Deltal基因转染人牙髓干细胞牙本质形成能力的研究

目的探讨Notch配体Delta1基因转导的人牙髓干细胞（dental pulp stem cell,DPSC）在裸鼠皮下牙本质的新生能力。方法以Delta1基因转染的第2代人DPSC作为实验组,空载体转染DPSC及单纯DPSC作为对照组,分别与纳米羟基磷灰石/胶原（nano-hydroxyapatite/collagen,nHAC）复合物载体材料复合,倒置荧光显微镜和扫描电镜观察细胞在材料表面的生长。将实验组和对照组细胞分化诱导后3d制成细胞悬液,按5×10^7/ml接种于材料表面（100μl/块）,体外培养48h。将细胞-材料复合物移植于8只SPF级8周龄BAL B/C-nu/nu雌性裸小鼠背部皮下,术后8周以组织学、免疫组织化学等方法检测牙本质生成情况。结果倒置荧光显微镜观察实验组材料表面及其孔隙内大量细胞生长,表达明亮绿色荧光。扫描电镜观察实验组细胞在载体材料的多孔间隙内大量增殖,细胞伸展良好,分泌大量细胞外基质;而对照组载体内细胞数量则较少。体内移植实验组均可见牙本质-牙髓复合物形成,新生成牙本质样细胞牙本质涎磷蛋白表达阳性;对照组均以纤维结缔组织为主,仅2块移植物有少量牙本质样物质形成。结论DPSC可作为Delta1基因的受体细胞,与nHAC复合后具有成牙本质能力。

ODONTOGENESIS OF DELTA1 GENE TRANSFECTED HUMAN DENTAL PULP STEM CELLS

OBJECTIVE: To investigate the heterotopic odontogenesis ability of Delta1 gene transfected human dental pulp stem cell (DPSC) and nano-hydroxyapatite/collagen (nHAC) composite scaffold. METHODS: The cultured human DPSC was transfected with Delta1-enhanced green fluorescent protein recombinant retrovirus supernatant,and was selected by puromycin to obtain the positive cell clone. The experimental group contained the Delta1 transfected DPSC; however, the control group did not contain the Delta1 transfected DPSC but contained DPSC transfected with vectors only. The cells were seeded into the nHAC carriers and were cultured in the odonto-inductive medium. The growth of the transduced cells in the carriers was observed by the fluorescent phase contrast microscope and the scanning electron microscope (SEM). The cell-carrier composites were subcutaneously transplanted into the Delta1 transfected 8 nude mice (female, 8 weeks old). Eight weeks after operation, the composites were taken out and tested with the histological and the immunohistological methods. RESULTS: Green fluorescence was observed in the cells in the experimental group, which were grown in the carriers by the fluorescent phase contrast microscope. Observed by SEM, great amounts of transduced DPSC were observed along the scaffold materials, even filling the porous structures of nHAC and secreting a lot of extracellular matrix. However, in the control group, much fewer cells were found in the carriers. All the 4 Delta1 transduced DPSC-nHAC composites produced dentin-like structures that lined the surfaces of some nHAC porous structures. The odontoblast-like cells extended the cytoplasmic processes into the dentinal matrix, which was interfaced with a pulp-like interstitial tissue infiltrated with the blood vessels. Dentin sialophosphoprotein was expressed in the odontoblast-like cells when immunohisochemistry was performed. The morphology of the control composite was a typical one of the fibrous connective tissue, and only a little dentin-like structure was found in 2 of the 8 control transplants. CONCLUSION: DPSC can be used as the recipient cell of the Delta1 gene for expression and secretion of the Delta1 protein. The composites of the transfected cells and nHAC can induce heterotopic odontogenesis, which indicates that Delta1 is a novel candidate for the gene enhanced dentin-pulp composite engineering.