CAJ | 학술논문

目的采用胶原酶预消化组织块贴壁法培养人退变椎间盘髓核细胞,与单纯Ⅱ型胶原酶消化法比较培养效果.方法收集腰椎间盘退变患者的髓核组织分为A、B两组进行培养.A组:体积分数0.025％Ⅱ型胶原酶预消化30 min后组织块接种;B组:体积分数0.025％Ⅱ型胶原酶消化5h后过滤、离心、接种培养.比较两种方法培养成功率和原代细胞融合时间.HE、甲苯胺蓝、免疫细胞荧光染色法观察细胞形态及Sox-9、Ⅱ型胶原和蛋白聚糖的表达.结果 A、B两组培养成功率差异无统计学意义(P＞0.05),融合时间A组显著短于B组(P＜0.01).A组培养的髓核细胞可被甲苯胺蓝染成天蓝色,免疫细胞荧光检测Sox-9、Ⅱ型胶原、蛋白聚糖呈阳性表达.结论与单纯Ⅱ形胶原酶消化法比较,Ⅱ型胶原酶预消化后贴壁培养法培养人退变椎间盘髓核细胞成功率高,短时间内可获大量细胞,且强表达Sox-9、Ⅱ型胶原和蛋白聚糖.
목적 채용효원매예소화조직괴첩벽법배양인퇴변추간반수핵세포,여단순Ⅱ형효원매소화법비교배양효과.방법 수집요추간반퇴변환자적수핵조직분위A、B량조진행배양.A조:체적분수0.025％Ⅱ형효원매예소화30 min후조직괴접충;B조:체적분수0.025％Ⅱ형효원매소화5h후과려、리심、접충배양.비교량충방법배양성공솔화원대세포융합시간.HE、갑분알람、면역세포형광염색법관찰세포형태급Sox-9、Ⅱ형효원화단백취당적표체.결과 A、B량조배양성공솔차이무통계학의의(P＞0.05),융합시간A조현저단우B조(P＜0.01).A조배양적수핵세포가피갑분알람염성천람색,면역세포형광검측Sox-9、Ⅱ형효원、단백취당정양성표체.결론 여단순Ⅱ형효원매소화법비교,Ⅱ형효원매예소화후첩벽배양법배양인퇴변추간반수핵세포성공솔고,단시간내가획대량세포,차강표체Sox-9、Ⅱ형효원화단백취당.
Objective To compare the efficiency of explants adherent after enzymatic pre-digestion and traditional typeⅡ?collagenase digestion methods for culturing human nucleus pulposus cells from degenerated intervertebral discs.Methods Human nucleus pulposus tissues collected from patients'degenerated intervertebral discs were divided into Groups A and B.In Group A,the nucleus pulposus tissues were cultured using explants adherent method directly after 0.025 ％ typeⅡ?collagenase digestion for 30 minutes.In Group B,the tissues were firstly digested with 0.025 ％ typeⅡ?collagenase for five hours,and then underwent filtration,centrifugation and inoculation successively Success rate and primary cell fusion time of the two culture methods were compared.Cell morphology and expressions of sex determining region Y-box 9(Sox-9),collagenⅡ?and aggrecan were observed by HE staining,toluidine blue staining and immunofluorescence cell staining.Results There was no significant difference in the successful culture rate between the two groups(P＞0.05).However,the average fusion time of primary passage in Group A was significantly shorter than that in Group B(P＜0.01).In Group A,the nucleus pulposus cells could be stained as azure by toluidine blue and immunofluorescence cell staining showed positive expressions of Sox-9,collagenⅡ?and aggrecan.Conclusions Compared with traditional typeⅡ?collagenase digestion,explants adherent method after typeⅡ?collagenase pre-digestion for the culture of human nucleus pulposus cells from degenerative intervertebral discs has a high success rate and obtains a large number of cells in a short time.Meanwhile,the cells have strong expressions of Sox-9,collagenⅡ?and aggrecan.