携带内皮抑素复制缺陷型腺病毒载体的构建及其应用

Construction and application of recombinant adenovirus vector expressing the mouse endostatin

目的构建鼠源性内皮抑素(mEndostatin)重组腺病毒真核表达载体,并将其转染胰腺癌细胞株,探讨其体外抑制血管形成的活性。方法 以mEndostatin质粒为模板通过PCR方法扩增回收mEndostatin,将mEndostatin连接到腺病毒载体pCA13中,构建pCA13-mEndo表达质粒。将此表达质粒与腺病毒重组质粒pBGHE3通过lipofectamine 2 000共同转染293细胞,经同源重组产生重组腺病毒Ad-mEndo,用氯化铯密度梯度超速离心法纯化,用50％组织培养感染剂量法测定病毒滴度。体外转染胰腺癌细胞株AsPC-1、BxPC-3,并测定感染的胰腺癌细胞上清液mEndostatin的表达及观察其抑制血管形成的活性。结果 扩增得到的mEndostatin经酶切鉴定、PCR扩增、DNA测序证实为鼠源性内皮抑素,重组腺病毒Ad-mEndo的滴度为6.3×1010pfu／ml,体外能高效转染胰腺癌细胞株,可分泌表达有抑制血管形成活性的mEndostatin。结论本实验所构建的mEndostatin重组腺病毒表达载体能有效表达具有生物活性的mEndostatin,为体内胰腺癌的基因治疗奠定了基础。

Objective To construct the recombinant adenovirus vector expressing the mouse en-dostatin and investigate where endostatin expressed by the infected pancreatic carcinoma cells(BxPC-3, AsPC-1)can inhibit angiogenesis. adenovirus. Methods The mEndostatin DNA extracted from vector containing mouse endostatin gene was successfully amplified using PCR and cloned into adenovirus vector pCA13,named pCA-mEndo. Recombinant adenovirus plasmid pCA13-mEndo was cotransfected with pBHG3 into 293 packaging cells by lipofectamine 2 000 and the replication-deficient recombinant adenovirus Ad-mEndo recombinant adenovirus was purified by CsCl density purification and the titer measured pancreatic adenocarcinoma cells(BxPC-3, AsPC-1)were infected with adenovirus Ad-mEndo in vitro. Supernatants were measured and the expression of endostatin proteinwas analyzed for their ability to inhibit angiogenesis. Results The mEndostatin DNA was confirmed by sequence meusure-ment. The virus titer was 6. 3 × 1010 pfu/ml. Ad-mEndo can efficiently infected pancrealic carcinoma cells and the expressed mEndoslatin can inhbit angiogenesis. Conclusion The conslrucled adenovirus Ad-mEndo can efficienlly express mEndoslalin, which can inhibit angiogenesis. This invesligation provides the basis for sludying pancrealic carcinoma gene therapy in vivo.