本文選題：髓核細(xì)胞 + Ⅱ型膠原酶消化法�。� 參考：《山東醫(yī)藥》2017年32期

【摘要】：目的建立一種體外分離、培養(yǎng)人髓核細(xì)胞(NPCs)的新方法,并探討其意義。方法采用0.2%Ⅱ型膠原酶消化法分離人正常NPCs,單層貼壁法進行傳代培養(yǎng),倒置顯微鏡下觀察細(xì)胞形態(tài)。取第1、2、3代NPCs,采用CCK-8法檢測細(xì)胞增殖能力(以吸光度值表示),連續(xù)檢測12天。取第2代NPCs,采用免疫組化法檢測Ⅱ型膠原(Col-2)、細(xì)胞角蛋白18(KRT-18)、KRT-19、低氧誘導(dǎo)因子1α(HIF-1α)、葡萄糖轉(zhuǎn)運子1(GLUT-1)、Sox-9、聚集蛋白聚糖(ACAN)、CD24陽性表達(dá)率。結(jié)果分離的NPCs呈多角形或短梭形,類似于軟骨細(xì)胞;第4代以后,NPCs細(xì)胞突起延長,呈長梭形,生長緩慢,出現(xiàn)老化現(xiàn)象。NPCs生長曲線呈S形:1~2天細(xì)胞生長緩慢;3~7天細(xì)胞生長迅速,進入對數(shù)生長期;8天后進入平臺期,細(xì)胞增殖緩慢。連續(xù)培養(yǎng)12天,第1、2、3代NPCs相同時間點吸光度值比較差異均無統(tǒng)計學(xué)意義(P均0.05)。第2代NPCs中Col-2、KRT-18、KRT-19、HIF-1α、GLUT-1、Sox-9、ACAN、CD24陽性表達(dá)率均≥85.6%(85.6%~91.2%)。結(jié)論單純0.2%Ⅱ型膠原酶消化法及單層貼壁法可分離、培養(yǎng)人NPCs,并經(jīng)免疫組化染色鑒定證實;傳代3代以內(nèi)的NPCs細(xì)胞形態(tài)及增殖能力均無明顯改變,可作為椎間盤退變相關(guān)研究的種子細(xì)胞。
[Abstract]:Objective to establish a new method for isolation and culture of human nucleus pulposus cells (NPCs) in vitro and to explore its significance. Methods normal NPCs were isolated by 0.2% collagenase digestion and cultured with monolayer adherent method. The morphology of NPCs was observed under inverted microscope. CCK-8 assay was used to detect the proliferative ability of NPCs in the 3rd generation of NPCs. The cell proliferation was measured by absorbance for 12 days. In the second generation of NPCs, immunohistochemical method was used to detect the positive expression rate of CD24 in type 鈪，

本文編號：1949107