基因导入途径与脑内表达的关系研究

Correlations Between Gene Transfer Approaches and Intracerebral Gene Expression

目的比较不同途径(股静脉、颈内动脉、侧脑室及鞘内)导入转铁蛋白靶向脂质体介导的LacZ基因(transferrintargeted LacZ gene pegylated liposomes,Tf-PLs)在大鼠脑内的表达,寻找出适合临床的安全有效的基因导入方法。方法 120只雄性SD大鼠随机分为静脉导入组、动脉导入组、侧脑室导入组、鞘内注射组和治疗组,其中治疗组分别将Tf-LacZ通过股静脉、颈内动脉、侧脑室立体定向及鞘内的方式注射入大鼠体内。术后24和48h分别取脑(每项检测指标每组随机选取8只大鼠),光学显微镜下观察LacZ基因编码的β-半乳糖苷酶(beta-gal)的表达,Real time-PCR比较不同组别LacZ mRNA的表达,试剂盒检测β-半乳糖苷酶的活性。结果免疫组化提示动脉导入组及静脉导入组可以实现β-半乳糖苷酶在脑内的广泛性表达,并且动脉导入组LacZ mRNA及β-半乳糖苷酶的表达高于静脉组(P<0.05)。侧脑室及鞘内注射组只能实现β-半乳糖苷酶的局限性表达,且LacZ mRNA及β-半乳糖苷酶的表达要低于动脉及静脉导入组(P<0.05)。结论动脉导入将外源基因转入大鼠脑内的效果最好,静脉导入的效果优于侧脑室注射和鞘内注射的方式。在可行性方面,经过静脉导入途径是较为方便、易行、有效。

Objective To compare intracerebral expression of transferrin targeted LacZ gene pegylated liposomes （Tf- PLs） trans- ferred by different approaches via the femorM vein, internal carotid artery （ICA）, lateral ventricle （LV） and intrathecal routes in rats, in an attempt to seek a safe and effective approach for gene transfer appropriate for clinical use. Methods Male SD rats were randomized in to four groups and Tf- LacZ was injected into the rats via the femoral vein, ICA, LV and intrathecal routes, respectively. Eight animals from each group were sacrificed and the brains were removed 24 and 48 h after Tf- LacZ injection. The expression of LacZ encoded 13 - galactosidase （13- gal） was observed under an optical microscope. LacZ mRNA expressions in different groups at the designated time points were detected by RT - PCR, and the expressions of 13 - gal were detected by Reagent kit. The results obtained were compared. Re- suits Extensive expression of 13 - gal was achieved in the femoral vein and ICA groups, and the expression of LacZ mRNA in the CA group was significantly high than that in the femoral vein group （ P 〈 0.05 ）. Only local expression was achieved in the LV and intrathecal groups, and the expressions of LacZ mRNA and f3 - gal activity in these two groups were significantly lower than those in the femoral vein and ICA groups （ P 〈 0.05 ）. Conclusion The effect of transferring exogenous gene into the rat brain was the best in the arterial injection group, and the effect in the venous injection group was better than that in the LV and intrathecal injection groups. The venous transfer route is easier, more convenient and more effective than the other routes in terms of practical feasibility.