The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r）...The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r） gene and enhanced green fluorescent protein （EGFP） gene linked through an inner ribosomal entry site （IRES） sequence directed by a Cytomegalovirus （CMV） promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells （BFF） cultured in vitro through electroporation （900 V/cm, 5 ms）. Transgenic bovine fibroblast cells （TBF） were enriched through addition of G418 in culture medium （800 μg/mL）. Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions （10% FBS） or treated with serum starvation （0.5% FBS for 2-4 days） followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFP-expressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells （23.2% VS 35.2%, P 〈 0.05）. No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed （23.2% VS 18.9%, P 〉 0.05）. Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients （21 embryos） was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.展开更多
AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a ...AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a "gene switch" system, which can induce gene expression by administration of tetracycline (Tet) derivatives such as doxcycline (Dox). Using this system, we established 293 cells to an artificial cell line secreting insulin in response to stimulation by Dox. METHODS: The mutated proinsulin cDNA was obtained from plasmid pcDNA3.1/C-mINS by the polymerase chain reaction (PCR), and was inserted downstream from the promoter on the expression vector pTRE2, to construct a recombined expression vector pTRE2mINS. The promoter on pTRE2 consists of the tetracycline-response element and the CMV minimal promoter and is thus activated by the reverse tetracycline-controlled transactivator (rtTA) when Dox is administrated. pTRE2mINS and plasmid pTK-Hyg encoding hygromycin were co-transfected in the tet293 cells, which express rtTA stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Dox was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of Dox in culture medium on the expression of proinsulin gene was estimated respectively with Northern blot, RT-PCR, and radioimmunoassay. RESULTS: From the 28 hygromycin-resistant cell strains, we selected one cell strain (tet293/Ins6) secreting insulin not only automatically, but in response to stimulation by Dox. The amount on insulin secretion was dependent on the Dox dose (0,10,100,200,400,800 and 1000 microg.L(-1)), the level of insulin secreted by the cells treated with Dox (1000 microg.L(-1)) was 241.0pU.d(-1).cell(-1) , which was 25-fold that of 9.7pU.d(-1).cell(-1) without Dox treatment. Northern blot analyses and RT-PCR further confirmed that the transcription of insulin gene had already been up-regulated after exposing tet293/Ins6 cells to Dox for 15 minutes, and was also induced in a dose-dependent manner. However, the concentration of insulin in the media did not increase significantly until 5 hours following the addition of Dox. CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability of gene therapy, suggesting that conditional establishment of artificial beta-cells may be a useful approach to develop cellular therapy for diabetes mellitus.展开更多
Islet transplantation as a promising treatment for type 1 diabetes has received widespread attention. Oxidative stress plays an essential role in cell injury during islet isolation and transplantation procedures. Anti...Islet transplantation as a promising treatment for type 1 diabetes has received widespread attention. Oxidative stress plays an essential role in cell injury during islet isolation and transplantation procedures. Antioxidants have been used in various studies to improve islet transplantation procedures. The present study reviews the role of oxidative stress and the benefits of antioxidants in islet transplantation procedures. The bibliographical databases Pubmed and Scopus were searched up to November 2008. All relevant human and animal in-vivo and in-vitro studies, which investigated antioxidants on islets, were included. Almost all the tested antioxidants used in the in-vitro studies enhanced islet viability and insulin secretion. Better control of blood glucose after transplantation was the major outcome of antioxidant therapy in all in-vivo studies. The data also indicated that antioxidants improved islet transplantation procedures. Although there is still insuffi cient evidence to draw definitive conclusions about the efficacy of individual supplements, the benefi ts of antioxidants in islet isolation procedures cannot be ignored.展开更多
If only at a small scale,islet transplantation has successfully addressed what ought to be the primary endpoint of any cell therapy:the functional replenishment of damaged tissue in patients.After years of less-thanop...If only at a small scale,islet transplantation has successfully addressed what ought to be the primary endpoint of any cell therapy:the functional replenishment of damaged tissue in patients.After years of less-thanoptimal approaches to immunosuppression,recent advances consistently yield long-term graft survival rates comparable to those of whole pancreas transplantation.Limited organ availability is the main hurdle that stands in the way of the widespread clinical utilization of this pioneering intervention.Progress in stem cell research over the past decade,coupled with our decades-long experience with islet transplantation,is shaping the future of cell therapies for the treatment of diabetes.Here we review the most promising avenues of research aimed at generating an inexhaustible supply of insulin-producing cells for islet regeneration,including the differentiation of pluripotent and multipotent stem cells of embryonic and adult origin along the beta cell lineage and the direct reprogramming of non-endocrine tissues into insulin-producing cells.展开更多
