摘要
肺癌是全球癌症死亡的首因，是一种常见的、高度恶化的肺部肿瘤，目前的治疗方法主要是化疗、放疗和手术治疗，但近年来肺癌病人5年生存率仅仅14%，因此积极寻找肺癌治疗的新策略，就成为摆在面前的迫切课题，基因治疗就是这种探索之一。
NKX2.1基因是核转录蛋白Nkx2基因家族成员之一，近年来被认为作为一个新的原癌基因参与到肺腺癌的生长中，其在相当部分肺腺癌中过表达大大增加肿瘤的致瘤性，被选择作为本课题分子治疗研究的靶标基因。hIL-24是新近发现的细胞因子水平的抑癌基因，具有抑制多种肿瘤细胞生长并诱导肿瘤细胞凋亡的作用，而对正常细胞尚未见毒副作用，也被选择作为本课题研究较为理想的抑癌基因。本研究运用宿主范围广，免疫原性低，并具有长期的外源基因表达潜力的重组AAV作为新一代载体，在典型的肺腺癌细胞中联合表达特异siRNA和重组hIL-24，在分子水平证实其抑制原癌基因表达，在细胞水平验证其抑制细胞生长和诱导细胞凋亡作用，并尝试外源基因设计、克隆、重组病毒载体和体内表达的相关工艺研究。
一方面，本研究设计三条靶向NKX2.1基因的特异性siRNA，将其DNA编码序列通过psiRNA-hH1neo质粒和pAAV-MCS质粒，组装rAAV-NX病毒；经感染NCI-H1975细胞，通过RT-QPCR和WB实验证实siRNA抑制NKX2.1的表达，其中siRNA3的抑制作用最强；通过MTT实验和凋亡实验证实rAAV-NX介导的siRNA下调NKX2.1基因的表达可以抑制肺腺癌NCI-H1975细胞的生长并诱导它的凋亡。
另一方面，本研究用PCR扩增pcDNA6.0质粒的hIL-24基因cDNA序列并克隆到pAAV-MCS载体上，组装rAAV-hIL-24病毒；通过RT-PCR实验和WB实验证实经rAAV-hIL-24介导的外源hIL-24能够在NCI-H1975细胞中表达；通过MTT实验、细胞周期实验和凋亡实验发现其抑制NCI-H1975细胞的生长、干扰细胞周期并诱导细胞凋亡。
更进一步，本研究将上述siRNA3编码序列和外源hIL-24，分别通过不同的酶切位点重组到同一重组病毒载体rAAV-N3-hIL-24，再次在NCI-H1975细胞中通过MTT实验、TUNEL实验发现联合表达抑制癌细胞的生长作用和促进癌细胞凋亡作用更为显著。
本研究利用Sepharose4FF和Plasmidselect Xtra层析柱获得纯度较高的超螺旋质粒，优化病毒包装条件，利用HiLoad16/10Q和HiLoad16/10SP层析柱得到较高纯度的的rAAV病毒颗粒，通过浓缩进一步提高了病毒滴度。
本实验尝试了应用rAAV载体分别和联合表达外源siRNA与蛋白基因获得了初步成果，为今后以AAV为载体的基因联合治疗肺腺癌提供参考。
Lung cancer is the first cause for cancer deaths in the world, which is a commonand highly deteriorating lung tumor, and its therapeutic methods still includechemotherapy, radiotherapy and operative therapy at present. In recent years,5-yearsurvival rate of lung cancer patients is only14%, so active searches of new lungcancer therapy strategy becomes urgent subject and gene therapy is one of the searchdirections.
As one of nuclear transcription protein Nkx2gene family members, NKX2.1geneis deemed as one new proto-oncogene that participates in the growth of lungadenocarcinoma and whose over-expression in lung adenocarcinoma considerablyincreases oncogenicity of tumour, so, it is selected as a target gene in the moleculartherapy. hIL-24is the cancer suppressor gene of cytokines level found recently, andplays a role in suppressing the growth of various tumor cells and inducing tumor cellapoptosis without toxic and side effects on normal cells. hIL-24may be used as idealcancer suppressor gene in this study. In this study, recombinant AAV is used as a newvector owing to its higher extensive host range, low immunogenicity and long-termexogenous gene expression potential, which is to co-express of the special siRNA andrecombinant hIL-24in the typical lung adenocarcinma cell, to prove its capacityinhibiting protooncogene expression in molecular level, to verify its functionsuppressing growth of cancer cell and induction of apoptosis. The related processstudy relating to exogenous gene design, cloning, and restructuring of virus vector andinternal expression is also included.
On the one hand, this study devises three special siRNAs targeting at NKX2.1gene, constructs psiRNA-hH1ne and pAAV-MCS plasmids by means of DNA codingsequence, and assemblies rAAV-NX virus. For the infected NCI-H1975cells, it isproved through RT-QPCR and WB experiments that siRNA inhibits the expressionof NKX2.1and siRNA3shows the significant inhibition effect. MTT and apoptosisexperiments prove that the expression of the NKX2.1gene down-regulated byrAAV-NX-mediated siRNA may suppress the growth of the lung adenocarcinomaNCI-H1975cells and induce their apoptosis.
On the other hand, in this study, the hIL-24gene cDNA sequence on pcDNA6.0 plasmid vector is amplified by PCR and cloned onto pAAV-MCS vector to assemblerAAV-hIL-24virus. RT-PCR and WB experiments prove that rAAV-hIL-24-mediatedexogenous hIL-24may express in NCI-H1975cells and it is found in MTT, cellcycle and apoptosis experiments that it inhibits the growth of NCI-H1975cells,disturb cell cycle, and induce their apoptosis.
This study further recombines the siRNA3coding sequence and exogenoushIL-24into the same recombinant virus vector rAAV-N3-hIL-24at differentrestriction enzyme cutting sites. It is also found in MTT and TUNEL experimentsthat in NCI-H1975cells joint expression inhibits the growth of cancer cells andpromotes significantly apoptosis of cancer cells.
In this study, the supercoiled plasmid is purified by Sepharose4FF andPlasmidselect Xtra plasmid purification chromatography columns. Then optimizevirus packaging conditions. By HiLoad16/10Q and HiLoad16/10SP plasmidpurification chromatography columns, rAAV virus of higher purity is obtained andvirus titer is increased after further concentration.
The experiment tries respective expression and co-expression of exogenoussiRNA and protein gene on rAAV vector, and has obtained initial achievements thatmay be used as references for lung adenocarcinoma therapy by gene collaborationtaking AAV as vector in the future.
引文
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