摘要
目的
子宫内膜异位症(Endometriosis,EM,内异症)是女性常见疾病,主要引起不孕、痛经、性交痛及慢性盆腔痛等症状。子宫内膜异位症虽是一种良性疾病,却具有类似肿瘤的恶性特征,例如局部浸润、远处器官受累和多发病灶等恶性行为。本病难以根治,复发率高,严重影响患者的身心健康。目前治疗应用的药物主要为激素类,然而这些药物容易出现肝功能损害、骨质疏松、潮热和心绪烦躁等严重副作用,连续使用不宜超过6个月。此类药物大都以雌激素作为治疗靶点,通过拮抗作用或抑制卵巢分泌等途径直接或间接降低体内雌激素水平,从而促使异位内膜病灶的萎缩来达到治疗目的,但这仅是针对病因的一方面进行治疗,而且一旦停药,随着雌激素水平的恢复,相当多患者的病情会复发。手术治疗子宫内膜异位症,对于病灶的祛除、盆腔解剖的恢复有一定作用,但在轻、中度内异症、不孕和复发患者,手术的价值仍存在一定争议。同时,手术后加用药物治疗虽在短期内可能提高治疗效果,但仍不能避免或减少术后复发。因此,疾病的复发仍然是目前子宫内膜异位症治疗的难点。其原因在于发病机制的不确定,缺乏以病因为靶点的治疗方法。因此,追踪内异症发病机制的最新研究进展,从中寻找一条新的治疗途径已经成为当前研究的迫切需要。
对于内异症的发生机理,目前大多数学者认可Sampson的经血逆流学说。经血逆流现象虽很普遍,发生率高达76%～90%,但与内异症的发生并无明显的因果关系,说明经血逆流可能只是诱因或基础。当脱落的子宫内膜组织附着在腹膜或其它部位时,异位内膜组织本身及其周围组织新的血供的建立和维持,才是异位子宫内膜组织种植、存活和内异症发生的基本条件。因此可以说,异位子宫内膜组织的转移、种植、生长等类似肿瘤转移的生物学行为,是内异症发病的关键。异位子宫内膜病灶的形成及生长必须依赖新生血管的形成,血管生成机制已成为内异症公认的重要发病机制。目前已有学者尝试用血管生成抑制因子针对内异症进行有关抗血管生成治疗的研究,初步显示了良好效果。因此,抗血管生成可能成为治疗内异症的有效途径。
人内皮抑素(Endostatin,Endo)是一种内源性血管生成抑制因子,具有强烈的抑制新生血管形成的能力,抑制效应和特异性都优于血管抑素。内皮抑素特异性地抑制血管内皮细胞增殖,而且只抑制新生血管内皮细胞,对平滑肌细胞和肿瘤细胞等非内皮起源的细胞无明显抑制作用,对正常的成熟血管内皮细胞的抑制作用也极弱。由于内皮细胞具有良好的遗传稳定性,不易发生突变,既大大提高了治疗的安全性,又减少了耐药性的发生。因此,内皮抑素具备了用于抑制肿瘤或其他疾病血管生成的诸多先天优势。目前动物实验显示体内注射内皮抑素蛋白或基因可抑制肿瘤的生长和转移,同时,治疗期间都没有发现任何毒副作用和耐药性,而且治疗停止后肿瘤仍一直处于休眠状态。内皮抑素抗血管形成治疗国外已进入到Ⅱ期临床阶段,国内也有用内皮抑素蛋白或基因抗血管生成治疗的动物实验研究。但内皮抑素应用研究局限于肿瘤治疗,尚未有用于子宫内膜异位症治疗方面的研究报道,因此,本课题就内皮抑素在内异症治疗中的效果及安全性进行了研究。
蛋白治疗和基因治疗是目前最常用的治疗方法。蛋白治疗具有一些难以克服的缺陷,如治疗剂量大、血药浓度维持时间短、需反复注射、所需疗程长以及可能传播毒性和感染颗粒等,造成蛋白治疗严重受限。而基因治疗则可克服这些缺点,与机体自然生理过程比较吻合,因此,基因治疗是未来治疗的重要趋势。选择理想的载体系统,将目的基因高效、安全地导入宿主细胞并使之高效表达,是基因治疗成功的关键。腺病毒载体是临床基因治疗研究中应用最为广泛的载体之一,与其他基因治疗载体相比,具有以下优点:①宿主范围广,对人致病性低,可广泛用于人类及非人类蛋白的表达;②能在增殖和非增殖细胞中感染和表达基因;③腺病毒载体能进行有效的增殖,产生较高的病毒滴度,非常适用于基因治疗;④其与人类基因同源,可使大多数人类蛋白都达到高水平表达并具有完全的功能;⑤不会像逆转录病毒那样随机整合到宿主染色体,进而导致基因失活或激活癌基因,它在染色体外表达,无插入致突变性。因此,腺病毒被极其广泛地应用于体外基因转导、体内接种疫苗和基因治疗等各个领域。
