本文選題：成牙本質(zhì)細胞 + ceRNA��； 參考：《武漢大學(xué)》2016年博士論文

【摘要】：1、Sp1 mRNA作為Klf4的ceRNA促進成牙本質(zhì)細胞分化研究目的：成牙本質(zhì)細胞分化是一種非常獨特且復(fù)雜的過程,有很多種分子機制參與調(diào)控成牙本質(zhì)細胞分化,包括生長因子,轉(zhuǎn)錄因子,microRNA和信號通路等,這些分子相互聯(lián)系形成網(wǎng)絡(luò)并從不同層面上調(diào)節(jié)成牙本質(zhì)細胞分化。CeRNA即競爭性內(nèi)源RNA,它是一種來源于生物體內(nèi)部的編碼或非編碼RNA,通過自身的microRNA反應(yīng)元件和相關(guān)的microRNA結(jié)合,從而競爭性的對受共同的microRNA調(diào)控的其他靶基因調(diào)控,在細胞分化,器官形成和疾病發(fā)生中有著重要作用。本研究試圖探索在成牙本質(zhì)細胞分化過程中以轉(zhuǎn)錄因子Klf4為中心的ceRNA調(diào)控網(wǎng)絡(luò)及其功能。研究方法：在本研究中,利用Targetscan數(shù)據(jù)庫預(yù)測Klf4的MRE及其結(jié)合的miRNA,根據(jù)共享miRNA的數(shù)量來預(yù)測KLF4的候選ceRNA。將體外誘導(dǎo)小鼠牙乳頭永生化細胞系(mDPC6T細胞)向成牙本質(zhì)細胞樣細胞分化作為模型。利用實時熒光定量PCR(qRT-PCR)檢測候選ceRNA中隨機挑選的六個基因在nDPC6T細胞分化過程中的表達模式；同時利用mRNA原位雜交技術(shù)(ISH)檢測目標(biāo)基因Sp1與Klf4的mRNA在出生后第二天小鼠下頜切牙中的表達模式。利用siRNA轉(zhuǎn)染的方法分別抑制Sp1與Klf4的表達,然后提取總RNA與總蛋白質(zhì),利用qRT-PCR與蛋白免疫印跡技術(shù)(western blot)檢測Sp1與Klf4在mRNA及蛋白水平表達的變化。利用雙熒光素酶報告實驗檢測Sp1與Klf4分別對于Klf4 3'UTR與Spl 3'UTR的作用。通過siRNA技術(shù)抑制Dicer酶的方法檢測Sp1與Klf4之間的相互調(diào)控作用是否具有microRNA依賴性。通過western blot技術(shù)檢測過表達共享microRNA對于Spl與Klf4的調(diào)控作用,并利用雙熒光素酶報告實驗驗證了mir-la, mir-7a-5p, mir-135a-5p和mir-29b與Spl與Klf4確實存在結(jié)合。最后,在mDPC6T細胞中過表達Spl 3'UTR,利用qRT-PCR和堿性磷酸酶活性檢測試驗驗證Sp1 3'UTR對成牙本質(zhì)細胞分化的影響。研究結(jié)果：以Klf4為中心的候選ceRNA有30個,隨機挑選的六個基因中,Spl, Qk和Tnrc6b的mRNA表達水平都顯著上升,這與Klf4 mRNA的表達趨勢一致。但是,Nfib,Nova1和Bc111a的mRNAb表達水平是顯著下降的,與Klf4 mRNA的表達趨勢相反；Spl和Klf4的mRNA在PN2小鼠的下頜切牙的成牙本質(zhì)細胞層中具有非常相似的表達模式。抑制Spl的表達能夠顯著下調(diào)Klf4的表達水平,同樣的,抑制Klf4的表達能顯著下調(diào)Spl的表達水平。下調(diào)Spl的表達能夠顯著抑制含有Klf4 3'UTR區(qū)的雙熒光素報告質(zhì)粒的熒光素酶活性,下調(diào)Klf4的表達能夠顯著抑制Spl 3'UTR區(qū)熒光素酶活性。當(dāng)Dicer的表達受到抑制時,Spl和Klf4的相互調(diào)控的microRNA依賴性消失；miR-la, miR-7a, miR-29b和miR-135a能同時與Spl和Klf4結(jié)合并能抑制Spl和Klf4的表達；Sp1 3'UTR能夠上調(diào)DMP1,DSPP和ALP的mRNA表達水平,并能上調(diào)堿性磷酸酶的活性。結(jié)論：SP1 mRNA作為KLF4的ceRNA能夠通過與Klf4競爭性的結(jié)合mir-1a, mir-7a-5p, mir-135a-5p和mir-29b米上調(diào)Klf4的表達,從而促進成牙本質(zhì)細胞分化。2、轉(zhuǎn)錄因子SP1通過激活DMP1啟動子的轉(zhuǎn)錄來促進成牙本質(zhì)細胞分化研究目的：成牙本質(zhì)細胞是一種神經(jīng)嵴來源的呈現(xiàn)極化特征的終末分化的細胞。牙乳頭細胞(DPCs)通常被認(rèn)為是成牙本質(zhì)細胞的祖細胞,他們具有分化為成牙本質(zhì)細胞的潛能。轉(zhuǎn)錄因子是一類能夠與基因啟動子區(qū)特異性的結(jié)合,從而確保目的基因以特定的強度在特定的時間與空間表達的蛋白質(zhì)分子。已有多種轉(zhuǎn)錄因子被證實參與了成牙本質(zhì)細胞的分化。SP1屬于specificity protein/Kruppel-like轉(zhuǎn)錄因子(SP/KLF)家族。SP1在很多生理過程中發(fā)揮了很重要的作用,包括細胞的分化,凋亡以及胚胎的形成等。在不同的組織器官中,SP1在細胞分化過程中都具有很重要的作用。本研究試圖探討轉(zhuǎn)錄因子SP1在成牙本質(zhì)細胞分化過程中的作用及其作用機制。研究方法：在本研究中,我們利用免疫組化的方法,檢測PN2小鼠切牙中SP1蛋白的表達模式。我們利用了在體外誘導(dǎo)小鼠牙乳頭細胞系(mDPC6T細胞),來模擬牙乳頭原代細胞向成牙本質(zhì)細胞分化的過程。mDPC6T細胞在礦化誘導(dǎo)液中連續(xù)培養(yǎng)0,1,5,7,11,14天,提取相應(yīng)天數(shù)的蛋白,通過Western blot檢測SP1蛋白的表達。同時,我們利用免疫組化檢測了SP1蛋白在mDPC6T細胞向成牙本質(zhì)細胞分化中的定位。為了探索SP1蛋白在小鼠成牙本質(zhì)細胞分化中的功能,在mDPC6T細胞中過表達SP1的蛋白編碼區(qū)(CDS)或者抑制SP1的表達,并通過qRT-PCR技術(shù)和蛋白免疫印跡技術(shù)來檢測成牙本質(zhì)細胞分化過程中標(biāo)志性基因DMP1, DSPP和ALP的表達水平。我們還應(yīng)用檢測堿性磷酸酶活性和茜素紅染色的方法來驗證過表達或者抑制SP1表達對于堿性磷酸酶活性和礦化結(jié)節(jié)形成量的影響。