Construction and Gene Silence Function of Gene Silence Adenovirus Vector Plasmid Targeting Both TG2 and Mertk Synchronously_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

 ZHENG Jianghua ¹ , CHEN Jing ² , CHEN Kai ¹ , ZHU Yanbin ¹ , WU Guo ¹

1.Department of Vascular Surgery， Affiliated Hospital of North Sichuan Medical College， Nanchong 637000， Sichuan Province， China;;
2.Biochemical Laboratory， North Sichuan Medical College， Nanchong 637000， Sichuan Province， China;

Corresponding author：

ZHENG Jianghua, Email: zhengjianghua@126.com

Keywords：

Transglutaminase 2; Mer receptor tyrosine kinase; RNA interference; Adenovirus; RAW264.7 cell

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Objective 　To construct gene silence adenovirus vector targeting both transglutaminase 2 （TG2） and Mer receptor tyrosine kinase （Mertk） synchronously and detect the gene silence function of it.
Methods 　The interfering plasmids targeting TG2 protein and Mertk protein were constructed firstly， then the H1 promoter and RNA interfering （RNAi） sequence were cut and ligated to pAdTrack for constructing pAdTrack/TG2/Mertk. The pAdTrack/TG2/Mertk was transfected into BJ5183 bacterial cells which contained pAdEasy-1， then the plasmid was detected by enzyme digestion after recovery. Adenovirus were harvested after that pAdTrack/TG2/Mertk was infected into HEK293 cells. The virus titer was measured after repeated amplification. The RAW264.7 cells were infected by pAdTrack/TG2/Mertk， pAdTrack/TG2， pAdTrack/Mertk， and pAdTrack/green fluorescent protein （GFP）， respectively. Then the expression levels of TG2 protein and Mertk protein of mouse macrophages were detected by Western blot after infection.
Results 　The virus titer of pAdTrack/TG2/Mertk plasmid was 6.13×1010GFU/mL. The pAdTrack/TG2/Mertk plasmid which contained 2 promoters and 2 RNAi sequences was identified successfully by enzyme digestion. Compared with pAdTrack/GFP group and pAdTrack/Mertk group （there was no significant differece between the 2 groups）， the expression levels of TG2 protein of mouse macrophages which infected with pAdTrack/TG2/Mertk or pAdTrack/TG2 decreased obviously （P＜0.01）， but there was no significant difference between the later 2 groups. Compared with pAdTrack/GFP group and pAdTrack/TG2 group （there was no significant difference between the 2 groups）， the expression levels of Mertk protein of mouse macrophages which infected with pAdTrack/TG2/Mertk or pAdTrack/Mertk decreased obviously too （P＜0.01）， but there was no significant difference between the later 2 groups.
Conclusion 　Gene silence adenovirus vector plasmid targeting both TG2 and Mertk synchronously is constructed successfully， and the pAdTrack/TG2/Mertk can reduce the expressions of TG2 protein and Mertk protein of mouse macrophages obviously.

Citation： ZHENG Jianghua,CHEN Jing,CHEN Kai,ZHU Yanbin,WU Guo. Construction and Gene Silence Function of Gene Silence Adenovirus Vector Plasmid Targeting Both TG2 and Mertk Synchronously. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2013, 20(4): 395-399. doi: Copy

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13.	Hall MO， Obin MS， Heeb MJ， et al. Both protein S and Gas6 stimulate outer segment phagocytosis by cultured rat retinal pigmentepithelial cells[J]. Exp Eye Res， 2005， 81(5)：581-591.
14.	Ait-Oufella H， Kinugawa K， Zoll J， et al. Lactadherin deficiency leads to apoptotic cell accumulation and accelerated atherosclerosisin mice[J]. Circulation， 2007， 115(16)：2168-2177.
15.	Platt N， Suzuki H， Kodama T， et al. Apoptotic thymocyte clearance in scavenger receptor class A-deficient mice is apparently normal[J]. J Immunol， 2000， 164(9)：4861-4867.
16.	Su HP， Nakada-Tsukui K， Tosello-Trampont AC， et al. Interaction of CED-6/GULP， an adapter protein involved in engulfment of apoptotic cells with CED-1 and CD91/low density lipoprotein receptor-related protein (LRP)[J]. J Biol Chem， 2002， 277(14)：11772-11779.
17.	Castanotto D， Rossi JJ. The promises and pitfalls of RNA-interference-based therapeutics[J]. Nature， 2009， 457(7228)：426-433.
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19.	Hannon GJ. RNA interference[J]. Nature， 2002， 418(6894)：244-251.
20.	刘启志，王烈. siRNA表达载体对肝素酶基因的沉默作用[J]. 中国普外基础与临床杂志， 2010， 17(2)：142-146.
21.	王群，余明华，王耕，等. RNAi沉默DLL4基因诱导人乳腺癌细胞凋亡及对多西他赛的增敏作用[J]. 中国普外基础与临床杂志， 2013， 20(1)：54-59.
22.	-215.

