PAMAM介导内皮抑素基因治疗对裸鼠人子宫内膜异位症的作用

Effect of PAMAM-mediated endostatin gene therapy on human endometriosis in nude mice

目的探讨聚酰胺-胺型树枝状高聚合物(PAMAM)介导内皮抑素(ES)基因抗血管生成对裸鼠人子宫内膜异位症(EMs)的治疗作用及时机。方法120只BALB/c雌性裸鼠经皮下移植法建立人子宫内膜异位症模型后,分为以下4组:ES转染组(n=33),病灶局部注射20μg pcDNA3.1(+)-hES+65μg PAMAM混合液85μl;PAMAM组(n=30),病灶局部注射65μg PAMAM+无菌蒸馏水混合液85μl;生理盐水组(n=27),病灶局部注射生理盐水85μl;空白组(n=30),病灶局部未做任何处理。每组又分为建模后第1天和第14天给药两个亚组。给药1周后处死动物,对裸鼠卵巢、子宫及EMs病灶进行称重,并测量病灶大小,计数卵泡数目、微血管密度(MVD),免疫组化法检测各组病灶中ES、血管内皮细胞生长因子(VEGF)的表达水平。结果在建模后第1天给药裸鼠中,ES转染组腺体中ES表达明显高于其余3组(P<0.05),但异位病灶重量和体积、VEGF表达水平及MVD与其余3组比较差异无统计学意义(P>0.05)。在建模后第14天给药裸鼠中,与其余3组比较,ES转染组腺体中ES表达增高、VEGF表达降低,异位病灶体积缩小、重量减轻,MVD降低(P<0.05)。在ES转染组中,与建模后第1天给药亚组比较,建模后第14天给药裸鼠VEGF表达水平及MVD明显降低(P<0.05)。在建模后第1天和第14天给药裸鼠中,与其余3组比较,ES转染组心、肝、肾、卵巢及子宫未见明显组织形态学改变,且卵巢、子宫重量及卵巢卵泡计数差别无统计学意义(P>0.05)。结论建模后14d采用PAMAM介导20μg ES基因病灶局部注射对裸鼠人EMs模型具有治疗作用,且对心、肝、肾及内生殖器无明显不良反应。

Objective To explore the effect of endostatin （ES） gene therapy mediated by polyamidoamine （PAMAM） on human endometriosis in nude mice. Methods One hundred and twenty nude female BALB/c mice were subcutaneously transplanted with human endometrium fragments for the establishment of endometriosis model. They were divided into four treatment groups： ES transfection group （n=33, a mixture of 65μg PAMAM and 20μg ES gene was injected into the lesion of nude mice）; PAMAM group （n=30, a mixture of 65μg PAMAM and 20μg sterile saline was injected into the lesion of nude mice）; normal saline group （n = 27, 85μl sterile saline was injected into the lesion of nude mice） ; blank group （n=30, nothing was injected）. Each group was then divided into 2 subgroups, i.e. day 1 treatment and day 14 treatment subgroups. All nude mice were sacrificed 1 week after treatment. Ovaries and uterus were weighed, number of ovarian follicles was obtained, and ES, VEGF protein and MVD in nude mice were analyzed by immunohistochemistry. Results The ES expression was significantly higher in day 1 subgroup of ES transfection group than in the day I subgroups of other three groups （P〈0. 05）, while no significant differences were found in lesion weight, VEGF expression and MVD when comparing the day I subgroups of the 4 groups （P〉0. 05）. The ES expression was higher, VEGF expression was lower, MVD was less, and the volume of lesions was smaller and weight was lighter in the day 14 subgroup of ES transfection group than those in the day 14 subgroups of other three groups （P〈0. 05）. The VEGF expression was lower and MVD was less in day 1 subgroups than in day 14 subgroups （P〈0. 05）. No pathological change was observed in heart, liver, kidney, ovaries and uterus in nude mice of all the 4 groups; and also, no significant difference existed in reproductive organ weight and ovarian follicle number among the 4 groups （P〉0. 05）. Conclusion PAMAM-mediated endostatin gene therapy is effective for endometriosis given 14 days after model establishment, without producing side effect on heart, liver, kidney and reproductive organ in nude mice.