慢病毒介导的Delta1-RNA干扰对人牙髓干细胞增殖及分化的影响

Effect of Notch ligand Deltal-RNA interference by lentivirus on proliferation and differentiation of human dental pulp stem cells

目的探讨Notch配体Delta1基因的特异性RNA干扰（RNA interference，RNAi）对人牙髓干细胞（dental pulp stern cell，DPSC）增殖及分化的影响。方法利用Delta1-RNAi慢病毒载体感染体外培养的DPSC获得稳定的Delta1—RNAi DPSC系；实验分3组：经Delta1—RNAi慢病毒感染的DPSC组（慢病毒组），经空慢病毒感染的阴性对照组和正常细胞的正常对照组，采用细胞生长曲线测定（cell counting kit-8，CCK-8）、流式细胞仪及免疫组化等方法检测细胞生长曲线、细胞周期、细胞核增殖抗原表达的变化情况；对各组细胞进行体外成牙本质分化诱导，采用茜素红染色法检测钙化结节数量的差别，并用碱性磷酸酶（ALP）活性检测ALP及蛋白质印迹法检测诱导后各组细胞中牙本质涎磷蛋白（dentin sialophosphoprotein，DSPP）表达量的区别。结果与正常对照及阴性对照组相比，慢病毒组DPSC增殖能力显著降低，其S期细胞比例及增殖指数分别由正常对照组的22．32±2．35和33．68±4．19显著降低至5．44±0．91和16．0±6．07（P〈0．05），细胞核增殖抗原的表达显著下降；慢病毒组细胞经诱导后形成钙化结节数量明显增多，ALP及DSPP表达含量较正常对照组及阴性对照组显著增高。结论Notch配体Delta1基因被干扰下调后，人DPSC的体外增殖受到抑制，在体外成牙本质诱导培养条件下，细胞向成牙本质细胞的分化加快，证明Notch—Delta1信号转导途径对人DPSC的自我更新及分化的调控起着重要作用，为牙髓损伤后修复提供了理论基础。

Objective To investigate the effects of specific RNA interference （RNAi） to Notch ligand Delta1 on proliferation and differentiation of human dental pulp stem cells （DPSC）. Methods DPSC were infected by lentivirus vectors carrying Delta1-RNAi. DPSC were divided into three groups, DPSC/ Deltal-RNAi group, DPSC/wt group and DPSC/vector group as control. Cell counting kit-8 （CCK-8） assay and flow cytometry were used to evaluate the proliferation of DPSC. Expression of proliferating cell nuclear antigen （PCNA） was examined with immunohistochemical staining. All groups were cultured in an odonto-inductive medium and were observed under microscope. The number of mineralization nodules was counted after Alizarin red staining. Alkaline phosphatase （ALP） activity and the expression of dentin sialophosphoprotein（DSPP） were detected by ALP activity assay and Western blotting. Results Compared with DPSC/wt or DPSC/vector separately, proliferating rate and S-cycle of DPSC/Delta1-RNAi was significantly lower. The S phase and proliferation index （PI） decreased markedly from 22.32± 2. 35 and 33.68 ± 4. 19 （DPSC/Delta1-RNAi） to 5.44± 0. 91 and 16. 00 ± 6. 07 （DPSC/wt）. The PCNA staining of DPSC/Delta1-RNAi was evidently weaker. DPSC/Delta1-RNAi group had more calcified cell nodules than the other two control groups, and ALP activity and DSPP expression of DPSC/Delta1-RNAi group incxeased markedly. Conclusions Delta1-RNAi induced by the lentivirus vectors may inhibit DPSC proliferation and differentiation. Notch-Delta signal pathway plays an important role in self-renewal and differentiation.