重组腺相关病毒介导的人血管内皮抑素基因治疗裸鼠子宫内膜异位症

Human endostatin antiangiogenic gene therapy mediated by recombinant adeno-associated virus vector in nude mouse with endometriosis

目的研究重组腺相关病毒介导的人血管内皮抑素（endostatin）基因对子宫内膜异位症（内异症）裸鼠模型的治疗作用。方法构建携带人血管内皮抑素基因和增强绿色荧光蛋白（EGFP）基因的重组腺相关病毒载体rAAV2-endostatin—EGFP。将2008年11—12月在天津医科大学第二医院妇科因子宫肌瘤行子宫全切除术的12例患者的子宫内膜种植于60只裸鼠的盆腹腔内，建立内异症裸鼠模型，1周后分为3组：治疗组20只，于异位病灶局部直接注射rAAV2-endostatin—EGFP；阴性对照组20只，于病灶局部直接注射空白载体rAAV2-EGFP；空白对照组20只，于病灶局部直接注射等体积的磷酸盐缓冲液。分别于给药后1、2、3周各开腹1次，观察裸鼠盆腹腔病灶，同时留取异位病灶组织，检测各组裸鼠异位病灶中人血管内皮抑素蛋白表达情况、腺体数、微血管密度（MVD）和血管内皮生长因子（VEGF）的表达情况；于给药后3周检测各组裸鼠血清中雌二醇、孕酮水平。结果（1）采用腹膜种植法建立内异症裸鼠模型，种植成功率达100％。给药1周后，治疗组裸鼠异位病灶平坦、中央下陷，光镜下可见腺体数减少，腺腔变窄，细胞稀疏、呈萎缩状态。（2）荧光显微镜下观察，给药后1、2、3周，治疗组裸鼠异位病灶组织腺体和间质内出现绿色荧光；而阴性对照组和空白对照组裸鼠异位病灶组织内未见绿色荧光。（3）各组裸鼠给药后1、2、3周的异位病灶内腺体数，治疗组分别为（7．8±1．9）、（7．0±1．5）和（5．54-1．7）个，均低于阴性对照组[分别为（10．1±1．7）、（10．2±2．0）和（9．8±2．4）个]和空白对照组[分别为（10．2±2．2）、（10．0±2．0）和（9．7±2．2）个]，差异均有统计学意义（P〈0．05）；治疗组裸鼠给药后1、2、3周的异位病灶内腺体数比较，差异也有统计学意义（P〈0．05）。（4）各组裸鼠给药后1、2、3周异位病灶内的MVD，治疗组分别为（12．2±1．5）、（11．4±2．1）、（9．0±1．4）条，均低于阴性对照组[分别为（16．5±1．7）、（16．5±1．9）、（16．9±1．9）条]和空白对照组[分别为（16．2±1．6）、（16．0±1.6）、（16．3±1．7）条]，差异均有统计学意义（P〈0．05）；治疗组裸鼠给药后1、2、3周异位病灶内MVD比较，差异也有统计学意义（P〈0．05）。（5）各组裸鼠给药后1、2、3周异位病灶中VEGF的阳性率及表达强度，治疗组分别为35％、30％、25％和1．60±0．43、1．33±0．30、1．03±0．36，均低于阴性对照组（分别为80％、75％、85％和2．43±0．53、2．43±0．29、2．66±0．45）和空白对照组（分别为85％、90％、90％和2．36±0．53、2．64±0．57、2．53±0．52），差异均有统计学意义（P〈0．05）；治疗组裸鼠给药后1、2、3周异位病灶中VEGF阳性率及表达强度比较，差异也有统计学意义（P〈0．05）。（6）给药后3周，治疗组裸鼠血清中的雌二醇、孕酮水平分别为（48±7）pmol/L、（61±8）nmol/L，分别与阴性对照组[分别为（50±9）pmol/L、（60±10）nmol／L]和空白对照组[分别为（48±7）pmol/L、（58±10）nmol/L]比较，差异均无统计学意义（P〉0．05）。结论携带人血管内皮抑素基因的重组腺相关病毒可抑制裸鼠内异症病灶的血管生成，从而抑制异位病灶的生长，而不影响体内生殖激素水平，抗血管生成基因治疗可能成为内异症治疗的新选择。

