新生大鼠原代软骨细胞分离和培养方法的改进  

Improvement of isolation and culture methods for primary chondrocytes of neonatal rats

作　　者：杨丹聃 陈骄阳 王馨珩 赵泽彤 潘莹[3] 薛百功[3] 高长曌 YANG Dandan;CHEN Jiaoyang;WANG Xinheng;ZHAO Zetong;PAN Ying;XUE Baigong;GAO Changzhao(Experimental Center of Pathogenobiology Immunology Cytobiology and Genetics,School of Basic Medical Sciences,Jilin University,Changchun 130021,China;Department of Dermatology,China-Japan Union Hospital,Jilin University,Changchun 130033,China;Department of Biology,School of Basic Medical Sciences,Jilin University,Changchun 130021,China;Department of Radiotherapy,China-Japan Union Hospital,Jilin University,Changchun 130033,China)

机构地区：[1]吉林大学基础医学院病原免疫细胞遗传学实验中心,吉林长春130021 [2]吉林大学中日联谊医院皮肤科,吉林长春130033 [3]吉林大学基础医学院细胞学系,吉林长春130021 [4]吉林大学中日联谊医院放疗科,吉林长春130033

出　　处：《吉林大学学报（医学版）》2024年第5期1438-1449,共12页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(8227091657);吉林省教育厅科研项目(JJKH20221050KJ)。

摘　　要：目的:探讨新生大鼠原代软骨细胞分离和培养的改进方法,以建立高效经济的体外软骨细胞培养体系。方法:从新生大鼠关节中分离原代软骨细胞,分为过夜消化(OD)组和快速消化(RD)组进行分离,OD组软骨细胞采用Ⅱ型胶原酶过夜消化,RD组软骨细胞采用预消化的物理化学消化相结合的手段分离细胞。采用含0%(空白组1)、1%、2%、4%和10%胎牛血清(FBS),0(空白组2)、0.1、0.2、0.4、0.8、1.0、2.0 g·L^(-1)维生素C(VC)和0(空白组3)、0.5、1.0、2.0、4.0、8.0、10.0μg·L^(-1)聚乳酸-羟基乙酸共聚体(PLGA)纳米粒子的改良培养液培养软骨细胞。将杜氏改良Eagle培养基F12营养混合液(DMEM/F12)与含不同浓度FBS、VC和PLGA的培养液分别混合,并按照各成分浓度进行相应分组。采用细胞计数仪计数各组细胞并检测各组细胞存活率和直径,采用甲苯胺蓝特异性染色法检测各组细胞形态表现,采用CCK-8法检测各组细胞增殖活性,采用细胞黏附实验检测各组细胞黏附率,采用Hoechst/碘化丙碇(PI)染色检测各组细胞凋亡情况,采用MTT法检测改良培养液培养后各组细胞增殖活性,将细胞分为DMEM/F12+10%FBS组(对照组)、DMEM/F12+1%FBS组、DMEM/F12+1%FBS+0.4 g·L^(-1)VC+1μg·L^(-1)PLGA组,采用实时荧光定量PCR(RT-qPCR)法检测改良培养液培养后各组细胞中性别决定区域Y框转录因子9(SOX9)、Ⅱ型胶原α1链(Col2A1)、Ⅹ型胶原α1链(Col10A1)和基质金属蛋白酶13(MMP13)mRNA表达水平,采用免疫荧光染色检测改良培养液培养后各组细胞中Ⅱ型胶原(COLⅡ)和SOX9表达情况。结果:OD组原代软骨细胞存活率小于RD组,细胞平均直径大于RD组。OD组原代软骨细胞形态较大,呈梭形,大多数细胞出现伪足;RD组原代软骨细胞形态较小,大多数细胞呈菱形,仅部分细胞出现伪足。2组原代软骨细胞经甲苯胺蓝特异性染色均显色明显,但RD组消化时间较短,软骨细胞实际培养时间较OObjective:To discuss the improved methods for the isolation and culture of primary chondrocytes from the neonatal rats,and to establish an efficient and economical in vitro chondrocyte culture system.Methods:The primary chondrocytes were isolated from the joints of neonatal rats and divided into overnight digestion(OD)group and rapid digestion(RD)group for separation.The chondrocytes in OD group were digested overnight by typeⅡcollagenase,while the chondrocytes in RD group were separated by the combination of pre-digestion with physical and chemical digestion methods.The chondrocytes were cultured in modified media containing 0%(blank group 1),1%,2%,4%,and 10%fetal bovine serum(FBS),0(blank group 2),0.1,0.2,0.4,0.8,1.0,and 2.0 g·L^(-1) vitamin C(VC),and 0(blank group 3),0.5,1.0,2.0,4.0,8.0,10.0μg·L^(-1) poly(lactic-co-glycolic acid)(PLGA)nanoparticles.The media containing different concentrations of FBS,VC,and PLGA were mixed with Dulbecco’s modified Eagle’s medium/nutrient mixture F-12(DMEM/F12),and were divided into related groups based on the concentrations of ingredients.Cell counter was used to count the chondrocytes in various groups and the survival rates and diameters of the chondrocytes in various groups were detected;Toluidine blue staining was used to detect the morphology of the chondrocytes in various groups;CCK-8 method was used to detect the proliferative activities of the chondrocytes in various groups;cell adhesion assay was used to detect the adhesion rates of the chondrocytes in various groups;Hoechst/propidium iodide(PI)staining was used to detect the apoptosis of the chondrocytes in various groups;MTT assay was used to detect the proliferation activities of the chondrocytes in various groups after treated with modified media.The cells were divided into DMEM/F12+10%FBS group,DMEM/F12+1%FBS group,and DMEM/F12+1%FBS+0.4 g·L^(-1) VC+1μg·L^(-1) PLGA group.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of sex-determining region Y-box 9(SO

关 键 词：软骨细胞 原代细胞培养 体外技术 条件培养基 血清 

分 类 号：Q813.1[生物学—生物工程]