【摘要】：目的 根據(jù)前期研究結(jié)果及文獻復(fù)習，我們得出如下假設(shè)：1. CXCR4陽性牙髓細胞（CXC chemokine receptor4-positive dental pulp cells，CXCR4+DPCs）可能是牙髓在形成修復(fù)性牙本質(zhì)過程中起重要作用的干/前體細胞；2.低氧誘導(dǎo)因子-1α（Hypoxia-inducible factor-1α，HIF-1α）可能是牙髓損傷后啟動牙髓修復(fù)反應(yīng)的重要啟動因子；3.去鐵胺（Deferoxamine, DFO）可能通過HIF-1α促進牙髓組織的修復(fù)能力。為驗證上述假設(shè)，我們采用免疫磁珠篩選CXCR4+DPCs并進行鑒定，然后考察CXCR4+DPCs的細胞克隆形成能力及多向分化能力，從而證明CXCR4+DPCs具有干細胞的特性；采用DFO處理DPCs，觀察其修復(fù)能力的變化，并結(jié)合RNA干擾（RNA interference，RNAi）技術(shù)，探討DFO通過HIF-1α調(diào)節(jié)修復(fù)性牙本質(zhì)形成的分子機理。 材料和方法 1.采用免疫磁珠篩選CXCR4+DPCs并進行鑒定，然后考察CXCR4+DPCs的細胞克隆形成能力及多向分化能力。 2.不同濃度DFO作用DPCs不同時間后，從對細胞活性、增殖及遷移等幾個方面的影響挑選出DFO的最佳作用濃度及時間。 3.以腺病毒作為載體，構(gòu)建HIF-1α shRNA，轉(zhuǎn)染DPCs，干擾HIF-1α基因的表達并進行驗證。 4.體外觀察DFO作用HIF-1α基因沉默前/后DPCs成牙本質(zhì)分化能力的變化。 5.觀察DFO預(yù)處理HIF-1α基因沉默前/后DPCs在體內(nèi)形成牙本質(zhì)樣組織的情況。 結(jié)果 1．成功分離和鑒定CXCR4+DPCs，證實其具有增殖能力強及多向分化能力特點。 2．10μM DFO處理DPCs48h，可促進其增殖和橫向遷移，并能提高其HIF-1α的表達，但是對其細胞活性和趨化作用的影響不明顯。 3. HIF-1α干擾腺病毒（pAd/pENTR/shRNA-/GFP-HIF-1α）在mRNA水平的干擾效率為73.4%，并在蛋白水平成功阻斷了DFO促進DPCs HIF-1α的表達效果。 4.10μM DFO處理DPCs48h，可以在體外促進其成牙本質(zhì)分化。幾個與成牙本質(zhì)分化相關(guān)的基因也被上調(diào)。沉默HIF-1α基因后，DFO成牙本質(zhì)分化的促進作用即被消除。 5.體內(nèi)實驗結(jié)果顯示10μM DFO預(yù)處理DPCs48h后，可在體內(nèi)形成更多的牙本質(zhì)樣組織，沉默HIF-1α基因后，DPCs幾乎不能形成牙本質(zhì)樣組織。 結(jié)論 1.本研究中我們成功分離CXCR4+DPCs，并證實其具有DPSCs的特性。 2. DFO處理DPCs的最佳作用濃度為10M，最佳處理時間為48h。 3.成功構(gòu)建HIF-1α干擾腺病毒（pAd/pENTR/shRNA-/GFP-HIF-1α）。 4. DFO可以在體外和體內(nèi)促進DPCs成牙本質(zhì)分化。沉默HIF-1α基因后，，DFO的促進作用即被消除。
[Abstract]:Objective according to the previous research results and literature review, we obtained the following assumptions: 1. CXCR4 positive pulpal cell (CXC chemokine receptor4-positive dental pulp cells,CXCR4 DPCs) may be a dry / precursor cell that plays an important role in the formation of repair dentin. 2. Hypoxia-inducible factor-1 偽 (HIF-1 偽) may be an important promoter of pulp repair after pulp injury. (Deferoxamine, DFO) may promote the repair ability of dental pulp tissue through HIF-1 偽. In order to verify the above hypothesis, we used immunomagnetic beads to screen CXCR4 DPCs and identify it. Then we investigated the ability of cell clone formation and multidirectional differentiation of CXCR4 DPCs, which proved that CXCR4 DPCs has the characteristics of stem cells. DFO was used to treat DPCs, to observe the change of repair ability, and RNA interference (RNA interference,RNAi) technique was used to explore the molecular mechanism of DFO regulating the formation of repaired dentin through HIF-1 偽. Materials and methods 1. CXCR4 DPCs was screened and identified by immunomagnetic beads, and then the cell clone forming ability and multidirectional differentiation ability of CXCR4 DPCs were investigated. 2. The optimal concentration and time of DFO were selected from the effects on cell activity, proliferation and migration of DPCs treated with different concentrations of DFO for different time. 3. Using adenovirus as vector, HIF-1 偽 shRNA, was constructed and transfected with DPCs, to interfere the expression of HIF-1 偽 gene. 4. The effect of DFO on DPCs dentin differentiation before and after HIF-1 偽 gene silencing was observed in vitro. 5. To observe the formation of dentin like tissue by DPCs before and after HIF-1 偽 gene silencing by DFO pretreatment in vivo. Results 1. CXCR4 DPCs, was successfully isolated and identified as having the characteristics of strong proliferative ability and multidirectional differentiation. 2. 2. 10 渭 M DFO could promote the proliferation and lateral migration of DPCs48h, and increase the expression of HIF-1 偽, but had no obvious effect on its cell activity and chemotaxis. 3. The interference efficiency of HIF-1 偽 interfering adenovirus (pAd/pENTR/shRNA-/GFP-HIF-1 偽) at the mRNA level was 73.4, and the effect of DFO on DPCs HIF-1 偽 expression was blocked at the protein level. DPCs48h, treated with 4.10 渭 M DFO could promote dentin differentiation in vitro. Several genes associated with dentin differentiation were also up-regulated. After silencing HIF-1 偽 gene, the promoting effect of DFO dentin differentiation was eliminated. 5. The results showed that 10 渭 M DFO pretreated with DPCs48h could form more dentin like tissues in vivo, and DPCs could hardly form dentin like tissue after HIF-1 偽 gene silencing. Conclusion 1. In this study, we successfully isolated CXCR4 DPCs, and confirmed that it has the characteristics of DPSCs. 2. The optimum concentration of DPCs treated with DFO was 10 m and the best treatment time was 48 h. 3. HIF-1 偽 interfering adenovirus (pAd/pENTR/shRNA-/GFP-HIF-1 偽) was successfully constructed. 4. DFO can promote DPCs dentin differentiation in vitro and in vivo. After silencing HIF-1 偽 gene, the promotion of DFO was eliminated.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R781.05

