| 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.
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