本课题拟通过分子生物学、实验动物学、病理学和免疫学的相关技术,利用改进的携带绿色荧光蛋白的AdEasy-1腺病毒载体系统,构建携带人内皮抑素基因的重组腺病毒,包装、扩增、纯化后,体外感染人脐静脉内皮细胞,诱导其发生凋亡;建立人内异症裸鼠皮下种植及腹腔注射模型,研究其血管生成特性,并用重组腺病毒局部注射裸鼠异位子宫内膜病灶,观察内皮抑素抑制异位内膜病灶生长情况以及可能出现的毒副反应,研究内皮抑素基因治疗内异症的可行性和安全性,为内异症的治疗寻找一种新方法,探索一条副作用小、便捷、经济的治疗新途径,解除患者痛苦,降低复发率,减少癌变,提高生育能力,改善临床治疗效果,提高患病妇女的生活质量。
方法:
1.利用改进的携带绿色荧光蛋白的AdEasy-1腺病毒载体系统,构建携带人内皮抑素基因的重组腺病毒质粒,并转染病毒的包装细胞以包装、大量扩增重组腺病毒,用上述相同的方法制备对照空载腺病毒。
2.重组腺病毒体外感染人脐静脉内皮细胞ECV 304,利用逆转录聚合酶链反应及免疫印迹等不同方法检测人内皮抑素基因的转录及其蛋白的表达情况。
3.利用多种方法检测重组腺病毒感染的不同时期,人脐静脉内皮细胞ECV304凋亡的情况。
4.应用子宫内膜异位症患者的在位内膜,建立人子宫内膜异位症裸鼠皮下种植及腹腔注射模型,建模后观察裸鼠皮色、食欲、活动情况及皮下种植结节的生长情况;2周后处死裸鼠,HE染色光镜观察及免疫组织化学法检测异位内膜病灶的形成情况及微血管生成情况;并测量分析裸鼠体重变化情况,评价造模的毒副作用。
5.采用人内异症裸鼠皮下种植模型,将重组腺病毒液、对照空载腺病毒液及生理盐水分别进行皮下异位内膜病灶局部注射,观察注射后异位内膜病灶的生长情况,并观察裸鼠活动、皮色、食欲等情况;注射后2周处死裸鼠,比较各组裸鼠异位病灶体积的变化,HE染色光镜观察异位病灶的形态,并应用免疫组织化学法进行微血管密度计数和血管内皮生长因子表达的检测;测量分析裸鼠体重变化情况,评价治疗的毒副作用。
结果:
1.以特异性真核表达质粒Pshuttle-Endostatin为模板,利用特异性上下游引物,应用聚合酶链反应(polymerase chain reaction,PCR)技术成功扩增得到编码人内皮抑素Endostatin蛋白的基因片段,大小约650pb,与预期的结果相符;将该基因片断大量扩增并纯化,经XbaI及KpnI两种特异性酶切后定向克隆到携带绿色荧光蛋白(green fluorecence protein,GFP)的AdEasy-1系统的穿梭质粒pAdTrack-CMV中,构建了重组穿梭质粒pAdTrack-Endo,经特异性酶切反应鉴定及DNA测定鉴定,证实重组穿梭质粒pAdTrack-Endo构建成功;将重组穿梭质粒pAdTrack-Endo大量扩增纯化后,经特异性核酸内切酶PmeI线形化,再与腺病毒基因组骨架质粒pAdEasy-1共转化大肠杆菌BJ5183,利用细菌内同源重组获得重组腺病毒质粒pAdEasy-Endo,经特异性酶切鉴定及DNA测序鉴定,证明携带人内皮抑素基因的重组腺病毒质粒pAdEasy-Endo构建正确;重组腺病毒质粒pAdEasy-Endo大量扩增纯化后,经特异性核酸内切酶PacI线形化,在体外利用脂质体2000成功转染病毒包装细胞AAV 293,最早在转染后16小时,在荧光显微镜下可见GFP的表达,后荧光逐渐增多增亮,约至转染后5～9天,细胞开始膨胀变圆,触角逐渐消失,荧光也不再增加,并有部分细胞开始悬浮,细胞呈串珠样改变,表明病毒的形成和扩增;约在转染后14天,大部分AAV 293细胞胞体固缩、悬浮,收集细胞裂解液进行PCR鉴定,得到预期650bp大小的Endostatin基因条带,证实重组腺病毒pAd-Endo已包装成功。利用终点稀释法(End-Point Dilution Assay)测得,扩增纯化后获得的重组腺病毒pAd-Endo及空载腺病毒pAd-Track的滴度(Titer)分别为2.06×10~(10)pfu/ml和1.73×10~(10)pfu/ml。
