含胎牛血清胶原酶消化法培养兔成骨细胞

背景:成骨细胞原代培养技术已经很成熟,然而各种消化酶单纯作用于原代组织时对成骨细胞膜蛋白有一定影响。目的:为尽可能避免酶对细胞的损害,用含胎牛血清的胶原酶消化胎兔颅盖骨骨组织,进行体外成骨细胞培养,观察加入胎牛血清后胶原酶对成骨细胞消化的效果。设计:对照实验。单位:华中科技大学同济医学院附属同济医院骨外科研究室。材料:实验于2004-03/12在华中科技大学同济医学院附属同济医院骨外科研究室完成。妊娠28d日本大耳白种孕兔2只。方法:将同一个批号的Ⅰ型胶原酶分别用含体积分数为0.15胎牛血清的培养基和DMEM溶液配制成0.1%的含胎牛血清的胶原酶和0.1%不含血清的胶原酶,分别作为实验组和对照组的酶消化液A液和B液。妊娠28d的孕兔,无菌条件下剖腹取出胎兔,然后取胎兔的颅盖骨,剔净、漂洗、剪碎。实验组和对照组分别加5mL37℃的A液和B液消化,两组均用含体积分数为0.15胎牛血清的培养液分别成梯度稀释为0倍、1倍、2倍和4倍后继续消化-培养成骨细胞。锥虫蓝染色测定细胞的存活率,细胞内碱性磷酸酶染色鉴定和测定成骨细胞的纯度,显微镜下观察细胞生长情况。主要观察指标:①用锥虫蓝染色来检测两组细胞的存活率。②以细胞内碱性磷酸酶染色来检测两组细胞的纯度。③用显微镜观察细胞形态来区别两组细胞的生长情况。结果:①细胞锥虫蓝染色结果:通过记数计算,实验组拒染率高达98%,细胞存活率高;对照组拒染率也高达95%,细胞存活率也较高。②细胞内碱性磷酸酶染色结果:实验组碱性磷酸酶染色阳性区域不少于95%,细胞纯度高;但对照组碱性磷酸酶染色阳性区域不超过75%细胞纯度较低。③显微镜下观察结果:实验组细胞饱满凸起,有立体感,细胞生长旺盛,营养充足;而对照组细胞凸起不明显,立体感较差,细胞生长营养较实验组差。结论:用含胎牛血清胶原酶消化胎兔颅盖骨骨组织,培养出的成骨细胞具有典型的成骨细胞特征,且成分单一,存活率高。

Culture of rabbit osteoblasts digested by collagenase in DMEM containing fetal bovine serum

BACKGROUND: The skill to culture osteoblasts primarily has been well developed, however, variousdigestive enzymes can affect membrane protein of osteoblasts when they are used on primary tissue separately.OBJECTIVE: To avoid the damage from enzyme to cell as best possible,digest cranial osseous tissue with collagenase in DMEM containing fetal bovine serum and carry out in vitro culture of osteoblasts, then observe the effect of collagenase in DMEM containing fetal bovine serum on the digestion of osteoblasts.DESIGN: Control experiment.SETTING: Department of Orthopaedic Surgery, Tongji Hospital Affiliated to Huazhong University of Science and Technology.MATERIALS: This experiment was carried out in the Department of Orthopaedic Surgery, Tongji Hospital Affiliated to Huazhong University of Science and Technology from March to December 2004. Two Japanese white rabbits with big ears that were pregnant for 28 days were used in the experiment.METHODS: Type I collagenase with the same batch number was prepared into 0.1% collagenase with fetal bovine serum and 0.1% collagenase without fetal bovine serum by using culture medium containing 0.15 volume fraction of fetal bovine serum and DMEM solution respectively, serving as the enzyme digestive juice A and B for experimental group and control group respectively. Abdominal delivery was performed to take ont the fetal rabbit which was at embryonic 28 days under aseptic condition.Then, craniums of fetal rabbits were dissected, rinsed and chipped into pieces. 5 mL solution A and 5 mL solution B were added into the experimental group and control group at 37℃, respectively. Culture solution containing 0.15 volume fraction of fetal bovine serum was gradiently diluted into 0,1,2 and 4 folds in each group, and continued to digest and culture osteoblasts. The cellular survival rate was measured with trypan blue staining, and the osteoblast and its purity were identified with alkaline phosphatase(ALP) staining. Cellular growth was observed under the microscope.MAIN OUTCOME MEASURES: ①Cellular survival rate of two groups was detected with trypan blue staining. ② Cellular purity of two groups was detected with ALP staining in the cells. ③Cellular growth was distinguished by observing cellular morphology under the microscope.RESULTS: ① Results of trypan blue staining: Through cell counting, the rate of cells, which refused to be stained, of the experimental group reached 98%, and the cellular survival rate was high; that of control group reached 95%, and the cellular survival rate was also very high. ② Results of ALP staining in the cells: The area of ALP staining positive of experimental group was not less than 95%, and cellular purity was very high; but that of control group was not more than 75%, and cellular purity was very low. ③ Observation results under the microscope: Cells of experimental group were well stacked, convex and stereoscopic, they grew prosperously and had enough nutrition; while those of control group were not significantly in comparison with experimental group.CONCLUSION: Collagenase containing fetal bovine serum is used to digest the osseous tissue of cranium of fetal rabbits, and the cultured osteoblasts have typical properties of osteoblasts with simple component and high survival rate.