方格星蟲多能性因子預(yù)測及其體表損傷修復的表達研究
發(fā)布時間：2018-10-24 09:47
【摘要】：干細胞多能性因子是一類能將體細胞誘導成多能干細胞的轉(zhuǎn)錄因子,誘導性多能干細胞與胚胎干細胞極其相似。多能性因子的概念一經(jīng)提出,其與損傷修復和再生的聯(lián)系就倍受關(guān)注。作為進入重編程的關(guān)鍵因素,其擁有的特質(zhì)與組織的再生關(guān)系密切。在各門類物種中,探索多能性因子與損傷修復和再生的聯(lián)系,有利于進一步確認多能性因子在再生過程中所處的重要地位,為再生醫(yī)學的研究提供理論依據(jù)。方格星蟲進化地位較低,然而其機體損傷修復的過程與高等生物非常相似。利用方格星蟲探索多能性因子與損傷修復和再生的聯(lián)系具有理論意義。本課題將立足于多能性因子,探索其與方格星蟲體表損傷修復之間的聯(lián)系。本課題基于方格星蟲轉(zhuǎn)錄組的測序結(jié)果,進行初步分析,發(fā)現(xiàn)24種候選干細胞多能性因子在方格星蟲中表達了17種,4種在誘導過程中發(fā)揮關(guān)鍵性作用的干細胞多能性因子,在方格星蟲中發(fā)現(xiàn)了3種,分別是Sox2、c-Myc和Klf4。另外Fthl處于所有組織內(nèi)的表達水平遠遠強于別的多能性因子。同時發(fā)現(xiàn)在方格星蟲中存在4條調(diào)控多能性的途徑。方格星蟲多能性因子同源基因與其再生相關(guān)同源基因在部分組織中的表達趨勢也存在相似性。根據(jù)預(yù)測結(jié)果從中選取出4種因子,Sox2、Klf4、c-Myc、Fthl以及WNT信號通路中的Wnt-7a、beta-Catenin,進行損傷修復的功能驗證。采用手術(shù)刀對方格星蟲肛門對側(cè)的體壁肌肉組織進行縱向劃傷,傷口長度約1 cm,深度約1-2 mm,損傷后分別在0 h、10 min、30 min、12 h、24 h、48 h、72 h采集損傷組織進行功能驗證,采用qPCR與免疫組化技術(shù)評估6種基因在損傷修復過程中表達量的變化以及組織細胞的增殖情況。實驗數(shù)據(jù)表明,在方格星蟲體表損傷修復過程中,這6種基因均能夠響應(yīng)損傷,并且Sox2、Klf4、Fthl、Wnt-7a和beta-Catenin在損傷修復過程中,能夠明顯的呈現(xiàn)出表達量的上調(diào)。從損傷后24 h開始,傷口附近的組織開始出現(xiàn)增殖的現(xiàn)象。根據(jù)實驗結(jié)果可以說明,在方格星蟲中,多能性因子與損傷修復確實存在著正相關(guān)的聯(lián)系。結(jié)合蚯蚓、蠑螈已有的研究結(jié)果推測,多能性因子與再生的聯(lián)系會隨著再生能力的增強而變得更加緊密。
[Abstract]:Stem cell pluripotent factor is a kind of transcription factor which can induce somatic cells into pluripotent stem cells. Induced pluripotent stem cells are very similar to embryonic stem cells. Once the concept of pluripotent factor is put forward, its relation with damage repair and regeneration has attracted much attention. As a key factor in reprogramming, its characteristics are closely related to organizational regeneration. To explore the relationship between pluripotent factors and injury repair and regeneration in various species is helpful to confirm the important position of pluripotent factors in regeneration process and to provide theoretical basis for the research of regenerative medicine. The phylogenetic status is low, but the process of damage repair is very similar to that of higher organisms. It is of theoretical significance to explore the relationship between pluripotent factors and damage repair and regeneration by using the starfish. Based on the pluripotent factor, this paper will explore the relationship between it and the repair of surface damage. Based on the sequencing results of the transcriptome, we found that 24 candidate stem cell pluripotent factors expressed 17 species and 4 stem cell pluripotent factors played a key role in the induction process. Three species, Sox2,c-Myc and Klf4., have been found in this field. In addition, the expression level of Fthl in all tissues is much stronger than other pluripotent factors. At the same time, there are 4 ways to regulate the pluripotency in the starfish. There was also similarity in the expression trend between the homologous genes of the multipotent factors and their regenerative homologous genes in some tissues. According to the predicted results, four factors, Sox2,Klf4,c-Myc,Fthl and Wnt-7a,beta-Catenin, in WNT signaling pathway, were selected to verify the function of damage repair. The muscle tissue of the opposite side of the anus of Scopodium oblongata was scratched longitudinally by scalpel. The wound length was about 1 cm, and the depth of injury was about 1-2 mm,. The injured tissues were collected at 0 h 10 min,30 min,12 h ~ 24 h and 48 h ~ 72 h for functional verification. QPCR and immunohistochemical techniques were used to evaluate the expression of six genes and the proliferation of histocytes in the process of injury and repair. The experimental data showed that the six genes were able to respond to the damage in the process of surface damage repair, and the expression of Sox2,Klf4,Fthl,Wnt-7a and beta-Catenin were up-regulated during the process of damage repair. From 24 hours after injury, the tissue around the wound began to proliferate. According to the experimental results, there is a positive correlation between pluripotent factors and damage repair. Combined with earthworm and salamander, it is speculated that the relationship between pluripotent factor and regeneration will become closer with the increase of regeneration ability.
