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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: Human alveolar osteoblast cell lines are cultured because alveolar bone cells originate from alveolar bone and are easily contaminated in the oral cavity. Laboratory studies often fail, and since the specimens are from adults, the cell division index is low and the success rate of culture is low. OBJECTIVE: To compare the biological characteristics of alveolar bone tissue from normal and chronic periodontitis patients with osteoporosis during aseptic surgery, and to establish the passage, cryopreservation and resuscitation cell lines after in vitro culture. The biological characteristics of both cells were compared. For the alveolar bone defects, repair, treatment theory and related experimental basis. Design: Controlled observation. SETTING: Department of Stomatology, Beijing Chaoyang Hospital Affiliated to Capital Medical University and PLA General Hospital of Orthopedics. Materials: Aseptic surgery was performed on alveolar bone from normal and clinically diagnosed patients with chronic periodontitis. Methods: The alveolar bone tissues from normal and chronic periodontitis patients with osteoporosis were cultured in vitro and cultured from four primary cell lines (H-171, H-258, 261, 262) Peripheral periodontitis patients cell line H-171, normal group H-258, with histochemistry, immunohistochemistry staining, observe the cell morphology. Cell doubling time was used to calculate both cell doubling time and dividing index. After many passages, cryopreservation, recovery, comparison of cell growth, aging laws. MAIN OUTCOME MEASURES: Passage and biological characteristics of two cell lines. Results: ① In primary alveolar bone group, the primary culture was successful at one time. The morphology of the alveolar bone was short fusiform, with three cryopreservation and three resuscitation survived with a doubling time of 53.4 h. The average split index was 4 ‰. After 20 passages, cells still grew well. ② In the normal alveolar bone group, 26 cases were primary cultured, and only 3 cases were passaged for a variety of reasons. They had been passaged 10 times in long fusiform form. After two cryopreservation and resuscitation, the survival rate and growth rate were higher H-171 slow, doubling time was 65.9h, the average cleavage index of 3.5 ‰. ③ Both cells adherently grew, with the characteristics of osteoblasts: AKP, toluidine blue, PAS, tetracycline-labeled mineralized nodules and type I collagen, BMP-2 immunohistochemical staining were positive. Conclusion: Both of the cultured cells have the characteristics of osteoblasts. H-171 grew faster than H-258 and passaged 20 times with no obvious variation. H-258 began to age and grew slowly at 8 passages.