[1]汤小康,应航,李敏,等.Ⅰ型与Ⅱ型胶原酶在原代成骨细胞培养中消化效果的对比研究[J].中医正骨,2013,25(06):6-9.
　TANG Xiao-kang*,YING Hang,LI Min,et al.Comparison study of type-Ⅰcollagenase and type-Ⅱcollagenase on digestion efficiency for primary osteoblast culture[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2013,25(06):6-9.
点击复制

Ⅰ型与Ⅱ型胶原酶在原代成骨细胞培养中消化效果的对比研究()
分享到：

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第25卷
期数:
2013年06期
页码:
6-9
栏目:
基础研究
出版日期:
2013-06-30

文章信息/Info

Title:
Comparison study of type-Ⅰcollagenase and type-Ⅱcollagenase on digestion efficiency for primary osteoblast culture
作者:
汤小康; 应航; 李敏; 包洁; 童培建
浙江中医药大学,浙江 杭州 310053
Author(s):
TANG Xiao-kang*; YING Hang; LI Min; BAO Jie; TONG Pei-jian.*
Zhejiang University of Traditional Chinese Medicine,Hangzhou 310053,Zhejiang,China
关键词:
成骨细胞 细胞培养技术 胶原酶类 动物实验
Keywords:
Osteoblasts;  Cell culture techniques;  Collagenases;  Animal experimentation
摘要:
目的:比较Ⅰ型与Ⅱ型胶原酶在原代成骨细胞培养中的消化效果。方法:取6只新生24 h内的SD大鼠,处死后取出颅盖骨并制成碎骨片。将碎骨片等分成2份,标记为Ⅰ组和Ⅱ组,分别用0.1%Ⅰ型胶原酶和0.1%Ⅱ型胶原酶在37 ℃消化2次,每次1 h 。将2次消化所得到的细胞悬液混匀后进行细胞培养,并对2组细胞分别进行细胞计数,观察细胞形态、细胞增殖情况及碱性磷酸酶染色情况,并测定碱性磷酸酶含量。结果:①细胞计数。Ⅰ组细胞3次细胞计数结果分别为6.20×105个·mL-1、6.00×105个·mL-1、5.90×105个·mL-1; Ⅱ组细胞3次细胞计数结果分别为3.20×105个·mL-1、3.40×105个·mL-1、2.90×105个·mL-1。Ⅰ组细胞总量为Ⅱ组细胞总量的2倍左右。②细胞形态。2组细胞多呈梭形,表面有2～3个突触与周围细胞相联系,2组细胞形态无明显差异。③细胞增殖。Ⅰ组光密度(0.492±0.001)与Ⅱ组光密度(0.491±0.002)比较,差异无统计学意义(t=0.379,P=0.713)。④碱性磷酸酶染色。2组细胞用苏木素染色后,胞浆内均可见明显的咖啡色和红棕色颗粒。⑤碱性磷酸酶含量。Ⅰ组碱性磷酸酶含量[(2.929±0.024)金氏单位]与Ⅱ组[(2.933±0.024)金氏单位]比较,差异无统计学意义(t=0.389,P=0.706)。结论:Ⅰ型胶原酶和Ⅱ型胶原酶均适用于成骨细胞的原代消化,但在消化1 h×2次条件下用Ⅰ型胶原酶容易获得更多的成骨细胞。
Abstract:
Objective:To compare type-Ⅰcollagenase with type-Ⅱcollagenase on digestion efficiency for primary osteoblast culture.Methods:Six neonatal(<24 hours old)SD rats were executed and their craniums were fetched out and made of bone fragments.The bone fragments were equally divided into 2 parts and marked as groupⅠand groupⅡ,then they were respectively digested by 0.1% type-Ⅰcollagenase and 0.1% type-Ⅱ collagenase at 37 ℃ two times,one hour at a time.The cell suspensions got from the two-time digestions were mixed and the cells were cultured.Subsequently,the cells in the 2 groups were respectively counted.The cell shape,cell proliferation and alkaline phosphatase(ALP)staining results were observed,then ALP contents were assayed.Results:Results from cell counting for groupⅠwere 6.20×105 cells·mL-1,6.00×105 cells·mL-1 and 5.90×105 cells·mL-1 respectively; while results from cell counting for groupⅡwere 3.20×105 cells·mL-1,3.40×105 cells·mL-1 and 2.90×105 cells·mL-1 respectively.GroupⅠhad about twice as many cells as compared with groupⅡ.Most cells in the 2 groups were in the shapes of fusiform,and cells were connected by 2-3 synapses.There was no significant difference in cell shape between the 2 groups.There was no statistical difference in optical density between groupⅠand groupⅡ[(0.492±0.001)vs(0.491±0.002); t=0.379,P=0.713].ALP staining results showed that coffee-colored granules and marron granules could be easily found in the cytoplasm in the 2 groups after hematoxylin staining.There was no statistical difference in ALP contents between groupⅠand groupⅡ[(2.929±0.024)vs(2.933±0.024)King unit; t=0.389,P=0.706].Conclusion:Although both type-Ⅰcollagenase and type-Ⅱcollagenase are suitable for digestions of primary osteoblasts,the former can get more osteoblasts after two-time digestions for one hour at a time.

参考文献/References:

[1] Lozupone E,Favia A,Grimaldi A.Effect of intermittent mechanical force on bone tissue in vitro:preliminary results[J].J Bone Miner Res,1992,7(Suppl 2):S407-S409.
[2] Sun DC,Li DH,Ji HC,et al.In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics[J].Braz J Med Biol Res,2012,45(6):502-509.
[3] 明磊国,葛宝丰,陈克明,等.蛇床子素对体外培养成骨细胞增殖与分化成熟的影响[J].中国骨伤,2010,23(9):688-691.
[4] 于波,崔宪春,谢进.复元活血汤含药血清对大鼠成骨细胞功能的影响[J].中医正骨,2011,23(1):17-21.
[5] 刘振峰,方锐,孟庆才.Ⅱ型胶原酶消化法可短时间获得大量纯化大鼠关节软骨细胞[J].中国组织工程研究与临床康复,2011,15(50):9323-9326.
[6] 刘明东,盛天金,王万宗.胰蛋白酶及Ⅱ型胶原酶消化获取关节软骨细胞[J].中国组织工程研究与临床康复,2010,14(46):8551-8554.
[7] Orriss IR,Taylor SE,Arnett TR.Rat osteoblast cultures[J].Methods Mol Biol,2012,816(1):31-41.
[8] Hasegawa Y,Shimada K,Suzuki N,et al.The in vitro osteogenetic characteristics of primary osteoblastic cells from a rabbit calvarium[J].J Oral Sci,2008,50(4):427-434.
[9] 程婉,许兵,应航,等.抗骨质疏松中药对成骨细胞作用的研究进展[J].中医正骨,2012,24(2):65-68.
[10] 许兵,方剑利,刘慧,等.补肾活血方对去势大鼠骨质疏松的影响[J].中华骨质疏松骨矿盐疾病杂志,2011,4(3):177-182.

备注/Memo

备注/Memo:
基金项目:浙江省自然基金(Y2110928),浙江省中医药科学研究基金计划(2010ZA020) 通讯作者:应航 E-mail:yh@zjtcm.net
更新日期/Last Update: 1900-01-01