【作者】 孙俊杰；

【导师】 糜若然； 尹利荣；

【作者基本信息】 天津医科大学 ， 妇产科学， 2010， 博士

【摘要】 第一部分携带人血管内皮抑素基因的重组腺相关病毒的制备和鉴定目的构建携带人血管内皮抑素(Endostatin)基因的腺相关病毒(Adeno-associated virus, AAV)载体包装质粒pSNAV-Endostatin-CMV-EGFP,进而包装携带人血管内皮抑素基因的2型重组腺相关病毒(rAAV2-Endostatin-EGFP),为下一步探讨其在抗血管生成基因治疗动物模型实验中的作用奠定基础。方法采用分子克隆技术,从pCD-sEndostatin质粒获得Endostatin cDNA,并将PCR扩增产物插入至AAV包装质粒pSNAV-CMV-EGFP上,构建pSNAV-Endostatin-CMV-EGFP的AAV重组包装质粒,经PCR、酶切及测序鉴定。用Lipofectamine 2000将此包装质粒转染BHK-21细胞,建立载体细胞株。在转瓶中培养,用HSV1-rc/ΔUL2感染后收集培养物,进行纯化并测定其滴度。用rAAV2-Endostatin-EGFP感染BHK-21细胞24、48小时后(MOI=1×105v.g./cell)在荧光显微镜观察目的基因Endostatin在细胞表面的表达,即转染效率；48小时后采用BCA法检测目的基因Endostatin在蛋白水平的表达。结果经PCR、酶切鉴定及基因测序证实AAV包装质粒pSNAV-Endostatin-CMV-EGFP构建成功,进而rAAV2-Endostatin-EGFP包装成功。所包装纯化的重组病毒滴度达5×1011v.g./ml。利用PCR反应进行鉴定,扩增得到Endostatin。rAAV2-Endostatin-EGFP能有效感染BHK-21细胞,24小时后,转染效率为50%；48小时后为70%,并检测培养液上清液中血管内皮抑素蛋白浓度达194μg/ml。结论构建的AAV包装质粒pSNAV-Endostatin-CMV-EGFP可作为rAAV2-Endostatin-EGFP表达载体的包装质粒。成功包装的rAAV2-Endostatin-EGFP病毒,可直接用于抗血管生成基因治疗的实验研究。第二部分子宫内膜异位症裸鼠模型的建立目的建立人子宫内膜异位症裸鼠模型,观察异位种植病灶新生血管生成并探讨周细胞在血管生成过程中作用。方法通过开腹手术方法将人子宫内膜组织块种植于裸鼠盆腹腔,1周后观察其生长情况,并进行病理检查,同时采用免疫组化S-P法检测异位病灶内微血管密度(MVD, CD34标记)和周细胞(平滑肌肌动蛋白,a-SMA标记)的情况。结果成功建立人子宫内膜异位症裸鼠模型,异位种植病灶能保持原有内膜组织的形态结构,并可见血管生成明显。内皮细胞CD34表达阳性,周细胞a-SMA表达阳性。高血管密度区的MVD为(16.20±1.64)条,阳性周细胞数为(22.40±7.23)个,低血管密度区MVD为(3.62±1.50)条,阳性周细胞数为(15.56±5.34)个,高血管密度区MVD及周细胞数均高于低血管密度区,差异具有统计学意义(P<0.05)。高血管密度区周细胞与MVD的比值为(1.38±0.13),低血管密度区则高达(4.30±0.92),两者差异具有统计学意义(P<0.05)。结论子宫内膜异位症裸鼠模型的建立是子宫内膜异位症早期临床研究的理想模型,而且异位种植病灶组织内血管新生的早期即有周细胞存在。周细胞可能参与异位种植病灶组织血管生成过程,周细胞在血管生成中可能起负向调节作用。第三部分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目的研究重组腺相关病毒介导的人血管内皮抑素基因对裸鼠子宫内膜异位症的治疗作用。方法建立子宫内膜异位症裸鼠模型1周后,取建模成功的60只裸鼠作为实验研究对象,分为三组：治疗组20只,于异位病灶局部直接注射rAAV2-Endostatin-EGFP;空载体对照组20只,于异位病灶局部直接注射rAAV2-EGFP;空白对照组20只,于异位病灶局部直接注射等体积的磷酸盐缓冲液(PBS)。分别于给药后1、2、3周各开腹一次,观察裸鼠盆腹腔病灶,同时留取异位病灶组织,观察各组裸鼠异位病灶中人血管内皮抑素蛋白表达情况和检测腺体数目；采用免疫组化S-P法检测病灶中微血管密度(MVD)和血管内皮生长因子(VEGF)的表达情况；同时称量体重观察脂肪沉积情况。于给药后3周猝死,采用ELISA法检测各组裸鼠血清中雌二醇(E2)和孕酮(P)的水平；并对各组裸鼠卵巢、子宫、心脏、肝脏、脾脏、肺脏、肾脏进行病理学检查。结果(1)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；而空载体对照组和空白对照组裸鼠异位病灶组织内未见绿色荧光。rAAV2-Endostatin-EGFP局部注射异位病灶后在异位病灶腺体和间质可见人血管内皮抑素蛋白表达。(2)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。(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±1.73)个,均低于空载体对照组[分别为(10.1±1.7)、(10.2±2.0)和(9.84±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)、(11.44±2.1)、(9.0±1.4)条,均低于空载体对照组[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,均低于空载体对照组[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)治疗组裸鼠体重[17.79±1.28、17.63±1.21和17.64±1.26 g]在给药后1、2、3周分别与空载体对照组[17.87±1.43、17.66±1.63和17.74±1.53 g]和空白对照组[17.62±1.57、17.54±1.54和17.55±1.59 g]比较,差异无统计学意义(P>0.05)。而且各组治疗前后比较差异均无统计学意义(P>0.05)。(7)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,与空载体对照组[分别为(50±9) pmol/L、(60±10) nmol/L]、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)。(8)给药后3周,对治疗组裸鼠的卵巢、子宫、心脏、肝脏、脾脏、肺脏、肾脏组织进行显微镜下观察,未发现缺血、坏死和炎症改变。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子宫内膜异位症病灶的血管生成,从而抑制异位病灶的生长,而不影响体内性激素水平和脂肪沉积,对卵巢、子宫和其他重要脏器也无影响,抗血管生成基因治疗可能成为子宫内膜异位症治疗的新选择。更多还原

