不同载体介导的绿色荧光蛋白基因在大鼠颌下淋巴结的表达

Expression of green fluorescent protein gene in rats submandibular lymph nodes mediated by vary vectors

目的探讨阳离子聚合物Sofast基因转染试剂(Sofast)和阳离子脂质体Lipofectamine2000(Lipo)经不同途径向大鼠转入绿色荧光蛋白基因(PEGFP-N2),观察其在颌下淋巴结(SMLN)的表达。方法45只Wistar大鼠随机分成9组:Lipo/DNA前房注射组(A)、结膜下注射组(B)、颞下方穹隆部注射组(C)和下睑皮下注射组(D);Sofast/DNA前房注射组(E)、结膜下注射组(F)、颞下方穹隆部注射组(G)、下睑皮下注射组(H)和阴性对照组(I)。分别于注射后48小时取同侧SMLN,通过荧光显微镜观查和流式细胞仪(FCM)检测各组的SMLN中PEG-FP-N2的表达。结果A-D、E-H各实验组的SMLN中均可观察到绿色荧光。相同载体不同部位注射DNA后颞下方穹隆部注射组和下睑皮下注射组可以获得相对较高的转染效率。相同部位不同载体注射DNA后Sofast/DNA注射组的转染效率高于Lipo/DNA注射组。结论Sofast在SMLN的基因转染中优于Lipo。DNA/Sofast可以通过颞下方穹隆部注射和下睑皮下注射在SMLN获得相对较高的表达。

Objective To explore the expression of Green Fluorescent Protein C, ene transduced into the rats via Sofast reagent （Sofast） and Lipofectamine2000 （Lipo） so as to select the better vector and feasible gene transfer route to submandibular lymph nodes （SMLN）. Methods Fortyfive Wistar rats were divided into 9 grous randomly： Lipo/DNA complex intra-anterior chamber injection group（A） ,intra-conjunctival sac injection group（B） ,intra-inferior temporal fornical injection group（ C）, intra-subcutaneous lower-lid injection group（D） and Sofast/DNA complex intra-anterior chamber injection group（E） ,intra-conjunctival sac injection group（F） ,intra-inferior temporal fornical injection group（ G）, intrasubcutaneous lower-lid injection group （ H）, negative contrast group （I）. The rats were sacrificed 48 hours after injection and their ipslateral SMLN were removed. Flow cytometer（FCM） and fluorescence micrcecopy were used to observe the expression of PEGFP-N2 in the SMLN. Results The expression of PEGFP-N2 in SMLN occurred in all the groups at 48 hours after injection. The rates of the GFP ＋ ceils in FCM were 3.30 ± 1.51,3.14 ± 0.71, 9.71 ± 1.02, 11.23 ± 2.04 （A-D）; 6.53 ±1.56, 5.74 ±1.59, 12.86 ±1.59, 13.83 ±2.61 （E-H）;0.84±0.26（I）. The expression levelofthe gene in the inferior temporal fornical injection groups and subcutaneous lower-lid groups were higher than the other groups after injection mediated by the sama vector （ P 〈 0. 001 ） ; The expression level of the SOfast/DNA injection groups were higher than the Lipo/DNA groups after injection through the same routes （ P 〈 0.05 ）. Conclusion As gene transducing vector in SMLN Sofast is superior to Lipo. High expression of foreign gene can be achieved by intra-inferior temporal fornical and intra-subcutaneous lower-lid injection with Sofast vectors. It may provide a potential strategy in treatment of corneal diseases associated with immunity.