国际输血及血液学杂志
國際輸血及血液學雜誌
국제수혈급혈액학잡지
INTERNATIONAL JOURNAL OF BLOOD TRANSFUSION AND HEMATOLOGY
2015年
3期
192-199
,共8页
杨娜%陈翀%秘红岭%李小莉%陈朝%褚培培%夏园%姚海娜%徐开林
楊娜%陳翀%祕紅嶺%李小莉%陳朝%褚培培%夏園%姚海娜%徐開林
양나%진충%비홍령%리소리%진조%저배배%하완%요해나%서개림
RNA,长链非编码%受体,Notch1%腺病毒,人%DNA,重组%肝细胞
RNA,長鏈非編碼%受體,Notch1%腺病毒,人%DNA,重組%肝細胞
RNA,장련비편마%수체,Notch1%선병독,인%DNA,중조%간세포
RNA,long noncoding%Receptor,Notch1%Adenovirus,human%DNA,recombinant%hepatocytes
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.
目的 構建可競爭抑製肝細胞內0610009E02Rik長鏈非編碼RNA(lncRNA)的重組腺病毒穿梭載體,為探索0610009E02Rik/Notch1在肝靜脈閉塞病(HVOD)肝細胞損傷脩複中的作用提供有效實驗方法.方法 採用基因組DNA提取試劑盒對截取的C57BL/6小鼠尾進行基因組DNA的提取,通過PCR擴增齣0610009E02Rik與Notch1基因第34箇外顯子重疊的1 000 bp基因序列,併連接入經BamHⅠ/Nhe Ⅰ雙酶切後的腺病毒穿梭載體GV359;競爭抑製0610009E02Rik lncRNA重組腺病毒穿梭載體與輔助包裝質粒pBHG共轉染HEK293細胞,併對競爭抑製0610009E02Rik lncRNA重組腺病毒進行包裝、擴增,採用氯化銫(CsCl)不連續密度梯度離心與連續密度梯度離心2箇步驟對重組腺病毒進行濃縮純化,併對重組腺病毒滴度進行測定;測定不同感染複數(MOI)競爭抑製0610009E02Rik lncRNA重組腺病毒感染肝細胞株H2.35的感染效率,併採用實時聚閤酶鏈反應(RT-PCR)與Western blotting對競爭抑製0610009E02Rik lncRNA重組腺病毒感染肝細胞株H2.35與同步培養的對照肝細胞中競爭抑製0610009E02Rik lncRNA片段、Notch1 mRNA及0610009E02Rik lncRNA相對錶達水平及其蛋白錶達水平,併進行統計學分析.結果 ①經基因序列比對分析髮現0610009E02Rik與Notch1的第34箇外顯子存在分子量為1 000 bp的重疊序列,依據0610009E02Rik與Notch1基因序列設計含BamH Ⅰ/Nhe Ⅰ酶切位點特異性引物,通過PCR擴增齣片段長度為1 000 bp的0610009E02Rik與Notch1基因第34箇外顯子重疊序列的競爭抑製0610009E02Rik lncRNA片段,DNA測序結果顯示競爭抑製0610009E02Rik lncRNA重組腺病毒穿梭載體與理論預期序列構建成功.②將競爭抑製0610009E02Rik lncRNA重組腺病毒穿梭載體轉染HEK293細胞後第12天,約90％細胞齣現細胞病變(CPE),收集細胞穫取病毒液.濃縮純化病毒液經梯度稀釋在感染HEK293細胞後第10天,于顯微鏡下觀察稀釋梯度1∶ (10～1010)均齣現CPE,病毒滴度約為5.01×109 PFU/mL.③噹競爭抑製0610009E02Rik lncRNA重組腺病毒MOI為80,感染肝細胞株H2.35 48 h後感染效率高達95％.收集經競爭抑製0610009E02Rik lncRNA重組腺病毒感染後肝細胞,通過RT-PCR檢測競爭抑製0610009E02Rik lncRNA片段相對錶達水平為同步培養肝細胞株H2.35對照的3.13±0.83倍,且差異有統計學意義(P=0.047);Notch1 mRNA相對錶達水平為對照細胞的(0.38±0.08)倍,且差異亦有統計學意義(P=0.010);0610009E02Rik lncRNA相對錶達水平為對照細胞的(1.04±0.26)倍,但差異無統計學意義(P＞0.05).經Western blotting檢測髮現,重組腺病毒感染後肝細胞Notch1蛋白錶達水平較對照肝細胞減低,與Notch1 mRNA相對錶達水平檢測結果相一緻.結論 成功構建競爭抑製0610009E02Rik lncRNA重組腺病毒穿梭載體,重組腺病毒感染肝細胞H2.35後,可下調肝細胞內Notch1 mRNA錶達水平及其蛋白錶達水平,這為HVOD肝細胞損傷脩複的深層機製研究奠定實驗數據基礎.
