竞争抑制0610009E02Rik长链非编码RNA重组腺病毒穿梭载体的构建与鉴定

Construction and identification of competitive 0610009E02Rik long noncoding RNA recombinant adenovirus shuttle vector

目的 构建可竞争抑制肝细胞内0610009E02Rik长链非编码RNA（lncRNA）的重组腺病毒穿梭载体,为探索0610009E02Rik/Notch1在肝静脉闭塞病（HVOD）肝细胞损伤修复中的作用提供有效实验方法.方法 采用基因组DNA提取试剂盒对截取的C57BL/6小鼠尾进行基因组DNA的提取,通过PCR扩增出0610009E02Rik与Notch1基因第34个外显子重叠的1 000 bp基因序列,并连接入经BamHⅠ/Nhe Ⅰ双酶切后的腺病毒穿梭载体GV359;竞争抑制0610009E02Rik lncRNA重组腺病毒穿梭载体与辅助包装质粒pBHG共转染HEK293细胞,并对竞争抑制0610009E02Rik lncRNA重组腺病毒进行包装、扩增,采用氯化铯（CsCl）不连续密度梯度离心与连续密度梯度离心2个步骤对重组腺病毒进行浓缩纯化,并对重组腺病毒滴度进行测定;测定不同感染复数（MOI）竞争抑制0610009E02Rik lncRNA重组腺病毒感染肝细胞株H2.35的感染效率,并采用实时聚合酶链反应（RT-PCR）与Western blotting对竞争抑制0610009E02Rik lncRNA重组腺病毒感染肝细胞株H2.35与同步培养的对照肝细胞中竞争抑制0610009E02Rik lncRNA片段、Notch1 mRNA及0610009E02Rik lncRNA相对表达水平及其蛋白表达水平,并进行统计学分析.结果 ①经基因序列比对分析发现0610009E02Rik与Notch1的第34个外显子存在分子量为1 000 bp的重叠序列,依据0610009E02Rik与Notch1基因序列设计含BamH Ⅰ/Nhe Ⅰ酶切位点特异性引物,通过PCR扩增出片段长度为1 000 bp的0610009E02Rik与Notch1基因第34个外显子重叠序列的竞争抑制0610009E02Rik lncRNA片段,DNA测序结果显示竞争抑制0610009E02Rik lncRNA重组腺病毒穿梭载体与理论预期序列构建成功.②将竞争抑制0610009E02Rik lncRNA重组腺病毒穿梭载体转染HEK293细胞后第12天,约90％细胞出现细胞病变（CPE）,收集细胞获取病毒液.浓缩纯化病毒液经梯度稀释在感染HEK293细胞后第10天,于显微镜下观察稀释梯度1∶ （10～1010）均出现CPE,病毒滴度约为5.01×109 PFU/mL.③当竞争抑制0610009E02Rik lncRNA重组腺病毒MOI为80,感染肝细胞株H2.35 48 h后感染效率高达95％.收集经竞争抑制0610009E02Rik lncRNA重组腺病毒感染后肝细胞,通过RT-PCR检测竞争抑制0610009E02Rik lncRNA片段相对表达水平为同步培养肝细胞株H2.35对照的3.13±0.83倍,且差异有统计学意义（P=0.047）;Notch1 mRNA相对表达水平为对照细胞的（0.38±0.08）倍,且差异亦有统计学意义（P=0.010）;0610009E02Rik lncRNA相对表达水平为对照细胞的（1.04±0.26）倍,但差异无统计学意义（P＞0.05）.经Western blotting检测发现,重组腺病毒感染后肝细胞Notch1蛋白表达水平较对照肝细胞减低,与Notch1 mRNA相对表达水平检测结果相一致.结论 成功构建竞争抑制0610009E02Rik lncRNA重组腺病毒穿梭载体,重组腺病毒感染肝细胞H2.35后,可下调肝细胞内Notch1 mRNA表达水平及其蛋白表达水平,这为HVOD肝细胞损伤修复的深层机制研究奠定实验数据基础.

Objective To construct a competitive adenovirus vector against 0610009E02Rik,a long noncoding RNA （lncRNA） for investigating its role in the repairment of injured hepatocytes during hepatic veno-occlusive disease （HVOD）.Methods Firstly,genome DNA was extracted from the tail of C57BL/6 mice,and amplified the overlapping region of the gene 0610009E02Rik and the 34th exon of Notch1 by polymerase chain reaction（RT-PCR）,then it was linked into the adenovirus shuttle plasmid GV359 which was digested by BamH Ⅰ/Nhe Ⅰ restriction enzyme.Secondly,the constructed shuttle plasmid and packaging plasmid pBHG were co-transfected into HEK293 cells for assembly of recombination adenovirus,and subsequent amplification,followed by cesium chloride （CsCl） gradient ultracentrifugation for purification and measurement of the viral titer in HEK 293 cells.Finally,the recombination adenovirus was transfected into hepatocyte cell line H2.35 with different multiplicity of infection （MOI）,and transfection efficiency was evaluated by fluorescence microscope.Meanwhile,real-time polymerase chain reaction （RT-PCR） was used to detect the expression of anti-lncRNA,Notch1 and 0610009E02Rik.Western blotting was used for the detection of the expression level of Notch1.Results ① It was discovered that the gene 0610009E02Rik and the 34th exon of Notch1 have the overlapping region （its fragment length is about 1 000 bp）,and then it was obtained by PCR with the primer which has the BamH Ⅰ/Nhe Ⅰ digestion sites,and DNA sequencing result confirmed that the competitive adenovirus shuttle plasmid against 0610009E02Rik was successfully constructed.② At the 12th day after transfection,about 90％ cells presented cytopathic effect （CPE）,then all the transfected cells were obtained a large amount of virus extract.Then HEK293 cells were transfected by the condensed and purified virus with a series of dilution,and cells transfected with dilution fold of 1 ∶ （10～1010） were all had CPE,and this is verifi the virus titers was 5.01x109 PFU/mL.③ Different MOI with different transfection efficiency in hepatocytes was observed and transfection efficiency of hepatocytes was about 95％ after 48 h with MOI of 80.Real-time PCR was adopted to detect the relative RNA expression levels.anti-0610009E02Rik lncRNA level were （3.13±0.83） folds higher than that of the control groups （P =0.047）.Notch1 mRNA relative expression level were （0.38±0.08） folds higher than that of the control group （P =0.010）,and 0610009E02Rik lncRNA expression level were （1.04 ± 0.26） folds higher than that of the control groups without significantly differences （P＞0.05）.Moreover,the protein level of Notch1 was lower than that of control group,and this is consistent with its mRNA level.Conclusion We successfully constructed the competitive adenovirus vector against 0610009E02Rik,which could down-regulate Notch1 mRMA and protein expression in hepatocyte cell line H2.35,providing a basis for further investigation of its role in the repairment of injury hepatocytes during HVOD.