论文部分内容阅读
目的:构建细胞周期蛋白D1(cyclin D1)的反义寡核苷酸真核表达载体,并检测其转染Tca8113/CDDP细胞系后的表达情况。方法:以Tca8113/CDDP细胞总RNA为模板,应用RT-PCR法扩增cyclin D1全长,通过克隆至pUCm-T载体中,测序完全正确;经IPTG诱导,可正确表达蛋白。上下游分别利用H ind III和EcoRI的酶切位点插入真核表达载体pcDNA3.1,测序鉴定;并构建表达cyclin D1反义寡核苷酸的2个重组载体,通过Lipofectam ine将其导入Tca8113/CDDP细胞系中,G418筛选获得阳性稳定表达细胞系;通过RT-PCR、免疫组化检测cyclin D1基因转录和蛋白表达。结果:成功构建pcDNA3.1-cyclin D1真核表达载体,分别命名为pcDNA3.1空载体(Co)、cyclin D1m RNA5’端为靶区的反义载体(C5’)、cyclin D1m RNA3’端为靶区的反义载体(C3’)、cyclin D1正义载体(CD1)。3’端反义寡核苷酸转染细胞后,cyclin D1m RNA表达水平降低58.8%,m RNA5’端反义寡核苷酸组无明显抑制。免疫组化结果显示,转导正义寡核苷酸组cyclin D1阳性表达为最高,转导m RNA3’端、m RNA5’端反义寡核苷酸组为弱阳性表达。结论:cyclin D1m RNA3’端反义寡核苷酸能明显抑制cyclin D1在Tca8113/CDDP中的表达,为进一步研究逆转Tca8113/CDDP耐药性提供了实验工具。
OBJECTIVE: To construct an antisense oligonucleotide eukaryotic expression vector for cyclin D1 and to detect its expression in Tca8113 / CDDP cell line. Methods: Total RNA of Tca8113 / CDDP cells was used as a template to amplify the full length of cyclin D1 by RT-PCR. The full-length cDNA of cyclin D1 was cloned into pUCm-T vector and sequenced. The protein was correctly expressed by IPTG induction. The upstream and downstream were respectively inserted into the eukaryotic expression vector pcDNA3.1 using H ind III and EcoRI restriction sites, and sequenced. Two recombinant vectors expressing the cyclin D1 antisense oligonucleotide were constructed and introduced into Tca8113 by Lipofectam ine / CDDP cell line, positive clones were screened by G418. The transcription and protein expression of cyclin D1 were detected by RT-PCR and immunohistochemistry. Results: The pcDNA3.1-cyclin D1 eukaryotic expression vector was successfully constructed and named as pcDNA3.1 empty vector (Co). The 5’-end of cyclin D1m RNA was antisense vector (C5 ’). The end of cyclin D1m RNA3 Target antisense vector (C3 ’), cyclin D1 sense vector (CD1). The expression of cyclin D1m RNA was reduced by 58.8% after transfection with 3 ’antisense oligonucleotide, but no significant inhibition was found at the 5’ antisense oligonucleotide group. The result of immunohistochemistry showed that the expression of cyclin D1 was the highest in transduced sense oligonucleotide group, which was weakly positive on the 3 ’end of transduced m RNA and antisense oligonucleotide on the 5’ end of m RNA. Conclusion: Cyclin D1m RNA3 ’antisense oligodeoxynucleotide can significantly inhibit the expression of cyclin D1 in Tca8113 / CDDP and provide experimental tools for further research on reversing the drug resistance of Tca8113 / CDDP.