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  

Comparison study of type-Ⅰcollagenase and type-Ⅱcollagenase on digestion efficiency for primary osteoblast culture

作　　者：汤小康[1] 应航[1] 李敏[1] 包洁[1] 童培建[1] 

机构地区：[1]浙江中医药大学,浙江杭州310053

出　　处：《中医正骨》2013年第6期6-9,共4页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：浙江省自然基金(Y2110928);浙江省中医药科学研究基金计划(2010ZA020)

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。Objective：To compare type- I collagenase with type- II collagenase 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 group I and group II , then they were respectively digested by 0.1% type- I col- lagenase 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 group 1 were 6.20 × 105 cells ·mL^-1 ,6.00 × 105 cells ·mL^-1and 5.90 x 105 cells·mL^-1respectively;while results from cell counting for group II were 3.20 × 105 cells ·mL^-1 ,3.40 × 105 cells·mL^-1 and 2.90 x 105 cells ·mL^-1 respectively. Group I had about twice as many cells as compared with group II. 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 group I and group II [ （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 con- tents between group I and group 11 [ （2. 929 ± 0. 024） vs （ 2. 933 ± 0. 024 ） King unit ; t = 0. 389, P = 0. 706 ]. Conclusion ： Although both type- I collagenase and type- II collagenase are suitable for digestions of primary osteoblasts, the former can get more osteoblasts ＇after two- time digestions for one hour at a time.

关 键 词：成骨细胞 细胞培养技术 胶原酶类 动物实验 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]