大鼠骨髓来源与前交叉韧带来源MSCs体外生物学特性比较研究
发布时间：2019-02-20 21:55
【摘要】：目的比较大鼠骨髓来源与前交叉韧带(anterior cruciate ligament,ACL)来源的MSCs体外增殖及成骨、成软骨、成脂分化潜能的差异。方法取SPF级6周龄雄性BN大鼠10只,体质量200~220 g,无菌条件下分别取大鼠骨髓及ACL,贴壁法培养获取BMSCs与ACL来源MSCs并进行传代,倒置相差显微镜下观察细胞形态变化。取生长良好的第3代细胞,流式细胞仪检测细胞免疫表型CD34、CD45、CD90和CD29表达;细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测细胞体外增殖能力,克隆形成实验检测细胞体外克隆形成能力;行成骨、成软骨及成脂体外诱导多向分化能力检测;实时荧光定量PCR检测成骨[ALP、骨钙蛋白、RUNX2、BMP-2、分泌性磷蛋白1(secreted phosphoprotein 1,Spp1)]、成软骨[Ⅱ型胶原α1(collagen typeⅡα1,Col2α1)、软骨聚糖蛋白(Aggrecan,Acan)、Sox9]及成脂[过氧化物酶体增殖物激活受体γ2(peroxisome proliferator activated receptorγ2,PPARγ2)、CCAAT/增强子结合蛋白α]相关基因mRNA相对表达量。结果第3代ACL来源MSCs与BMSCs形态相似,均表现为贴壁生长的长梭形细胞。流式细胞仪检测示,两种细胞均表达CD29、CD90,基本不表达CD45及CD34。CCK-8法检测示,ACL来源MSCs的吸光度(A)值(1.11±0.08)显著高于BMSCs(0.78±0.05),差异有统计学意义(t=3.599,P=0.023);ACL来源MSCs的细胞集落数[(53.00±5.51)个/孔]亦明显多于BMSCs[(30.67±4.84)个/孔](t=3.045,P=0.038)。经成骨、成软骨、成脂体外诱导培养21d后,BMSCs及ACL来源MSCs均表现为茜素红、甲苯胺蓝及油红O染色阳性。实时荧光定量PCR检测示,ACL来源MSCs的BMP-2、Spp1、Col2α1、Acan、Sox9及PPARγ2 mRNA相对表达量显著高于BMSCs,差异均有统计学意义(P0.01)。结论 ACL来源MSCs比BMSCs具有更强的体外增殖能力,在相同体外条件下更易向软骨分化,是一种有潜力的促进腱骨愈合的种子细胞。
[Abstract]:Objective to compare the proliferation, osteogenesis, cartilage formation and adipogenic potential of MSCs derived from rat bone marrow and anterior cruciate ligament (anterior cruciate ligament,ACL) in vitro. Methods 10 male BN rats of SPF grade 6 weeks old, weighing 200 ~ 220g, were harvested from bone marrow of rats under aseptic condition and cultured with ACL, adherent method to obtain MSCs derived from BMSCs and ACL. The morphologic changes of cells were observed under inverted phase contrast microscope. The expression of CD34,CD45,CD90 and CD29 was detected by flow cytometry. Cell count kit 8 (cell counting kit 8 CCK-8) was used to detect the ability of cell proliferation in vitro, clone formation test to detect the ability of cell clone formation in vitro, osteogenesis, cartilage formation and fat-forming ability to induce multidirectional differentiation in vitro. Osteogenesis [ALP, osteocalcin, RUNX2,BMP-2, secreted phosphoprotein 1 (Spp1)], chondrogenic cartilage (type 鈪，
本文编号：2427324
[Abstract]:Objective to compare the proliferation, osteogenesis, cartilage formation and adipogenic potential of MSCs derived from rat bone marrow and anterior cruciate ligament (anterior cruciate ligament,ACL) in vitro. Methods 10 male BN rats of SPF grade 6 weeks old, weighing 200 ~ 220g, were harvested from bone marrow of rats under aseptic condition and cultured with ACL, adherent method to obtain MSCs derived from BMSCs and ACL. The morphologic changes of cells were observed under inverted phase contrast microscope. The expression of CD34,CD45,CD90 and CD29 was detected by flow cytometry. Cell count kit 8 (cell counting kit 8 CCK-8) was used to detect the ability of cell proliferation in vitro, clone formation test to detect the ability of cell clone formation in vitro, osteogenesis, cartilage formation and fat-forming ability to induce multidirectional differentiation in vitro. Osteogenesis [ALP, osteocalcin, RUNX2,BMP-2, secreted phosphoprotein 1 (Spp1)], chondrogenic cartilage (type 鈪，
本文编号：2427324
本文链接：https://www.wllwen.com/yixuelunwen/jjyx/2427324.html
最近更新
教材专著
