本文選題：SFN + 創(chuàng)面愈合　； 參考：《西北大學(xué)》2016年碩士論文

【摘要】：研究背景創(chuàng)面愈合是外科學(xué)的主要問(wèn)題之一。近期有研究表明通過(guò)對(duì)Hippo通路中關(guān)鍵組成元件Yap的調(diào)控可以促進(jìn)創(chuàng)面愈合。SFN作為14-3-3家族成員之一,調(diào)節(jié)信號(hào)轉(zhuǎn)導(dǎo)通路及細(xì)胞周期,調(diào)節(jié)細(xì)胞增殖,分化,存活,凋亡。已有報(bào)道證明SFN蛋白通過(guò)與家族其他成員發(fā)生同源或是異源二聚化影響Yap的核漿穿梭。因此,我們推測(cè)截短型SFN可以通過(guò)控制Hippo通路中Yap的核漿穿梭調(diào)節(jié)表皮細(xì)胞增殖及肉芽組織形成�；谶@些研究,我們?cè)O(shè)計(jì)了缺少C端的40個(gè)氨基酸的截短型SFN,它與14-3-3家族成員形成異源二聚體影響了該家族與Yap的結(jié)合,導(dǎo)致Yap發(fā)生核轉(zhuǎn)位,Yap與PP2A結(jié)合進(jìn)入到細(xì)胞核,與TEAD發(fā)生作用,共同起始轉(zhuǎn)錄,因而,表皮細(xì)胞持續(xù)增殖,促進(jìn)創(chuàng)面愈合。實(shí)驗(yàn)?zāi)康奶剿饕吧秃徒囟绦蚐FN基因治療對(duì)創(chuàng)面愈合的影響,分析截短型SFN體內(nèi)影響Yap胞漿分布來(lái)調(diào)節(jié)Hippo信號(hào)通路促進(jìn)表皮細(xì)胞增殖的分子機(jī)制,為基因治療創(chuàng)面愈合的高效性提供科學(xué)依據(jù)。實(shí)驗(yàn)方法1)利用RT-PCR擴(kuò)增目的基因,分子克隆技術(shù)對(duì)野生型及截短型真核表達(dá)載體進(jìn)行構(gòu)建；2)使用Si02法大提質(zhì)粒并使用TritonX-114等溫相分離法去除內(nèi)毒素；3)將40只8w的健康雌性C57小鼠隨機(jī)分為5組,每只小鼠麻醉后背部脫毛,在脊柱中線兩側(cè)構(gòu)建小鼠全層皮膚缺損模型,PEI體內(nèi)介導(dǎo)F-127,空載體pSecX,野生型pSecX-SFN1和截短型pSecX-SFN2的基因治療,在各組小鼠治療第3d,第5d,第7d傷口組織取材,測(cè)量傷口愈合面積,分析血管化,對(duì)組織包埋切片,進(jìn)行HE染色和Masson染色,進(jìn)一步通過(guò)再上皮化及再生膠原纖維探討該基因?qū)?chuàng)面愈合的治療效果。實(shí)驗(yàn)結(jié)果1)成功構(gòu)建截短型和野生型真核表達(dá)載體,通過(guò)雙酶切及測(cè)序鑒定了其正確性；2)使用Si02等溫相內(nèi)毒素去除法提取到高純度質(zhì)粒；3)基因治療小鼠創(chuàng)面愈合。第3d時(shí),截短型SFN2治療組殘余傷口面積64.43±1.18mm2,傷口邊緣有少量較厚再上皮化和膠原纖維生成,各組均有肉芽組織形成。治療第5d時(shí),治療組與對(duì)照組統(tǒng)計(jì)學(xué)均有顯著性差異P0.05,野生型SFN1治療組殘留傷口面積為70.94±3.09mm2,再上皮化和膠原纖維最少,傷口附近的血管化較為豐富,截短型SFN2治療組的愈合速率遠(yuǎn)遠(yuǎn)超過(guò)其他組,殘留傷口面積僅剩16.02±2.59mm2,增殖的表皮細(xì)胞數(shù)量及沉積的膠原纖維最多,傷口及其周?chē)芑^少。治療第7d時(shí),各組表皮細(xì)胞由邊緣向中心位置增殖遷移,截短型SFN2治療組與野生型SFN1治療組統(tǒng)計(jì)學(xué)差異顯著P0.05,截短型SFN2治療組完全再上皮化,大量膠原纖維排列在表皮細(xì)胞下,血管化較少,結(jié)果顯示截短型pSecX-SFN2促進(jìn)傷口愈。
[Abstract]:Background wound healing is one of the major problems in surgery. Recent studies have shown that regulation of Yap, a key component of Hippo pathway, can promote wound healing as a member of 14-3-3 family, regulate signal transduction pathway and cell cycle, regulate cell proliferation, differentiation, survival and apoptosis. It has been reported that SFN protein affects the nuclear and cytoplasmic shuttle of Yap by homology or heterodimerization with other family members. Therefore, we speculate that truncated SFN can regulate epidermal cell proliferation and granulation tissue formation by controlling the nuclear and cytoplasmic shuttle of Yap in Hippo pathway. Based on these studies, we designed 40 truncated SFNs lacking C-terminal amino acids, which formed heterodimers with 14-3-3 family members to affect the binding of the family to Yap, resulting in the nuclear translocation of Yap, Yap and PP2A binding into the nucleus. It acts with TEAD and begins transcription together. Therefore, epidermal cells proliferate continuously and promote wound healing. Objective to investigate the effects of wild-type and truncated SFN gene therapy on wound healing, and to analyze the molecular mechanism of