单纯疱疹病毒-胸苷激酶基因转染骨髓基质干细胞治疗大鼠脑胶质瘤

Anti-glioma activity of treatment by bone marrow stromal cells transfected with HSVotk in the rat

目的 研究转染单纯疱疹病毒-胸苷激酶（HSV-tk）基因的骨髓基质干细胞（BMSCs）对大鼠脑胶质瘤的治疗作用。方法 原代培养BMSCs，AdCMV-tk转染BMSCs。逆转录-多聚酶链反应（RT-PCR）检测BMSCs／tk对tk基因的转录。以BrdU标记BMSCs／tk，观察其趋瘤性。四甲基偶氮唑盐（MTr）法检测其对C6细胞的旁观者效应。将BMSCs／tk注入脑胶质瘤大鼠肿瘤对侧脑组织，原位末端标记（TUNEL）法检测脑内肿瘤细胞凋亡，动态MRI监测肿瘤体积的变化，观察荷瘤鼠生存期。结果全骨髓细胞培养法可获得纯化的BMSCs。感染AdCMV-tk 21d后，BMSCs仍有明显的tk基因表达。转染tk基因后的BMSCs仍具有明显的趋瘤性。BMSCs／tk不仅在体外可产生明显的旁观者效应，在荷瘤鼠脑内也显示了明显的诱导肿瘤细胞凋亡的旁观者效应，凋亡阳性率为20．38％±2．57％，与BMSCs组（2．56％±0．52％）和对照组（2．74％±0．38％）相比，差异均有统计学意义（P值分别为0．023和0．025）。BMSCs／tk移植3周时，BMSCs／tk组、BMSCs组和对照组肿瘤体积分别为（8．28±2．64）、（134．51±16．37）和（147．22±31．05）mm^3，BMSCs／tk组远小于BMSCs组（P=0．001）和对照组（P〈0．01），并且部分大鼠脑内肿瘤消失。BMSCs／tk组大鼠生存期为（52．60±13．11）d，与对照组相比明显延长（P=0．01）。结论 HSV-tk转染的BMSCs可能成为脑胶质瘤治疗的有效手段。

Objective To study the anti-glioma activity of treatment by bone marrow stromal cells （BMSCs） transfected with AdCMV-tk containing HSV-tk gene in rats. Methods Primary cultured BMSCs were obtained and transfected with HSV-tk （BMSCs/tk） and were injected into contralateral brain of gliomabearing rats to observe their tropism for glioma cells. RT-PCR was performed to examine the transduct of tk gene after it was transduced into BMSCs. C6 glioma cells were co-cultured with BMSCs transfected with HSV-tk. MTY test was performed to examine its antitumor activity. BMSCs, after being transfected with HSV-tk, were injected into contralateral brain tissue of glioma-bearing rats to show their in vivo antitumor activity. Dynamic MRI was performed to monitor the development of intracranial glioma. Results Purified BMSCs were obtained by primary cultured bone marrow cells. After being transfected with HSV-TK, the cells still stably displayed extensive tropism for intracranial glioma and transcripted tk gene. RT-PCR showed that BMSCa/tk were transduced tk gene obviously at 21 days after AdCMV-tk transfection. BMSCs/tk showed a clear bystander effect after being co-cultured with C6 glioma cells in vitro. TUNEL assay showed that BMSCa/tk could obviously show bystander effect and induce apoptosis of glioma cells in vivo with an apoptosis positive ratio of 20.38% ± 2.57%, showing a statistically significant difference in comparison with BMSCs group （2.56%±0. 52%, P =0.023） and control group （2.74% ±0.38%, P =0.025）. Compared with the control group （21.40 ± 1.63 days）, BMSCs/tk transplantation significantly prolonged the survival time of glioma-bearing rats （52.60 ± 13.11 days, P =0. 000）. MBI detection showed that the least volume of intracranial glioma in BMSCs/tk group （ 8.28 ± 2.64 mm^3 ）, significantly smaller than that in BMSCs group （ 134.51 ± 16.37 mm3, P = 0.001 ） and control group （ 147.22± 31.05 mm^3, P = 0.001 ）. Some of the intracranial gliomaa disappeared after transplantation of BMSCs/tk. Conclusion BMSCs transfected with AdCMV-tk may become an effective therapy method in the treatment for glioma.