体内逆转录病毒介导的sFlt-1抑制小鼠骨肉瘤生长的实验研究

Retros Flt-1 decelerates the growth of a murine experimental osteosarcoma

目的调查逆转录病毒介导的sFlt-1基因针对高血管内皮生长因子（VEGF）表达的人骨肉瘤G-292细胞进行体外转导的效率，并构建重度联合免疫缺陷（SCID）小鼠的骨肉瘤模型，评估sFh-1转基因治疗对于骨肉瘤生长的抑制作用。方法2010年3月至2010年10月应用逆转录病毒介导的sFlt-1基因（sFlt-1转导组，n=10）和LacZ基因（LacZ转导组，n=10）体外转染人骨肉瘤G-292细胞（G-292组，n=10），种植到重度联合免疫缺陷小鼠胫骨近端，建立骨肉瘤动物模型。应用显微CT监测骨肉瘤的生长发育，8周后取出骨肉瘤标本进行组织学以及分子生物学分析。结果酶联免疫吸附试验测定证实sFlt-1基因转导成功。细胞种植后2周时所有小鼠胫骨近端均出现了骨肉瘤的发生、发育，sFlt-1转导组骨肉瘤体积小于G-292组和LacZ转导组，差异无统计学意义（P〉0．05）；在细胞种植后4、6、8周时，sFh．1转导组骨肉瘤体积明显小于G-292组和LacZ转导组，差异具有统计学意义（P〈0．05）。组织学表现显示了典型的骨肉瘤特征，包括重度的细胞多型现象、骨质破坏和新生血管形成。实时定量PCR测定结果显示sFlt-1转导组的sFlt-1基因相对定量表达为（4．6±1．3）倍，高于其他两组，差异具有统计学意义（P〈0．05）。结论逆转录病毒介导的sFlt-1基因抑制了小鼠骨肉瘤的生长。

Objective To examine the influence of vascular endothelial growth factors （VEGF） in controlling the growth of an experimental osteosarcoma in mice by performing retrovirus-mediated sFlt-1 gene modification. Methods From March to October 2010 human osteosarcoma G-292 ceils were in vitro infected with retroviral vectors encoding soluble Flt-1 or LacZ gene before transplanted into proximal tibiae of immune deficient SCID mice to establish experimental orthotopic osteosarcoma. Daily observation and biweekly microCT were performed to monitor tumor development and progression till sacrifice at 8 weeks after tumor cell inoculation for histological and molecular analyses. Results Successful transgene expression was confirmed in the culture media of sFlt-1 transduced G-292 cells using ELISA, and with positive X-gal staining of the LacZ transduced cells. Noteworthy tumors were grown in all mice on the tibiae receiving G-292 cell inoculation, with clear detection on microCT images starting 2 weeks after inoculation. Over the time period, tumors derived from sFlt-1 transduced G-292 cells were distinctively smaller in size compared to the ones from wide-type G-292 and G-292-LacZ cells. Histology showed typical osteosarcoma characteristics including severe cellular pleomorphism, bone erosions, and neo-vascularization. Real-time polymerase chain reaction indicated significantly higher sFlt-1 expression in sFlt-1 transduced groups than the wild-type G-292 or LacZ treated groups. Strong expression of oncogenes c-myc and c-fos were also obvious, along with the expression of VEGF in the primary tumor tissue. Conclusion Retrovirus-mediated sFLT-1 gene modification decelerates the osteosarcoma tumor growth in this murine model.