā)現(xiàn),P311在早期增生性瘢痕肌成纖維細(xì)胞和創(chuàng)面肉芽組織活化的成纖維細(xì)胞中表達(dá)增高,可以通過促進(jìn)TGF-β1表達(dá)在增生性瘢痕的形成過程中發(fā)揮一定作用。但目前P311在皮膚創(chuàng)面再上皮化過程是否發(fā)揮一定作用尚不清楚。在此基礎(chǔ)上,我們對P311在皮膚創(chuàng)面再上皮化過程中的作用進(jìn)行了研究,主要涉及以下三個方面:1)P311在皮膚創(chuàng)面新生表皮和正常皮膚表皮中的表達(dá)和分布;2)P311在表皮干細(xì)胞遷移中的作用;3)P311促進(jìn)表皮干細(xì)胞遷移的分子機(jī)制。結(jié)果:1、P311在人和小鼠皮膚創(chuàng)面新生表皮中表達(dá)增高。我們收集了5例人皮膚創(chuàng)面組織標(biāo)本和同體正常皮膚對照,同時構(gòu)建了小鼠全層皮膚缺損模型,檢測樣本中P311基因表達(dá)。免疫組織化學(xué)技術(shù)檢測發(fā)現(xiàn)P311主要表達(dá)于人和小鼠皮膚創(chuàng)面新生表皮細(xì)胞胞漿中,而正常皮膚表皮細(xì)胞中無P311表達(dá)。2、P311可以促進(jìn)表皮干細(xì)胞遷移。利用Ⅳ型膠原快速黏附法分離了健康青年男性包皮中表皮干細(xì)胞,應(yīng)用細(xì)胞免疫熒光和流式細(xì)胞技術(shù)檢測了分離細(xì)胞中表皮干細(xì)胞分子標(biāo)記物表達(dá)。細(xì)胞免疫熒光發(fā)現(xiàn)分離細(xì)胞中角蛋白K19和β1整合素高表達(dá),并且二者于培養(yǎng)細(xì)胞中共定位表達(dá)。流式細(xì)胞技術(shù)檢測發(fā)現(xiàn)培養(yǎng)細(xì)胞高表達(dá)α6整合素,而陰性標(biāo)志物CD71表達(dá)陰性。我們進(jìn)一步利用細(xì)胞體外創(chuàng)傷模型檢測了P311腺病毒載體轉(zhuǎn)染表皮干細(xì)胞后對細(xì)胞遷移功能的影響。結(jié)果發(fā)現(xiàn)P311增強(qiáng)表達(dá)后可以促進(jìn)表皮干細(xì)胞遷移。我們分離了P311-/-和P311+/+新生小鼠皮膚表皮干細(xì)胞并應(yīng)用流式細(xì)胞技術(shù)進(jìn)行分子鑒定,結(jié)果發(fā)現(xiàn)培養(yǎng)細(xì)胞高表達(dá)α6整合素,而陰性標(biāo)志物CD71表達(dá)陰性。進(jìn)一步利用細(xì)胞體外創(chuàng)傷模型檢測了P311敲除后對表皮干細(xì)胞遷移功能的影響,結(jié)果發(fā)現(xiàn)P311敲除后可以表皮干細(xì)胞遷移明顯下降。我們構(gòu)建了小鼠全層皮膚缺損模型檢測P311敲除后對皮膚創(chuàng)面再上皮化的影響。結(jié)果發(fā)現(xiàn)小鼠全層皮膚缺損模型可以顯著抑制小鼠皮膚創(chuàng)面收縮,P311+/+小鼠皮膚創(chuàng)面再上皮化速度明顯快于P311-/-小鼠,P311敲除后小鼠皮膚創(chuàng)面再上皮化能力減弱。3、Rho GTPases在P311促進(jìn)表皮干細(xì)胞遷移中的作用研究。為了探索P311促進(jìn)表皮干細(xì)胞遷移的作用機(jī)制,我們應(yīng)用Pulldown技術(shù)檢測了Rho GTPases Rho A、Rac1和Cdc42活性。結(jié)果發(fā)現(xiàn):P311增強(qiáng)表達(dá)后Rho A、Rac1和Cdc42活性明顯增高,同時P311敲除后Rho A、Rac1和Cdc42活性下降。我們應(yīng)用細(xì)胞體外創(chuàng)傷模型檢測了Rho A、Rac1和Cdc42特異性抑制劑對P311促進(jìn)表皮干細(xì)胞的影響。結(jié)果發(fā)現(xiàn)Rho A和Rac1抑制劑可以顯著抑制P311對表皮干細(xì)胞遷移能力的增強(qiáng)作用,而Cdc42抑制劑無明顯影響。