Dissertation > Excellent graduate degree dissertation topics show

Transplantation of Islets Transduced with Heme Oxygenase-1 Gene Using Adenovirus Vector

Author: LiYongXiang
Tutor: TanJianMing;LuDaRu;XiaQiang;DongWeiPing
School: Fudan University
Course: Organ Transplant Surgery
Keywords: Islets of Langerhans Transplantation Adenovirus Diabetes Apoptosis Rejection Gene Human
CLC: R657.5
Type: PhD thesis
Year: 2006
Downloads: 138
Quote: 0
Read: Download Dissertation


Objectives:1. Established techniques for gene transducion with adenovirus vector containinghuman HO-1 (heme oxygenase-1) gene.2. Established reliable protocol of isolation for adult rat islets and adult human islets in order to provide hight-grade islets for experiments in vitro and vivo.3. Established techniques for infecting islet cells with adenovirus vector containing human HO-1 gene and searched for optimal MOI in transduction.4. To investigate effects of human heme oxygenase-1 gene (hHO-1) on islet cells, andto confirm expression of HO-1 gene in transduced islet cells.5. Allogenic islet transplantation model was established using portal vein as transplant site, in order to investigate whether human HO-1 gene has the function of antiapoptosis, enhancing islets survival, depressing lymphocytic infiltration, and further to look for good gene therapy method in clinical islet transplantation.Methods:1. Using sequenator to detect the sequence of plasmid vector pobtt, comparing the sequence with human HO-1 gene map, and using polymerase chain reaction experiment to identify the constructed adenovirus vector (Ad-HO-1) containing human HO-1 gene. Amplified, purified and tittered the adenovirus vector containing human HO-1 gene (Ad-HO-1) .2. Adult human islets were isolated and purified using the method reported by Ricordi. Islets of Langerhans were isolated from outbred male Sprague-Dawley rat pancreata by a collagenase digestion technique and discontinuous Ficoll density gradient centrifugation. A algorithm was used for the calculation of the 150-μm- diameter islet equivalent number. Islet cell viability was assessed using fluorescence staining with acridine orange and propidium iodide. Insulin releasing tests were performed to investigate the secreting function of isolated islets.3. Transduction efficacy of islets was examined by infecting islets with Ad-EGFP at MOIs of 2, 5, 10 and 20. Infection efficacy was examined both by EGFP fluorescence and flow cytometery. Combined the results of EGFP gene transduction and refered other reports to determine optimal MOI for studies in vitro and vivo.4. New isolated islets were divided into three groups: HO-1 group (islets were transduced with Ad-HO-1, MOI=20) ; EGFP group (islets were transduced with Ad-EGFP, MOI=20); Control group (islets were not transduced with any vector). New isolated rat islets were used as negative control in western blot assays for detecting wether transduced human islets and transduced rat islets expressing human HO-1 after transduction for 48 hours.5. After cultured for 7 days in vitro, islets in EGFP group, HO-1 group, and control group were stimulated with low glucose (1.67 mmol/L) and high glucose (16.7 mmol/L), supernatant was collected for detecting concentration of insulin using radioimmunoassay kit. Stimulation ratios were calculated by dividing the insulin response to high glucose incubation by the insulin response to low glucose incubation to investigate insulin secreting function in each group.6. After infection with Ad-HO-1 or Ad-EGFP for 24 hours (rat islets infected and cultured for 7 days, MOI=20) , islets cultures were incubated with recombinant tumor necrosis factor- α (rTNF α , 5000units/ml) and cyclohexmide (CHX, Sigma, 50 μ g/ml) for 48 hours, islets were dispersed into single-cell suspention by a brief incubation in trypsin/EDTA. Cells were analyzed by flow cytometery using a BD FACSCalibur CellSorting System to detect apoptotic ratio of each group.7. Outbred SD rats, body weight 200-250 g, were rendered diabetic by intra-abdominal cavity injection of streptozotocin at a dose of 65 mg/kg. Before transplantation, diabetes was confirmed by blood glucose levels of greater than 16.7 mmol/l on 2 separate days at least 14 days after injection of streptozotocin. The diabetic rats were divided into three groups: Control group (received mock infected islets), HO-1 group (received islets infected with Ad-HO-1); Phosphate buffered saline (PBS) group (only received 0.8ml PBS). One thousand two hundred IEQ (islet equivalent) suspended in 0.6-0.8ml of PBS and collected in a 1-ml syringe were injected through a 22-gauge needle into the portal vein exposed with an abdominal incision. Observed the changes of blood glucose and weight of each group.8. Livers of HO-1 group and control group were removed from recipients after islets transplantation for 7 days (when the recipients’ blood glucose levels were normal) and 21 days (when engrafted islets lost function). Liver specimens were fixed in a 10% buffered formalin solution and embedded in paraffin. Sections were made at 4 μ m and deparaffinized, and after washing and blocking, incubated with primary rabbit anti-rat insulin monoclonal antibody (Santa Cruz), mouse anti-HO-1 monoclonal antibody or mouse anti-rat CD3 antibody at 4-8 ℃ for 18 hours. Anti-mouse antibody-HRP or Anti-rabbit antibody-HRP (Antibody Dignostica) was applied corresponding to the primary antibodies. Finally, the