本文選題：無(wú)機(jī)三氧化聚合物 + 根尖牙乳頭干細(xì)胞�。� 參考：《南京醫(yī)科大學(xué)》2012年碩士論文

【摘要】：生物活性材料無(wú)機(jī)三氧化聚合物（mineral trioxide aggregate, MTA）現(xiàn)已廣泛應(yīng)用于根尖誘導(dǎo)成形術(shù)的臨床治療，并取得了較好的效果，但其對(duì)牙根發(fā)育及再生過(guò)程中的重要種子細(xì)胞——根尖牙乳頭干細(xì)胞（stem cells from apicalpapilla, SCAPs）增殖分化能力的影響，以及其中的作用機(jī)制尚不清楚。本文將分三個(gè)部分就這一問(wèn)題進(jìn)行探討分析： 第一部分MTA條件培養(yǎng)基的制備和誘導(dǎo)濃度篩選 研究目的：制備MTA條件培養(yǎng)基，篩選最佳誘導(dǎo)濃度，觀察其對(duì)SCAPs增殖能力的影響。 研究方法：制備MTA條件培養(yǎng)基，堿性磷酸酶（alkaline phosphatase, ALP）活性檢測(cè)實(shí)驗(yàn)篩選誘導(dǎo)SCAPs礦化的最佳濃度，通過(guò)茜素紅染色實(shí)驗(yàn)及10%氯代十六烷基吡啶（cetylpyridinium chloride, CPC）溶液溶解鈣離子定量實(shí)驗(yàn)佐證。另外，采用四甲基偶氮噻唑鹽（methyl-thiazolyl-tetrazolium, MTT）法繪制SCAPs生長(zhǎng)曲線，計(jì)算細(xì)胞群體倍增時(shí)間（population doubling time, PDT），以及流式細(xì)胞術(shù)（flow cytometry, FCM）檢測(cè)細(xì)胞周期分布，計(jì)算細(xì)胞的增殖指數(shù)（proliferation index, PI），檢測(cè)MTA條件培養(yǎng)基對(duì)SCAPs的增殖能力的影響。 實(shí)驗(yàn)結(jié)果： ALP活性檢測(cè)結(jié)果顯示，MTA條件培養(yǎng)基誘導(dǎo)3d時(shí)，2mg/mL組的SCAPs細(xì)胞ALP活性最高（P 0.01）。采用該濃度MTA誘導(dǎo)14d時(shí)，茜素紅染色結(jié)果顯示，誘導(dǎo)組礦化結(jié)節(jié)形成能力明顯高于未誘導(dǎo)組（P 0.01）。PDT和細(xì)胞周期分析后的PI結(jié)果均顯示，兩組SCAPs的增殖能力無(wú)明顯差異（P0.05）。 結(jié)論：2mg/mL MTA不影響SCAP細(xì)胞的增殖能力，但對(duì)SCAPs的礦化能力有明顯的促進(jìn)作用。 第二部分MTA對(duì)SCAPs分化以及形態(tài)發(fā)生能力的影響 研究目的：進(jìn)一步明確MTA對(duì)SCAPs牙向/骨向分化以及形態(tài)發(fā)生能力的影響。 研究方法：提取MTA誘導(dǎo)3d和7d時(shí)的細(xì)胞總RNA和總蛋白，通過(guò)實(shí)時(shí)定量逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)實(shí)驗(yàn)（real-time reverse transcription polymerase chainreaction, real-time RT-PCR）檢測(cè)牙向/骨向分化相關(guān)標(biāo)志基因（ALP, DSPP,RUNX2, OCN等）的表達(dá)情況，蛋白免疫印跡實(shí)驗(yàn)（Western blot）檢測(cè)牙向/骨向分化相關(guān)標(biāo)志蛋白（DSP, RUNX2, OCN等）的表達(dá)情況。另外，將細(xì)胞團(tuán)復(fù)合根管支架材料，體內(nèi)移植后，觀測(cè)誘導(dǎo)后SCAPs的形態(tài)發(fā)生能力。 