摘要
目的
构建含绿色荧光蛋白(GFP)标记、β-连环蛋白(β-catenin)特异的RNAi重组腺病毒表达载体,利用包装好的重组腺病毒感染羊膜间充质干细胞(AMSC),在转录后水平有效的抑制β-catenin的表达,探讨siRNA干扰(β-catenin对AMSC增殖的影响。
方法
利用脂质体将设计合成好的三个β-catenin siRNA干扰片段瞬时转染AMSC,利用免疫印迹(Western Blot)筛选最优的β-catenin siRNA干扰片段；然后编码针对最优干扰片段,即CTNNB1短发卡RNA (short hairpin RNA, shRNA)的单链DNA,并通过退火反应获得目的基因双链DNA,将目的基因连接至腺病毒穿梭质粒中构建阳性重组质粒pAdTrack-si-β-catenin,经PmeI酶切线性化后使其在BJ5183菌中与腺病毒骨架质粒进行同源重组从而获得重组腺病毒载体pAd-si-β-catenin,利用脂质体将经Pac I酶切线性化后的重组腺病毒质粒转染至HEK293细胞,包装重组腺病毒、空斑实验测定病毒滴度。重组腺病毒感染AMSC, Western Blot法检测AMSC中β-catenin及GSK-3β的表达情况,CKK-8法检测β-catenin沉默表达对AMSC增殖的影响。
结果
1. β-catenin siRNA干扰片段瞬时转染AMSC可以有效抑制β-catenin的表达,通过Western Blot成功的筛选出了最优的目的基因靶序列,设计并合成了shRNA的DNA双链,并成功构建出重组腺病毒表达载体p Ad-si-β-catenin;成功的包装、扩增出重组腺病毒,获得高滴度重组腺病毒。
2.重组腺病毒感染AMSC后,在荧光倒置显微镜下可以观察到GFP表达,通过Western Blot检测到重组腺病毒感染AMSC后可以有效的抑制β-catenin基因的表达,感染组β-catenin明显少于对照组。
3.重组腺病毒感染AMSC后,通过CKK-8法检测细胞生长曲线,与对照组相比,感染组细胞的增殖受到明显抑制。
结论
构建的特异RNAi重组腺病毒能够有效的抑制β-catenin基因的表达,减少了细胞内β-catenin的水平,且β-catenin的沉默表达可以明显抑制AMSC的增殖。
OBJECTIVE
To construct recombinant adenovirus containing Green fluorescent protein and P-catenin silenced specifically by RNAi, to inhibit the expression of P-catenin in the post-transcriptional, to investigate the effects of β-catenin siRNA on proliferation of amniotic mesenchymal stem cell (AMSC).
METHODS
In the first part, we designed and synthesized three interference fragment, P-catenin siRNA was transfected instantaneously into AMSC by liposome, selected the most efficient interference fragment by Western blot. Then encoded single-stranded DNA matching targeting genes CTNNB1short hairpin RNA (shRNA) and acquired targeting genes double-stranded DNA. Then cutted targeting genes by two endonucleases and directional linked to the pAd track-cmv vector. Afterward, the correct recombinant was linearized by Pmel, following co-transformation with the backbone vector pAdEasy-1in E.coli BJ5183. The homologous recombinant was linearized by Pad, and then transfected into HEK293cell line to pack the adenovirus. And detected the viral titer by plaque assay. In the second part, the adenovirus infected AMSC. The expression of β-catenin and GSK-3P were assayed by Western blot. The change of cell proliferation was determined by CKK-8.
RESULTS
1. Transfection of β-catenin siRNA to AMSC could effectively inhibit the expression of P-catenin, we selected the best target gene sequence successfully by Western blot, designed and synthesized targeting genes double-stranded DNA. And we constructed recombinant adenovirus vector pAd-si-β-catenin successfully. We packaged and amplified recombinant adenovirus successfully, and we obtained the adenovirus supernatant of high titer.
2. After AMSC infected by the adenovirus, we could observed the expression of GFP in infection group, and by Western blot we could detected that the expression of P-catenin was inhibited effectively, and the expression of β-catenin in infection group was less than control group obviously.
3. After AMSC infected by the adenovirus, we could detected cell growth curve. And compared with control group, the cell proliferation of infection group was inhibited effectively.
CONCLUSION
The β-catenin-targeting RNAi recombination adenovirus is efficient to inhibit the expression of P-catenin gene in AMSC and to decrease the expression of β-catenin in AMSC. And P-catenin silenced could inhibit effectively cell proliferation in AMSC.
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