构建FGFR3基因沉默慢病毒载体及其对ATDC5细胞增殖和分化的影响
发布时间：2018-12-31 08:08
【摘要】：目的通过构建慢病毒介导的FGFR3RNAi,观察FGFR3对小鼠前软骨细胞系ATDC5增殖和分化的影响。方法针对FGFR3基因的有效靶序构建FGFR3RNAi慢病毒载体,并转染293T细胞进行病毒包装。用包装成功的慢病毒转染ATDC5细胞,Real-time PCR和Western blot检测ATDC5中FGFR3RNAi效率,细胞计数及MTT检测ATDC5的增殖变化,Real-time PCR检测ATDC5中软骨分化相关分子Ⅱ型胶原(collagenⅡ,ColⅡ)、Ⅹ型胶原(collagenⅩ,ColⅩ)和基质金属蛋白酶-13(MMP-13)的表达与变化。结果FGFR3RNAi慢病毒载体构建成功,并包装出相应的慢病毒,滴度为5×108TU/mL。FGFR3RNAi慢病毒转染ATDC5后FGFR3mRNA水平分别较空白组和阴性对照(NC)组下降了65.2%和68.8%(P0.01)。Western blot结果显示,与空白组和NC组相比,FGFR3RNAi组FGFR3蛋白水平显著降低(P0.01)。细胞计数及MTT检测结果显示,FGFR3RNAi组细胞增殖能力较空白组和NC组增强(P0.05,P0.01)。Real-time PCR结果显示,经向软骨诱导分化后,FGFR3RNAi组细胞中ColⅡ、ColⅩ和MMP-13的表达水平较空白组和NC组显著增加(P0.01)。结论成功包装的FGFR3RNAi慢病毒能有效降低ATDC5细胞中FGFR3基因的表达。FGFR3表达水平降低后对ATDC5细胞增殖和分化的抑制作用明显减弱。
[Abstract]:Aim to investigate the effect of FGFR3 on the proliferation and differentiation of mouse precursor cartilage cell line ATDC5 by constructing lentivirus-mediated FGFR3RNAi,. Methods FGFR3RNAi lentivirus vector was constructed according to the effective target sequence of FGFR3 gene and transfected into 293T cells for viral packaging. ATDC5 cells were transfected with lentivirus, FGFR3RNAi efficiency in ATDC5 was detected by Real-time PCR and Western blot, cell count and ATDC5 proliferation were detected by MTT, collagen 鈪，
本文编号：2396300
[Abstract]:Aim to investigate the effect of FGFR3 on the proliferation and differentiation of mouse precursor cartilage cell line ATDC5 by constructing lentivirus-mediated FGFR3RNAi,. Methods FGFR3RNAi lentivirus vector was constructed according to the effective target sequence of FGFR3 gene and transfected into 293T cells for viral packaging. ATDC5 cells were transfected with lentivirus, FGFR3RNAi efficiency in ATDC5 was detected by Real-time PCR and Western blot, cell count and ATDC5 proliferation were detected by MTT, collagen 鈪，
本文编号：2396300
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