2.采用不同MOI的病毒液感染ECV 304细胞,感染后培养48h在荧光显微镜下观察ECV 304细胞的感染效率,结果可见:当MOI为1时,仅有10%左右的细胞有荧光表达;当MOI为100时,约有95%以上的细胞GFP表达阳性。用逆转录聚合酶链反应(Reverse Transcript Polymerase Chain Reaction,RT-PCR)检测到被感染的ECV 304细胞中Endostatin mRNA的表达,而阴性对照中无表达;免疫印迹法(Western blot)检测到被感染的ECV 304细胞中有分子量约为20KD的蛋白质表达,而阴性对照中无表达,表明重组腺病毒感染ECV 304细胞后,Endostatin基因进行了有效的转录及蛋白的表达。
3.应用流式细胞仪、Hoechest 33258染色、DNA Ladder等检测方法,均发现感染重组腺病毒后,ECV 304细胞发生了显著的凋亡,并随着感染时间的延长,凋亡率呈上升趋势。
4.将6～10周龄的BALB/c雌性裸鼠20只,随机分为两组,利用卵巢巧克力囊肿或子宫腺肌症患者的在位内膜,经皮下种植及腹腔注射两种方法成功建立人子宫内膜异位症裸鼠模型,两组均无死亡例数。两组均于造模后14日处死,探查腹腔注射组有8只成模,每只约1～2个病灶不等,成功率为80.0%,大体观察可见病灶多位于盆腹腔的腹壁、肠系膜,肠管和肝表面,大小约2.0mm×2.0mm×2.0mm左右,呈清亮水疱样结构,表面有小血管爬行,部分可见粘连带与腹壁相连;皮下种植组有9只成模,成功率为90%,腹部切口均于术后3天愈合,每3天观察一次种植病灶结节,约于第12天形成大小约2.0mm×2.0mm×2.0mm结节样突起结构。两种方法造模成功率比较,其差异无显著性意义(P=1.000)。腹腔注射及皮下接种两种造模方法的异位病灶标本HE染色光镜下均可见类似子宫内膜样的腺体和问质结构,腺上皮细胞呈柱状或扁平状,病灶边缘与裸鼠肌细胞层相延续,部分切片中可见间质细胞侵入肌层的现象;血管内皮细胞生长因子(Vascular Endothelial cell Growth Factor,VEGF)蛋白阳性表达表现为胞浆棕色颗粒,在两组共17个异位病灶组织的腺上皮、间质及血管内皮中均有表达;微血管计数应用免疫组化法,CD34阳性染色定位于血管内皮细胞,呈棕黄色,微血管有的形成管腔,有的仅为单个内皮细胞或内皮细胞簇;两组共17个异位病灶组织均有较多的微血管生成,两种造模方法均未观察到明显的毒副作用。
5.将造模后4周的30只皮下种植裸鼠模型随机分为三组:治疗组(pAd-Endo)、阴性对照组(pAd-Track)及空白对照组(生理盐水)。病灶局部注射后2周处死全部裸鼠,分别测量异位病灶的体积,经重复测量方差分析,结果显示各组间差异有显著性意义(F=612.717,P=0.000),治疗组病灶体积低于阴性对照及空白对照组;治疗组病灶体积减小,其差异有显著性意义(F=817.754,P=0.000),阴性及空白对照组体积变化均无显著性意义(P均＞0.05),治疗方法同体积之间有交互作用(F=753.460,P=0.000),提示不同组别治疗前后,体积变化趋势不同。标本HE染色,光镜下可见治疗组腺体萎缩明显,部分结构不完整,间质伴有不同程度的坏死。经单向方差分析,结果显示治疗组的VEGF表达低于阴性对照及空白对照组,其差异均有显著性意义(P均=0.000),阴性对照与空白对照组VEGF表达的差异无显著性意义(P=0.120);治疗组的MVD值低于阴性对照及空白对照组,其差异均有显著性意义(P均=0.000),阴性对照与空白对照组MVD值的差异无显著性意义(P=0.952)。各组实验裸鼠均未观察到明显的毒副作用。
结论:
1.改进的AdEasy-1腺病毒载体是一种高效、安全、便捷的基因载体系统,其拥有多个特异性酶切位点可供选择,实验操作简单方便;其携带的绿色荧光蛋白便于观察追踪;腺病毒感染目的细胞的效率很高,并且能进行有效的增殖,产生较高的病毒滴度。
2.重组腺病毒感染人脐静脉内皮细胞后,人内皮抑素基因有效的进行了的转录和蛋白表达,证实了病毒功能的完整性,也完成了内皮抑素从基因到蛋白水平的功能转化。
3.内皮抑素在体外可以成功诱导人脐静脉内皮细胞ECV 304发生凋亡,随着感染时间的延长,细胞凋亡率呈上升趋势,这可能是内皮抑素能够拮抗新生血管生成的机理之一。
