转染PF4 cDNA的GRC-1细胞培养液对于ECV-304细胞生长及VEGF表达的影响

The effects of culture supernatant of human GRC-1 cells transfected with PF4 cDNA on the VEGF expression and the growth of ECV304 cells

目的 :构建真核表达载体pcDNA3 PF4 SS ,检测稳定转染后的GRC 1细胞的培养上清对于血管内皮细胞ECV30 4的生长以及转染细胞中血管内皮生长因子 (VEGF)表达的影响。方法 :构建真核表达载体pcDNA3 PF4 SS ,并经BglⅡ/BamHI双酶切鉴定。通过脂质体介导 ,以该载体稳定转染GRC 1细胞 ,转染细胞中VEGF因子的表达情况用免疫组化染色法检测 ,转染细胞的培养上清对于ECV30 4细胞生长的影响用MTT法检测。结果 :经BglⅡ /BamHI双酶切证实 ,hPF4cDNA已成功地克隆到真核表达载体pcDNA3 CD34 SS中。RT PCR法检测证实 ,hPF4cDNA已成功地转染GRC 1细胞。免疫组化染色检测显示 ,转染PF4基因的GRC 1细胞的胞浆及胞膜均有VEGF表达 ,但较转染前明显减弱。细胞计数及MTT法显示 ,转染后GRC 1细胞的培养上清对ECV30 4细胞的生长具有抑制作用 ,吸光度值 (A)明显降低。结论 :构建了真核表达载体pcDNA3 PF4 SS ,并稳定转染GRC 1细胞。转染细胞的培养上清对ECV30 4细胞的生长及VEGF的表达 ,均具有明显的抑制作用 ,为探讨抗肿瘤生长机制和肿瘤疫苗的研制奠定了一定的基础。

AIM: To construct the eukaryotic expression vector pcDNA3 PF4 SS, and to detect the effects of the culture supernatant of transfected GRC 1 cells on the VEGF expression in transfected GRC 1 cells and the growth of ECV304 cells. METHODS: The eukaryotic expression vector pcDNA3 PF4 SS was constructed and identified with Bgl Ⅱ/ Bam H I digestion. The pcDNA3 PF4 SS was tranfected stably into GRC 1 cells with lipofectamine mediation. The VEGF expression in transfected GRC 1 cells was detected by immunohistochemical staining, and the effect of the culture supernatant of transfected GRC 1 cells on ECV304 cells was detected by MTT colorimetry. RESULTS: Restrictive enzyme ( Bgl Ⅱ/ Bam H I)digestion analysis showed that the recombinant expression vector pcDNA3 PF4 SS had been constructed successfully. RT PCR detection proved that hPF4 cDNA had been transfected into GRC 1 cells. The result of immunohistochemical staining showed that the VEGF expression could be seen in the cytoplasm and on cytomembrane of GRC 1 cells transfected with pcDNA3 PF4 SS, but the expression obviously weakened as comparison with that before transfection. Cell counting and MTT colorimetry manifested that the culture supernatant of transfected GRC 1 cells could inhibit markedly growth of ECV304 cells. CONCLUSION: The eukaryotic expression vector pcDNA3 PF4 SS has been constructed successfully, and stably transfected into the GRC 1 cells. The culture supernatant of transfected GRC 1 cells has obviously inhibitory effect on the growth of ECV304 cells and the VEGF expression in the GRC 1 cells, which lays some foundation for exploring the mechanism for anti tumor growth and developing tumor vaccine for kidney neoplasms.