Chen Chuangui, Sun Jinjin, Liu Geng, Chen Jianqiu
Department of Surgery, The Second Hospital of TianJin Medical University, Tianjin, 300211, China.
Pathol Oncol Res. 2009 Mar;15(1):109-14. doi: 10.1007/s12253-008-9063-7. Epub 2008 May 29.
To study the effect of recombinant adeno-associated virus (rAAV) vector bearing small inference RNA (siRNA) targeting hypoxia inducible factor 1alpha (HIF-1alpha) combined L: -ascorbate on pancreatic tumors in athymic mice primarily. A cassette encoding siRNA targeting HIF-1alpha mediated by rAAV was constructed, giving rAAV-siHIF. In vitro, rAAV-hrGFP, rAAV-siHIF and L: -ascorbate which were used alone or in combination were delivered to exponentially growing MiaPaCa2 cells. Then, we examined the expression of HIF-1alpha mRNA and protein, the secretion of VEGF in MiaPaCa2 cells under hypoxic condition with Real-time PCR, Western Blot, ELISA, respectively. In vivo, MiaPaCa2 cells were inoculated subcutaneously on the back of nude mice. Nude mice with xenograft tumor were randomly divided into equal groups and were injected with rAAV-hrGFP or rAAV-siHIF or were fed with L: -ascorbate. Then, we measured the size of tumor every 3 days and drew a tumor growth curve. After 30 days, all mice were sacrificed and the tumors were dissected. At last, we examined the expression of HIF-1alpha, VEGF and CD34 by immunohistochemistry and counted micro-vessel density (MVD). In vitro, we found that rAAV-siHIF could inhibit the expression of HIF-1alpha mRNA and protein in MiaPaCa2 human pancreatic cancer cells but L: -ascorbate could only restrain the expression of HIF-1alpha protein. Moreover, rAAV-siHIF and L: -ascorbate could all inhibit the secretion of vascular VEGF. In vivo, we found that rAAV-siHIF could inhibit the growth of nude mice xenograft tumor and the expression of HIF-1alpha and VEGF and MVD while L: -ascorbate can only inhibit the growth of xenograft tumor in the early and middle stage. These results suggest that rAAV-siHIF and L: -ascorbate can inhibit the growth of nude mice xenograft tumor and HIF-1alpha could be a target of pancreatic cancer genetic and pharmacological therapy.
主要研究携带靶向缺氧诱导因子1α（HIF-1α）的小干扰RNA（siRNA）的重组腺相关病毒（rAAV）载体联合L-抗坏血酸对无胸腺小鼠胰腺肿瘤的影响。构建由rAAV介导的编码靶向HIF-1α的siRNA的盒式结构，得到rAAV-siHIF。在体外，将单独或联合使用的rAAV-hrGFP、rAAV-siHIF和L-抗坏血酸递送至指数生长的MiaPaCa2细胞。然后，我们分别用实时定量PCR、蛋白质免疫印迹法、酶联免疫吸附测定法检测缺氧条件下MiaPaCa2细胞中HIF-1α mRNA和蛋白的表达、VEGF的分泌。在体内，将MiaPaCa2细胞皮下接种于裸鼠背部。将移植瘤裸鼠随机分成相等的组，分别注射rAAV-hrGFP或rAAV-siHIF或喂食L-抗坏血酸。然后，我们每3天测量一次肿瘤大小并绘制肿瘤生长曲线。30天后，处死所有小鼠并解剖肿瘤。最后，我们通过免疫组织化学检测HIF-1α、VEGF和CD34的表达并计数微血管密度（MVD）。在体外，我们发现rAAV-siHIF可抑制MiaPaCa2人胰腺癌细胞中HIF-1α mRNA和蛋白的表达，但L-抗坏血酸只能抑制HIF-1α蛋白的表达。此外，rAAV-siHIF和L-抗坏血酸均可抑制血管VEGF的分泌。在体内，我们发现rAAV-siHIF可抑制裸鼠移植瘤的生长以及HIF-1α、VEGF的表达和MVD，而L-抗坏血酸仅在早期和中期抑制移植瘤的生长。这些结果表明，rAAV-siHIF和L-抗坏血酸可抑制裸鼠移植瘤的生长，HIF-1α可能是胰腺癌基因治疗和药物治疗的靶点。
