大鼠乳鼠成骨细胞建立及其生物学活性的研究

Establish of neonatal rat osteoblasts and research of osteoblasts biological activies

关键词:  成骨细胞  骨形态发生蛋白  碱性磷酸酶  细胞增值  骨钙素

Key words:  [Key words]　osteoblast  bone morphogenetic protein  alkalinity phos phatase  cell increment  bone calciumelement

摘要:

[摘要]　目的　建立一种简单、快速、成活率和纯度高的原代培养方法，以获得大量成骨细胞，并对成骨细胞生物学活性进行研究。　方法　无菌取5日龄SD大鼠的颅盖骨，采用改良的二次酶消化法分离成骨细胞，通过倒置相差显微镜观察，并用碱性磷酸酶和矿化结节染色鉴定,利用微量滴定(MTT)法测定一个培养周期的成骨细胞活性分布；培养的成骨细胞单独应用BMP-2和BMP-7以及联合应用BMP-2和BMP-7作用48 h后，用MTT法检测成骨细胞增殖，同时测定成骨细胞裂解液中碱性磷酸酶（ALP）活性和骨钙素（OCN）含量。　结果　较为快速获得大量成骨细胞,成骨细胞培养至第10天时活性最强。单独应用BMP-2和BMP-7，联合应用BMP-2和BMP-7作用48 h后,均能刺激大鼠乳鼠成骨细胞的增殖，增加成骨细胞ALP活性和OCN含量,但单独应用BMP-2和BMP-7组与对照组比较差异无显著性意义（P>0.05）。联合应用BMP-2和BMP-7作用后，与对照组、单独应用BMP-2和BMP-7组比较，能明显增强成骨细胞生物学活性，差异具有显著性意义（P<0.05）。　结论　用改良的二次酶消化法能够在短时间内获得大量成骨细胞。联合应用BMP-2和BMP-7能使成骨细胞生物学活性得到显著增强。

Abstract:

[Abstract] Objective　To culture a large number of pure osteoblasts with high survival rate，which would be a foundation of bone tissue engineering. The effects of osteoblast`s biology activity were observed with using incorporation of BMP-2 and BMP-7.　Methods　5 days old SD rats received an aseptic surgery to get its calvaria bone.An improved second enzymatic digestion method were used to separate osteoblasts. To observe the osteoblast activity distribution,the inverted phase contrast microscope observation, alkaline phosphatase staining, mineralized nodule identification and micro-titration (MTT) method were used after a culture cycle. Cells were cultured with BMP-2 and BMP-7 respectively and bothly.After 48 h,MTT was used to assay cell proliferation,and the ALP activity and OCN content were determined from cell lysate.〖WTHZ〗Results　MTT method and morphological observation showed that 10 d was the strongest activity time after osteoblasts were cultured. The neonatal rat osteoblast proliferation was stimulated with using BMP-2 and BMP-7,so was the incorporation of BMP-2 and BMP-7.osteoblast ALP activity and OCN contents were significantly increased after using BMP-2 and BMP-7 respectively and bothly. However, using BMP-2 and BMP-7 respectively are not significant. Treatment with BMP-2 and BMP-7HA resulted in signifcant improvement when compared to BMP-2 and BMP-7 respectively treatmented group (P<0.05).　Conclusion　These results suggest that the modified secondary enzymatic digestion can be able to access a large number of osteoblasts in a short time. The biological activity of osteoblasts was significantly enhanced with incorporation of BMP-2 and BMP-7.