4.子宫内膜异位症患者的在位内膜具有较强的异位种植及促新生血管生成的能力,可以作为建立人子宫内膜异位症动物模型的原料,增殖期及分泌期子宫内膜均可成功建立裸鼠模型;腹腔注射及皮下种植均是成功率较高的造模方式,且未观察到明显的毒副作用,具有较高的安全性。
5.人内异症裸鼠皮下种植模型更加直观方便,有利于病灶体积变化及治疗效果的观察;采用病灶局部注射的治疗方法,操作简单,治疗靶向性强、治疗作用集中,全身的毒副反应轻微;综合病灶体积分析、VEGF表达及微血管密度计数三个因素可以较全面的评价新生血管的生成及抑制情况。
以上研究表明:人内皮抑素抗血管生成基因治疗可能成为子宫内膜异位症安全有效的治疗途径。
Objective
The incidence of endometriosis(EM for short) is nearly 10% in child-bearing age women,and it is increasingly common.It often causes infertility,dysmenorrhea, dyspareunia and chronic pelvic pain.Although endometriosis is a kind of benign disease,it has many similar characters to malignant disease,such as cellular proliferation,infiltration and recidivation.Endometriosis is hard to be cured because of its recrudescence,it is very harmful to the patients.Sexual hormone is frequently used to control the disease,which have so many side effcets,such as damage of hepatic function,osteoporosis,hectic fever and restlessness,the time of using sexual hormone is often no more than 6 months.Using sexual hormone is aimed at inhibition ectopic lesions by depressing the ovarial function to redcuse the level of estrogen directly or indirectly,but aimed at the cause of a disease,so,with the recovery of estrogen after discontinuation,recidivation is inevitable.Operation is good for cleaning the fucuses,reconstitution pelvic anatomical construction,but is ambiguous for mild or moderate cases, infertility and recidivation. Also it is ambiguous that using drugs after operation is help for improving pregnancy rate and delaying recurrence. So far,recurrence is the key point to treatment of endometriosis because the true cause of the disease is ambiguous,all of the therapies are lack of the exact target. It is very urgent to find out the pathogenesis of endometriosis in oder to get a effective pathway for treatment.
