基因枪注射BALB/c小鼠制备Graves病模型

Establishment of a mouse model for Graves disease by injection with gene gun

目的：探讨基因枪注射和普通质粒肌肉注射两种不同方法对甲亢动物模型的差异。方法：将45只小鼠随机分成4组，分别用基因枪金颗粒包裹质粒免疫、普通质粒肌肉注射免疫。加强免疫后第4周后处死小鼠，RIA法测定血清FL、FT4、TSH水平。RIA法测定血清与hTSHR-CHO孵育后上清cAMP浓度以计算TRAb活性，RIA法测定脾脏培养后IL-4的浓度，ELISA法测定血清TSAb浓度，取甲状腺做石蜡HE切片观察组织学变化。结果：基因枪免疫组（GGI）动物4周后血清FT4（0．34±0．11）pg／ml高于质粒肌肉注射组（PLS）（0．21±0．06）pg／ml和基因枪免疫空质粒组（GG0）（0．17±0．08）pg/ml（F=14．34，P=0．002）。GGI组TRAb活性（167．27±117．37）％高于PLS组（113．81±65．50）％和GG0组（98．27±14．80）％（Hc=35．198，P=0．007）。GGI组脾脏培养上清液IL-4产量（0．244±0．092）pmol／L明显高于PLS组（0．173±0．039）pmol／L和GG0组（0．151±0．041）pmol／L（F=7．0403，P=0．005）。GGI组TRAb浓度（29．3±1．2）U／L明显高于GG0组（13．3±0．2）U／L（F=9．02，P〈0．001），但和PLS组（24．3±0．9）U／L比无显著性差异（q=-1．32，P=0．423）。HE切片观察造模成功小鼠甲状腺滤泡细胞高柱状排列，未出现甲状腺细胞破坏甲状腺炎的嗜酸性变。结论：基因枪免疫雌性BALB／c鼠是一种可行的复制Gmves动物模型，在免疫第2～4周可以观察到FT4升高和自身抗体改变等理想的免疫效果。

Objective：To explore the difference between injection with gene gun and normal muscle injection of the plasmid in establishing an animal model for Graves disease.Methods：45 mice were randomized into three groups and then immunized with either gene gun gold particles（GG1 group） or the plasmid muscularly （PLS group）. An empty plasmid was injected by gone gun into mice. which was designed as another group for the control（GG0 group）. After 4 weeks the mice were killed and blood samples were taken to determine. FT3, FF4 and TSH with RIA, TSAb concentrations with ELISA. Splenecytes were cultured to determine IL-4 concentration with RIA. By RIA, TPAb activity was determined through the production of cAMP in hTSHR-CHO cells cultttred with the mouse sara. Thyroid HE sections were applied to observe the thyroid gland histology. Results： It was shown that the mice immunized with gene gun gold particles resulted in significant increase of FT4 （0.34 ±0.11） pg/ml compared with the other two groups（0.21 ± 0.06） pg/ml in PLS and （0.17 ± 0.08） pg/ml in GG0, F = 14.34, P = 0. 002）. TRAb activity （ 167.27 ±117.37） % was obviously increased in the mice of gone gun injection than in PLS（ 113.81± 65.50） % and GG0（98. 27 ± 14.80）%, with statistically significant difference（ Hc = 35. 198, P = 0.007）. IL-4 production （0. 244± 0. 092） pmol/L in spleen cells was higher than in GG0 group （0. 173 ±0.039） pmol/L and in GG0（0. 151± 0.041） pmol/L（ F = 7. 040 3, P = 0.005） .TRAb concentration was higher in gene gun group（29.3 ± 1.2） U/L than in GG0（ 13.3 ±0.2） U/L（ F = 9.02, P 〈 0.001 ） but no difference between GG1 group and PLS group（24.3± 0.9） U/L, （ q = - 1.32, P = 0.423）. HE sections showed that mice immunized successfully with gone gun had thyroid gland follicular cells hyperplasic. However, there was not any sign which indicated for cellular destruction or lymphocyte infihratin, a phenomenon of inflammation. Conclusion： Female BALB/c mice immunized with gene gun gold particles are an feasible way to establish the animal model of Graves disease. Disease eccurrs in the second to fourth week featured by increase of FT4 autoimmune antibody and pathology.