DCX和SPAPC共表达对胶质瘤细胞生长、侵袭和放射敏感性的影响及其机制研究
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摘要
目的
构建DCX和SPARC双基因共表达腺病毒载体（Ad-DCX-SPARC），研究其对胶质瘤细胞生长、侵袭和放射敏感性的影响及其机制研究。
方法
1. PCR扩增获得DCX和SPARC目的基因，构建针对DCX和SPARC的pAdTrack-CMV-DCX-polyA+promoter-SPARC双基因共表达重组转移质粒；
2.将构建正确的pAdTrack-CMV-DCX-polyA+promoter-SPARC重组转移质粒经PmeI酶切后与pAdEasy-1腺病毒骨架质粒在BJ5183大肠杆菌中同源重组；
3.同源重组质粒pAdEasy-1-pAdTrack-CMV-DCX-polyA+promoter-SPARC(简称为pAd-DCX-polyA+promoter-SPARC)经PacI酶切后再用脂质体转染QBI-293A细胞，经多轮感染和扩增后获得Ad-DCX-polyA+promoter-SPARC(简称Ad-DCX-SPARC)双基因共表达重组腺病毒载体；
4.将Ad-DCX-SPARC重组腺病毒体外感染胶质瘤细胞U251和A172，筛选出Ad-DCX-SPARC的最佳感染剂量；
5.通过一系列检测细胞增殖、侵袭和放射敏感性的实验方法，研究增加DCX和SPARC双基因共表达对胶质瘤细胞生长、侵袭和放射敏感性的影响，并分析其可能的作用机制。
结果
1.成功构建了双启动子介导的DCX和SPARC双基因共表达腺病毒载体Ad-DCX-SPARC，其能高效感染胶质瘤细胞U251和A172，最佳感染剂量分别为20MOI和100MOI；2.腺病毒介导的DCX和SPARC双基因共表达能明显抑制胶质瘤细胞的生长，削弱SPARC促胶质瘤侵袭的作用；3.增加DCX-SPARC双基因共表达能使U251和A172胶质瘤细胞照射后克隆形成能力明显降低；4.单纯增加DCX和SPARC双基因共表达对胶质瘤细胞凋亡无明显影响，但联合辐照后能明显促进胶质瘤细胞的凋亡；5. DCX-SPARC双基因共表达提高了照前G2期细胞的比例，联合X射线照射后能减少照后G2期细胞的阻滞，从而减少了DNA损伤的修复，最终促进了胶质瘤细胞的死亡；6.分子机制检测结果表明Ad-DCX-SPARC联合X射线照射能明显上调U251和A172胶质瘤细胞bax，bad的表达和下调bcl-2的表达。
结论
1.成功获得了DCX和SPARC双基因共表达腺病毒载体Ad-DCX-SPARC，能明显抑制胶质瘤细胞的生长，削弱SPARC促胶质瘤侵袭的作用；
2. DCX和SPARC双基因共表达联合X射线照射能通过抑制胶质瘤细胞的克隆形成、提高照射前G2期细胞阻滞、增加辐照引起的细胞凋亡、使细胞照后G2期的阻滞得到减弱从而减少照后DNA损伤的修复等机制提高脑胶质瘤细胞的放射敏感性；
3. Ad-DCX-SPARC联合X射线照射可通过调节U251和A172细胞中bax，bad和bcl-2等细胞凋亡相关基因诱导胶质瘤细胞的凋亡。
Objective
To construct a recombinant adenoviral vector co-expressing Doublecortin(DCX)and Secreted protein acidic and rich in cysteine(SPARC) and study its enhancedanti-tumor and radiosensitivity effects.
Methods
1. DCX and SPARC were amplified by PCR using cDNA as template.pAdTrack-CMV-DCX-polyA+promoter-SPARC double genes co-expression transfervector was constructed and identified by PCR, double endonuclease digestion, andDNA sequencing;
2. pAdTrack-CMV-DCX-polyA+promoter-SPARC transfer vector was linearizedwith PmeI digestion and further co-transfected with pAdEasy-1backbone vector intobacteria BJ5183competent cells for homologous recombination;
3. The production of pAdEasy-1-pAdTrack-CMV-DCX-polyA+promoter-SPARC(pAd-DCX-polyA+promoter-SPARC) homologous recombinant plasmid purified fromthe above BJ5183cells was transfected into the bacterial DH5α cells to abundantlyamplify pAd-DCX-polyA+promoter-SPARC plasmids, then they were linearized withPacI digestion and transfected into the human embryonic kidney293(QBI-293A) cellsby Lipofectamine2000, leading to packaging of the recombinant adenovirusesAd-DCX-polyA+promoter-SPARC(Ad-DCX-SPARC).First generation virus-containingsupernatants was used for obtaining large quantities of recombinant adenoviral vectorsafter several cycles of transfection and amplifying;
4. The best MOIs of Ad-DCX-SPARC were chosen by Ad-DCX-SPARC infectingU251and A172glioma cells;
5. A series experimental methods which tested cell proliferation、invasion andradiation sensitivity were carried out to study the effects of increased DCX and SPARCco-expression for glioma cell growth, invasion and radiosensitivity, and analyzed itspossible mechanism.
