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(5.5±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.
目的 研究重組腺相關病毒介導的人血管內皮抑素(endostatin)基因對子宮內膜異位癥(內異癥)裸鼠模型的治療作用.方法 構建攜帶人血管內皮抑素基因和增彊綠色熒光蛋白(EGFP)基因的重組腺相關病毒載體rAAV2-endostatin-EGFP,,將2008年11-12月在天津醫科大學第二醫院婦科因子宮肌瘤行子宮全切除術的12例患者的子宮內膜種植于60隻裸鼠的盆腹腔內,建立內異癥裸鼠模型,1週後分為3組:治療組20隻,于異位病竈跼部直接註射rAAV2-endostatin-EGFP;陰性對照組20隻,于病竈跼部直接註射空白載體rAAV2-EGFP;空白對照組20隻,于病竈跼部直接註射等體積的燐痠鹽緩遲液.分彆于給藥後1、2、3週各開腹1次,觀察裸鼠盆腹腔病竈,同時留取異位病竈組織,檢測各組裸鼠異位病竈中人血管內皮抑素蛋白錶達情況、腺體數、微血管密度(MVD)和血管內皮生長因子(VEGF)的錶達情況;于給藥後3週檢測各組裸鼠血清中雌二醇、孕酮水平.結果(1)採用腹膜種植法建立內異癥裸鼠模型,種植成功率達100%.給藥1週後,治療組裸鼠異位病竈平坦、中央下陷,光鏡下可見腺體數減少,腺腔變窄,細胞稀疏、呈萎縮狀態.(2)熒光顯微鏡下觀察,給藥後1、2、3週,治療組裸鼠異位病竈組織腺體和間質內齣現綠色熒光;而陰性對照組和空白對照組裸鼠異位病竈組織內未見綠色熒光.(3)各組裸鼠給藥後1、2、3週的異位病竈內腺體數,治療組分彆為(7.8±1.9)、(7.0±1.5)和(5.5±1.7)箇,均低于陰性對照組[分彆為(10.1±1.7)、(10.2±2.0)和(9.8±2.4)箇]和空白對照組[分彆為(10.2±2.2)、(10.0±2.0)和(9.7±2.2)箇],差異均有統計學意義(P<0.05);治療組裸鼠給藥後1、2、3週的異位病竈內腺體數比較,差異也有統計學意義(P<0.05).(4)各組裸鼠給藥後1、2、3週異位病竈內的MVD,治療組分彆為(12.2±1.5)、(11.4±2.1)、(9.0±1.4)條,均低于陰性對照組[分彆為(16.5±1.7)、(16.5±1.9)、(16.9±1.9)條]和空白對照組[分彆為(16.2±1.6)、(16.0±1.6)、(16.3±1.7)條],差異均有統計學意義(P<0.05);治療組裸鼠給藥後1、2、3週異位病竈內MVD比較,差異也有統計學意義(P<0.05).(5)各組裸鼠給藥後1、2、3週異位病竈中VEGF的暘性率及錶達彊度,治療組分彆為35%、30%、25%和1.60±0.43、1.33±0.30、1.03±0.36,均低于陰性對照組(分彆為80%、75%、85%和2.43±0.53、2.43±0.29、2.66±0.45)和空白對照組(分彆為85%、90%、90%和2.36±0.53、2.64±0.57、2.53±0.52),差異均有統計學意義(P<0.05);治療組裸鼠給藥後1、2、3週異位病竈中VEGF暘性率及錶達彊度比較,差異也有統計學意義(P<0.05).(6)給藥後3週,治療組裸鼠血清中的雌二醇、孕酮水平分彆為(48±7)pmol/L、(61±8)nmol/L,分彆與陰性對照組[分彆為(50±9)pmol/L、(60±10)nmol/L]和空白對照組[分彆為(48±7)pmol/L、(58±10)nmol/L]比較,差異均無統計學意義(P>0.05).結論 攜帶人血管內皮抑素基因的重組腺相關病毒可抑製裸鼠內異癥病竈的血管生成,從而抑製異位病竈的生長,而不影響體內生殖激素水平,抗血管生成基因治療可能成為內異癥治療的新選擇.
