正常人与慢性牙周炎伴有骨质疏松患者牙槽骨组织体外培养细胞系的生物学特性(英文)

Biological characteristics of cell lines cultured in vitro from alveolar bone tissue in normal persons and patients with chronic periodontitis complicated by osteoporosis

背景:培养人牙槽骨细胞系,因细胞来源于牙槽骨,取材于口腔很容易污染,常导致实验室研究失败,又因为标本来自成年人,细胞分裂指数低,培养的成功率低。目的:比较无菌手术中取正常人和慢性牙周炎伴骨质疏松患者的牙槽骨组织,经体外培养,建立的传代、冻存、复苏成活的细胞系的生物学特性。比较两种细胞的生物学特性。为牙槽骨的缺损、修复、治疗提供理论和相关实验依据。设计:对照观察。单位:首都医科大学附属北京朝阳医院口腔科和解放军总医院骨科研究所。材料:无菌手术中取正常人和临床诊断为慢性牙周炎患者的牙槽骨组织。方法:由正常人和慢性牙周炎伴骨质疏松患者的牙槽骨组织经体外培养,从原代培养出来的4个细胞系(H-171、H-258、261、262)中,选出牙周炎患者组细胞系H-171,正常组H-258,用组化、免疫组化等染色,观察细胞形态。用细胞计数法计算出两种细胞倍增时间和分裂指数。经过多次传代、冻存、复苏,比较细胞的生长、老化规律。主要观察指标:两组细胞系的传代情况及生物学特性。结果:①异常牙槽骨组,原代培养一次成功,细胞形态为短梭形,3次冻存,3次复苏均存活,其倍增时间为53.4h。平均分裂指数为4‰。细胞传代20次后,仍生长良好。②正常牙槽骨组原代培养26例,因多种原因能传代的细胞系仅有3例,已传代10次,形态为长梭形,经两次冻存复苏,细胞存活、生长速度较H-171慢,倍增时间为65.9h,平均分裂指数3.5‰。③两种细胞均贴壁生长,具有成骨细胞的特点:AKP、甲苯胺兰、PAS、四环素标记矿化结节组化染色及Ⅰ型胶原、BMP-2免疫组化染色均为阳性。结论:培养出的两种细胞均具有成骨细胞特点,其中H-171生长速度较H-258快,传代20次,无明显变异,H-258传到8代开始老化,生长速度减慢。

BACKGROUND：Because human cells for culturing alveolar bone cell line are from alveolar bone, which is in oral cavity,and easily polluted, so laboratory study is often unsuccessful. Because the samples are from adults, so cell division index and the successful rate of culture are low.OBJECTIVE： To compare the biological characteristics of survived cell line established through passage,cryopreservation and revitalization following in vitro culturing the alveolar bone tissue obtained from normal persons and patients with chronic periodontitis accompanied with osteoporosis in aseptic operation; To compare the biological characteristics of two kinds of cells so as to provide theoretical and related experimental evidence for defect, repair and treatment of alveolar bone.DESIGN： Controlled observation.SETTING： Department of Stomatology, Beijing Chaoyang Hospital, Capital Medical University; Institute of Orthopaedics,General Hospital of Chinese PLA.MATERIALS： Alveolar bone tissue obtained from normal persons and patients with chronic periodontitis confirmed in clinic was used in aseptic operation.METHODS： Alveolar bone tissue from normal persons and chronic periodontitis accompanied with osteoporosis were cultured in vitro. In the four cell lines （H-171, H-258, 261, 262） cultured primarily, cell lines H-171 and H-258 were chosen from periodonitis patients group and normal group respectively, and stained with histochemical and immunohistochemical methods. Cell morphology was observed. Doubling time and division index of two kinds of cells were calculated with cytometry. After several circles of passage, cryopreservation and revitalization, growth and aging rule of cells were compared.MAIN OUTCOME MEASURES： Passage and biological characteristics of two groups of cell lines.RESULTS： ①In the abnormal alveolar bone group, there was one successful primary culture and cells presented short-spindle shape. There were 3 times of cryopreservation and 3 times of revitalization. Its doubling time was 53.4 hours. The average division index was about 4‰. Cells well grew after 20 times of passages. ②In the normal alveolar bone group, there were 26 cases of cell lines cultured primarily, but passage was found in only 3 cases of cell lines due to various causes. There were 10 passages and the cells presented long-spindle shape. After two circles of cryopreservation and revitalization, the survival and growth rate of cells were inferior as compared with cell line H-171.Doubling time was 65.9 hours and the average division index was 3.5‰. ③Both two kinds of cells adhered the wall, with the characteristics of osteoblasts： AKP, toluidine blue, PAS, tetracycline-labeled mineralized nodus, type Ⅰ collagen and BMP-2 immunohistochemical staining all presented positive.CONCLUSION： Both two kinds of cultured cells have the characteristics of osteoblasts. The growth speed of cell line H-171 is faster than that of cell line H-258. No obvious mutation is found in 20 passages. In the 8th generation of H-258,aging appears and growth speed becomes slow.