外源基因经静脉途径跨血脑屏障在大鼠脑内的特异性表达

目的 构建脑特异性启动子GFAP启动的由转铁蛋白受体抗体(OX26)靶向的免疫脂质体0X26-pGFAP-IL,研究其携带外源基因LaeZ在大鼠脑内的表达情况.方法 将pCMV-LacZ脂质体(pCMV-Liposome)、OX26-pCMV-LacZ免疫脂质体(0X26-pCMV-IL)、0X26-pGFAP-LacZ免疫脂质体(OX26-pGFAP-IL)和空白脂质体,分别通过股静脉注射至大鼠体内.24 h后Q-PCR检测LacZ基因mRNA在脑及周围器官的表达相对量,48 h后检测LacZ基因在脑及周围器官的蛋白表达情况.结果 药物注射24 h后OX26-pCMV-IL组和OX26-pGFAP-IL组脑中LacZ基因mRNA表达相对量分别为49.2 x 10~(-6)和44.9×10~(-6),显著高于pCMV-Liposome组和空白脂质体组(P<0.05).OX26-pCMV-IL组在周围脏器的LacZ基因mRNA相对量显著高于OX26-pGFAP-IL组(P<0.05).48 h后OX26-pCMV-IL组和OX26-pGFAP-IL组脑中β-半乳糖苷酶活性分别为0.67 pg/mg和0.92 pg/mg,显著高于pCMV-Liposome组和空白脂质体组(P<0.05).OX26-pCMV-IL组和OX26-pGFAP-IL组在脑中β-半乳糖苷酶活性差异无统计学意义.组织化学染色OX26-pGFAP-IL组可以实现在脑内的特异性阳性表达,减少在周围脏器的阳性表达.结论 OX26-pGFAP-IL通过静脉途径注射后可以透过血脑屏障,在GFAP启动子的作用下实现外源基因脑内特异性的表达,同时减少在周围脏器的非特异表达,是实现颅内疾病的非病毒载体基因治疗的一种可行的方法.

Specific expression in brain of exogenous gene by transpassing blood-brain barrier after intravenous injection

Objective To study the expression of exogenous LacZ gene in brain via a delivery of OX26-pGFAP-IL.Methods pCMV-Liposome,OX26-pCMV-IL,OX26-pGFAP-IL and blank liposome were injected into rats via femoral vein.At 24 h post-injection,the method of Q-PCR was adopted to calculate the relative quantities of LaeZ gene mRNA in brain and peripheral organs.At 48 h post-injection, the protein expression of LacZ gene waS detected by the activity of β-galactosidase and the method of histochemical stain.Results The result of Q-PCR showed that,at 24 h post-injection,the relative quantities of LacZ mRNA in OX26-pCMV-IL group (49.2×10~(-6)) and OX26-pGFAP-IL group (44.9×10~(-6)) were significantly hisher than pCMV-liposome and blank liposome groups (P<0.05).In peripheral organs,the relative quantity of LacZ mRNA in OX26-pCMV-IL group were significantly higher than that in OX26-pGFAP-IL group(P<0.05).At 48 h post-injection,the activity of β-galactosidase in OX26-pCMV-Ⅱ.(0.67 pg/mg) and OX26-pGFAP-IL groups (0.92 pg/mg) were significantly higher than pCMV-liposome and blank liposome groups (P<0.05).There was no significant difference between OX26-pCMV-IL group and OX26-pGFAP-IL group in terms of the expression of β-galactosidase.The result of histochemical stain showed that OX26-pGFAP-IL achieved a specifically positive expression in brain and had a decreased expression in peripheral organs.Conclusion OX26-pGFAP-IL injected via femoral vein can cross the brain-blood barrier and achieve a specific expression in brain under the control of GFAP promoter.OX26-pGFAP-Ⅱ,decreases the non-specific expression in peripheral organs and it may be used as an non-viral gene therapy for intra-cranial diseases.