CAJ | 학술논문

目的：基因敲除小鼠已广泛用于关节疾病的研究中。文中探讨小鼠关节处理方式、消化酶种类及胶原酶消化时间3种因素对获取原代成纤维样滑膜细胞（ fibroblast-like synoviocytes ， FLSs）数量的影响。方法将12只雄性SPF级小鼠，采用打开关节（6只）与不打开关节（6只）的方法处理双后肢，使用Ⅳ、Ⅱ型胶原酶消化分离原代细胞，差速消化法逐代纯化FLSs。倒置显微镜观察细胞形态，流式细胞仪检测细胞种类、活力与纯度。结果不打开关节处理获得的FLSs数量[（24933±503）个]显著高于打开关节处理获得的数量[（19133±115）个]，差异有统计学意义（ P＜0．05）；连续收集消化单支后肢1～7 h细胞悬液， FLSs数分别为（700±300）、（600±100）、（15200±900）、（5100±800）、（2700±300）、（900±200）、（300±100）个，以3 h的FLSs 数量最多。Ⅳ型胶原酶消化所得FLSs 细胞总数[（24900±500）个]明显高于Ⅱ型胶原酶[（18100±400）个]，差异有统计学意义（P＜0．05）。结论消化培养小鼠原代FLSs，建议不打开关节，使用Ⅳ型胶原酶消化，取2～6h消化的细胞悬液进行后续培养。
목적：기인고제소서이엄범용우관절질병적연구중。문중탐토소서관절처리방식、소화매충류급효원매소화시간3충인소대획취원대성섬유양활막세포（ fibroblast-like synoviocytes ， FLSs）수량적영향。방법장12지웅성SPF급소서，채용타개관절（6지）여불타개관절（6지）적방법처리쌍후지，사용Ⅳ、Ⅱ형효원매소화분리원대세포，차속소화법축대순화FLSs。도치현미경관찰세포형태，류식세포의검측세포충류、활력여순도。결과불타개관절처리획득적FLSs수량[（24933±503）개]현저고우타개관절처리획득적수량[（19133±115）개]，차이유통계학의의（ P＜0．05）；련속수집소화단지후지1～7 h세포현액， FLSs수분별위（700±300）、（600±100）、（15200±900）、（5100±800）、（2700±300）、（900±200）、（300±100）개，이3 h적FLSs 수량최다。Ⅳ형효원매소화소득FLSs 세포총수[（24900±500）개]명현고우Ⅱ형효원매[（18100±400）개]，차이유통계학의의（P＜0．05）。결론소화배양소서원대FLSs，건의불타개관절，사용Ⅳ형효원매소화，취2～6h소화적세포현액진행후속배양。
Obej ctive Knockout mice are widely used in the studies of joint diseases .This article investigated the effects of joint processing methods , collagenase types ,and collagenase digestion time on the number of primary fibroblast -like synoviocytes (FLSs) obtained from mice. Methods The hind legs of 6 of the 12 male mice were cut open from the hip joints , but not those of the other 6.FLSs were isolated using the type-Ⅳcollagenase digestion method and purified by differential digestion .Cell morphology was observed under the inverted microscope .The type, viability, and purity of the cells were determined by flow cytometry . Rse ults Significantly fewer FLSs were obtained from the mice with the hind legs cut open ( 19 133 ±115 ) than from those without (24 933 ± 503) (P<0.05).The numbers of FLSs collected from the cell suspension at 1, 2, 3, 4, 5,6 , and 7 hours after digestion were 700 ±300 , 600 ±100 , 15 200 ±900 , 5100 ±800 ,2700 ±300 , 900 ±200, and 300 ±100, respectively, the highest at 3 hours. There were statistically significant differences in the total number of FLSs obtained by type-Ⅳ and type-Ⅱ collagenase digestions (24900 ±500v s 18 100 ±400, P<0.05). Conclusion For in virt o culture of primary mouse FLSs, it is recommended that the hip joints be not cut open, and type-Ⅳcollagenase be used with cell sus-pension at 2-6hours after digestion .