腺相关病毒载体介导乳腺癌BA46基因转移的实验研究

Adeno-associated virus mediate the breast cancer BA46 gene transfer into dendri tic cells

目的 评价腺相关病毒 (AAV)为载体介导乳腺癌BA4 6基因转导树突状细胞 (DC)的可行性。方法 构建重组质粒d16 95 /BA4 6 p5/NeoSV40 (简称rAAV/BA4 6 /Neo) ,并制备该病毒。分离外周血单核细胞 (DC前体细胞 ) ,采用GM CSF、IL 4、TNF α诱导 ,以rAAV/BA4 6 /Neo病毒感染DC前体细胞 ,感染后 72h ,流式细胞仪检测rAAV/BA4 6 /Neo病毒感染效率及BA4 6表达情况 ,6d后 ,RT PCR分析BA4 6mRNA转录情况 ,PCR/Southernblot分析BA4 6基因染色体整合情况。结果 AAV成功介导BA4 6基因转导DC ,BA4 6在DC内良好表达 ,病毒基因整合入DC基因组内。结论 AAV载体成功介导BA4 6基因转导DC ,为以DC为基础的乳腺癌的基因治疗打下基础。

Objective To evaluate the possibility of the a dn o-associated virus(AAV) as vector mediated the breast cancer BA46 gene transfer into the dendritic cells(DC). Methods We constructed the recombinant plasmid d16-95/BA46 p5 /Neo SV40 (rAAV/BA46/Neo), and pre pared the rAAV/BA46/Neo virus stock using the pSH3 packaging system. We also iso lated the mononuclear cells(DC precursor) from PMBC, induced with GM-CSF, IL-4 and TNF-α, infected the mononuclear cells with rAAV/BA46/Neo virus stock and analyzed the infected efficiency with FACS after 72 h, and the BA46 mRNA transla tion with RT-PCR and detected viral integration by PCR/Southern blot analysis a fter 6 d was analyzed. Results AAV medicated the BA46 gen e transfer into DC successfully, DC expressed the BA46 well, viral gene was inte grated into the DC chromosomal. Conclusion AAV vector med iated BA46 gene tranducing into DC, which made a base for breast cancer immunoth erapy with dendritic cells gene transfer. [