Results
1. The double promoters mediated DCX and SPARC gene co-expressionadenoviral vector Ad-DCX-SPARC was successfully constructed. It could infect gliomacells U251and A172with high efficiency at the best infection doses of20MOI and100MOI respectively;2. Adenovirus-mediated DCX and SPARC gene co-expressionsignificantly inhibited the growth of glioma cells, weakening the role of SPARCpromoting glioma invasion in vitro;3. DCX and SPARC double gene co-expressioncould remarkably inhibit cell colony formation of U251and A172glioma cells postirradiation;4. DCX and SPARC double gene co-expression showed little effect onapoptosis in glioma cells, but it could promote apoptosis significantly after combinationwith radiation;5. Increasement of DCX and SPARC expression could enhanceproportion of G2phase cells before radiation. When combined with X-ray radiation, itwould decrease G2phase cell cycle arrest after irradiation to reduce repairment of DNAdamage with a result of more death of cells ultimately;6. Molecular mechanismanalysis showed that Ad-DCX-SPARC and radiation combination could remarkablyup-regulated the expressions of bax and bad, and down-regulated the expression ofbcl-2.
Conclusion:
1. Our study successfully constructed DCX and SPARC gene co-expressionadenovirus vector Ad-DCX-SPARC, it could significantly inhibit the growth of gliomacells and weakening the role of SPARC promoting glioma invasion in vitro;
2. Ad-DCX-SPARC combined with X-ray radiation inhibited glioma cell colony formation, increased G2phase cells arrest before irradiation and enhancedirradiation-induced apoptosis. In addition, it could also weaken the G2phase cell arrestafter glioma irradiation, so that DNA damage repair was decreased with more gliomacells death. All the above results increased cell radiosensitivity of glioma;
3. Ad-DCX-SPARC and radiation combination induced glioma cell apoptosisthrough up-regulating bax、bad, down-regulating bcl-2and so on apoptosis relatedgenes.
构建DCX和SPARC双基因共表达腺病毒载体（Ad-DCX-SPARC），研究其对胶质瘤细胞生长、侵袭和放射敏感性的影响及其机制研究。
方法
1. PCR扩增获得DCX和SPARC目的基因，构建针对DCX和SPARC的pAdTrack-CMV-DCX-polyA+promoter-SPARC双基因共表达重组转移质粒；
2.将构建正确的pAdTrack-CMV-DCX-polyA+promoter-SPARC重组转移质粒经PmeI酶切后与pAdEasy-1腺病毒骨架质粒在BJ5183大肠杆菌中同源重组；
3.同源重组质粒pAdEasy-1-pAdTrack-CMV-DCX-polyA+promoter-SPARC(简称为pAd-DCX-polyA+promoter-SPARC)经PacI酶切后再用脂质体转染QBI-293A细胞，经多轮感染和扩增后获得Ad-DCX-polyA+promoter-SPARC(简称Ad-DCX-SPARC)双基因共表达重组腺病毒载体；
4.将Ad-DCX-SPARC重组腺病毒体外感染胶质瘤细胞U251和A172，筛选出Ad-DCX-SPARC的最佳感染剂量；
5.通过一系列检测细胞增殖、侵袭和放射敏感性的实验方法，研究增加DCX和SPARC双基因共表达对胶质瘤细胞生长、侵袭和放射敏感性的影响，并分析其可能的作用机制。
结果
1.成功构建了双启动子介导的DCX和SPARC双基因共表达腺病毒载体Ad-DCX-SPARC，其能高效感染胶质瘤细胞U251和A172，最佳感染剂量分别为20MOI和100MOI；2.腺病毒介导的DCX和SPARC双基因共表达能明显抑制胶质瘤细胞的生长，削弱SPARC促胶质瘤侵袭的作用；3.增加DCX-SPARC双基因共表达能使U251和A172胶质瘤细胞照射后克隆形成能力明显降低；4.单纯增加DCX和SPARC双基因共表达对胶质瘤细胞凋亡无明显影响，但联合辐照后能明显促进胶质瘤细胞的凋亡；5. DCX-SPARC双基因共表达提高了照前G2期细胞的比例，联合X射线照射后能减少照后G2期细胞的阻滞，从而减少了DNA损伤的修复，最终促进了胶质瘤细胞的死亡；6.分子机制检测结果表明Ad-DCX-SPARC联合X射线照射能明显上调U251和A172胶质瘤细胞bax，bad的表达和下调bcl-2的表达。
结论
1.成功获得了DCX和SPARC双基因共表达腺病毒载体Ad-DCX-SPARC，能明显抑制胶质瘤细胞的生长，削弱SPARC促胶质瘤侵袭的作用；
2. DCX和SPARC双基因共表达联合X射线照射能通过抑制胶质瘤细胞的克隆形成、提高照射前G2期细胞阻滞、增加辐照引起的细胞凋亡、使细胞照后G2期的阻滞得到减弱从而减少照后DNA损伤的修复等机制提高脑胶质瘤细胞的放射敏感性；
3. Ad-DCX-SPARC联合X射线照射可通过调节U251和A172细胞中bax，bad和bcl-2等细胞凋亡相关基因诱导胶质瘤细胞的凋亡。
Objective
To construct a recombinant adenoviral vector co-expressing Doublecortin(DCX)and Secreted protein acidic and rich in cysteine(SPARC) and study its enhancedanti-tumor and radiosensitivity effects.
