摘要
背景:人牙髓组织较少,对酶解消化的耐受性较差,经胰蛋白酶和胶原酶分步消化处理后,细胞贴壁数量少,生长缓慢,难以成功传代,组织块酶解法获得的牙髓细胞种类少,活性差。目的:探索组织块培养法在人牙髓细胞体外培养中的应用。设计、时间及地点:细胞观察实验,于2005/2007年在吉林大学和平校区及口腔医学院进行。材料:健康人正畸或阻生拔除的牙齿。方法:选取第三磨牙,取出牙髓,在少量DMEM培养液浸润下,将牙髓组织剪碎至1.0mm×1.0mm×0.5mm组织块,将牙髓组织块移入含培养液的6孔板中,调整组织块密度至3～6块每孔,间距0.5cm均匀摆置。将6孔板倒置放入37℃、体积分数为5%CO2、饱和湿度条件的培养箱中,3h后翻转培养板,保持组织块贴壁生长,加培养液至2mL后继续培养。4～6d换液1次,细胞6～15d从组织块边缘游出,在组织块周围形成生长晕。细胞渐进汇合时,以用0.25%的胰蛋白酶、0.02%EDTA的混合液消化2.0～3.0min,按1∶(2～3)的比例传代培养至25mL培养瓶。原代培养细胞通过反复消化传代的方式逐渐获得纯化的成纤维样细胞。主要观察指标:采用免疫组织化学方法鉴定细胞形态,碱性磷酸酶值测定牙髓细胞分泌。MTT法绘制牙髓组织细胞生长曲线。结果:相差显微镜下,获得的牙髓细胞主要是典型成纤维细胞,形态多呈星形、长梭形,胞体丰满,胞浆均匀,核圆形或卵圆,核仁清晰。免疫组织化学检测显示,细胞表面波丝蛋白阳性、角蛋白阴性,碱性磷酸酶阳性。在接种于96孔平底培养板1d后,细胞数量有所下降,但在接种2d后数量又有所增加,此时细胞增加不很明显。4d开始进入对数生长期,细胞增加显著,8d进入平台期,9d开始减少,整个细胞曲线呈"S"形。结论:采用组织块培养法从成体人牙髓组织中可以分离培养出细胞,在体外能有效增殖并保持低分化状态。
BACKGROUND： Human pulp tissue has been known to be less, and exhibit poor tolerance to enzymatic digestion and less adherent cells after step-by-step digestion of trypsin and collagenase, thereby often leading to a failure of passage. Only several kinds of dental pulp cells with poor activity can be obtained by the tissue explant-collagenase digestion. OBJECTIVE： To investigate human dental pulp cells cultured in vitro by tissue explant method. DESIGN, TIME AND SETTING： A cytological observation was performed at Heping Campus and School of Stomatology, Jilin University from 2005 to 2007. MATERIALS： Healthy young human teeth extracted for orthodontic correction or impaction. METHODS： Pulp tissue from the third molar teeth was collected, cut into small blocks with a size of 1.0 mm×1.0 mm×0.5 mm under the infiltration of small amount of Dulbecco＇s modified eagle＇s medium, and then transferred into a 6-well plate containing culture medium for incubation in a 5% CO2 and saturated humidity atmosphere at 37 ℃. During the process of incubation, pulp tissue was adjusted at a density of 3 6 blocks/well, with an equal spacing of 0.5 cm and the 6-well plate was kept inverted. Three hours later, the 6-well plate was turned over to make tissue blocks adhering to the plate wall. Culture was continued after addition of 2 mL of culture medium. Culture medium was renewed every 4-6 days. After 6-15 days, cells emigrated from the edge of tissue blocks and cell outgrowth appeared around each tissue block. When cells closed to confluency, a digestion procedure of 2.0 3.0 minutes （0.25% trypsin and 0.02% ethylenediamine tetraacetic acid） was followed by passage culture at a proportion of 1：（2 3） in 25 mL of culture flasks. Purified fibroblast-like cells were gradually obtained from primarily cultured cells by repeated digestion and passage. MAIN OUTCOME MEASURES： Cellular morphology was identified by immunohistochemistry; secreted dental pulp cells were determined using alkaline phosphatase activity; the growth curves of human pulp tissue cells were depicted by MTT assay. RESULTS： Under an inverted phase contrast microscope, the obtained dental pulp cells were primarily typical flbroblasts with a long-shuttled appearance, well-rounded cell body, uniform cytoplasm, round or oval nucleus, and clear nucleolus. Immunohistochemistry results showed cell surface vimentin-positive, pan cytokeratin-negative, and alkaline phosphatase-positive. These cells were decreased after culturing 1 day, were slightly increased after 2 days, entered the logarithmic growth period and were markedly increased after 4 days, entered a platform period after 8 days, and began to decrease again after 9 days. The whole growth curve of cells appeared in ＂S＂ shape. CONCLUSION： The dental pulp cells isolated from human pulp tissue by tissue explant method can effectively proliferate and retain a poorly differentiated state in vitro.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第28期5416-5420,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
吉林省科技厅基金资助课题(200705351)
长春市科技局基金资助课题(2008109)~~
