Study Of An Antiangiogenesis Gene Therapy With Endostatin Of Endometriosis

ObjectiveThe 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.Methods1.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.Results1.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 2.06×10¹⁰pfu/ml and 1.73×10¹⁰pfu/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.Conclusions1.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 endostatin4.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 angiogenesisIt is demonstratated: Antiangiopoiesis of endostatin is potentially to be used as a candidate way for therapy of endometriosis.