【Abstract】 Part 1 The packaging of recombinant adeno-associated virus type-2 vector encoding human endostatin geneObjective To construct the packaging plasmid pSNAV-Endostatin-CMV-EGFP of adeno-associated virus vector encoding human Endostatin cDNA. Then to construct the recombinant adeno-associated virus type-2(rAAV-2) vector encoding human Endostatin. Methods The human Endostatin cDNA obtained from plasmid pCD-sEndostatin was subcloned into the packaging plasmid pSNAV-CMV-EGFP of AAV by molecular clone ways. The recombinant plasmid pSNAV-Endostatin-CMV-EGFP was identified by PCR analysis,restriction enzymes analysis and sequencing analysis.The pSNAV-Endostatin-CMV-EGFP was transfected into BHK-21 cells with Lipofectamine 2000,then we obtained the BHK/Endostatin-EGFP cells.Infecting BHK/Endostatin-EGFP cells with HSV1-rc/ΔUL2 at an MOI of 0.1 resulted in the optimal yields of recombinant adeno-associated virus type-2 vector,then was purified and the titer of the virus was determined.Human Endostatin gene expression be detected through fluorescent microscopy in BHK-21 cells after 24 and 48 hours of infection by recombinant adeno-associated virus type-2 vector at MOI=1×105 v.g./cell.Human Endostatin gene expression in protein level could be detected by BCA method in BHK-21 cells after 48 hours of infection at MOI=1×105 v.g./cell. Results The recombinant pSNAV-Endostatin-CMV-EGFP was correctly constructed and confirmed by PCR analysis,restriction enzymes analysis and sequencing analysis.The recombinant adeno-associated virus type-2 vector encoding human Endostatin(rAAV2-Endostatin-EGFP)was successfully constructed.The resulted virus titer reached 5×1011v.g./ml and the amplified human Endostatin cDNA was confirmed by PCR analysis.Endostatin proteins can be expressed at 24 hours (infection ratio 50% cells) and 48 hours (infection ratio 70% cells) after rAAV2-Endostatin-EGFP infected BHK-21 cells in vitro.The level of human endostatin protein in supernate was 194μg/ml at 48 hours after rAAV2-Endostatin-EGFP infected BHK-21 cells.Conclusions The constructed AAV packaging plasmid pSNAV-Endostatin-CMV-EGFP could be the packaging plasmid of rAAV2-Endostatin-EGFP. The successfully constructed rAAV2-Endostatin-EGFP can be used in antiangiogenesis gene therapy research.Part 2 Establishment of human endometriosis model in nude mouseObjective To establish the human endometriosis model in nude mouse and to investigate angiogenesis in endometriotic lesions and pericyte cells of vessels during the transplants development.Methods Endometriosis models of nude mice were established by transplanting human endometrial fragments into peritoneal surface.We observed the growth of the transplants in nude mice and removed them to be examined by light microscope at 1 week after transplantation. The endothelial cells or microvessel density(MVD) was tracted by CD34 and the pericytes were tracted by a-SMA of endometriotic lesions through immunohistochemical staining(IHC).Results The human endometriosis model in nude mouse was successfully established.We can see the glands of endometrium under the microscope and the angiogenesis around and in the transplants.The positive cells for CD34 were endothelial cells situated inside the vascular wall.The positive cells for a-SMA were pericytes.Quantitative morphological analysis showed that the MVD and pericytes in the high-microvascular density areas (16.20±1.64,22.40±7.23) are much higher than that in the low-microvascular density areas (3.62±1.50,15.56±5.34) (P<0.05).The ratio of pericytes and MVD in the low-microvascular density areas (4.30±0.92) are much higher than that in the high-microvascular density areas (1.38±0.13) (P<0. 