【參考文獻】

相關(guān)期刊論文 前10條

1 付士波;楊英;王冠軍;何平;;Hif-1α RNA干擾表達載體的構(gòu)建及鑒定[J];吉林大學(xué)學(xué)報(醫(yī)學(xué)版);2007年05期

2 凌亦凌,劉艷梅;低氧血癥與組織缺氧時細胞的損傷及其機制[J];國外醫(yī)學(xué).呼吸系統(tǒng)分冊;2004年02期

3 江龍;朱亞琴;;第三期牙本質(zhì)基質(zhì)形成的研究進展[J];國際口腔醫(yī)學(xué)雜志;2008年03期

4 韋盛豪;;活髓治療中蓋髓材料的研究現(xiàn)狀及方向[J];廣西醫(yī)學(xué);2007年09期

5 張曉英,孫善珍;人牙髓干細胞向成牙本質(zhì)細胞分化的誘導(dǎo)及鑒定[J];口腔醫(yī)學(xué);2004年01期

6 賀慧霞,金巖,史俊南,劉源,王亦菁,周澤淵,王新文;人牙髓干細胞的體外分離、培養(yǎng)及鑒定[J];臨床口腔醫(yī)學(xué)雜志;2004年09期

7 江龍;朱亞琴;;SDF-1-CXCR4軸與組織損傷后修復(fù)[J];上海交通大學(xué)學(xué)報(醫(yī)學(xué)版);2008年11期

8 張蓉,肖明振,趙守亮,顧淑萍,岳玲,鄭欣娟,汪平;小鼠牙胚、軟骨組織中牙本質(zhì)涎磷蛋白基因的克隆[J];牙體牙髓牙周病學(xué)雜志;2001年04期

9 郭紅延,吳補領(lǐng),郭希民,王常勇;大鼠牙髓干細胞的培養(yǎng)和鑒定[J];牙體牙髓牙周病學(xué)雜志;2004年05期

10 韓劍麗;活髓保存治療中生物材料的研究和應(yīng)用[J];牙體牙髓牙周病學(xué)雜志;2004年05期

本文編號：2355085