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低氧诱导型VEGF真核表达载体的构建及其体外递送的应用与功能鉴定
作者:林彦霞 白睿 刘志强 杨锡琴 金至赓 田鹏 刘惠亮 
单位:100039  北京 锦州医科大学解放军总医院第三医学中心研究生培养基地(林彦霞) 100039  北京 解放军总医院第三医学中心心内科(白睿、金至赓) 心脏病研究所(刘惠亮) 100850  北京 军事医学研究院军事认知与脑科学研究所(刘志强、杨锡琴、田鹏) 
关键词:VEGF165基因 真核表达 DNA重组技术 PLGA纳米粒 
分类号:R310.17
出版年,卷(期):页码:2019,44(2):98-106
摘要:

[摘要]  目的  构建低氧诱导型的血管内皮生长因子(VEGF)真核表达载体,建立体外递送方法并对其效能进行鉴定。方法  应用基因重组技术,在真核表达载体pGL4.73[hRluc/SV40](pSV)启动子上游插入促红细胞生成素(EPO) 增强子,构建低氧诱导型表达载体(pEPO-SV),以海肾荧光素酶(Rluc)为下游报告基因;随后以VEGF165基因取代Rluc 插入到pEPO-SV质粒,同时将其插入到pSV质粒作为对照,分别获得pEPO-SV-VEGFpSV-VEGF表达载体;在体外分别将表达RlucVEGF165的质粒转染人胚肾293T细胞,在正常和低氧条件下处理24h48h后,通过RlucVEGF165 的表达对所构建载体的低氧诱导功能进行鉴定;然后基于聚乳酸-羟基乙酸共聚物(PLGA)纳米颗粒建立质粒的细胞内递送方法,并在体外细胞缺氧模型中对上述低氧诱导型真核表达质粒的效能进行鉴定。结果  在质粒构建中,分别通过酶切、PCR扩增和DNA测序证实了EPO增强子和VEGF165基因的成功插入与正确性。表达RlucVEGF165的质粒分别转染293T细胞后,正常培养条件下报告基因Rluc(其一质粒pSVpEPO-SV荧光表达值分别为2448.24±158.51 3173.97±379.92,其二质粒pSVpEPO-SV荧光表达值分别为55 500.00±3237.0551 193.18±866.32)及目的基因VEGF165表达差异无统计学意义(P>0.05),而在低氧情况下Rluc(氯化钴低氧下,质粒pSVpEPO-SV荧光表达值分别为4857.70±1223.2816 432.64±1618.73;低氧培养箱中,质粒pSVpEPO-SV荧光表达值分别为2504.45±213.2017 274.35±685.60)VEGF165表达明显高于对照组,差异有统计学意义(P<0.01),表明所构建的pEPO-SVpEPO-SV-VEGF质粒具有典型的低氧诱导表达活性。利用PLGA纳米颗粒在体外293T细胞中进行pEPO-SVpSV递送后,在正常培养条件下与低氧条件下报告基因Rluc的表达变化与上述一致,即正常培养条件下,处理24h48hpSVpEPO-SV质粒的荧光表达值分别为149.44±4.01127.09±15.051074.91±114.781064.56±137.48;低氧条件下,处理24h48h pSVpEPO-SV质粒的荧光表达值分别为3265.34±440.008828.87±637.033202.06±33.439114.75±292.06结论  成功建立了典型的低氧诱导型VEGF真核表达系统,并建立了有效的体外递送方法;这一低氧诱导型表达载体及递送方法在缺血、缺氧等组织损伤疾病中可能具有重要的应用前景。

[Abstract]  Objective  To construct the eukaryotic expression vector of hypoxia inducible vascular endothelial growth factor (VEGF), and establish its in vitro delivery method. Methods  Erythropoietin (EPO) enhancer was inserted into eukaryotic expression vector pGL4.73 [hRluc/SV40] (pSV) promoter by gene recombination technique to construct hypoxia inducible expression system (pEPO-SV). Renilla luciferase (Rluc) was used as downstream reporter gene. Then the VEGF165 gene was inserted into the pEPO-SV plasmid instead of Rluc, and the pEPO-SV-VEGF and pSV-VEGF expression vectors were obtained by inserting the pEPO-SV-VEGF gene into pSV as control. The pSV plasmid expressing Rluc or VEGF165 and pEPO-SV plasmid were transfected in vitro into human embryonic kidney 293T cells. The expression of Rluc or VEGF165 was used to identify the hypoxia induction function of the constructed vector after being treated under normal and hypoxic conditions for 24h and 48h. The intracellular delivery method of plasmids was then established based on poly (lactic acid-glycolic acid) copolymer (PLGA) nanoparticles as carrier, and the efficiency of the eukaryotic expression plasmids induced by hypoxia was evaluated under the in vitro hypoxia model. Results  In the construction of plasmid, the successful insertion and correctness of EPO enhancer and VEGF165 gene were confirmed by restriction endonuclease digestion, PCR amplification and DNA sequencing. The plasmid expressing Rluc or VEGF165 was transfected into 293T cells respectively. There was no significant difference in the expression of reporter gene Rluc (one, plasmid pSV and pEPO-SV fluorescence expression values were 2448.24±158.51 and 3173.97±379.92, the second, plasmid pSV and pEPO-SV fluorescence expression values were 55 500.00±3237.05 and 51 193.18±866.32, respectively) or target gene VEGF165 in normal culture (P>0.05). But the expression of Rluc (In the cobalt chloride of hypoxia, the fluorescence expression values of pSV and pEPO-SV were 4857.70±1223.28 and 16 432.64±1618.73, respectively. In the hypoxia incubator, the fluorescence expression values of pSV and pEPO-SV were 2504.45±213.20 and 17 274.35±685.60, respectively) or VEGF165 in hypoxia was significantly higher than that in control group (P<0.01). The results showed that the constructed pEPO-SV and pEPO-SV-VEGF plasmids had typical hypoxia inducible expression activity. PLGA nanoparticles were used to in vitro deliver pEPO-SV and pSV in 293T cells. The results of detecting the reporter gene Rluc in normal culture and hypoxic conditions were consistent with those mentioned above, that is, under normal conditions, the 24h and 48h fluorescence expression values of plasmids pSV and pEPO-SV were 149.44±4.01 and 127.09±15.05, 1074.91±114.78 and 1064.56±137.48, respectively; under hypoxic conditions, the 24h and 48h fluorescence expression values of pSV and pEPO-SV were 3265.34±440.00 and 8828.87±637.03, 3202.06±33.43 and 9114.75±292.06, respectively. Conclusion  A typical hypoxia inducible VEGF eukaryotic expression system has been successfully established, and an in vitro effective delivery method is also established, which may have an important application prospect in ischemia, hypoxia and other tissue injury diseases.

基金项目:
国家自然科学基金(81500077,81672607);全军医学科技青年拔尖项目(16QNP129);北京市科技新星计划项目(Z171100001117115)
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