辐射诱导表达载体pEgr-hPTEN的构建及其体外抗肿瘤作用的研究

目的 克隆人野生型抑癌基因PTEN／MMAC1 cDNA序列、构建辐射诱导表达载体pEgr-hPTEN并研究其体外稳定转染联合X-射线照射对恶性胶质瘤细胞SHG-44增殖的抑制作用。方法 利用RT—nested PCR法从正常人胎盘组织中扩增出约1200bp的PTEN片段，并将其重组人pcDNA3．1-Egr载体中，构建为表达质粒pEgr-hPTEN，体外转染肿瘤细胞并经G418筛选稳定转染细胞株SHG-44-sPTEN，接受不同剂量X射线照射，观察基因-放射联合作用对肿瘤细胞生长的影响。结果 经全自动测序证明克隆得到了野生型PTEN基因；辐射诱导表达载体pEgr—hPTEN构建正确；稳定转染联合X-射线照射可明显抑制肿瘤细胞的恶性增殖，5Gy以内随吸收剂量的增加，肿瘤抑制作用更明显。结论 本研究成功克隆了人野生型抑癌基因PTEN／MMAC1 cDNA序列并构建了辐射诱导表达载体pEgr-hPTEN，体外基因-放射联合治疗具有明显的肿瘤抑制作用。本研究为提高临床恶性肿瘤放疗疗效奠定了理论和实验基础。

Study on construction of pEgr-hPTEN expression vector induced by irradiation and its anti-tumor effect in vitro

Objective To clone the cDNA of human tumor suppressor gene-PTEN ,construct pEgr-hPTEN e xpression vector induced by irradiation and study its inhibitory effect on proli feration of malignant glioma cell line SHG-44 transfected steadily with pEgr-h PTEN after different doses of X-ray irradiation. Methods A DNA fragment about 1 200 bp ,PTEN, was amplified from human placenta tissues by using RT-nes ted PCR and was cloned into pUCm-T vector after automatic sequencing, the n the fragment was inserted into a vector pcDNA3 1-Egr to construct an express i on vector pEgr-hPTEN. pEgr-hPTEN was transfected into SHG-44 cells in vitro . Stably transfected cell line SHG-44-sPTEN was selected through G418. The inhi bitor effect on SHG-44-sPTEN was observed after different doses of X-ray irra diation in vitro. Results The PTEN cDNA has been cloned correctly and its expre ssion vector pEgr-hPTEN was also constructed. Growth of SHG-44 cells was inhi bited significantly by stable pEgr-hPTEN transfection combined with X-ray irra diation. With the increase of dose , the inhibitory effect was enhanced within 5 Gy. Conclusion Human tumor suppressor gene-PTEN cDNA has been cloned and its e xpression vector has been constructed. The tumor was inhibited significantly by gene-radiotherapy in vitro. The result provides the theoretical and experim ental basis for improvement of clinical radiotherapeutic effect on tumors .;