In machine-made EM morbidity research, the recipient at present common viewpoint is that Sampson prefers in 1927 propose that the "cultivating theory",but the incidence of cultivating is up to 76%～90% which is not to accord with the incidence of endometriosis,it is suggested that the cultivating only was the inducement,pointing out ectopia has to fall in newborn blood vessel support,then it is possible maintain the development and maintenance of endometriosis.A lots of researches shows that the malignant characters,such as cellular proliferation, infiltration and recidivation,are the key point to formation of endometriosis.The blood vessel generates mechanism already becoming one of universally accepted important EM morbidity mechanism.At the same time,many studies indicated that angiogenesis play a important role in formation and development of endometriosis,the formation and growth of ectopic lesions is depend on the new blood vessels.Some scientises are trying to treat endometriosis by using antiangiogenesis,which showing a good result.In brief,antiangiogenesis might to be the effectiv way for treatment of endometriosis.
Endostatin is a kind of the most potent endogenous angiogenic inhibitor at present,which has more intensive effect to inhibit the new blood vessels than angiostatin.Endostatin can inhibit the proliferation of vascular endothelial cells specificly but has no action to unendothelial cells,such as tumor cell and smooth muscle cell.Endostatin always inhibits the new vascular endothelial cells rather than the normal mature vascular endothelial cells.Using endostatin was safe and was hard to induce drug tolerance because of the genetic stability of vascular endothelial cell.Endostatin has so many adventages in depressing angiogenesis of tumor and other diseases.There are several researches show that injecting in animals of gene or protein of endostatin can inhibit the growth and metastasis of tumor,which has none side effect and drug tolerance was observed during treatment,and the tumor has been in dormancy after treatment.The abroad clinic research of antiangiogenesis by endostatin is on phase II,some chinases scientises are also using endostatin protein or gene in the antiangiogenesis therapy for tumor in animals, while its application in the treatment of endometriosis is seldom reported.
Nevertheless,using protein for treatment has many disadvantages so that its application was limited,such as high effectiv dose,short time of blood drug level,multiple injections,spreading toxicity and infectiou particles.Gene therapy is the important way because of its own predominances than protein therapy.
Our study intent to structure the defect type recombination adenovirus vector carring endostatin gene by using molecular biology,experimental zoology,pathology and immunology methods,which can infect the human umbilical vein endothelial ECV 304 cell and induce the cells to apoptosis.Establishing the nude mouse endometriosis model by subcutaneous implantation and abdominal cavity injection,injecting in local focus with recombination adenovirus to investigate the feasibility and security of antiangiogenesis therapy by endostatin,in oder to find out a new therapy way for endometriosis which is cheap, convenient and safe,releasing the pain of patients,degrading the rate of recurrce and cancerization,increasing rate of pregnance,improving the effects of treatment,making more profits for all the women.