綜上所述,我們的研究首次發(fā)現(xiàn)P311可能通過增強(qiáng)Rho A和Rac1活性促進(jìn)表皮干細(xì)胞遷移和創(chuàng)面再上皮化。本研究拓展了P311在創(chuàng)面愈合中的認(rèn)識,可能為今后創(chuàng)面愈合的研究和治療提供新的線索。第二部分:P311在腎纖維化中的作用及機(jī)制研究背景:腎纖維化是腎臟組織在多種損傷因素的刺激下自我修復(fù)過程失調(diào)的結(jié)果,臨床上,有很大部分比例的腎纖維化病人最終將發(fā)展為腎功能衰竭,需要終生進(jìn)行血液透析或腎臟移植治療維持生命。大量流行病學(xué)研究表明,腎功能衰竭病人的患病率在世界范圍內(nèi)正逐漸增加。腎纖維化疾病現(xiàn)在已發(fā)展成為全球范圍內(nèi)的公共健康問題,給患者個人、家庭及社會造成了巨大的社會經(jīng)濟(jì)負(fù)擔(dān)。目前,臨床上針對腎纖維化疾病尚無十分有效的治療方法。鑒于此,深入研究腎纖維化形成的病理機(jī)制,尋找腎纖維化形成的相關(guān)特異性基因是目前該領(lǐng)域研究的熱點、重點和難點問題之一。腎臟纖維化是多種慢性腎臟疾病中不可逆的、進(jìn)行性動態(tài)發(fā)展的病理過程,以腎臟小球及小管間質(zhì)中大量的細(xì)胞外基質(zhì)的合成和積聚為主要特征,該過程需要腎臟多種類型細(xì)胞的參與。基質(zhì)產(chǎn)生細(xì)胞的活化被認(rèn)為是腎纖維化病理過程中的關(guān)鍵,而基質(zhì)產(chǎn)生細(xì)胞的來源主要包括成纖維細(xì)胞,腎小管上皮細(xì)胞、血管平滑肌細(xì)胞和巨噬細(xì)胞。腎小管上皮細(xì)胞是腎纖維化晚期階段不可逆發(fā)展過程中最主要的效應(yīng)細(xì)胞,在多種促纖維化因子(尤其是TGF-β1)的作用下,腎小管上皮細(xì)胞經(jīng)歷上皮細(xì)胞間質(zhì)轉(zhuǎn)型(epithelial-mesenchymal transition,EMT)過程,細(xì)胞表型發(fā)生改變,轉(zhuǎn)分化成為肌成纖維細(xì)胞。TGF-β1是腎小管上皮細(xì)胞EMT過程中關(guān)鍵的調(diào)控分子,可以介導(dǎo)多種慢性腎臟疾病的病理發(fā)展過程,可以誘導(dǎo)腎小管上皮細(xì)胞表達(dá)波形蛋白(vimentin,一種成纖維細(xì)胞標(biāo)志)和α肌動蛋白(α-smooth muscle actin,α-SMA,一種肌成纖維細(xì)胞細(xì)胞標(biāo)志)。P311最初在小鼠胚胎大腦中發(fā)現(xiàn),在成年小鼠小腦、海馬和嗅球中保持較高水平表達(dá)。P311基因定位于人5號染色體、小鼠18號染色體,編碼分子量約8-k Da胞內(nèi)蛋白。P311蛋白包含68個氨基酸,尚不屬于任何已知蛋白質(zhì)家族。目前研究發(fā)現(xiàn)P311主要在神經(jīng)和肺泡發(fā)育、神經(jīng)膠質(zhì)瘤細(xì)胞侵襲及遷移、血壓穩(wěn)態(tài)維持、肌成纖維細(xì)胞分化和運動中發(fā)揮重要作用。本課題組研究發(fā)現(xiàn),P311基因在早期增生性瘢痕中高表達(dá),可以通過調(diào)節(jié)TGF-β1和α-SMA表達(dá)促進(jìn)成纖維細(xì)胞向肌成纖維細(xì)胞分化,在增生性瘢痕形成中發(fā)揮一定的促進(jìn)作用。在此基礎(chǔ)上,我們對P311在腎纖維化中的作用進(jìn)行了研究,主要涉及以下三個方面:1)P311在腎纖維化組織和正常腎臟組織中的表達(dá)和分布;2)P311敲除后對腎纖維化的影響;3)P311促進(jìn)腎臟纖維化的分子機(jī)制。結(jié)果：1、P311在人和小鼠腎纖維化組織表達(dá)增高。我們收集了22例人腎纖維化組織標(biāo)本和2例人正常腎臟組織標(biāo)本,同時構(gòu)建了小鼠單側(cè)輸尿管結(jié)扎模型成功地誘導(dǎo)了小鼠腎臟組織纖維化,檢測樣本中P311基因表達(dá)。