specimens were colored with substrate DAB (3, 3’-diaminobenzidine) solution and counterstained with hematoxylin. Histological grades of lymphocytic infiltration were evaluated for individual islets. TUNNEL assays were performed to detect apoptosis of engrafted islet cells.Results:1. The total number of purified islets from one donor rat was 629 ± 102 IEQ. Thepurity of islets was more than 85%. The viability of islet cells was more than 90%.The secretion amount of insulin was 3.70±0.49 mlU/L and 10.48 ± 2.23 mlU/L at 1.67mmol/L and 16.7mmol/L concentration of glucose respectively, the stimulation index was 2.84±0.10. Islet cells began to cohere after 24 hours of culturing, and nearly the entire islet cells adhered on the floor of plate after cultured for 7 days. After cultured for one month the islet cell still remained in good condition.2. The result of PCR for adenovirus vector Ad-HO-1 manifested that this vectorcontained human HO-1 gene order. The result of sequencing plasmid vector pobtt conformed that this vector contained open-reading frame of human HO-1 gene. EGFP continued to express in adult human islets and rat islets for more than four weeks. Fluorecence in transduced islets was more intense after transduction for 7 days.3. In vitro experiments of adult human islets: In control group of human islets (noinfected), the insulin release wasl82.36±58.96mIU/L after stimulation by glucose at high (16.7mmol/L) levels. The insulin release of HO-1 group and EGFP group were 270.09±89.37mIU/L and 175.95±75.05mIU/L respectively. Compared to control group and EGFP group, the insulin release of HO-1 group increased significantly (P<0.05). Compared to control group ( 90.86% ± 11.25% )apoptotic ratio of HO-1 group (63.08% ± 10.86%) was decreased significantly (P<0.01) after treatment with rTNF a and CHX; In vitro studies of adult rat islets: After treatment with rTNF a and CHX apoptotic ratio of islet cells was 4.22% ±2.38% in HO-1 group (MOI=20), significantly lower than 23.81% ± 8.51% in control group (P<0.05), and 28.76%±14.76% in EGFP group (MOI=20), (P<0.05). Although the apoptotic ratio in EGFP group was higher than that in control group, the statistic difference between the two group was not distinguished (P > 0.05).4. After observation for two months the blood glucose levels of rat diabetic modelwere always above 16.7 mmol /L. Diabetic rats had typical symptoms of diabetes.5. Nonfasting blood glucose levels diabetic rats before transplantation were: Controlgroup, 23.67± 2.57mmol/L; HO-1 group, 22.05 ± 2.57 mmol/L; PBS group, 24.06 ± 3.57 mmol/L (P > 0.05). In control group, the mean survival time of the islet grafts was 5.33 ± 4.18days; in HO-1 group, the mean survival time of the islet grafts was 10.56±4.33 days, significantly longer than that in control group (P < 0.05); None of the 6 diabetic rats in PBS group became normoglycemic after transplantation. Body weight levels at the day of operation were: control group, 222.70 ± 23.46g; HO-1 group, 230.00± 20.67g; PBS group, 210.15 ± 10.82g, differences between every two of the three groups were not distinguished (P > 0.05). By 3 weeks posttransplantation, the weight levels of the three groups were: control group, 237.29 + 41.11g; HO-1 group, 274.33 ± 30.28g; PBS group, 209.00 ± 13.84 g. The body weight gain of 19.82%±12.4% in HO-1 group rats at three-weeks posttransplantation was greater than 0.66% ± 6.24% in the control group rats and 0.25%± 9.16% in PBS group rats (P < 0.05).6. The outcome of immunohistochemical staining on specimens from recipients’livers on day 7 posttransplantation showed that engrafted islets in both HO-1 group and control group consisted of numerous well-granulated insulin containing cells. The engrafted islets infected with Ad-HO-1 demonstrated positive staining of HO-1 protein. Most islets in control group exhibited lymphocytic infiltration of grade 2 or 3; in contrast, most islets in HO-1 group exhibited little or mild lymphocytic infiltration of grade 0 or 1. TUNNEL staining of engrafted islet cells revealed that 4.2%± 2.3% apoptotic islet cellswere found in the islets of control group and no obvious apoptotic cells were found in the engrafted islets in HO-1 group. After 3 weeks granulated insulin containing cells were not found in engrafted islets in both HO-1 group and control group, most islets in the two groups exhibited lymphocytic infiltration of grade 3. Conclusion:1. Collagenase P digestion technique and discontinuous Ficoll density gradient centrifugation have high yield and high purity of islets in rat islet isolation.2. The adenovirus vector containing human HO-1 gene had been successfully constructed. Human HO-1 protein was expressed in the cultured islet cells transduced with Ad-HO-1.3. Adenovirus vectors have high efficency in gene transduction in adult islet cells and rat islet cells. EGFP expressed in cultured islet cells for more than four weeks in vitro.4. Transduction human islets with Ad-HO-1 improved anti-apoptotic function in cultured human islets and rat islets, HO-1 gene act as the role of stimulating insulin release on human islets.5. HO-1 gene had cytoprotective effects on engrafted islets, which could prolong engrafted islets survival in allogenic transplantation model, and diminish the lymphocytic infiltration degree in islet grafts.6. Transduction donor islets with adenovirus vector containing HO-1 gene has potential value in clinical islet transplantation.

