修复性牙本质形成机理的相关实验研究

发布时间：2018-11-24 21:13

【摘要】：目的根据前期研究结果及文献复习，我们得出如下假设：1. CXCR4阳性牙髓细胞（CXC chemokine receptor4-positive dental pulp cells，CXCR4+DPCs）可能是牙髓在形成修复性牙本质过程中起重要作用的干/前体细胞；2.低氧诱导因子-1α（Hypoxia-inducible factor-1α，HIF-1α）可能是牙髓损伤后启动牙髓修复反应的重要启动因子；3.去铁胺（Deferoxamine, DFO）可能通过HIF-1α促进牙髓组织的修复能力。为验证上述假设，我们采用免疫磁珠筛选CXCR4+DPCs并进行鉴定，然后考察CXCR4+DPCs的细胞克隆形成能力及多向分化能力，从而证明CXCR4+DPCs具有干细胞的特性；采用DFO处理DPCs，观察其修复能力的变化，并结合RNA干扰（RNA interference，RNAi）技术，探讨DFO通过HIF-1α调节修复性牙本质形成的分子机理。材料和方法 1.采用免疫磁珠筛选CXCR4+DPCs并进行鉴定，然后考察CXCR4+DPCs的细胞克隆形成能力及多向分化能力。 2.不同浓度DFO作用DPCs不同时间后，从对细胞活性、增殖及迁移等几个方面的影响挑选出DFO的最佳作用浓度及时间。 3.以腺病毒作为载体，构建HIF-1α shRNA，转染DPCs，干扰HIF-1α基因的表达并进行验证。 4.体外观察DFO作用HIF-1α基因沉默前/后DPCs成牙本质分化能力的变化。 5.观察DFO预处理HIF-1α基因沉默前/后DPCs在体内形成牙本质样组织的情况。结果 1．成功分离和鉴定CXCR4+DPCs，证实其具有增殖能力强及多向分化能力特点。 2．10μM DFO处理DPCs48h，可促进其增殖和横向迁移，并能提高其HIF-1α的表达，但是对其细胞活性和趋化作用的影响不明显。 3. HIF-1α干扰腺病毒（pAd/pENTR/shRNA-/GFP-HIF-1α）在mRNA水平的干扰效率为73.4%，并在蛋白水平成功阻断了DFO促进DPCs HIF-1α的表达效果。 4.10μM DFO处理DPCs48h，可以在体外促进其成牙本质分化。几个与成牙本质分化相关的基因也被上调。沉默HIF-1α基因后，DFO成牙本质分化的促进作用即被消除。 5.体内实验结果显示10μM DFO预处理DPCs48h后，可在体内形成更多的牙本质样组织，沉默HIF-1α基因后，DPCs几乎不能形成牙本质样组织。结论 1.本研究中我们成功分离CXCR4+DPCs，并证实其具有DPSCs的特性。 2. DFO处理DPCs的最佳作用浓度为10M，最佳处理时间为48h。 3.成功构建HIF-1α干扰腺病毒（pAd/pENTR/shRNA-/GFP-HIF-1α）。 4. DFO可以在体外和体内促进DPCs成牙本质分化。沉默HIF-1α基因后，，DFO的促进作用即被消除。
[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 伪 (HIF-1 伪) 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 伪. 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 伪. 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 伪 shRNA, was constructed and transfected with DPCs, to interfere the expression of HIF-1 伪 gene. 4. The effect of DFO on DPCs dentin differentiation before and after HIF-1 伪 gene silencing was observed in vitro. 5. To observe the formation of dentin like tissue by DPCs before and after HIF-1 伪 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 伪, but had no obvious effect on its cell activity and chemotaxis. 3. The interference efficiency of HIF-1 伪 interfering adenovirus (pAd/pENTR/shRNA-/GFP-HIF-1 伪) at the mRNA level was 73.4, and the effect of DFO on DPCs HIF-1 伪 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 伪 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 伪 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 伪 interfering adenovirus (pAd/pENTR/shRNA-/GFP-HIF-1 伪) was successfully constructed. 4. DFO can promote DPCs dentin differentiation in vitro and in vivo. After silencing HIF-1 伪 gene, the promotion of DFO was eliminated.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R781.05

【参考文献】