Transplantation of pancreatic tissue, as either the intact whole pancreas or isolated pancreatic islets has become a clinical option to be considered in the treatment of patients with type 1 insulin-dependant diabetes...Transplantation of pancreatic tissue, as either the intact whole pancreas or isolated pancreatic islets has become a clinical option to be considered in the treatment of patients with type 1 insulin-dependant diabetes mellitus. A successful whole pancreas or islet transplant offers the advantages of attaining normal or near normal blood glucose control and normal hemoglobin Alc levels without the risks of severe hypoglycemia associate with intensive insulin therapy. Both forms of transplants are also effective at eliminating the occurrence of significant hypoglycemic events （even with only partial islet function evident）. Whereas whole pancreas transplantation has also been shown to be very effective at maintaining a euglycemic state over a sustained period of time, thus providing an opportunity for a recipient to benefit from improvement of their blood glucose control, it is associated with a significant risk of surgical and post-operative complications. Islet transplantation is attractive as a less invasive alternative to whole pancreas transplant and offers the future promise of immunosuppression-free transplantation through pretransplant culture. Islet transplantation however, may not always achieve the sustained level of tight glucose control necessary for reducing the risk of secondary diabetic complications and exposes the patient to the adverse effects of immunosuppression. Although recent advances have led to an increased rate of obtaining insulin-independence following islet transplantation, further developments are needed to improve the longterm viability and function of the graft to maintain improved glucose control over time.展开更多
Although allogeneic islet transplantation can successfully cure type 1 diabetes,it has limited applicability.For example,organs are in short supply;several human pancreas donors are often needed to treat one diabetic ...Although allogeneic islet transplantation can successfully cure type 1 diabetes,it has limited applicability.For example,organs are in short supply;several human pancreas donors are often needed to treat one diabetic recipient;the intrahepatic site may not be the most appropriate site for islet implantation;and immunosuppressive regimens,which are associated with side effects,are often required to prolong survival of the islet graft.An alternative source of insulinproducing cells would therefore be of major interest.Pigs represent a possible alternative source of beta cells.Grafting of pig islets may appear difficult because of the immunologic species barrier,but pig islets have been shown to function in primates for at least 6 mo with clinically incompatible immunosuppression.Therefore,a bioartificial pancreas made of encapsulated pig islets may resolve issues associated with islet allotransplantation.Although several groups have shown that encapsulated pig islets are functional in small-animal models,less is known about the use of bioartificial pancreases in large-animal models.In this review,we summarize current knowledge of encapsulated pig islets,to determine obstacles to implantation in humans and possible solutions to overcome these obstacles.展开更多
OBJECTIVE: To investigate the immune privilege induced by the Fas ligand (FasL) expressed by cotransplanted testicular Sertoli cells in islet allografts, and the effect of FasL gene transfection on islet cells in panc...OBJECTIVE: To investigate the immune privilege induced by the Fas ligand (FasL) expressed by cotransplanted testicular Sertoli cells in islet allografts, and the effect of FasL gene transfection on islet cells in pancreatic islet allografts. METHODS: Allogeneic islets and testicular cells were cotransplanted into diabetic recipients.Pancreatic islets were infected with the recombinant adenovirus, AdV-FasL, and transplanted into diabetic recipients. Allograft survival, islet function, apoptosis of infiltrative lymphocytes in allografts and gene transfected islet allografts were analyzed. RESULTS: All animals receiving islet allograft alone returned to a diabetic state in a few days (mean survival time 6.3 +/- 0.6 days). When the quantity of testicular cells cotransplanted with islets increased to 1 x 10(7), all animals remained normoglycemic throughout the follow-up period (60 days). FasL expression by cotransplanted Sertoli cells induced apoptosis of activated lymphocytes. Rejection of allografts in the FasL gene transfer group was accelerated and allograft survival was shortened to 3.4 +/- 0.2 days (P展开更多
Objective To induce islet allograft long-term survival through cotransplantation of islet cells with sertoli cells.Methods Testicular sertoli cells were prepared by digestion with collagenase, trypsin and DNase, and ...Objective To induce islet allograft long-term survival through cotransplantation of islet cells with sertoli cells.Methods Testicular sertoli cells were prepared by digestion with collagenase, trypsin and DNase, and were cultured for 48 hours. Collagenase digested and Ficoll purified donor (Wistar rat) islets were cotransplanted with allogeneic sertoli cells in the absence of systemic immunosuppression. Terminal leoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) was used to label apoptosis of lymphocytes surrounding the islet graft.Results Cotransplantation of islets and 1 × 107 sertoli cells reversed the diabetic state for more than 60days in 100% (6/6) of the chemically diabetic Sprague Dawley rats. Grafts consisting of islets alone or islets plus 1 × 105 sertoli cells survived only for 5 - 6 days. Apoptosis of lymphocytes surrounding the islets was quite clear.Conclusion Cotransplantation of islets with FasL+ sertoli cells induces local immune privilege and allows long-term graft survival without systemic immunosuppression.展开更多