Methods
1.The endostatin gene encoding for protein was amplified from plasmid Pshuttle-Endostatin by polymerase chain reaction (PCR) with specific upriver and downstream primers,after purification and reactiong with restricted enzyme,which called XbaI and KpnI,then cloned into the pAdTrack-CMV which carring the green fluorecence protein (GFP).The new recombinant transfer vector plasmid is named pAdTrack-Endo,after amplification,purification,restricted analysis and DNA sequencing,the new recombinant transfer vector plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E.coli strain BJ5183.Taking the advantage of the high efficient homologous recombinant machinery presented in bacteria,the new recombinant adenoviral backbone plasmid was generated in BJ5183,which named pAd-Endo.After amplification and purification,the new recombinant adenoviral backbone plasmid then was transfected into AAV 293 cells by liposome 2000.The recombinant adenovirus pAd-Endo was packaged and propagated in AAV 293 cells with high titers,purification by cesium chloride gradient density centrifugalization.We have also constructed the control recombinant adenovirus pAd-Track in the same way.
2.The titer was detected by the End-Point Dilution Assay.The human umbilical vein endothelial ECV 304 cells are infected with the recombinant adenovirus.The transcription of endostatin and expression of endostatin protein were detected by reverse transcript polymerase chain reaction (RT-PCR) and Western blot in ECV 304 cells infected with pAd-Endo.
3.The apoptosis were detected by flow cytometer (FCM),Hoechest staining and DNA ladder in ECV 304 cells infected with pAd-Endo for dfferent time.
4.Establishing the nude mouse endometriosis model by subcutaneous implantation and abdominal cavity injection with the endometria of endometriosis patients,killing the nude mouse 14days after establishment,the ectopic focus are detected by microscopy after HE staining.The expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD) were detected by immunohistochemistry.
5.Injecting in local focus with the recombination adenovirus,physiologic saline and the control recombination adenovirus,observing the activity and appetite of the nude mouse,the nude mouse were killed 14 days after injection,the volume of the ectopic focus were measured and the tissues were detected by microscopy after HE staining.The expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD) were detected by immunohistochemistry to investigate the feasibility and security of antiangiogenesis by endostatin.
Results
1.The full-length cDNA from the Pshuttle-Endostatin plasmid was amplified with ad hoc primer and high fidelity Taq DNA polymerase to obtain the expected 650bp gene segment,and the amplification was analyzed by agarose gel electrophoresis.The pAdTrack-Endo containing full-length cDNA of endostatin was amplified and purified,then detected by Kpn I and Xba I.And two specific bands exhibiting 9200bp and 650bp fragments were observed in the agarose gel,which represented the pAdTrack-CMV shuttle vector and the full-length endostatin cDNA.Then,the resultant pAdTrack-Endo was linearized with Pme I and transformed together with the pAdEasy-1 backbone vector into competent E. coli strain BJ5183. Recombinants were screened by restriction endonuclease digestion with Pac I and identified by agarose gel electrophoresis.The recombinants were digested into 23 kb and 4.5 kb segments as previously described.The results suggested that an adenoviral plasmid with a full-length cDNA encoding endostatin,which was called pAd-Endo,was obtained.The pAd-Endo was digested with Pac I to liberate linear adenoviral genomes,then transfected into AAV 293 cells.To assess whether the packaged viral particles could be observed,the transfected cells were monitored daily for GFP expression by a fluorescence microscope.GFP expression was visible 16 hours after transfection and became apparent at 5-9 days,by which part of the cells looked like string-of-beads,swelling and floating. About 14 days post-transfection,most of the cells bacame pycnosis and floating.The cells were lysed with three consecutive freeze-thaw cycles,and the virus (pAd-Endo) was collected from supernatant and proved by PCR. The titer of pAd-Endo and pAd-Track was 2.06×10~(10)pfu/ml and 1.73×10~(10)pfu/ml respectively.The amplification of pAd-Endo cultured in ECV304 cells was electrophoresed.
