【摘要】：目的通過構(gòu)建慢病毒介導(dǎo)的FGFR3RNAi,觀察FGFR3對小鼠前軟骨細(xì)胞系A(chǔ)TDC5增殖和分化的影響。方法針對FGFR3基因的有效靶序構(gòu)建FGFR3RNAi慢病毒載體,并轉(zhuǎn)染293T細(xì)胞進(jìn)行病毒包裝。用包裝成功的慢病毒轉(zhuǎn)染ATDC5細(xì)胞,Real-time PCR和Western blot檢測ATDC5中FGFR3RNAi效率,細(xì)胞計(jì)數(shù)及MTT檢測ATDC5的增殖變化,Real-time PCR檢測ATDC5中軟骨分化相關(guān)分子Ⅱ型膠原(collagenⅡ,ColⅡ)、Ⅹ型膠原(collagenⅩ,ColⅩ)和基質(zhì)金屬蛋白酶-13(MMP-13)的表達(dá)與變化。結(jié)果FGFR3RNAi慢病毒載體構(gòu)建成功,并包裝出相應(yīng)的慢病毒,滴度為5×108TU/mL。FGFR3RNAi慢病毒轉(zhuǎn)染ATDC5后FGFR3mRNA水平分別較空白組和陰性對照(NC)組下降了65.2%和68.8%(P0.01)。Western blot結(jié)果顯示,與空白組和NC組相比,FGFR3RNAi組FGFR3蛋白水平顯著降低(P0.01)。細(xì)胞計(jì)數(shù)及MTT檢測結(jié)果顯示,FGFR3RNAi組細(xì)胞增殖能力較空白組和NC組增強(qiáng)(P0.05,P0.01)。Real-time PCR結(jié)果顯示,經(jīng)向軟骨誘導(dǎo)分化后,FGFR3RNAi組細(xì)胞中ColⅡ、ColⅩ和MMP-13的表達(dá)水平較空白組和NC組顯著增加(P0.01)。結(jié)論成功包裝的FGFR3RNAi慢病毒能有效降低ATDC5細(xì)胞中FGFR3基因的表達(dá)。FGFR3表達(dá)水平降低后對ATDC5細(xì)胞增殖和分化的抑制作用明顯減弱。
[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，

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