中华肝胆外科杂志
中華肝膽外科雜誌
중화간담외과잡지
CHINESE JOURNAL OF HEPATOBILIARY SURGERY
2012年
2期
145-149
,共5页
贾岩峰%崔云峰%李东华%崔乃强%彭雁飞%宁召臣%张琚
賈巖峰%崔雲峰%李東華%崔迺彊%彭雁飛%寧召臣%張琚
가암봉%최운봉%리동화%최내강%팽안비%저소신%장거
腺病毒科%胆结石%基因
腺病毒科%膽結石%基因
선병독과%담결석%기인
Adenoviridae%Cholelithiasis%Genes
目的 构建小鼠SCP2基因shRNA重组腺病毒载体,进一步研究SCP2基因与胆囊胆固醇结石形成机制.方法 (1)两步PCR法合成针对小鼠SCP2基因shRNA,并连接到质粒PDC312上；(2)采用AdMax TM Adenoviru5 Vector系统包装病毒,CsCl法纯化病毒、TCID50法测定滴度；(3)Western印迹检测SCP2蛋白验证重组腺病毒沉默效果,RT-PCR检测重组腺病毒在小鼠hepa-1～6细胞中SCP2、HMGCR、CYP7A1基因mRNA的表达.结果 (1)腺病毒载体Adsh1RNA和Adsh2RNA对SCP2均有抑制作用,在SCP2mRNA水平,与AdsshRNA组比较,Adsh1RNA tD1=9.056,P＜0.05,Adsh2RNA tD2=7.777,P＜0.05,差异均有统计学意义,蛋白水平与mRNA一致；(2)SCP2基因低表达时,CYP7A1 mRNA表达升高,t=10.68,P＜0.05,HMGCR mRNA表达下降,t=10.10,P＜0.05,差异有统计学意义.结论 成功构建小鼠SCP2基因的shRNA重组腺病毒载体.SCP2低表达时可能影响HMGCoA还原酶和CYP7-α1羟化酶的活性,导致胆固醇代谢的变化,从而减少胆固醇结石的形成.
目的 構建小鼠SCP2基因shRNA重組腺病毒載體,進一步研究SCP2基因與膽囊膽固醇結石形成機製.方法 (1)兩步PCR法閤成針對小鼠SCP2基因shRNA,併連接到質粒PDC312上；(2)採用AdMax TM Adenoviru5 Vector繫統包裝病毒,CsCl法純化病毒、TCID50法測定滴度；(3)Western印跡檢測SCP2蛋白驗證重組腺病毒沉默效果,RT-PCR檢測重組腺病毒在小鼠hepa-1～6細胞中SCP2、HMGCR、CYP7A1基因mRNA的錶達.結果 (1)腺病毒載體Adsh1RNA和Adsh2RNA對SCP2均有抑製作用,在SCP2mRNA水平,與AdsshRNA組比較,Adsh1RNA tD1=9.056,P＜0.05,Adsh2RNA tD2=7.777,P＜0.05,差異均有統計學意義,蛋白水平與mRNA一緻；(2)SCP2基因低錶達時,CYP7A1 mRNA錶達升高,t=10.68,P＜0.05,HMGCR mRNA錶達下降,t=10.10,P＜0.05,差異有統計學意義.結論 成功構建小鼠SCP2基因的shRNA重組腺病毒載體.SCP2低錶達時可能影響HMGCoA還原酶和CYP7-α1羥化酶的活性,導緻膽固醇代謝的變化,從而減少膽固醇結石的形成.
목적 구건소서SCP2기인shRNA중조선병독재체,진일보연구SCP2기인여담낭담고순결석형성궤제.방법 (1)량보PCR법합성침대소서SCP2기인shRNA,병련접도질립PDC312상；(2)채용AdMax TM Adenoviru5 Vector계통포장병독,CsCl법순화병독、TCID50법측정적도；(3)Western인적검측SCP2단백험증중조선병독침묵효과,RT-PCR검측중조선병독재소서hepa-1～6세포중SCP2、HMGCR、CYP7A1기인mRNA적표체.결과 (1)선병독재체Adsh1RNA화Adsh2RNA대SCP2균유억제작용,재SCP2mRNA수평,여AdsshRNA조비교,Adsh1RNA tD1=9.056,P＜0.05,Adsh2RNA tD2=7.777,P＜0.05,차이균유통계학의의,단백수평여mRNA일치；(2)SCP2기인저표체시,CYP7A1 mRNA표체승고,t=10.68,P＜0.05,HMGCR mRNA표체하강,t=10.10,P＜0.05,차이유통계학의의.결론 성공구건소서SCP2기인적shRNA중조선병독재체.SCP2저표체시가능영향HMGCoA환원매화CYP7-α1간화매적활성,도치담고순대사적변화,종이감소담고순결석적형성.
Objectives It was constructed that the replication defective adenoviral vectors carried the short hairpin sequences of mouse SCP2.And we will make a further study of mechanism between SCP2 gene and cholesterol stone in gallbladder.Methods The short hairpin sequences of mouse SCP2 were cloned by two-step PCR,and connected together with the plasmid pDC312.The Admax Adenoviral Vector System was used to generate the replication defective adenoviral vectors,which were purified by CsCl method.The processes of TCID50 were applied to detect the titers of the adenoviral vectors.Furthermore,Protein levels of SCP2 were determined by Western blot analysis,and the levels of SCP2、CYP7A1、HMGCR mRNA from the hepa1-6 cell of mouse were measured by the usage of RT-PCR.Results SCP2mRNA and SCP2 protein were down-regulated by the replication defective adenoviral vectors carried the SCP2-shRNA.With the decreasing SCP2mRNA,the levels of HMGCRmRNA were down-regulated at same the time,while CYP7A1mRNA were up-regulated.Conclusions The replication defective adenoviral vectors carried SCP2-shRNA were constructed successfully.The lower levels of SCP2 could affect the activities of HMG-CoA reductase and CYP7-a enzyme,which caused the variations of cholesterol metabolism and then decreased the formation of cholesterol stone.
