PAX2稳转细胞系构建及对细胞迁移和侵袭能力的作用研究

Study on the Construction of PAX2 Stably Transfected Cell Lines and Their Effects on Cell Migration and Invasion Ability

目的探讨PAX2稳转细胞系构建及生物学作用。方法选择处于对数生长期大鼠肾小管上皮细胞,实验分为稳转组、空载组、对照组。稳转组采用阳离子脂质体转导将p EGFP-PAX2转入大鼠肾小管上皮细胞,空载组采用同样方法配置空载质粒转染溶液,对照组不添加质粒转染溶液。经G418筛选,建立稳定转染细胞系。通过细胞划痕实验及Transwell实验检测PAX2稳转细胞系的细胞迁移率及细胞侵袭作用。结果应用G418筛选14 d,稳转组大鼠肾小管上皮细胞有明显克隆长出,建立PAX2基因稳定转染肾小管上皮细胞系。稳转组在荧光显微镜下可见绿色荧光,基因融合表达获得成功。稳转组细胞PAX2条带密度与β-action吸光度比值为(1.00±0.04),空载组为(0.83±0.03),对照组为(0.85±0.04),3组吸光度比值比较,差异有统计学意义(F=398.7,P<0.05);其中稳转组吸光度比值高于空载组和对照组,差异均有统计学意义(P<0.05)。细胞划痕后18 h,对照组、空载组和稳转组细胞迁移率分别为(39.34±5.34)%、(40.56±7.54)%和(83.72±7.12)%,3组细胞迁移率比较,差异有统计学意义(F=431.8,P<0.05);其中稳转组细胞迁移率高于对照组和空载组,差异均有统计学意义(P<0.05)。各组细胞培养48 h后,对照组、空载组和稳转组迁移细胞分别为(23.33±3.63)、(22.00±8.14)、(45.67±7.14),3组迁移细胞数比较,差异有统计学意义(F=248.5,P<0.05);其中稳转组迁移细胞数高于对照组和空载组,差异均有统计学意义(P<0.05)。结论 p EGFP-PAX2质粒成功转染大鼠肾小管上皮细胞,并成功筛选出高效稳定表达PAX2的稳转细胞系。PAX2稳转增加了肾小管上皮细胞的迁移及侵袭能力。

Objective To investigate the construction and biological effects of PAX2 stably transfected cell lines.Methods Rat renal tubular epithelial cells at exponential phase were selected,and the cells were divided into stably transfected group,empty-loading group and control group. For stably transfected group,cationic liposome transduction was conducted to transfer p EGFP-PAX2 into rat renal tubular epithelial cells; for empty-loading group,cationic liposome transduction was also conducted with empty-loading plasmid transfection solution; for control group,no plasmid transfection solution was added. By G418 selection, stably transfected cell lines were constructed. Cell wound scratch assay and Tanswell test were conducted to measure cell migration rate and cell invasion of PAX2 stably transfected cell lines. Results G418 selection was undertaken for14 days, the cells of stably transfected group had obvious cloning growth, and PAX2 stably transfected cell lines were constructed. Green fluorescence was observed under fluorescence microscope in stably transfected group, and successful gene fusion expression was made. The ratio of the PAX2 strip density to the absorbancy of β-action was（ 1. 00 ± 0. 04） for stably transfected group,（ 0. 83 ± 0. 03） for empty-loading group and（ 0. 85 ± 0. 04） for control group. The three groups were significantly different in optical density ratio（ F = 398. 7,P 0. 05）; the stably transfected group was higher than empty-loading group in optical density ratio（ P 0. 05）. At 18 hours after cell scratch was made,the cell migration rates of control group,empty-loading group and stably transfected group were（ 39. 34 ± 5. 34） %,（ 40. 56 ± 7. 54） % and（ 83. 72± 7. 12） %,with significant differences among the three groups（ F = 431. 8,P 0. 05） and the stably transfected group higher than control group and empty-loading group（ P 0. 05）. At 48 hours of culture, the numbers of migrated cells of control group,empty-loading group and stably transfected group were（ 23. 33 ± 3. 63）,（ 22. 00 ± 8. 14） and（ 45. 67 ± 7. 14）,with significant differences among the three groups（ F = 248. 5,P 0. 05） and stably transfected group higher than control group and empty-loading group（ P 0. 05）. Conclusion p EGFP-PAX2 plasmid is successfully transfected into rat renal tubular epithelial cells, and stably transfected cell lines with highly effective and stable expression of PAX2 are successfully constructed. The stable transfection of PAX2 increases migration and invasion of renal tubular epithelial cells.