Methods
1. DCX and SPARC were amplified by PCR using cDNA as template.pAdTrack-CMV-DCX-polyA+promoter-SPARC double genes co-expression transfervector was constructed and identified by PCR, double endonuclease digestion, andDNA sequencing;
2. pAdTrack-CMV-DCX-polyA+promoter-SPARC transfer vector was linearizedwith PmeI digestion and further co-transfected with pAdEasy-1backbone vector intobacteria BJ5183competent cells for homologous recombination;
3. The production of pAdEasy-1-pAdTrack-CMV-DCX-polyA+promoter-SPARC(pAd-DCX-polyA+promoter-SPARC) homologous recombinant plasmid purified fromthe above BJ5183cells was transfected into the bacterial DH5α cells to abundantlyamplify pAd-DCX-polyA+promoter-SPARC plasmids, then they were linearized withPacI digestion and transfected into the human embryonic kidney293(QBI-293A) cellsby Lipofectamine2000, leading to packaging of the recombinant adenovirusesAd-DCX-polyA+promoter-SPARC(Ad-DCX-SPARC).First generation virus-containingsupernatants was used for obtaining large quantities of recombinant adenoviral vectorsafter several cycles of transfection and amplifying;
4. The best MOIs of Ad-DCX-SPARC were chosen by Ad-DCX-SPARC infectingU251and A172glioma cells;
5. A series experimental methods which tested cell proliferation、invasion andradiation sensitivity were carried out to study the effects of increased DCX and SPARCco-expression for glioma cell growth, invasion and radiosensitivity, and analyzed itspossible mechanism.
Results
1. The double promoters mediated DCX and SPARC gene co-expressionadenoviral vector Ad-DCX-SPARC was successfully constructed. It could infect gliomacells U251and A172with high efficiency at the best infection doses of20MOI and100MOI respectively;2. Adenovirus-mediated DCX and SPARC gene co-expressionsignificantly inhibited the growth of glioma cells, weakening the role of SPARCpromoting glioma invasion in vitro;3. DCX and SPARC double gene co-expressioncould remarkably inhibit cell colony formation of U251and A172glioma cells postirradiation;4. DCX and SPARC double gene co-expression showed little effect onapoptosis in glioma cells, but it could promote apoptosis significantly after combinationwith radiation;5. Increasement of DCX and SPARC expression could enhanceproportion of G2phase cells before radiation. When combined with X-ray radiation, itwould decrease G2phase cell cycle arrest after irradiation to reduce repairment of DNAdamage with a result of more death of cells ultimately;6. Molecular mechanismanalysis showed that Ad-DCX-SPARC and radiation combination could remarkablyup-regulated the expressions of bax and bad, and down-regulated the expression ofbcl-2.
Conclusion:
1. Our study successfully constructed DCX and SPARC gene co-expressionadenovirus vector Ad-DCX-SPARC, it could significantly inhibit the growth of gliomacells and weakening the role of SPARC promoting glioma invasion in vitro;
2. Ad-DCX-SPARC combined with X-ray radiation inhibited glioma cell colony formation, increased G2phase cells arrest before irradiation and enhancedirradiation-induced apoptosis. In addition, it could also weaken the G2phase cell arrestafter glioma irradiation, so that DNA damage repair was decreased with more gliomacells death. All the above results increased cell radiosensitivity of glioma;
3. Ad-DCX-SPARC and radiation combination induced glioma cell apoptosisthrough up-regulating bax、bad, down-regulating bcl-2and so on apoptosis relatedgenes.
引文
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