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[1]湯小康,應(yīng)航,李敏,等.Ⅰ型與Ⅱ型膠原酶在原代成骨細(xì)胞培養(yǎng)中消化效果的對(duì)比研究[J].中醫(yī)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.
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Ⅰ型與Ⅱ型膠原酶在原代成骨細(xì)胞培養(yǎng)中消化效果的對(duì)比研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第25卷
期數(shù):
2013年06期
頁(yè)碼:
6-9
欄目:
基礎(chǔ)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:
湯小康; 應(yīng)航; 李敏; 包潔; 童培建
浙江中醫(yī)藥大學(xué),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
關(guān)鍵詞:
成骨細(xì)胞 細(xì)胞培養(yǎng)技術(shù) 膠原酶類 動(dòng)物實(shí)驗(yàn)
Keywords:
Osteoblasts;  Cell culture techniques;  Collagenases;  Animal experimentation
摘要:
目的:比較Ⅰ型與Ⅱ型膠原酶在原代成骨細(xì)胞培養(yǎng)中的消化效果。方法:取6只新生24 h內(nèi)的SD大鼠,處死后取出顱蓋骨并制成碎骨片。將碎骨片等分成2份,標(biāo)記為Ⅰ組和Ⅱ組,分別用0.1%Ⅰ型膠原酶和0.1%Ⅱ型膠原酶在37 ℃消化2次,每次1 h 。將2次消化所得到的細(xì)胞懸液混勻后進(jìn)行細(xì)胞培養(yǎng),并對(duì)2組細(xì)胞分別進(jìn)行細(xì)胞計(jì)數(shù),觀察細(xì)胞形態(tài)、細(xì)胞增殖情況及堿性磷酸酶染色情況,并測(cè)定堿性磷酸酶含量。結(jié)果:①細(xì)胞計(jì)數(shù)。Ⅰ組細(xì)胞3次細(xì)胞計(jì)數(shù)結(jié)果分別為6.20×105個(gè)·mL-1、6.00×105個(gè)·mL-1、5.90×105個(gè)·mL-1; Ⅱ組細(xì)胞3次細(xì)胞計(jì)數(shù)結(jié)果分別為3.20×105個(gè)·mL-1、3.40×105個(gè)·mL-1、2.90×105個(gè)·mL-1。Ⅰ組細(xì)胞總量為Ⅱ組細(xì)胞總量的2倍左右。②細(xì)胞形態(tài)。2組細(xì)胞多呈梭形,表面有2～3個(gè)突觸與周圍細(xì)胞相聯(lián)系,2組細(xì)胞形態(tài)無(wú)明顯差異。③細(xì)胞增殖。Ⅰ組光密度(0.492±0.001)與Ⅱ組光密度(0.491±0.002)比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(t=0.379,P=0.713)。④堿性磷酸酶染色。2組細(xì)胞用蘇木素染色后,胞漿內(nèi)均可見明顯的咖啡色和紅棕色顆粒。⑤堿性磷酸酶含量。Ⅰ組堿性磷酸酶含量[(2.929±0.024)金氏單位]與Ⅱ組[(2.933±0.024)金氏單位]比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(t=0.389,P=0.706)。結(jié)論:Ⅰ型膠原酶和Ⅱ型膠原酶均適用于成骨細(xì)胞的原代消化,但在消化1 h×2次條件下用Ⅰ型膠原酶容易獲得更多的成骨細(xì)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.

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備注/Memo

備注/Memo:
基金項(xiàng)目:浙江省自然基金(Y2110928),浙江省中醫(yī)藥科學(xué)研究基金計(jì)劃(2010ZA020) 通訊作者:應(yīng)航 E-mail:[email protected]
更新日期/Last Update: 1900-01-01