CAJ | 학술논문

背景：成骨细胞获取的方法较多，如何简便而迅速的获得高纯度的成骨细胞成为研究的热点。 　　目的：比较组织块法、胶原酶消化法、改良胶原酶和胰酶分段消化法体外培养纯化乳兔颅骨来源的成骨细胞结果及其细胞的生物学特点。 　　方法：取新生24 h内新西兰大白兔乳兔颅盖骨，采用组织块法、胶原酶消化法和改良胶原酶和胰酶分段消化法分离获取兔原代成骨细胞，并进行传代培养。通过倒置显微镜下形态学观察、锥虫蓝排斥法计数活细胞率及MTT法绘制细胞生长曲线、茜素红染色、细胞培养上清液碱性磷酸酶和骨钙素检测、Ⅰ型胶原和Ⅲ型胶原免疫组织化学法和RT-PCR检测骨钙素和Ⅰ型胶原mRNA的表达等对成骨细胞进行鉴定。 　　结果与结论：分离培养的成骨细胞均一性好、增殖能力强，具备成骨细胞的典型特征，茜素红染色阳性，Ⅰ型胶原免疫组织化学染色阳性，Ⅲ型胶原免疫组织化学染色阴性，细胞培养上清液碱性磷酸酶、骨钙素均有表达，PT-PCR结果有Ⅰ型胶原蛋白和骨钙素mRNA表达。改良胶原酶和胰酶分段消化法较传统胶原酶消化法有更高的细胞获取率，更好的细胞活性，且比传统胶原酶消化法耗时短(P<0.05)；组织块法操作方法最为简单，细胞活性最高，但是细胞产出率最低、耗时最长，不适合用于成骨细胞大规模培养。用改良的胶原酶和胰酶分段消化法可获得数量较多且纯度较高的成骨细胞，可以成为一种相对可靠、有效的原代成骨细胞的体外培养方法。
배경：성골세포획취적방법교다，여하간편이신속적획득고순도적성골세포성위연구적열점。 　　목적：비교조직괴법、효원매소화법、개량효원매화이매분단소화법체외배양순화유토로골래원적성골세포결과급기세포적생물학특점。 　　방법：취신생24 h내신서란대백토유토로개골，채용조직괴법、효원매소화법화개량효원매화이매분단소화법분리획취토원대성골세포，병진행전대배양。통과도치현미경하형태학관찰、추충람배척법계수활세포솔급MTT법회제세포생장곡선、천소홍염색、세포배양상청액감성린산매화골개소검측、Ⅰ형효원화Ⅲ형효원면역조직화학법화RT-PCR검측골개소화Ⅰ형효원mRNA적표체등대성골세포진행감정。 　　결과여결론：분리배양적성골세포균일성호、증식능력강，구비성골세포적전형특정，천소홍염색양성，Ⅰ형효원면역조직화학염색양성，Ⅲ형효원면역조직화학염색음성，세포배양상청액감성린산매、골개소균유표체，PT-PCR결과유Ⅰ형효원단백화골개소mRNA표체。개량효원매화이매분단소화법교전통효원매소화법유경고적세포획취솔，경호적세포활성，차비전통효원매소화법모시단(P<0.05)；조직괴법조작방법최위간단，세포활성최고，단시세포산출솔최저、모시최장，불괄합용우성골세포대규모배양。용개량적효원매화이매분단소화법가획득수량교다차순도교고적성골세포，가이성위일충상대가고、유효적원대성골세포적체외배양방법。
BACKGROUND:There are many kinds of ways to obtain osteoblasts at present, but how to get high-purity osteoblasts in a easy and fast way has become a hot research. OBJECTIVE:To explore a method to get massive and high purified osteoblasts effectively by comparing three common primary osteoblast culture methods, and to observe the biological characteristics of the osteoblasts from the skul of neonatal rabbit. METHODCalvarias were dissected from newborn New Zealand white rabbits within 24 hours, and osteoblasts were isolated with bone tissue method, col agenase digestion method and modified tryptase and col agenase sequential digestion method respectively, then the cells were subcultured in vitro. Osteoblast proliferation and osteogenic activity were identified by inverted microscope for morphology observation. The rate of living osteobalsts was counted with trypan blue staining. The growth curve of the cells was drawn with MTT method. Alizarin red staining was applied to detect alkaline phosphatase and osteocalcin protein in the cellculture supernatants. Col agen I and col agen III immunohistochemical staining was also performed. RT-PCR was used to determine the expression of osteocalcin and col agen I mRNA expression. RESULTS AND CONCLUSION:The cultured cells showed highly homogeneous appearance with active proliferation, and they had the typical features of osteoblasts. Alizarin red staining and col agen I immunohistochemical staining were both positive, while col agen III immunohistochemical staining was negative. Alkaline phosphatase and osteocalcin protein expression in the cellculture supernatants can be detected. The expression of osteocalcin and col agen I mRNA was positive in the RT-PCR test. Compared with col agenase digestion method, the modified tryptase and col agenase I sequential digestion method cost less time, presented higher production of osteoblasts and higher cellsurvival rate (P<0.05). Bone tissue method was the easiest method and did the least damage to osteoblasts, but it presented lowest production of osteoblasts and cost the maximum time among the three methods. So it cannot be used in large-scale osteoblast culture. A large quantity of high purity osteoblasts were obtained by modified trypsase and col agenase I sequential digestion method, which can be used as a reliable and efficient way to obtain the original generation osteoblasts in vitro.