利用雙熒光素酶報告實驗來驗證SP1蛋白是否可激活DMP1啟動子活性,通過在mDPC6T細胞中抑制SP1的表達的同時過表達DMP1來檢測DMP1是否能挽救抑制SP1導(dǎo)致的成牙本質(zhì)細胞分化受阻。研究結(jié)果：SP1蛋白在前期成牙本質(zhì)細胞中沒有表達,高表達于極化期的成牙本質(zhì)細胞中；在分泌期的成牙本質(zhì)細胞中SP1蛋白信號強度較弱；僅有極少數(shù)的成熟期的成牙本質(zhì)細胞表達SP1蛋白。在mDPC6T細胞分化過程中,SP1蛋白水平在第7天出現(xiàn)顯著上調(diào),在第11天與第14天時,SP1的蛋白一直維持高表達水平。免疫熒光顯示在第0天時,SP1蛋白表達在mDPC6T細胞的胞漿和胞核里,且在胞核表達較少；在誘導(dǎo)第7天時,SP1蛋白信號的亮度明顯增強,且SP1蛋白主要集中在胞核中,在胞漿中也有微弱表達。過表達SP1可上調(diào)DMP1,DSPP和ALP mRNA和蛋白表達水平,并可促進礦化結(jié)節(jié)的形成；抑制SP1表達可下調(diào)DMP1, DSPP和ALP mRNA和蛋白表達水平,并可抑制礦化結(jié)節(jié)的形成。雙熒光素酶報告實驗發(fā)現(xiàn)SP1可以激活DMP1的啟動子,并可促進DMP1的表達,并且過表達DMP1可在一定程度上挽救由于抑制SP1表達而引起的成牙本質(zhì)細胞分化受阻現(xiàn)象。結(jié)論：轉(zhuǎn)錄因子SP1在小鼠牙乳頭細胞向成牙本質(zhì)細胞分化過程中表達上調(diào),并可以通過上調(diào)DMP 1啟動子活性來促進成牙本質(zhì)細胞分化。
[Abstract]:1, Sp1 mRNA as Klf4 ceRNA promotes odontoblast differentiation. The differentiation of odontoblast cells is a very unique and complex process. There are many molecular mechanisms involved in the regulation of odontoblast differentiation, including growth factors, transcription factors, microRNA and signal transduction pathways. These molecules interact with each other to form a network and form a network. Modulating the differentiation of odontoblast cells at different levels,.CeRNA, a competitive endogenous RNA, is a encoding or non coding RNA derived from the internal organism, combining its own microRNA reaction element with the associated microRNA, so as to compete for the regulation of its target gene regulated by common microRNA, in cell differentiation, organ formation and This study attempts to explore the ceRNA regulatory network and its function centered on the transcription factor Klf4 in the process of odontoblast differentiation. In this study, the Targetscan database is used to predict the MRE of Klf4 and the miRNA of its binding, and to predict the candidate ceRNA. of KLF4 based on the number of shared miRNA. In vitro induction of mouse dental papilla immortalized cell line (mDPC6T cells) to odontoid cell like cell differentiation as a model. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression pattern of six randomly selected genes in the candidate ceRNA in nDPC6T cell differentiation; the target gene was detected by mRNA in situ hybridization (ISH) at the same time. The expression pattern of Sp1 and Klf4 mRNA in the mandibular incisor of mice at second days after birth. The expression of Sp1 and Klf4 was inhibited by siRNA transfection, then the total RNA and total protein were extracted, and the changes in the expression of Sp1 and Klf4 were detected by qRT-PCR and protein immunoblotting (Western blot). The double luciferase reporter was used. The effect of Sp1 and Klf4 on Klf4 3'UTR and Spl 3'UTR respectively. The interaction between Sp1 and Klf4 was detected by siRNA technology to detect the mutual regulation between Sp1 and Klf4. The experiment verified that mir-la, mir-7a-5p, mir-135a-5p