Researches About Bioluminescent Imaging And The Function Of Insulin Synthesis Of Pancreatic Islets Based On Gene Transfer Technique

Clinical pancreatic islet transplantation has developed rapidly these several years. This development makes the cure of diabetes mellitus hopeful. The relative researches of islet transplantation have developed quickly in these several years too. Using transgenic technology to modify the function of pancreatic islets to promote the clinical performance of islet transplantation is an important field in these researches. This dissertation is on the researches about islet bioluminescent imaging and the function of insulin synthesis of insulin gene transduced islets. Islet bioluminescent imaging could achieve in vivo monitor of islet graft after islet transplantation and provide timely, straight and corresponding information of the graft to clinical treatment and relative researches. Insulin gene transfer could enhance the function of insulin synthesis and excretion of isolated pancreatic islets. This technique may archive curing diabetes with much less islets so that save islet donors and alleviate the critical shortage of organ donors to some extent. These two gene transfer techniques may be contributive to the development of clinical performance of islet transplantation.Part 1: Monitoring the islet graft using bioluminescent imaging after islet transplantation in murine modelObjective: How to monitor the islet graft safely and effectively after islet transplantation is still a big problem in present clinical islet transplantation. We tried to use bioluminescent imaging to monitor the living condition of islet graft after islet transplantation in mouse models, and discussed the superiority and the feasibility of using this technique clinically in the future. Furthermore, we designed a method to overcome the obstacle of performing this technique clinically that the bioluminescent information will attenuate when photons pass thick tissues in human body so that no imaging could be obtained. We tried to use luciferase gene transduced islets transplanted into the subcutaneous fat as a symbol of the islet graft transplanted into deeper organ to solve this problem so that to prove that this technique is a good choice to clinical islet transplantation.Methods: Islets were isolated from brain-dead organ donors and normal B6 mice and Bclb/c mice and transduced by Luciferase gene. Diabetic mouse models were induced through chemical method. The transduced islets were transplanted into these models. Islets were injected into subcutaneous fat (sf models) or subcutaneous fat and kidney simultaneously (sf-k models). The recipients were divided into groups without immune rejection and groups with immune rejection by different arrangement of honors and recipients. We scanned the sf models on designed time points with CCD camera after transplantation with different islet dose. In sf models without immune rejection, we measured the pixel intensity in the region of interest (ROI) and calculated the correlation between the intensity and islet dose. In sf models with immune rejection, we observed the variety of the ROI pixel intensity along with time passing after transplantation and compared the time of it with that of the random blood sugar of these mice. In sf-k models, we harvested the subcutaneous fat and the kidney bearing islet graft 60 days after transplantation in group without immune rejection but 7 days and 10 days after transplantation in group with immune rejection. Histological analysis was carried on through common HE or AF staining. We observed the structure of the islet graft, quantity of the granules in islet B cells and inflammatory reaction in the graft area through these sections. And, the time of the histological variety was compared with that of the ROI pixel intensity and random blood sugar of sf models with immune rejection.Results: In sf models without immune rejection, the ROI pixel intensity has positive correlation with the dose of injected islets. In sf models with immune rejection, the ROI pixel intensity rose in the first several days and reduced in the following days after transplantation, while the random blood sugar of these recipient mice reduced first and rose again after that. And the reduction of the ROI pixel intensity occurred earlier than the rise of the random blood sugar statistically. In histological analysis, healthy islet structure with B cells containing abundant granules was shown and no obvious infiltration of inflammatory cells to the grafts was found at both kidney and subcutaneous sites in sf-k models without immune rejection on 60th day after transplantation. However, evident infiltration of inflammatory cells surrounding islet graft and reduced granules in B cells was shown on 7th day and eventually inflammatory cell infiltration into the islets graft area with destruction of islet structure was shown on 10th day at both subcutaneous and kidney sites in sf-k models with immune rejection. The histological appearances were at the same level at both transplantation sites on the same time points and the luciferase gene transduced islet graft transplanted in subcutaneous fat could be a corresponding symbol of that in the deeper organ. The time of the histological variety conformed to that of the ROI pixel intensity and random blood sugar in sf models with immune rejection.Conclusion: Bioluminescent imaging achieved through gene transfer technique can monitor the living condition of the islet graft promptly, straightly and correspondingly after transplantation in living recipients. The luciferase gene transduced islet graft transplanted in subcutaneous fat could be a corresponding symbol of that in the deeper organ. This method could solve the critical problem of performing this technology clinically. Our research confirmed that this technology is a good choice to be applied in clinical islet transplantation and it also provides a very useful measure to the relevant researches of islet transplantation on living objects.Part 2: The insulin synthesizing mode of insulin gene transduced isletsObjective: It has been confirmed that human pro-insulin gene transduced human islets can secrete more insulin than normal islets do when they are under the condition of high concentration of glucose. And it has also been confirmed that this kind of gene transduced human islets were more competent to cure the hyperglycemia of the recipient mice in islet transplantation models. We designed two-insulin system islet model and simulated the injury factor after islet transplantation in vitro. We tried to confer the insulin synthesizing mode of this kind of gene transduced islets through insulin content measurement and to find whether the function of insulin synthesis of these islets have more resistance to injury factor in culture so to reveal the functional character of these islets more deeply.Methods: Islets were isolated from normal C57/B6(B6) mice and transduced by human pro-insulin gene to make two-insulin system islet model. Then these islets were cultured under normal condition or in medium with pro-inflammatory cytokines. On designed time points, islet samples were obtained and fragmented by ultrasound to measure the insulin content in them. We observed the variety of the external and internal insulin content in these islets along with time passing. And the total insulin content in these islets was calculated and compared with that in normal islets under the same condition and on the same time points.Results: Under normal culture condition, the total insulin quantity in the islets had no statistical difference in normal mouse islets and human pro-insulin gene transduced mouse islets, and kept stable in the process of the experiment (9 days) while the quantity of human insulin in tranduced islets increased gradually in the same process. In the culture with pro-inflammatory cytokines, the total insulin content began to reduce from 5th to 6th day after the cytokines were added into the medium either in normal mouse islets or transduced mouse islets without statistical difference on the same time points, and the quantity of human insulin in the transdued islets had the same varying mode.Conclusion: The enhanced capability to reverse hyperglycemia of recipient mice of insulin gene transduced islets in islet transplantation models, which is confirmed in previous research, is not because of more insulin content in the islets cells but the enhanced capability of insulin synthesis and secretion under high glucose concentration. The function of insulin gene transduced islets to synthesize insulin has no more resistance to pro-inflammatory cytokines. It performed the same variety just as normal islets did when pro-inflammatory cytokines existed. The researches about insulin gene transduced islets may supply a new way to save islet donors.