中文摘要：

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,Col Ⅱ）、Ⅹ型胶原（collagen Ⅹ, Col Ⅹ）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FGFR3 mRNA水平分别较空白组和阴性对照（NC）组下降了65.2%和68.8%（P〈0.01）。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（P〈0.01）。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（P〈0.05，P〈0.01）。Real-time PCR结果显示，经向软骨诱导分化后，FGFR3RNAi组细胞中Col Ⅱ、Col Ⅹ和MMP-13的表达水平较空白组和NC组显著增加（P〈0.01）。 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。

英文摘要：

Objective To construct a lentivirus vector targeting fibroblast growth factor receptor 3 （FGFR3） gene and determine its effect on the proliferation and differentiation of prechondrogenic ATDC5 cells. Methods The lentivirus vector targeting FGFR3 gene was constructed and then transfected into 293T cells. The recombinant lentivirus vector was transfected into ATDC5 cells. The expression of FGFR3 was detected by real-time PCR and Western blotting. The effect of FGFR3RNAi vector on the proliferation of ATDC5 cells was measured by cell count assay and MTT assay. The effects of FGFR3RNAi vector on the makers of chondrogenic differentiation, including collagenⅡ, collagen Ⅹ and MMP-13, were detected by real-time PCR. Results The recombinant lentivirus vector targeting FGFR3 gene was successfully constructed. The lentivirus titer was 5×108TU/mL. Real-time PCR showed that the FGFR3 mRNA level was decreased by 65.2% in FGFR3RNAi transfected cells compared with the cells transfected by the blank vector （P〈0.01）, and lower by 68.8% than the negative control （P〈0.01）. Western blotting indicated that the expression of FGFR3 was also significantly reduced in FGFR3RNAi transfected cells than the blank vector-transfected cells and negative control （P〈0.01）. Cell count assay and MTT assay demonstrated that FGFR3RNAi vector resulted in strongly inhibited proliferation in ATDC5 cells than the blank vector and negative control （P〈0.05, P〈0.01）. Real-time PCR indicated that FGFR3 induced the mRNA expression of collagenⅡ, collagenⅩ and MMP-13 than the other 2 kinds of cells （P〈0.01）. Conclusion The recombinant lentivirus vector targeting FGFR3 gene is successfully constructed. It efficiently down-regulates FGFR3 in ATDC5 cells and alleviates the FGFR3-induced inhibition on the proliferation and differentiation in ATDC5 cells.