The potential use of al ogeneic islet transplantation in curing type 1 diabetes mel itus has been adequately demonstrated, but its large-scale application is limited by the short supply of donor islets and the need fo...The potential use of al ogeneic islet transplantation in curing type 1 diabetes mel itus has been adequately demonstrated, but its large-scale application is limited by the short supply of donor islets and the need for sustained and heavy immunosuppressive therapy. Encapsulation of pig islets was therefore suggested with a view to providing a possible alternative source of islet grafts and avoiding chronic immunosuppression and associated adverse or toxic effects. Nevertheless, several vital elements should be taken into account before this therapy becomes a clinical reality, including cell sources, encapsulation approaches, and implantation sites. This paper provides a comprehensive review of xenotransplantation of encapsulated pig islets for the treatment of type 1 diabetes mel itus, including current re-search findings and suggestions for future studies.展开更多
Islet transplantation is an attractive treatment of type 1 diabetes mellitus. Xenotransplantation, using the pig as a donor, offers the possibility of an unlimited supply of islet grafts. Published studies demonstrate...Islet transplantation is an attractive treatment of type 1 diabetes mellitus. Xenotransplantation, using the pig as a donor, offers the possibility of an unlimited supply of islet grafts. Published studies demonstrated that pig islets could function in diabetic primates for a long time （〉6 months）. However, pig-islet xenotransplantation must overcome the selection of an optimal pig donor to obtain an adequate supply of islets with high-quality, to reduce xeno- antigenicity of islet and prolong xenograft survival, and to translate experimental findings into clinical application. This review discusses the suitable pig donor for islet xenotransplantation in terms of pig age, strain, structure/function of islet, and genetically modified pig.展开更多
文摘The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r） gene and enhanced green fluorescent protein （EGFP） gene linked through an inner ribosomal entry site （IRES） sequence directed by a Cytomegalovirus （CMV） promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells （BFF） cultured in vitro through electroporation （900 V/cm, 5 ms）. Transgenic bovine fibroblast cells （TBF） were enriched through addition of G418 in culture medium （800 μg/mL）. Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions （10% FBS） or treated with serum starvation （0.5% FBS for 2-4 days） followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFP-expressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells （23.2% VS 35.2%, P 〈 0.05）. No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed （23.2% VS 18.9%, P 〉 0.05）. Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients （21 embryos） was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.
基金the"Hundred Talents"Program of Shanghai Municipal Government,No.98BR018
文摘AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a "gene switch" system, which can induce gene expression by administration of tetracycline (Tet) derivatives such as doxcycline (Dox). Using this system, we established 293 cells to an artificial cell line secreting insulin in response to stimulation by Dox. METHODS: The mutated proinsulin cDNA was obtained from plasmid pcDNA3.1/C-mINS by the polymerase chain reaction (PCR), and was inserted downstream from the promoter on the expression vector pTRE2, to construct a recombined expression vector pTRE2mINS. The promoter on pTRE2 consists of the tetracycline-response element and the CMV minimal promoter and is thus activated by the reverse tetracycline-controlled transactivator (rtTA) when Dox is administrated. pTRE2mINS and plasmid pTK-Hyg encoding hygromycin were co-transfected in the tet293 cells, which express rtTA stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Dox was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of Dox in culture medium on the expression of proinsulin gene was estimated respectively with Northern blot, RT-PCR, and radioimmunoassay. RESULTS: From the 28 hygromycin-resistant cell strains, we selected one cell strain (tet293/Ins6) secreting insulin not only automatically, but in response to stimulation by Dox. The amount on insulin secretion was dependent on the Dox dose (0,10,100,200,400,800 and 1000 microg.L(-1)), the level of insulin secreted by the cells treated with Dox (1000 microg.L(-1)) was 241.0pU.d(-1).cell(-1) , which was 25-fold that of 9.7pU.d(-1).cell(-1) without Dox treatment. Northern blot analyses and RT-PCR further confirmed that the transcription of insulin gene had already been up-regulated after exposing tet293/Ins6 cells to Dox for 15 minutes, and was also induced in a dose-dependent manner. However, the concentration of insulin in the media did not increase significantly until 5 hours following the addition of Dox. CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability of gene therapy, suggesting that conditional establishment of artificial beta-cells may be a useful approach to develop cellular therapy for diabetes mellitus.