1. Boisvert WA， Rose DM， Boullier A， et al. Leukocyte transglutaminase 2 expression limits atherosclerotic lesion size[J]. Arterioscler Thromb Vasc Biol， 2006， 26(3)：563-569.
2. Li Y， Gerbod-Giannone MC， Seitz H， et al. Cholesterol-induced apoptotic macrophages elicit an inflammatory response in phagocytes， which is partially attenuated by the Mer receptor[J]. J Biol Chem， 2006， 281(10)：6707-6717.
3. Thorp E， Li G， Seimon TA， et al. Reduced apoptosis and plaque necrosis in advanced atherosclerotic lesions of Apoe-/- and Ldlr-/- mice lacking CHOP[J]. Cell Metab， 2009， 9(5)：474-481.
4. Thorp E， Cui D， Schrijvers DM， et al. Mertk receptor mutation reduces efferocytosis efficiency and promotes apoptotic cell accum-ulation and plaque necrosis in atherosclerotic lesions of apoe-/- mice[J]. Arterioscler Thromb Vasc Biol， 2008， 28(8)：1421-1428.
5. Ait-Oufella H， Pouresmail V， Simon T， et al. Defective mer receptor tyrosine kinase signaling in bone marrow cells promotes apoptotic cell accumulation and accelerates atherosclerosis[J]. Arterioscler Thromb Vasc Biol， 2008， 28(8)：1429-1431.
6. Adam LH. Phagocytosis of dying cells：from molecular mechanisms to human diseases[M]. 2th ed， Netherlands：Springer， 2009：.
7. 孙鹏宇，张艳玲，荆玉明，等. 腺病毒滴度不同测定方法比较[J]. 南方医科大学学报， 2011， 31(2)：234-238.
8. Tabas I. Macrophage death and defective inflammation resolution in atherosclerosis[J]. Nat Rev Immunol， 2010， 10(1)：36-46.
9. Thorp E， Tabas I. Mechanisms and consequences of efferocytosisin advanced atherosclerosis[J]. J Leukoc Biol， 2009， 86(5)：1089-1095.
10. 汪海飞，陈开，郑江华. 凋亡细胞清除机制在动脉粥样硬化中的研究进展[J]. 中国普外基础与临床杂志， 2012， 19(2)：224-227.
11. Tóth B， Garabuczi E， Sarang Z， et al. Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells[J]. J Immunol， 2009， 182(4)：2084-2092.
12. Scott RS， Mcmahon EJ， Pop SM， et al. Phagocytosis and clearance of apoptotic cells is mediated by MER[J]. Nature， 2001， 411(6834)：207-211.
13. Hall MO， Obin MS， Heeb MJ， et al. Both protein S and Gas6 stimulate outer segment phagocytosis by cultured rat retinal pigmentepithelial cells[J]. Exp Eye Res， 2005， 81(5)：581-591.
14. Ait-Oufella H， Kinugawa K， Zoll J， et al. Lactadherin deficiency leads to apoptotic cell accumulation and accelerated atherosclerosisin mice[J]. Circulation， 2007， 115(16)：2168-2177.
15. Platt N， Suzuki H， Kodama T， et al. Apoptotic thymocyte clearance in scavenger receptor class A-deficient mice is apparently normal[J]. J Immunol， 2000， 164(9)：4861-4867.
16. Su HP， Nakada-Tsukui K， Tosello-Trampont AC， et al. Interaction of CED-6/GULP， an adapter protein involved in engulfment of apoptotic cells with CED-1 and CD91/low density lipoprotein receptor-related protein (LRP)[J]. J Biol Chem， 2002， 277(14)：11772-11779.
17. Castanotto D， Rossi JJ. The promises and pitfalls of RNA-interference-based therapeutics[J]. Nature， 2009， 457(7228)：426-433.
18. Olejniczak M， Galka P， Krzyzosiak WJ. Sequence-non-specific effects of RNA interference triggers and microRNA regulators[J].Nucleic Acids Res， 2010， 38(1)：1-16.
19. Hannon GJ. RNA interference[J]. Nature， 2002， 418(6894)：244-251.
20. 刘启志，王烈. siRNA表达载体对肝素酶基因的沉默作用[J]. 中国普外基础与临床杂志， 2010， 17(2)：142-146.
21. 王群，余明华，王耕，等. RNAi沉默DLL4基因诱导人乳腺癌细胞凋亡及对多西他赛的增敏作用[J]. 中国普外基础与临床杂志， 2013， 20(1)：54-59.
22. -215.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction and Gene Silence Function of Gene Silence Adenovirus Vector Plasmid Targeting Both TG2 and Mertk Synchronously

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