實(shí)驗(yàn)結(jié)果：Real-time RT-PCR和Western blot結(jié)果顯示，MTA誘導(dǎo)組OCN、RUNX2、DSP等基因/蛋白表達(dá)水平明顯高于未誘導(dǎo)組，提示MTA可增強(qiáng)SCAPs的牙向/骨向分化能力。體內(nèi)移植實(shí)驗(yàn)發(fā)現(xiàn)，與未誘導(dǎo)組相比，MTA誘導(dǎo)后的SCAPs可以形成類牙本質(zhì)牙髓復(fù)合體結(jié)構(gòu)，并且表達(dá)較高水平的DSP、RUNX2、OCN等礦化相關(guān)蛋白。 結(jié)論：MTA明顯促進(jìn)SCAPs的牙/骨向分化、牙本質(zhì)牙髓復(fù)合體形成能力。 第三部分MTA調(diào)控SCAPs分化的NF-κB通路機(jī)制 研究目的：探討核因子κB（nuclear factor kappa B, NF-κB）通路在MTA誘導(dǎo)的SCAPs分化過(guò)程中的調(diào)控作用。 研究方法：選取MTA條件培養(yǎng)基刺激0h、0.25h、0.5h、3h、6h和12h，分別提取各時(shí)間點(diǎn)的SCAPs胞漿蛋白和胞核蛋白，Western blot檢測(cè)通路相關(guān)蛋白（NF-κB P65、IκB-α、磷酸化NF-κB P65、磷酸化IκB-α）的表達(dá)，檢測(cè)NF-κB通路的激活情況。另外，制作細(xì)胞爬片，將上述六個(gè)時(shí)間點(diǎn)的SCAPs固定后，對(duì)NF-κB P65進(jìn)行免疫熒光染色，觀察P65的核轉(zhuǎn)位情況，，間接反映NF-κB通路的活性狀態(tài)。 實(shí)驗(yàn)結(jié)果：NF-κB通路關(guān)鍵蛋白的Western blot和熒光染色檢測(cè)結(jié)果表明，P65核轉(zhuǎn)位明顯。表達(dá)結(jié)果顯示：P65逐漸由胞漿轉(zhuǎn)入核內(nèi)；p-P65在0.25h表達(dá)已顯著升高，并在0.5h時(shí)繼續(xù)上升，但隨后開始下降；IκB-α先略有下降，然后恢復(fù)；p-IκB-α在0.25h表達(dá)即到最高，0.5h時(shí)雖開始下降但仍為高表達(dá)。提示NF-κB通路被激活。NF-κB P65的熒光染色檢測(cè)亦表明，P65轉(zhuǎn)入細(xì)胞核內(nèi)啟動(dòng)相關(guān)轉(zhuǎn)錄程序。 結(jié)論：MTA誘導(dǎo)SCAPs的牙/骨向分化和形態(tài)發(fā)生，可能是通過(guò)激活NF-κB通路來(lái)實(shí)現(xiàn)的。
[Abstract]:Mineral trioxide aggregate (MTA), a bioactive material, has been widely used in the clinical treatment of apex inducement and has achieved good results. However, it is an important seed cell in the process of root development and regeneration (stem cells from apicalpapilla, SCAPs) in root development and regeneration. The influence of chemical capacity and the mechanism of action are not yet clear. This article will discuss and analyze the problem in three parts.
Part one preparation of MTA conditioned medium and screening of induced concentration
Objective: to prepare MTA conditioned medium and screen the best induction concentration and observe its effect on SCAPs proliferation.
