采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株。将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接，构建包含TRPV1基因的过表达载体pCDH-TRPV1。将过表达载体pCDH-TRPV1转化DH 5α感受态细菌，大量扩繁后提取过表达载体pCDH-TRPV1的质粒，与psPAX2和pMD两种含有慢病毒包装所必需元件的质粒混合，再与脂质体混合制备脂质体-载体混合液。将脂质体-载体混合液转染至单层的293T细胞中，培养48h进行病毒包装。收集富含慢病毒颗粒的293T细胞上清液，超离心纯化成浓缩病毒，然后再与polybrene 一起感染单层Caco-2细胞，通过GFP绿荧光信号来筛选获得TRPV1基因过表达的稳定细胞株。通过Realtime PCR方法和Western-blot检测TRPV1的mRNA表达量及蛋白表达量，结果表明，Caco-2-TRPV1重组细胞株的TRPV1的mRNA表达量及蛋白表达量均显著高于Caco-2-GFP对照细胞(P＜0。05)。成功构建了TRPV1基因过表达的稳定细胞株，为后续辣椒素降脂机理的研究提供了正向调控细胞模型。
Construction of Caco-2 stable cell line overexpressing TRPV1 gene
A stable recombinant strain of the human colorectal adenocarcinoma cell line Caco-2,overexpressing the TRPV1 gene,was successfully established using a lentiviral vector system.To construct the overexpression vector pCDH-TRPV1,the TR-PV1 PCR product and the empty lentiviral vector pCDH were digested and ligated using T4 DNA ligase.This vector was then transformed into DH5α competent cells for propagation.The plasmid of the overexpression vector pCDH-TRPV1 was extracted and co-transfected with psPAX2 and pMD plasmids,essential for lentivirus packaging,into 293T cells using a liposome-mediated method.After 48 hours of culture,the supernatant containing lentiviral particles was harvested and concentrated via ultracentrif-ugation.Caco-2 cells were then infected with the concentrated virus in the presence of polybrene.Stable TRPV1-overexpressing cell lines were selected based on GFP fluorescence.Real-time PCR and Western blot analysis confirmed that TRPV1 mRNA and protein expression levels were significantly elevated in Caco-2-TRPV1 cells compared to Caco-2-GFP control cells(P＜0.05).This work successfully generated a stable TRPV1-overexpressing cell line,offering a valuable model for studying the lipid-lower-ing effects of capsaicin.
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维普