05).Conclusions Endometriosis model in nude mouse is an appropriate model for the study of the early phase of endometriosis.Vessels wih pericytes were shown to supply endometriotic lesions in nude mice at the early phase. Pericytes involve in angiogenesis of endometriotic lesions. The results of this study imply that pericytes may possibly inhibit angiogenesis.Part 3 Experimental study on endostatin gene therapy mediated by recombinant adeno-associated virus vector on endometriosis of animal modelObjective To study the therapeutic effect of recombinant adeno-associated virus carrying human endostatin gene therapy on endometriosis in nude mice model.Methods 60 endometriosis models of nude 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 when ectopic lesion formated after transplanting one week. At 1,2 and 3 weeks after treatment, those mice underwent laparotomy to observe the ectopic lesions in abdominal cavity.The expression of human endostatin in ectopic lesions was detected by fluorescent label by fluorescent microscope; glands number of ectopic lesions by light microscope; the microvessel density(MVD) was tracted by CD34 and vascular endothelial growth factor(VEGF) in ectopic lesions through immunohistochemical staining(IHC) and the body weight was measured. All of these mice were killed at three weeks after the treatment,the serum level of estradiol and progesterone were detected in nude mice among every groups through Enzyme-Linked Immunosorbnent Assay (ELISA) and observe ovaries,uterus,heart,liver,lung,spleen and kidney of rAAV2-Endostatin-EGFP group by light microscope.Results (1)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 (EGFP) in ectopic lesion of treatment group.EGFP was not observed in ectopic lesion of rAAV2-EGFP control group and blank control group.(2)After 1 week’s treatment, flat lesion nodes, decreased gland number and narrow and atrophy glandular cavity were observed by light microscope. (3)Glands number of ectopic lesion in treatment group[7.8±1.9,7.0±1.5 and 5.5±1.73] was 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 blank control group at 1,2 and 3 weeks after treatment(all P<0.05).Glands number of ectopic lesion in treatment 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 treatment 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 blank 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 treatment 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 treatment 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 blank control group [85%,90%,90% and 2.36±0.53,2.64±0.57,2.53±0.52] at 1,2 and 3 weeks after treatment (all P<0.05).The expression of VEGF at ectopic lesion in treatment group at 3 weeks was significantly less than those at 1 and 2 weeks after treatment (P< 0.05). (6)Nude mice weight of treatment group [17.79±1.28,17.63±1.21 and 17.64±1.26 g] did not reach statistical difference when compared with those at rAAV2-EGFP control group[17.87±1.43,17.66±1.63 and 17.74±1.53 g] and blank control group[17.62±1.57,17.54±1.54 and 17.55±1.59 g] at 1,2 and 3 weeks after treatment (all P>0.05).There was no difference of nude mice weight between before and after treatment among every groups (all P>0.05).(7)The level of estradial and progesterone in serum of nude mice of treatment 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 blank control group [E2:(48±7)pmol/L,P:(58±10)nmol/L,P>0.05].(8)There were no inflammatory,degeneration and necrosis in pathological slices of ovaries,uterus,heart,liver,lung,spleen and kidney of treatment group at 3 weeks after treatment.Conclusions The recombinant adeno-associated virus carrying human endostatin gene therapy could inhibit angiogenesis at endometriotic lesions and not influence steroid level,fatty deposition and ovaries,uterus,other important organs.The antiangiogenic gene therapy might become a novel option for endometriosis.更多还原