He Fei, Yang Zhengrong, Tan Yinghui
Department of Stomatology, Xinqiao Hospital, Third Military Medical University, Chongqing 400038, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2007 Oct;21(10):1133-6.
To investigate the heterotopic odontogenesis ability of Delta1 gene transfected human dental pulp stem cell (DPSC) and nano-hydroxyapatite/collagen (nHAC) composite scaffold.
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.
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.
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.
研究Delta1基因转染的人牙髓干细胞（DPSC）与纳米羟基磷灰石/胶原（nHAC）复合支架的异位牙发生能力。
用Delta1-增强绿色荧光蛋白重组逆转录病毒上清转染培养的人DPSC，并用嘌呤霉素筛选获得阳性细胞克隆。实验组包含Delta1转染的DPSC；然而，对照组不包含Delta1转染的DPSC，而是仅包含用载体转染的DPSC。将细胞接种到nHAC载体中，并在成牙诱导培养基中培养。通过荧光相差显微镜和扫描电子显微镜（SEM）观察转导细胞在载体中的生长情况。将细胞-载体复合物皮下移植到8只Delta1转染的裸鼠（雌性，8周龄）体内。术后8周，取出复合物，用组织学和免疫组织学方法进行检测。
实验组细胞在荧光相差显微镜下观察到在载体中生长，并发出绿色荧光。通过SEM观察，沿支架材料观察到大量转导的DPSC，甚至填充了nHAC的多孔结构并分泌大量细胞外基质。然而，在对照组中，载体中发现的细胞要少得多。所有4个Delta1转导的DPSC-nHAC复合物均产生了排列在一些nHAC多孔结构表面的牙本质样结构。成牙本质样细胞将细胞质突起延伸到牙本质基质中，该基质与充满血管的牙髓样间质组织相连。进行免疫组织化学时，成牙本质样细胞中表达牙本质涎磷蛋白。对照复合物的形态是典型的纤维结缔组织形态，在8个对照移植物中的2个中仅发现少量牙本质样结构。
DPSC可作为Delta1基因的受体细胞用于Delta1蛋白的表达和分泌。转染细胞与nHAC的复合物可诱导异位牙发生，这表明Delta1是基因增强牙本质-牙髓复合工程的新候选基因。
