本文選題：受體 + Notch��； 參考：《北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)》2014年01期

【摘要】：目的:建立Notch信號(hào)通路激活的人牙髓細(xì)胞模型,探討激活Notch信號(hào)通路對(duì)人牙髓細(xì)胞老化的影響。方法:體外培養(yǎng)人牙髓細(xì)胞,從第4代開(kāi)始實(shí)驗(yàn),將牙髓細(xì)胞分為激活組和陰性對(duì)照組。激活組采用10 mg/L的Jagged1蛋白包被培養(yǎng)皿的方法激活Notch信號(hào)通路,陰性對(duì)照組的培養(yǎng)皿不做處理。以實(shí)時(shí)定量PCR(real-time quantitative PCR,RT-qPCR)檢測(cè)第4代、第8代、第10代Notch信號(hào)通路下游基因Hes1的表達(dá)水平。分三個(gè)水平觀察Notch信號(hào)通路激活后人牙髓細(xì)胞的變化:(1)細(xì)胞水平,用倒置顯微鏡觀察細(xì)胞形態(tài)的變化,用MTT法檢測(cè)細(xì)胞活力改變;(2)代謝酶水平,用堿性磷酸酶(alkaline phosphatase,ALP)染色及活性測(cè)定試劑盒檢測(cè)ALP的表達(dá)和活性,用衰老相關(guān)β-半乳糖苷酶染色方法檢測(cè)陽(yáng)性細(xì)胞百分率;(3)基因水平,用RT-qPCR檢測(cè)老化相關(guān)基因p53和p16的表達(dá)。t檢驗(yàn)比較激活組和陰性對(duì)照組各指標(biāo)的差異。結(jié)果:采用Jagged1蛋白包被培養(yǎng)皿后,Notch信號(hào)通路下游基因Hes1表達(dá)增高,表明建立了Notch信號(hào)通路激活的人牙髓細(xì)胞模型。激活組牙髓細(xì)胞與陰性對(duì)照組牙髓細(xì)胞相比,較晚出現(xiàn)形態(tài)顯現(xiàn)衰老的細(xì)胞,第8代、第10代細(xì)胞活力升高,ALP表達(dá)量升高;第10代ALP活性升高,β-半乳糖苷酶表達(dá)下降;各代p16基因表達(dá)水平均降低,第8代、第10代p53基因表達(dá)水平降低。結(jié)論:Jagged1蛋白可有效激活Notch信號(hào)通路,激活Notch信號(hào)通路后,人牙髓細(xì)胞在細(xì)胞水平、代謝酶水平、基因水平均表現(xiàn)出老化延遲的趨勢(shì)。
[Abstract]:Aim: to establish a human dental pulp cell model with Notch signaling pathway activation and to investigate the effect of Notch signal pathway activation on human dental pulp cell aging. Methods: human dental pulp cells were cultured in vitro. Dental pulp cells were divided into activated group and negative control group. In the activation group, the Notch signaling pathway was activated by using a 10 mg / L JaggeD1 protein coated with a petri dish, but not in the negative control group. The expression of Hes1 gene downstream of Notch signaling pathway in the 4th, 8th and 10th generation was detected by real-time quantitative PCR RT-qPCR. The changes of human dental pulp cells after Notch signal pathway activation were observed at three levels: (1) cell level, cell morphology by inverted microscope, cell viability by MTT assay, and (2) metabolic enzyme level. The expression and activity of alkaline were detected by alkaline phosphatase staining and activity assay kit, and the percentage of positive cells was detected by senescence associated 尾 -galactosidase staining. (3) Gene level. RT qPCR was used to detect the expression of p53 and p16 genes. T test was used to compare the differences between the activation group and the negative control group. Results: the expression of Hes1 gene downstream of Notch signaling pathway was increased after coated with Jagged1 protein, which indicated that the human dental pulp cell model was established with Notch signaling pathway activated. Compared with the dental pulp cells of the negative control group, the activated dental pulp cells appeared the aging cells later, the activity of ALP increased and the expression of 尾 -galactosidase decreased in the 8th generation, the 10th generation and the 10th generation respectively, and the activity of ALP increased and the expression of 尾 -galactosidase decreased. The expression level of p16 gene decreased in all generations, but decreased in the 8th generation and 10th generation. Conclusion the cell level, metabolic enzyme level and gene level of human dental pulp cells showed a tendency of delayed aging after the activation of Notch signaling pathway by 1: Jagged1 protein.
【作者單位】： 北京大學(xué)口腔醫(yī)學(xué)院·口腔醫(yī)院牙體牙髓科;
【基金】：國(guó)家自然科學(xué)基金(30801289)資助~~
【分類(lèi)號(hào)】：R329.26

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本文編號(hào)：2052167