Research methods: preparation of MTA conditioned medium, alkaline phosphatase (alkaline phosphatase, ALP) activity detection test to screen the best concentration of SCAPs mineralization, by alizarin red staining experiment and 10% Chloroalkyl sixteen alkyl pyridine (cetylpyridinium chloride, CPC) solution calcium ion quantitative test evidence. In addition, the use of four methyl azothiazide. The growth curve of SCAPs was plotted by methyl-thiazolyl-tetrazolium (MTT), and the cell multiplication time (population doubling time, PDT) was calculated and the cell cycle distribution was detected by flow cytometry (flow cytometry, FCM). The proliferation index (proliferation index) was calculated and the proliferation ability of the conditioned medium was detected. Influence.
The results of ALP activity detection showed that the ALP activity of SCAPs cells in 2mg/mL group was the highest (P 0.01) when MTA conditioned medium induced 3D. The results of alizarin red staining showed that the formation ability of the mineralized nodules in the induced group was significantly higher than that of the uninduced group (P 0.01).PDT and the PI results after cell cycle analysis were all displayed, two There was no significant difference in the proliferation ability of group SCAPs (P0.05).
Conclusion: 2mg/mL MTA does not affect the proliferation of SCAP cells, but has a significant effect on SCAPs mineralization.
The second part is the effect of MTA on SCAPs differentiation and morphogenesis.
Objective: to further clarify the effect of MTA on SCAPs's odontogenic / osteogenic differentiation and morphogenesis.
Methods: to extract the total RNA and total protein of 3D and 7d induced by MTA, and to detect the expression of tooth / bone differentiation related genes by real-time quantitative reverse transcriptase polymerase chain reaction (real-time reverse transcription polymerase chainreaction, real-time RT-PCR), protein immuno printing The trace test (Western blot) was used to detect the expression of DSP, RUNX2, OCN and so on. In addition, after transplantation, the morphogenetic ability of SCAPs was observed after transplantation in vivo.
The results of Real-time RT-PCR and Western blot showed that the expression level of OCN, RUNX2, DSP and other genes and proteins in MTA induced group was significantly higher than that in the uninduced group, suggesting that MTA could enhance the tooth / bone differentiation ability of SCAPs. In vivo transplantation experiment found that SCAPs after MTA inducement could form the dentinal pulp complex knot of the dentin like group compared with the uninduced group. It also expressed higher levels of mineralization related proteins such as DSP, RUNX2 and OCN.
Conclusion: MTA can significantly promote the SCAPs / odontoblast differentiation and dentin pulp complex formation ability.
The third part is the mechanism of NF- kappa B pathway regulated by MTA in SCAPs differentiation.
Objective: To investigate the regulatory role of B nuclear factor kappa B (NF- B) pathway in MTA induced SCAPs differentiation.
Methods: MTA conditioned medium was selected to stimulate 0h, 0.25h, 0.5h, 3h, 6h and 12h, respectively, to extract SCAPs cytoplasmic protein and nucleoprotein at each time point, and Western blot to detect the expression of pathway related proteins. After fixation with SCAPs at six time points, immunofluorescence staining of NF- kappa B P65 was performed to observe the nuclear translocation of P65 and indirectly reflect the activity of NF- kappa B pathway.
The results showed that the Western blot and fluorescence staining of the key protein of the NF- kappa B pathway showed that the transposition of the P65 nucleus was obvious. The expression results showed that P65 was gradually transferred from the cytoplasm to the nucleus; the expression of p-P65 in 0.25h increased significantly, and continued to rise at 0.5h, but then began to decline; I kappa B- alpha decreased slightly and then recovered; p-I kappa B- alpha was 0.2 The expression of 5h is the highest, while 0.5h begins to decline but is still high, suggesting that the NF- kappa B pathway is activated by the fluorescence staining of.NF- kappa B P65 and indicates that P65 is transferred into the nucleus to start the related transcriptional program.
Conclusion: MTA induced SCAPs tooth / bone differentiation and morphogenesis may be achieved by activating NF- kappa B pathway.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R783.1

【共引文獻(xiàn)】

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