双病毒共感染的人脐带间充质干细胞多重靶向肝癌细胞的治疗体系的建立

Double virus co-infected human umbilical cord mesenchymal stem cells as vehicles: A multiple targeted therapy against hepatocarcinoma

目的基于间充质干细胞(MSC)向肿瘤部位归巢的能力,利用E1A基因修饰的人脐带间充质干细胞(HUMSC)包装出携带人端粒酶逆转录启动子(hTERTp),以驱动抑癌基因的腺病毒,并感染HepG2肝癌细胞系。方法利用组织块贴壁法从脐带Wharton’s jelly(WJ)分离培养HUMSC。以pGL3-basic、pCDH1-MSC1-EF1-Ds Red、pAd-Track等载体为基础,用聚合酶链反应(PCR)、overlap PCR、酶切、连接等技术构建pGL3-hTERTp报告基因载体、pLentiR.E1A慢病毒表达载体、pAd-hTERTp腺病毒表达载体,经测序验证其正确性。利用双荧光素酶的方法鉴定hTERTp的特异性。利用实时定量PCR的方法分别检测MSC.pLentiR+pAd-Track(共转染腺病毒及对照慢病毒的MSC)和MSC.pLentiR.E1A+pAdTrack中不同时间点的病毒相对拷贝数,用电子显微镜观察HUMSC中病毒颗粒的产生。流式细胞术检测由双病毒共感染的HUMSC包装出的腺病毒对HepG2细胞系的感染效率。Transwell方法研究双病毒感染对HUMSC向HepG2细胞趋化的影响。结果成功从脐带WJ组织中分离培养HUMSC。成功构建载体pGL3-hTERTp、pLentiR.E1A、pAd-hTERTp。双荧光素酶方法证明hTERTp在不同肿瘤细胞中具有较高的转录活性,而在正常细胞中转录活性极低。Ad-Track及LentiR.E1A共同感染HUMSC后,随着腺病毒早期复制基因E1A逐渐表达,Ad-Track启动复制程序,最终将MSC裂解并释放具有感染能力的病毒颗粒,进一步感染HepG2细胞。感染双病毒的HUMSC与对照组一致在LentiR.E1A感染后32 h内可以穿过Transwell小室,向培养HepG2细胞的下室迁移。结论经过E1A修饰的HUMSC可包装出携带hTERTp肿瘤特异启动子的复制缺陷型腺病毒,并进一步感染肿瘤细胞的多重靶向治疗系统,为肿瘤的靶向基因治疗提供了新思路。

Objective To study replicate and assemble new viruses carrying therapeutic gene driven by human telomerase reverse transcriptase promoter（hTERTp）, and consequently infect HepG2 cell based on tropism of mesenchymal stem cells（MSC）in tumor by E1A gene modified human umbilical cord derived mesenchymal stem cells（HUMSC）. Method HUMSC were isolated from gelatinous Wharton＇s jelly（WJ） by tissue adherent method. Based on vectors of pGL3-basic, pCDH1-MSC1-EF1-Ds Red and pAd-Track, the report gene vectors of pGL3- hTERTp, pLentiR.E1A lentivirus and pAd-hTERTp adenoviral were constructed by polymerase chain reaction（PCR）, overlap PCR, restriction enzyme cleavage and linkage, then verified correctness by sequencing. Dual luciferase assay was used to verify specific activity of hTERTp promoter. The relative viral copies in MSC.pLentiR ＋ pAd-Track or MSC.pLentiR. E1A ＋ pAd-Track were detected by real time PCR and analyzed by standard curve.The production of adenovirus were detected by electron microscope. The infection efficiency of adenovirus assembled by HUMSC, which were co-infected by LentiR.E1A and Ad-Track was detected by flow cytometry. The migratory ability of virusloaded HUMSC in vitro was determined by Transwell in cell culture inserts. Results HUMSC were successfully obtained from WJ within umbilical cord. The reporter gene vector pGL3-hTERTp, lentiviral expression vector pLentiR.E1A and adenoviral expression vector pAd-hTERTp were successfully constructed. The hTERTp was confirmed with specific transcriptional activity in tumor cells but without in normal cells. As E1A was expressed gradually, and concentration of viral DNA was observed to increase sharply and synchronously after infection in HUMSC co-infected by LentiR.E1A and Ad-Track. Consequently, adenovirus were replicated and assembled by E1A, and infected HepG2 cells. The virus-loaded HUMSC migrated to culture of HepG2 in similar pattern to unmodified HUMSC within 32-hour after infected by LentiR.E1A in vitro. Conclusion It is demonstrated that an exploration of a promising targeted therapeutic strategy used E1A modified HUMSC to deliver the replication-deficient adenovirus, replicate and assemble into new viruses against hepatocarcinoma. The therapeutic strategy pro-vides new effective and safe administration route for replication-deficient adenovirus.