Related Dissertations

  1. Research and Implementation on Knowledge Representation of Gene Regulatory Network,Q78
  2. Research on Combinatorial Regulation of Multiple Transcription Factors,Q78
  3. Attitude Control of a Three-Axis Stabilized Satellite,V448.22
  4. Research on Sparse Image Representation and Coding Model,TP391.41
  5. The Research of 3D Human Motion Capture Based on Reference Points,TP391.41
  6. The Research on Paper Currency Classification Method Based on Harr-Like Feature and Minimal Ball Including Samples,TP391.41
  7. Multi-Sensor Information Fusion and Its Applications on Wearable Computer,TP202
  8. Study of Oridonin on SGC-7901 Cell Proliferation by Inhibitting Cell Cycle Proteins,R285
  9. Cloning and Expression of CHS and CHI Genes and Their Regulation on the Accumulation of Flavonoids in ’Cara Cara’ Navel Orange (Citrus Sinensis Osbeck) and ’Guoqing NO.4’ Satsuma Mandarin (Citrus Unshiu Marcow),S666.4
  10. The Expression of Let-7 microRNA during the Process of the Mouse Embryonic Lung Development and the Construction of the Adenovirus Shuttle Plasmid,Q78
  11. Chemical Structures and Mechanism Research of Anti-aging Leading Compounds from Natural Products,R285.5
  12. Serum A-FABP Levels and Its Expression in Atherosclerotic Plaques of Diabetes Mellitus,R587.1
  13. Expression of Prolyl Isomerase Pin1 in Osteosarcoma and the Effect of Regulation on Cell Cycle,R738.1
  14. Study of Intracerebral Transplantation of Umbilical Cord Blood Mesenchymal Stem Cells in a 6-Hydropamine Rat Model of Pakinson’s Disease,R742.5
  15. CYP2C19 Gene Polymorphism in Patients with Ischemic Cerebrovascular,R743
  16. The Expression Survey of BMP Signalling Pathway in the Human Embryonic Tooth Germ,R78
  17. The Study on Cryopreservation and Mechanism of Freezing Injury on the Spermatozoa of Coelomactra Antiquate,S968.3
  18. The Anti-tumor Effect of CADPE and Inducing Apoptosis in Human Gastric Cancer Cells,R735.2
  19. Cloth and β- elemene combined administration of anti-tumor effect and mechanism of celecoxib,R96
  20. Yunnan Province, human capital, the network structure of critical efficiency factor,F249.27
  21. Cloning and Expression of Growth Hormone Gene in Cobia (Rachycentron Canadus),Q786

CLC: > Medicine, health > Surgery > Of surgery > Abdominal surgery > Pancreas
© 2012 www.DissertationTopic.Net  Mobile