文摘Islet transplantation as a promising treatment for type 1 diabetes has received widespread attention. Oxidative stress plays an essential role in cell injury during islet isolation and transplantation procedures. Antioxidants have been used in various studies to improve islet transplantation procedures. The present study reviews the role of oxidative stress and the benefits of antioxidants in islet transplantation procedures. The bibliographical databases Pubmed and Scopus were searched up to November 2008. All relevant human and animal in-vivo and in-vitro studies, which investigated antioxidants on islets, were included. Almost all the tested antioxidants used in the in-vitro studies enhanced islet viability and insulin secretion. Better control of blood glucose after transplantation was the major outcome of antioxidant therapy in all in-vivo studies. The data also indicated that antioxidants improved islet transplantation procedures. Although there is still insuffi cient evidence to draw definitive conclusions about the efficacy of individual supplements, the benefi ts of antioxidants in islet isolation procedures cannot be ignored.
基金Supported by Funding of the National Institutes of Healththe Juvenile Diabetes Research Foundation+2 种基金the American Diabetes Associationthe Foundation for Diabetes Researchthe Diabetes Research Institute Foundation
文摘If only at a small scale,islet transplantation has successfully addressed what ought to be the primary endpoint of any cell therapy:the functional replenishment of damaged tissue in patients.After years of less-thanoptimal approaches to immunosuppression,recent advances consistently yield long-term graft survival rates comparable to those of whole pancreas transplantation.Limited organ availability is the main hurdle that stands in the way of the widespread clinical utilization of this pioneering intervention.Progress in stem cell research over the past decade,coupled with our decades-long experience with islet transplantation,is shaping the future of cell therapies for the treatment of diabetes.Here we review the most promising avenues of research aimed at generating an inexhaustible supply of insulin-producing cells for islet regeneration,including the differentiation of pluripotent and multipotent stem cells of embryonic and adult origin along the beta cell lineage and the direct reprogramming of non-endocrine tissues into insulin-producing cells.
文摘Transplantation of pancreatic tissue, as either the intact whole pancreas or isolated pancreatic islets has become a clinical option to be considered in the treatment of patients with type 1 insulin-dependant diabetes mellitus. A successful whole pancreas or islet transplant offers the advantages of attaining normal or near normal blood glucose control and normal hemoglobin Alc levels without the risks of severe hypoglycemia associate with intensive insulin therapy. Both forms of transplants are also effective at eliminating the occurrence of significant hypoglycemic events （even with only partial islet function evident）. Whereas whole pancreas transplantation has also been shown to be very effective at maintaining a euglycemic state over a sustained period of time, thus providing an opportunity for a recipient to benefit from improvement of their blood glucose control, it is associated with a significant risk of surgical and post-operative complications. Islet transplantation is attractive as a less invasive alternative to whole pancreas transplant and offers the future promise of immunosuppression-free transplantation through pretransplant culture. Islet transplantation however, may not always achieve the sustained level of tight glucose control necessary for reducing the risk of secondary diabetic complications and exposes the patient to the adverse effects of immunosuppression. Although recent advances have led to an increased rate of obtaining insulin-independence following islet transplantation, further developments are needed to improve the longterm viability and function of the graft to maintain improved glucose control over time.