and mir-29b have a real combination with Spl and Klf4. Finally, the over expression of Spl 3'UTR in mDPC6T cells, and the effect of qRT-PCR and alkaline phosphatase activity test to verify the effect of Sp1 3'UTR on the differentiation of dentin cells. Of the six genes, the mRNA expression level of Spl, Qk and Tnrc6b increased significantly, which was in accordance with the expression trend of Klf4 mRNA. However, the mRNAb expression level of Nfib, Nova1 and Bc111a decreased significantly, contrary to the expression trend of Klf4 mRNA, and the expressions were very similar in the dentinal cell layer of the lower jaw incisors of the mice. The expression of inhibition of Spl can significantly downregulate the expression level of Klf4. Similarly, inhibition of the expression of Klf4 can significantly downregulate the expression level of Spl. Down regulation of Spl can significantly inhibit the luciferase activity of the double luciferase reporter plasmid containing Klf4 3'UTR region, and the down-regulation of Klf4 expression can significantly inhibit the Spl 3'UTR region fluorescein. Enzyme activity. When the expression of Dicer is inhibited, the microRNA dependence of the mutual regulation of Spl and Klf4 disappears; miR-la, miR-7a, miR-29b and miR-135a can simultaneously be combined with Spl and Klf4 and can inhibit the expression of Spl and Klf4. MRNA as the ceRNA of KLF4 can increase the expression of Klf4 by competing with Klf4, mir-1a, mir-7a-5p, mir-135a-5p and mir-29b meters, thus promoting the differentiation of odontoblast cells.2. The transcription factor SP1 activates the transcription of the DMP1 promoter to promote the development of odontoblast differentiation: the odontoblast is a neural crest. The cells of the terminal differentiation of the origin of polarization. Dental papilla cells (DPCs) are usually considered as progenitor cells of odontoblast cells, and they have the potential to differentiate into odontoblast cells. Protein molecules expressed in space. Many transcriptional factors have been confirmed to be involved in the differentiation of odontoblast cells.SP1 belongs to the specificity protein/Kruppel-like transcription factor (SP/KLF) family.SP1, which plays an important role in many physiological processes, including cell differentiation, apoptosis, and embryo formation. In different tissues SP1 plays an important role in the process of cell differentiation. This study attempts to explore the role and mechanism of the transcription factor SP1 in the process of odontoblast differentiation. In this study, we used immunohistochemical method to detect the expression pattern of SP1 protein in the incisor of PN2 mice. The mouse dental papilla cell line (mDPC6T cell) was induced to simulate the differentiation of dental papilla cells to odontoblasts..mDPC6T cells were