【學位授予單位】：華僑大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R318;Q78
本文編號：2291035
[Abstract]:Stem cell pluripotent factor is a kind of transcription factor which can induce somatic cells into pluripotent stem cells. Induced pluripotent stem cells are very similar to embryonic stem cells. Once the concept of pluripotent factor is put forward, its relation with damage repair and regeneration has attracted much attention. As a key factor in reprogramming, its characteristics are closely related to organizational regeneration. To explore the relationship between pluripotent factors and injury repair and regeneration in various species is helpful to confirm the important position of pluripotent factors in regeneration process and to provide theoretical basis for the research of regenerative medicine. The phylogenetic status is low, but the process of damage repair is very similar to that of higher organisms. It is of theoretical significance to explore the relationship between pluripotent factors and damage repair and regeneration by using the starfish. Based on the pluripotent factor, this paper will explore the relationship between it and the repair of surface damage. Based on the sequencing results of the transcriptome, we found that 24 candidate stem cell pluripotent factors expressed 17 species and 4 stem cell pluripotent factors played a key role in the induction process. Three species, Sox2,c-Myc and Klf4., have been found in this field. In addition, the expression level of Fthl in all tissues is much stronger than other pluripotent factors. At the same time, there are 4 ways to regulate the pluripotency in the starfish. There was also similarity in the expression trend between the homologous genes of the multipotent factors and their regenerative homologous genes in some tissues. According to the predicted results, four factors, Sox2,Klf4,c-Myc,Fthl and Wnt-7a,beta-Catenin, in WNT signaling pathway, were selected to verify the function of damage repair. The muscle tissue of the opposite side of the anus of Scopodium oblongata was scratched longitudinally by scalpel. The wound length was about 1 cm, and the depth of injury was about 1-2 mm,. The injured tissues were collected at 0 h 10 min,30 min,12 h ~ 24 h and 48 h ~ 72 h for functional verification. QPCR and immunohistochemical techniques were used to evaluate the expression of six genes and the proliferation of histocytes in the process of injury and repair. The experimental data showed that the six genes were able to respond to the damage in the process of surface damage repair, and the expression of Sox2,Klf4,Fthl,Wnt-7a and beta-Catenin were up-regulated during the process of damage repair. From 24 hours after injury, the tissue around the wound began to proliferate. According to the experimental results, there is a positive correlation between pluripotent factors and damage repair. Combined with earthworm and salamander, it is speculated that the relationship between pluripotent factor and regeneration will become closer with the increase of regeneration ability.
【學位授予單位】：華僑大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R318;Q78
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