小鼠增強(qiáng)型綠色熒光蛋白-過(guò)氧化物酶體增長(zhǎng)因子活化受體γ2融合表達(dá)重組腺病毒的構(gòu)建及表達(dá)

廖麗姿 肖金剛 楊苗苗 孔子任 孫欽策 田衛(wèi)東

（1.口腔疾病研究國(guó)家重點(diǎn)實(shí)驗(yàn)室，四川大學(xué)，四川成都610041；2.四川大學(xué)生命科學(xué)學(xué)院，四川成都610064）

小鼠增強(qiáng)型綠色熒光蛋白-過(guò)氧化物酶體增長(zhǎng)因子活化受體γ2融合表達(dá)重組腺病毒的構(gòu)建及表達(dá)

廖麗姿1肖金剛1楊苗苗1孔子任1孫欽策2田衛(wèi)東1

（1.口腔疾病研究國(guó)家重點(diǎn)實(shí)驗(yàn)室，四川大學(xué)，四川成都610041；2.四川大學(xué)生命科學(xué)學(xué)院，四川成都610064）

目的構(gòu)建小鼠增強(qiáng)型綠色熒光蛋白（EGFP）-過(guò)氧化物酶體增長(zhǎng)因子活化受體（PPAR）γ2基因的腺病毒重組體，并檢測(cè)其在感染病毒的小鼠骨髓間充質(zhì)干細(xì)胞（BMSC）中的表達(dá)。方法從pcDNA flag PPARγ質(zhì)粒上切取目的片段PPARγ2，克隆入pEGFP-C1和pEGFP-N1，以pEGFP-C1-PPARγ2為模板通過(guò)PCR技術(shù)獲得EGFP-PPARγ2基因，酶切DC315質(zhì)粒，獲得DC315-EGFP-PPARγ2質(zhì)粒，在脂質(zhì)體介導(dǎo)下與腺病毒輔助大質(zhì)粒pBHGlox△E1、3Cre共轉(zhuǎn)染HEK293細(xì)胞，包裝產(chǎn)生復(fù)制缺陷型重組腺病毒Ad-EGFP-PPARγ2，酶切鑒定證實(shí)構(gòu)建成功。轉(zhuǎn)染HEK293細(xì)胞并檢測(cè)其體外表達(dá)情況，經(jīng)HEK293細(xì)胞擴(kuò)增后，測(cè)定病毒滴度，轉(zhuǎn)染小鼠BMSC，72 h后鑒定其成脂分化情況。結(jié)果將pEGFP-C1-PPARγ2和pEGFP-N1-PPARγ2通過(guò)脂質(zhì)體轉(zhuǎn)染入HEK293細(xì)胞后，在倒置相差顯微鏡下觀察，前者在細(xì)胞核內(nèi)有較強(qiáng)的綠色熒光，后者的熒光強(qiáng)度則極低。對(duì)重組腺病毒進(jìn)行酶切和PCR鑒定，證實(shí)含小鼠EGFP-PPARγ2的Ad5腺病毒載體構(gòu)建成功。在倒置相差顯微鏡下觀察到BMSC細(xì)胞核內(nèi)有綠色熒光。結(jié)論成功構(gòu)建含小鼠EGFP-PPARγ2的重組Ad5腺病毒載體，為基因輔助的脂肪組織工程技術(shù)、抗腫瘤研究等提供了安全有效的轉(zhuǎn)基因載體。

過(guò)氧化物酶體增長(zhǎng)因子活化受體；重組腺病毒；骨髓間充質(zhì)干細(xì)胞

Construction of recombinant gene adenovirus encoding enhanced green fluorecence protein-peroxisome proliferator-activated receptor γ2 fusion protein and its expression in bone marrow mesenchymal stem cel ls

LIAO Li-zi1,XIAO Jin-gang1,YANG Miao-miao1,KONG Zi-ren1,SUN Qin-ce2,TIAN Wei-dong1.
（1.State Key Laboratory of Oral Diseases,Sichuan University,Chengdu 610041,China;2.College of Life Science,Sichuan University,Chengdu 610064,China）

Objective To construct mouse enhanced green fluorecence protein（EGFP）-peroxisome proliferatoractivated receptor（PPAR）γ2,and to detect EGFP-PPARγ2 expression in infected mouse bone marrow mesenchymal stem cells（BMSC）.M ethods Cut the fragment of PPARγ2 from the expression plasmid pcDNA flag PPARγ2, then cloned the gene fragment into pEGFP-C1 and pEGFP-N1 vector.Subsequently,subclone the fragment EGFPPPARγ2 from pEGFP-C1-PPARγ2 into the shuttle plasmid DC315.HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid DC315-EGFP-PPARγ2 and large adenovirus helper plasmid pBHGlox△E1,3Cre in mediation of liposome.The obtained replication-defective recombinant adenovirus Ad-EGFP-PPARγ2 was confirmed.Then it was propagated in HEK293 cells.After the BMSC were transfected for 72 h,adipogenic differentiation was demonstrated.Results HEK293 cells were transfected with the pEGFP-C1-PPARγ2 or pEGFP-N1-PPARγ2 in mediation of liposome.The former green fluorescence protein was better than the latter by fluorescence microscope. The recombinant plasmids were digested and identified.Western blot analysis showed the expression of EGFPPPARγ2 in vitro.EGFP-PPARγ2 protein was detectable in the nucleus of BMSC.Conclusion The recombinant adenovirus encoding EGFP-PPARγ2 fusion protein was successfully constructed,which provided a basis for applica

tion of EGFP-PPARγ2 gene to adenovirus-mediated gene therapy.

peroxisome proliferator-activated receptor；recombinant adenovirus；bone marrow mesenchymal stem cell

2009-12-22；

2010-05-25

國(guó)家自然科學(xué)基金資助項(xiàng)目（30973348）；高等學(xué)校博士學(xué)科點(diǎn)專項(xiàng)科研基金資助項(xiàng)目（20070610064）

廖麗姿（1983—），女，湖南人，碩士

田衛(wèi)東，Tel：028-85501445

Q 81

A

10.3969/j.issn.1000-1182.2010.04.022

1000－1182（2010）04－0430-05