Hippo signaling pathway in regulating the cytoplasmic distribution of Yap in truncated SFN. To provide scientific basis for the high efficiency of gene therapy wound healing. Method 1) the target gene was amplified by RT-PCR. The wild and truncated eukaryotic expression vectors were constructed by molecular cloning technique. The plasmid was extracted by Si02 method and the endotoxin was removed by TritonX-114 isothermal phase separation method. 40 healthy female C57 mice were randomly divided into 5 groups. After anesthesia, each mouse was treated with hair loss on the back, and the full-thickness skin defect model was constructed on both sides of the midline of the spine. The gene therapy of F-127, pSecX, wild type pSecX-SFN1 and truncated pSecX-SFN2 was mediated in vivo. The wound tissue was taken from each group on the 3rd, 5th and 7th day of treatment. The wound healing area was measured, vascularization was analyzed, tissue embedded sections were stained with HE and Masson staining, and the therapeutic effect of the gene on wound healing was further studied by re-epithelialization and regeneration of collagen fibers. Results 1) truncated eukaryotic expression vectors and wild type eukaryotic expression vectors were successfully constructed, and their correctness was confirmed by double enzyme digestion and sequencing. The high purity plasmids were extracted by Si02 isothermal phase endotoxin removal method. On the 3rd day, the area of residual wound in truncated SFN2 group was 64.43 鹵1.18mm-2, there was a little thicker reepithelialization and collagen fiber formation at the edge of the wound, and granulation tissue was formed in each group. At the 5th day of treatment, there was significant difference between the treatment group and the control group (P 0.05). The area of residual wound in the wild-type SFN1 treatment group was 70.94 鹵3.09m2, and the reepithelialization and collagen fiber were the least, and the vascularization near the wound was abundant. The healing rate of truncated SFN2 group was much higher than that of other groups. The area of residual wound was only 16.02 鹵2.59mm2.The number of proliferative epidermis cells and collagen fibers deposited were the most, and the vascularization of wound and its surroundings was less. At the 7th day of treatment, the epidermal cells proliferated and migrated from the edge to the center in each group. There was a significant difference between the truncated SFN2 treatment group and the wild-type SFN1 treatment group (P0.05). The truncated SFN2 treatment group was completely re-epithelialized and a large number of collagen fibers were arranged under the epidermal cells. The results showed that truncated pSecX-SFN2 promoted wound healing.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R450;R64

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1 邵蕾;SFN基因治療促進(jìn)創(chuàng)面愈合的在體研究[D];西北大學(xué);2016年

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本文編號(hào)：1891287