Objective To study the therapeutic effect of recombinant adeno-associated virus carrying human endostatin gene therapy on endometriosis in mice model. Methods Recombinant adenoassociated virus vector carrying human endostatin gene and enhanced green fluorescent proteins gene （rAAV2-endostatin-EGFP） was constructed. Endometrium was from 12 patients with leiomyoma undergoing hysterectomy in Second Hospital, Tianjin Medical University between November and December 2008. Endometriosis models of nude mice were established by transplanting human endometrial fragments intooperitoneal surface. After 1 week, those 60 mice were divided into 3 groups： treatment group including 20 mice injected with rAAV2-endostatin-EGFP to ectopic lesion, control group including 20 mice injected with rAAV2-EGFP to ectopic lesion and blank control group including 20 mice injected with phosphate buffered saline （PBS） to the ectopic lesion. At 1, 2 and 3 weeks after treatment, those mice underwent laparotomy to observe the location and size of ectopic lesion in abdominal cavity. The expression of endostain protein, number of gland, microvessel density （MVD） and vascular endothelial growth factor （VEGF） were measured in ectopic lesions. The serum level of estradiol and progesterone were detected in nude mice among every groups. Results （ 1 ） All endometriosis of nude mice models were established successfully through peritoneum transplanting. After 1 week＇s treatment, flat lesion nodes, decreased gland number and narrow and atrophy glandular cavity were observed by light microscope. （2） The endostatin gene was transferred into nude mice successfully and expressed effectively. It was observed that endostatin protein expression was shown with enhanced green fluorescent proteins in ectopic lesion. （3） Glands number of ectopic lesion in rAAV2-endostatin-EGFP group（7. 8 ± 1.9,7. 0 ± 1.5 and 5.5 ± 1.7） were significantly less than 10. 1 ±1.7, 10. 2 ±2. 0 and 9.8 ±2.4 in rAAV2-EGFP control group and 10. 2± 2. 2, 10. 0 ± 2. 0 and 9.7 ± 2.2 in PBS control group at 1,2 and 3 weeks after treatment（ all P 〈 0. 05 ）. Glands number of ectopie lesion in rAAV2-endostatin-EGFP group at 3 weeks was significantly less than those at 1 and 2 weeks after treatment （P 〈0. 05）. （4） MVD of ectopic lesion in rAAV2-endostatin-EGFP group （ 12. 2 ± 1.5,11.4 ± 2. 1 and 9. 0 ±1.4） was significantly less than those at rAAV2-EGFP control group （ 16. 5± 1.7,16. 5 ± 1.9 and 16.9±1.9） and PBS control group （16.2 ±1.6,16.0 ±1.6 and 16.3 ±1.7） at 1,2 and 3 weeks after treatment （all P 〈 0. 05 ）. MVD of ectopie lesion in rAAV2-endostatin-EGFP group at 3 weeks was significantly less than those at 1 and 2 weeks after treatment （ P 〈 0. 05 ）. （5） The rate and density of VEGF expression at ectopic lesion in rAAV2-endostatin-EGFP group （35 %, 30%, 25 % and 1.60 ± 0.43,1.33± 0. 30,1.03 ±0. 36） were significantly less than those at rAAV2-EGFP control group （ 80% ,75 % ,85 % and 2.43 20.53,2.43 20.29,2.66 20.45） and PBS control group （85% ,90% ,90% and 2. 36 20.53,2. 64 ±0.57,2.53 ＋0.52） at one 1,2 and 3 weeks after treatment （all P 〈0.05）. The expression of VEGF at eetopic lesion in rAAV2-endostatin-EGFP group at 3 weeks was significantly less than those at 1 and 2 weeks after treatment （ P 〈 0. 05 ）. （6） The level of estradial and progesterone in serum of nude mice of rAAV2- endostatin-EGFP group [ E2 ： （48 ± 7 ） pmol/L, P ： （ 61± 8 ） nmol/L ] did not reach statistical difference when compared with those at rAAV2-EGFP control group [ E2 ： （50 ±9 ）pmol/L, P：（60± 10）nmol/L] and PBS control group [E2：（48 ±7）pmol/L,P：（58±10）nmol/L,P〉0.05]. Conclusions The recombinant adeno-associated virus carrying human endostatin gene therapy could inhibit angiogenesis at endometriotic lesions and not influence steroid level. The antiangiogenic gene therapy might become a novel option for endometriosis.