CAJ | 학술논문

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。
목적：건립일충간단、쾌속、성활솔화순도고적원대배양방법，이획득대량성골세포，병대성골세포생물학활성진행연구。방법무균취5일령SD대서적로개골，채용개량적이차매소화법분리성골세포，통과도치상차현미경관찰，병용감성린산매화광화결절염색감정，이용미량적정（ MTT）법측정일개배양주기적성골세포활성분포；배양적성골세포단독응용BMP-2화BMP-7이급연합응용BMP-2화BMP-7작용48 h후，용MTT법검측성골세포증식，동시측정성골세포렬해액중감성린산매（ALP）활성화골개소（OCN）함량。결과교위쾌속획득대량성골세포，성골세포배양지제10천시활성최강。단독응용BMP-2화BMP-7，연합응용BMP-2화BMP-7작용48 h후，균능자격대서유서성골세포적증식，증가성골세포ALP활성화OCN함량，단단독응용BMP-2화BMP-7조여대조조비교차이무현저성의의（P＞0．05）。연합응용BMP-2화BMP-7작용후，여대조조、단독응용BMP-2화BMP-7조비교，능명현증강성골세포생물학활성，차이구유현저성의의（P＜0．05）。결론용개량적이차매소화법능구재단시간내획득대량성골세포。연합응용BMP-2화BMP-7능사성골세포생물학활성득도현저증강。
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 con-trast 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 .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-7re-spectively 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 -7respectively 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 incorpora-tion of BMP-2 and BMP-7.