基金Supported by European Grant Titled Xenome,UE LSHBCT-2006-037377
文摘Although allogeneic islet transplantation can successfully cure type 1 diabetes,it has limited applicability.For example,organs are in short supply;several human pancreas donors are often needed to treat one diabetic recipient;the intrahepatic site may not be the most appropriate site for islet implantation;and immunosuppressive regimens,which are associated with side effects,are often required to prolong survival of the islet graft.An alternative source of insulinproducing cells would therefore be of major interest.Pigs represent a possible alternative source of beta cells.Grafting of pig islets may appear difficult because of the immunologic species barrier,but pig islets have been shown to function in primates for at least 6 mo with clinically incompatible immunosuppression.Therefore,a bioartificial pancreas made of encapsulated pig islets may resolve issues associated with islet allotransplantation.Although several groups have shown that encapsulated pig islets are functional in small-animal models,less is known about the use of bioartificial pancreases in large-animal models.In this review,we summarize current knowledge of encapsulated pig islets,to determine obstacles to implantation in humans and possible solutions to overcome these obstacles.
基金ThisstudywassupportedbytheNationalNaturalScienceFundationofChina (No 3 9770 72 6)
文摘OBJECTIVE: To investigate the immune privilege induced by the Fas ligand (FasL) expressed by cotransplanted testicular Sertoli cells in islet allografts, and the effect of FasL gene transfection on islet cells in pancreatic islet allografts. METHODS: Allogeneic islets and testicular cells were cotransplanted into diabetic recipients.Pancreatic islets were infected with the recombinant adenovirus, AdV-FasL, and transplanted into diabetic recipients. Allograft survival, islet function, apoptosis of infiltrative lymphocytes in allografts and gene transfected islet allografts were analyzed. RESULTS: All animals receiving islet allograft alone returned to a diabetic state in a few days (mean survival time 6.3 +/- 0.6 days). When the quantity of testicular cells cotransplanted with islets increased to 1 x 10(7), all animals remained normoglycemic throughout the follow-up period (60 days). FasL expression by cotransplanted Sertoli cells induced apoptosis of activated lymphocytes. Rejection of allografts in the FasL gene transfer group was accelerated and allograft survival was shortened to 3.4 +/- 0.2 days (P
基金ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina (No 39770 72 6and 3970 0 14 5 )
文摘Objective To induce islet allograft long-term survival through cotransplantation of islet cells with sertoli cells.Methods Testicular sertoli cells were prepared by digestion with collagenase, trypsin and DNase, and were cultured for 48 hours. Collagenase digested and Ficoll purified donor (Wistar rat) islets were cotransplanted with allogeneic sertoli cells in the absence of systemic immunosuppression. Terminal leoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) was used to label apoptosis of lymphocytes surrounding the islet graft.Results Cotransplantation of islets and 1 × 107 sertoli cells reversed the diabetic state for more than 60days in 100% (6/6) of the chemically diabetic Sprague Dawley rats. Grafts consisting of islets alone or islets plus 1 × 105 sertoli cells survived only for 5 - 6 days. Apoptosis of lymphocytes surrounding the islets was quite clear.Conclusion Cotransplantation of islets with FasL+ sertoli cells induces local immune privilege and allows long-term graft survival without systemic immunosuppression.
基金Project supported by the National Natural Science Foundation of China(No.30700772)
文摘The potential use of al ogeneic islet transplantation in curing type 1 diabetes mel itus has been adequately demonstrated, but its large-scale application is limited by the short supply of donor islets and the need for sustained and heavy immunosuppressive therapy. Encapsulation of pig islets was therefore suggested with a view to providing a possible alternative source of islet grafts and avoiding chronic immunosuppression and associated adverse or toxic effects. Nevertheless, several vital elements should be taken into account before this therapy becomes a clinical reality, including cell sources, encapsulation approaches, and implantation sites. This paper provides a comprehensive review of xenotransplantation of encapsulated pig islets for the treatment of type 1 diabetes mel itus, including current re-search findings and suggestions for future studies.
基金supported by the National Natural Science Foundation of China(No.30700772)
文摘Islet transplantation is an attractive treatment of type 1 diabetes mellitus. Xenotransplantation, using the pig as a donor, offers the possibility of an unlimited supply of islet grafts. Published studies demonstrated that pig islets could function in diabetic primates for a long time （〉6 months）. However, pig-islet xenotransplantation must overcome the selection of an optimal pig donor to obtain an adequate supply of islets with high-quality, to reduce xeno- antigenicity of islet and prolong xenograft survival, and to translate experimental findings into clinical application. This review discusses the suitable pig donor for islet xenotransplantation in terms of pig age, strain, structure/function of islet, and genetically modified pig.