continuously cultured for 0,1,5,7,11,14 days in the mineralized inducer, and the protein of the corresponding days was extracted, and the expression of SP1 protein was detected by Western blot. At the same time, we detected the SP1 protein by immunohistochemistry. Localization of mDPC6T cells to odontoblast differentiation. In order to explore the function of SP1 protein in mouse odontoblast differentiation, the expression of SP1 protein coding region (CDS) or inhibition of SP1 expression in mDPC6T cells, and the identification of the differentiation process of odontoblast by qRT-PCR technique and protein immunoblotting technique The expression level of sex genes DMP1, DSPP and ALP. We also tested the effects of alkaline phosphatase activity and alizarin red staining to verify the effect of overexpression or inhibition of SP1 expression on the activity of alkaline phosphatase and the formation of mineralized nodules. The double luciferase reporter assay was used to verify whether the SP1 protein could activate the activity of DMP1 promoter. Over expression of SP1 in mDPC6T cells and over expression of DMP1 to detect whether DMP1 can save the differentiation of odontoblast induced by inhibition of SP1. The results: SP1 protein is not expressed in early dentin cells, highly expressed in the dentin cells of polarization stage, and SP1 protein in the odontoblasts of the secretory phase. The signal intensity was weak; only a few mature dentin cells expressed SP1 protein. In the process of mDPC6T cell differentiation, the level of SP1 protein increased significantly on seventh days, and the protein of SP1 remained high at the eleventh and fourteenth days. The immunofluorescence showed that the SP1 protein was expressed in the cytoplasm of mDPC6T cells at the time of immunofluorescence. The expression of SP1 protein signal was obviously enhanced at seventh days, and the SP1 protein was mainly concentrated in the nucleus and weak expression in the cytoplasm. Overexpression of SP1 could increase the level of DMP1, DSPP and ALP mRNA and protein expression, and promote the formation of mineralized nodules, and reduce the expression of SP1 to reduce DMP1, DSPP. And ALP mRNA and protein expression levels, and inhibit the formation of mineralized nodules. Double luciferase reporter experiment found that SP1 activates the promoter of DMP1 and promotes the expression of DMP1, and overexpression of DMP1 can save the differentiation of dentin cells caused by inhibition of SP1 expression to a certain extent. Conclusion: transcription factor SP1 It was upregulated in odontoblast differentiation of mouse dental papilla cells and could promote odontoblast differentiation by up regulating the activity of DMP 1 promoter.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R78

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