HE染色顯示腎纖維化組織可見典型的腎小管擴(kuò)張、萎縮塌陷和大量炎癥細(xì)胞浸潤,Masson染色可見腎臟間質(zhì)大量膠原纖維沉積和積聚。免疫組織化學(xué)技術(shù)檢測發(fā)現(xiàn)P311主要表達(dá)于人和小鼠腎纖維化組織部分腎小管上皮細(xì)胞胞漿中,而正常腎臟組織中無P311表達(dá)。2、P311可能通過誘導(dǎo)腎小管上皮細(xì)胞EMT促進(jìn)腎臟組織纖維化。我們應(yīng)用masson染色、Real-Time PCR和western bloting技術(shù)檢測了P311+/+和P311-/-小鼠腎纖維化組織中膠原含量和波形蛋白的表達(dá)。Masson染色結(jié)果顯示P311-/-小鼠腎纖維化組織中膠原含量明顯低于P311+/+小鼠。Real-Time PCR結(jié)果顯示P311-/-小鼠腎纖維化組織中Ⅰ膠原m RNA水平明顯低于P311+/+小鼠。Western Bloting技術(shù)檢測結(jié)果顯示P311敲除后,小鼠腎纖維化組織中波形蛋白含量明顯下降。α-SMA是組織纖維化過程中肌成纖維細(xì)胞活化的典型標(biāo)志物,因此我們利用免疫組織化學(xué)、Real-Time PCR和western bloting檢測了P311+/+和P311-/-小鼠腎纖維化組織中α-SMA表達(dá)。免疫組織化學(xué)結(jié)果顯示α-SMA蛋白主要表達(dá)于腎纖維化組織部分腎小管上皮細(xì)胞胞漿中,并且于P311陽性的細(xì)胞共定位表達(dá)。Real-Time PCR和Western Bloting結(jié)果顯示α-SMA m RNA和蛋白在P311-/-小鼠腎纖維化組織中表達(dá)明顯低于P311+/+小鼠。3、P311敲除后抑制了腎纖維化組織中TGF-β/Smad信號通路。TGF-β1是腎纖維化過程中腎小管上皮細(xì)胞EMT最關(guān)鍵的細(xì)胞因子,可以被損傷腎小管上皮細(xì)胞和浸潤的巨噬細(xì)胞大量分泌,因此我們利用免疫組織化學(xué)、Real-Time PCR和western bloting檢測了P311+/+和P311-/-小鼠腎纖維化組織中TGF-β1表達(dá)和巨噬細(xì)胞數(shù)量。免疫組織化學(xué)結(jié)果顯示TGF-β1蛋白主要表達(dá)于腎纖維化組織部分腎小管上皮細(xì)胞胞漿中,并且于P311和α-SMA陽性的細(xì)胞共定位表達(dá)。Real-Time PCR結(jié)果顯示TGF-β1 m RNA表達(dá)水平在P311+/+和P311-/-小鼠腎纖維化組織中無顯著性差異,western bloting結(jié)果顯示TGF-β1蛋白在P311-/-小鼠腎纖維化組織中表達(dá)明顯低于P311+/+小鼠。TGF-β/Smad信號在腎纖維化病理發(fā)展過程中其中是否重要的作用,因此我們進(jìn)一步利用Real-Time PCR和western bloting技術(shù)檢測TGF-β1相關(guān)受體和Smad蛋白的表達(dá)。Real-Time PCR結(jié)果顯示與P311+/+小鼠相比,TGF-β1Ⅰ型受體和TGF-β1Ⅱ型結(jié)果:受體m RNA表達(dá)水平在P311-/-小鼠腎纖維化組織明顯下降。并且p-Smad2,Smad2,p-Smad3,Smad3,Smad4和Smad7蛋白P311-/-小鼠腎纖維化組織中表達(dá)明顯低于P311+/+小鼠。綜上所述,我們的研究發(fā)現(xiàn)P311可能通過增強(qiáng)TGF-β/Smad信號通路促進(jìn)腎小管上皮細(xì)胞EMT和腎臟纖維化。本研究拓展了P311在纖維化疾病中的認(rèn)識,可能為今后腎纖維病理過程的研究和治療提供新的線索。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R641;R692

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