목적 연구중조선상관병독개도적인혈관내피억소(endostatin)기인대자궁내막이위증(내이증)라서모형적치료작용.방법 구건휴대인혈관내피억소기인화증강록색형광단백(EGFP)기인적중조선상관병독재체rAAV2-endostatin-EGFP,,장2008년11-12월재천진의과대학제이의원부과인자궁기류행자궁전절제술적12례환자적자궁내막충식우60지라서적분복강내,건립내이증라서모형,1주후분위3조:치료조20지,우이위병조국부직접주사rAAV2-endostatin-EGFP;음성대조조20지,우병조국부직접주사공백재체rAAV2-EGFP;공백대조조20지,우병조국부직접주사등체적적린산염완충액.분별우급약후1、2、3주각개복1차,관찰라서분복강병조,동시류취이위병조조직,검측각조라서이위병조중인혈관내피억소단백표체정황、선체수、미혈관밀도(MVD)화혈관내피생장인자(VEGF)적표체정황;우급약후3주검측각조라서혈청중자이순、잉동수평.결과(1)채용복막충식법건립내이증라서모형,충식성공솔체100%.급약1주후,치료조라서이위병조평탄、중앙하함,광경하가견선체수감소,선강변착,세포희소、정위축상태.(2)형광현미경하관찰,급약후1、2、3주,치료조라서이위병조조직선체화간질내출현록색형광;이음성대조조화공백대조조라서이위병조조직내미견록색형광.(3)각조라서급약후1、2、3주적이위병조내선체수,치료조분별위(7.8±1.9)、(7.0±1.5)화(5.5±1.7)개,균저우음성대조조[분별위(10.1±1.7)、(10.2±2.0)화(9.8±2.4)개]화공백대조조[분별위(10.2±2.2)、(10.0±2.0)화(9.7±2.2)개],차이균유통계학의의(P<0.05);치료조라서급약후1、2、3주적이위병조내선체수비교,차이야유통계학의의(P<0.05).(4)각조라서급약후1、2、3주이위병조내적MVD,치료조분별위(12.2±1.5)、(11.4±2.1)、(9.0±1.4)조,균저우음성대조조[분별위(16.5±1.7)、(16.5±1.9)、(16.9±1.9)조]화공백대조조[분별위(16.2±1.6)、(16.0±1.6)、(16.3±1.7)조],차이균유통계학의의(P<0.05);치료조라서급약후1、2、3주이위병조내MVD비교,차이야유통계학의의(P<0.05).(5)각조라서급약후1、2、3주이위병조중VEGF적양성솔급표체강도,치료조분별위35%、30%、25%화1.60±0.43、1.33±0.30、1.03±0.36,균저우음성대조조(분별위80%、75%、85%화2.43±0.53、2.43±0.29、2.66±0.45)화공백대조조(분별위85%、90%、90%화2.36±0.53、2.64±0.57、2.53±0.52),차이균유통계학의의(P<0.05);치료조라서급약후1、2、3주이위병조중VEGF양성솔급표체강도비교,차이야유통계학의의(P<0.05).(6)급약후3주,치료조라서혈청중적자이순、잉동수평분별위(48±7)pmol/L、(61±8)nmol/L,분별여음성대조조[분별위(50±9)pmol/L、(60±10)nmol/L]화공백대조조[분별위(48±7)pmol/L、(58±10)nmol/L]비교,차이균무통계학의의(P>0.05).결론 휴대인혈관내피억소기인적중조선상관병독가억제라서내이증병조적혈관생성,종이억제이위병조적생장,이불영향체내생식격소수평,항혈관생성기인치료가능성위내이증치료적신선택.
Objective To study the therapeutic effect of recombinant adeno-associated virus carrying human endostatin gene therapy on endometriosis in mice model. Methods Recombinant adeno-associated 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 laparotemy 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 ectopic 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 ectopic 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±0.53,2.43±0.29,2.66±0.45) and PBS control group (85% ,90% ,90% and 2.36±0.53,2.64± 0.57,2.53±0.52) at one 1, 2 and 3 ,weeks after treatment (all P<0.05). The expression of VEGF at ectopic 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 [ E_(2)> : (48±7 ) pmol/L, P: (61±8 ) nmol/L ] did not reach statistical difference when compared with those at rAAV2-EGFP control group [ E_(2): (50±9) pmol/L, P: (60±10) nmol/L] and PBS control group [E_(2):(48±7)pmol/L,P: (58±10)nmol/L,P>0.05]. Conclusions The recombinant adeno-asseciated 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.
