可溶性转化生长因子βⅡ型受体对LoVo细胞侵袭能力的影响

Effects of soluble type Ⅱ transforming growth factor β receptor on invasive ability of the colorectal carcinoma LoVo cells

目的构建人转化生长因子βⅡ型受体细胞外区基因的真核表达载体，初步探讨可溶性转化生长因子βⅡ型受体（sTGFβRⅡ）对结直肠癌LoVo细胞侵袭能力的影响。方法采用DNA重组技术构建sTGFβRⅡ的表达载体pcDNA3．1（＋）／sTGFβRⅡ，用脂质体转染技术转染人结直肠癌LoVo细胞，以RT-PCR、Western印迹和细胞免疫荧光技术分别检测目的基因及蛋白表达；Matrigel^TM侵袭试剂盒检测sTGFβRⅡ对LoVo细胞侵袭能力和人脐静脉内皮细胞（HUVEC）血管生成能力的影响，RT-PCR检测sTGFβRⅡ对LoVo细胞基质金属蛋白酶-9（MMP-9）基因和血管内皮生长因子（VEGF）基因表达的影响。结果sTGFβRⅡ在LoVo细胞中获得表达，转染sTGFβRⅡ基因的LoVo细胞侵袭能力（穿膜细胞数为111．0±10．1）明显低于空载体转染组（171．3±3．2）和对照组（177．0±2．7，F=100．1，P〈0．01），HUVEC在人工膜上形成管腔的能力显著下降，转染组管腔数目（1．3±0．6）低于空载体转染组（4．7±1．5）和对照组（5．0±1．0，F=13．3，P〈0．01）。pcDNA3．1（＋）／sTGFβRⅡ转染的LoVo细胞MMP-9和VEGF基因表达明显下降（P〈0．01）。结论sTGFβRⅡ能拮抗TGFβⅡ1的生物活性，可能通过抑制MMP-9和VEGF表达，进而抑制LoVo细胞侵袭和诱导血管生成能力，达到抗肿瘤作用。

Objective To construct a eukaryotic expression vector encoding the gene of extra cellular region of type Ⅱ transforming growth factor β receptor（sTGFβRⅡ ） and to investigate its effect on invasion potential of LoVo cells. Methods The pcDNA3. 1 （＋）/sTGFβRⅡ expression vector was constructed by DNA recombinant technique and was introduced into LoVo cells by lipofectactin-mediated DNA transfection. RT-PCR, Western blot and immunofluorescent assay were used to determine sTGFβRⅡ expression. The invasion of LoVo cells and the angiogenesis of human umbilical vein endothelial celIs（HUVEC） were determined by BD BioCoat^TM Matrigel^TM invasion chambers. The effect of sTGFβRⅡ on expression of the matrix metalloproteinase-9 （MMP-9） and vascular endothelial growth factor（VEGF） genes were measured by RT-PCR. Results sTGFβRⅡ mRNA and protein expressed in LoVo cells. The invasive ability of LoVo cells that transfected sTGFβRⅡ genes was decreased significantly. The number of the cells that could cut through membrane was markedly lower than that of the un-transfected group（ 111.0± 10.1 vs 177.0 ±2.7, P 〈 0.01 ）. The ability of HUVEC proliferation and tube formation was decreased in artificial membrane. The lumens number of transfected group was lower than that of the un-transfected group（1.3 ± 0.6 vs 5.0 ± 1.0, P 〈 0.01）. The gene expressions of MMP-9 and VEGF in LoVo cells which were transfected by pcDNA3. 1 （＋）/sTGFβRⅡ were significantly decreased. Conclusions sTGFβRⅡ can inhibit TGFβ1 signaling and resist the biological activity of TGFβ1 in LoVo cells in vitro, sTGFβRⅡ can attenuate tumor invasion and metastasis, possibly through suppresses the expression of MMP-9 and VEGF, and thus supresses the invasion and angiogenesis of LoVo cell.