2.ECV304 cells were infected by the recombinant adenovirus with MOI of 1 and 100,10% and 95% of the cells expressing the GFP.The endostatin specificity 650bp gene fragment was noted in the gene-transfer group,but not in the empty vector group,suggesting a successful tansfection of pAd-Endo in ECV304 cells and a specific expression of endostatin mRNA.The endostatin expression product was electrophoresed by SDS-PAGE and transferred to the nylon membrane for Western blot analysis.A major band exhibiting a molecular weight of 20 kDa was specifically detected in the pAd-Endo transfer group,but not in the empty vector group,indicating that endostatin protein was successfully expressed in the ECV304 cells that transfected with Endostatin.
3. Apoptosis of ECV 304 cells induced by pAd-Endo for different time. The ECV 304 cells were analysed by DNA Ladder ,Hochest 33258 and flow cytometer.
4. 20 female BALB/c nude mice were divided in 2 groups,modeling by subcutaneous implantation and abdominal cavity injection with the endometria of endometriosis patients respectively,none was died.Killing the nude mouse 14 days after establishment, 8 and 9 mouse were accoplished in subcutaneous implantation group and in abdominal cavity injection respectively.In abdominal cavity injection group ,there are 1-2 ectopic lesions in each mice,which is located in abdominal wall, mesentery,intestinal canal and suface of liver.The volume of the ectopic lesions is about 2.0×2.0×2.0mm,bubble-like with new blood vessels on its surface.In subcutaneous implantation group,the incisions were healed 3 days after operation.The ectopic focuses were becoming more and more small and were in a nodus-shape which about 2.0×2.0×2.0mm 12 days after operation.The survival rate between the two group was not statistically significant(P=1.000).After HE staining,the ectopic lesions were observed the endometrium-liked structrue with gland and substance in microscope.The microvessel density was demonstrated by CD34 positive cells using immunohistochemistry.All of the 17 ectopic lesions were VEGF positive and were to form many microvessels.
5.30 nude mouse model of endometriosis by subcutaneoues implantation 4weeks were divided in 3 groups :pAd-Endo, pAd-Track and physiologic saline.All rats were then executed two week following the injection.The volume of implants were measured.Pathological changes were detected in the ectopic endometrium.
Microscopic examination showed the bulk of the endometrial tissue decreased and glands depauperated.The glandular epithelium had partially degenerated,and necroti debris was presented in the endometrial stroma. The differences were statistically significant for the volumes of endometriotic lesions before and after pAd-Endo injection (F=817.754,P=0.000).The differences were statistically significant for the volumes of endometriotic lesions after treatment by endostatin compared with the other two control groups (F=612.717,P=0.000). The differences were not statistically significant for the volumes of endometriotic lesions after treatment in the two control groups (P>0.05).There is interaction between each therapy method and volume(F=753.460,P=0.000). MVD (P=0.000) and the expression of VEGF (P=0.000) was decreased significantly in the group using pAd-Endo compared with the two control groups. MVD(P=0.952) and the expression of VEGF(P=0.120) was not statistically significant the two control groups.None apparente side effect was found in any group.
Conclusions
1.The recombinant adenoviral vector AdEasy-1 is a effective,safe and convenient,which has so many restricted enzyme point.It is convenient to observed because of GFP.It can infect the target cell with high effeciency and high viral titer.
2. The endostatin mRNA and protein were successfully expressed after infection of pAd-Endo in ECV304 cells,which confirmed the integrality of the virus and function transformation from mRNA to protien of endostatin.
3. Apoptosis of ECV 304 cells can induced by pAd-Endo,which may be one of the angiogenesis mechanism of endostatin
4.The endometrium of endometriosis patients has especial ability of transplantation and angiogenesis,it is a kind of ideal materials to endometriosis animal model. The endometriosis mice model of subcutaneous implantation and abdominal cavity are also safe and available.
5. The endometriosis mice model of subcutaneous implantation is more convenient to observe ;The local injection has many advantages,such as convenient, high specificity, concentrated action and slight side effect.To unite lesion volume,VEGF and MVD is a integrative way to evaluate the angiogenesis and angiogenesis
It is demonstratated: Antiangiopoiesis of endostatin is potentially to be used as a candidate way for therapy of endometriosis.
引文
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