목적 구건가경쟁억제간세포내0610009E02Rik장련비편마RNA(lncRNA)적중조선병독천사재체,위탐색0610009E02Rik/Notch1재간정맥폐새병(HVOD)간세포손상수복중적작용제공유효실험방법.방법 채용기인조DNA제취시제합대절취적C57BL/6소서미진행기인조DNA적제취,통과PCR확증출0610009E02Rik여Notch1기인제34개외현자중첩적1 000 bp기인서렬,병련접입경BamHⅠ/Nhe Ⅰ쌍매절후적선병독천사재체GV359;경쟁억제0610009E02Rik lncRNA중조선병독천사재체여보조포장질립pBHG공전염HEK293세포,병대경쟁억제0610009E02Rik lncRNA중조선병독진행포장、확증,채용록화색(CsCl)불련속밀도제도리심여련속밀도제도리심2개보취대중조선병독진행농축순화,병대중조선병독적도진행측정;측정불동감염복수(MOI)경쟁억제0610009E02Rik lncRNA중조선병독감염간세포주H2.35적감염효솔,병채용실시취합매련반응(RT-PCR)여Western blotting대경쟁억제0610009E02Rik lncRNA중조선병독감염간세포주H2.35여동보배양적대조간세포중경쟁억제0610009E02Rik lncRNA편단、Notch1 mRNA급0610009E02Rik lncRNA상대표체수평급기단백표체수평,병진행통계학분석.결과 ①경기인서렬비대분석발현0610009E02Rik여Notch1적제34개외현자존재분자량위1 000 bp적중첩서렬,의거0610009E02Rik여Notch1기인서렬설계함BamH Ⅰ/Nhe Ⅰ매절위점특이성인물,통과PCR확증출편단장도위1 000 bp적0610009E02Rik여Notch1기인제34개외현자중첩서렬적경쟁억제0610009E02Rik lncRNA편단,DNA측서결과현시경쟁억제0610009E02Rik lncRNA중조선병독천사재체여이론예기서렬구건성공.②장경쟁억제0610009E02Rik lncRNA중조선병독천사재체전염HEK293세포후제12천,약90％세포출현세포병변(CPE),수집세포획취병독액.농축순화병독액경제도희석재감염HEK293세포후제10천,우현미경하관찰희석제도1∶ (10～1010)균출현CPE,병독적도약위5.01×109 PFU/mL.③당경쟁억제0610009E02Rik lncRNA중조선병독MOI위80,감염간세포주H2.35 48 h후감염효솔고체95％.수집경경쟁억제0610009E02Rik lncRNA중조선병독감염후간세포,통과RT-PCR검측경쟁억제0610009E02Rik lncRNA편단상대표체수평위동보배양간세포주H2.35대조적3.13±0.83배,차차이유통계학의의(P=0.047);Notch1 mRNA상대표체수평위대조세포적(0.38±0.08)배,차차이역유통계학의의(P=0.010);0610009E02Rik lncRNA상대표체수평위대조세포적(1.04±0.26)배,단차이무통계학의의(P＞0.05).경Western blotting검측발현,중조선병독감염후간세포Notch1단백표체수평교대조간세포감저,여Notch1 mRNA상대표체수평검측결과상일치.결론 성공구건경쟁억제0610009E02Rik lncRNA중조선병독천사재체,중조선병독감염간세포H2.35후,가하조간세포내Notch1 mRNA표체수평급기단백표체수평,저위HVOD간세포손상수복적심층궤제연구전정실험수거기출.
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.
