Xenotransplantation of caprine pancreatic islets in diabetic mice
Diabetes is one of the aggressive metabolic syndromes, which allied with high levels of blood glucose subsequent of imperfections in insulin production that causes glucose to increase in the body. For decades, excellent improvement has been obtained in clinical transplantation of pancreatic is...
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| Format: | Thesis |
| Language: | English |
| Published: |
2013
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/67603/1/IB%202013%2027%20IR.pdf |
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| Summary: | Diabetes is one of the aggressive metabolic syndromes, which allied with high levels of
blood glucose subsequent of imperfections in insulin production that causes glucose to
increase in the body. For decades, excellent improvement has been obtained in clinical
transplantation of pancreatic islet. Because of that, islet transplantation has become the
main alternative cure for type 1 diabetics. However, important obstacles still remain to
use of islet transplants routinely as a treatment choice. The deficiency of human islet
donors makes the search for alternative islet sources mandatory for future developments
in pancreatic islet transplantation. Venturing into xenotransplantation provides
tremendous extensive sources of islet cells. The improvement of genetically engineered
pigs expressing human complement regulatory proteins to defeat immune damaging
pathways has been promising; however, the necessity of massive immunosuppressive
and the concern for zoonotic viral transmission are some of the constraints. The present
investigation unravels the potential of caprine islets as an alternative islet source for
xenotransplantation. One of the main reasons for performing this preliminary study was
that religious and cultural factors in countries including Malaysia may favor goats over
pigs as xenograft donors. However, potential limitations associated with the use of
caprine donors must be recognized. First, the mean yield of 120,000 islets per adult goat
is somewhat lower than the reported yield of up to 360,000 islets obtained from adult
pigs. Second, the efficacy of goat insulin in humans is not known. The amino acid
sequence of goat insulin differs from that of human insulin at four residues, compared to
pig insulin which differs at only one. However, bovine insulin, which differs at three of
the same four residues as goat insulin, has been shown to control diabetes in humans.
Furthermore, should there be a problem with goat insulin, it would be feasible to
genetically modify goats to produce human insulin. This study aimed i) to characterize
viable caprine islets, ii) to inspect the impact of antioxidant and secretagogues factors in
vitro maintenance of caprine islets and their viability in culture media iii) to scrutinize
the in vitro enhancing factors for insulin promoter gene and protein expression in
caprine islets, iv) to assess the functionality of grafted caprine islets in immunized
diabetic murine models.
Microscopic characterization of viable caprine islets was carried out to evaluate
endocrine cell types. Caprine islets were successfully obtained using a collagenase based digestion, isolation and Euro-Ficoll density gradient purification technique at
optimum pH (= 7.4). Purity and viability of islets were determined by dithizone and
FDA/PI staining respectively. The viability of purified islet cells exceeded 90%.
Caprine islet morphological assessment and cyto-architectural study were carried out
using single and multiple immunostaining for insulin, glucagon and somatostatin and
then assessed by confocal microscopy and flow cytometry. Under the confocal
microscope, the mean percentage of β-cell, α-cell and δ-cell in different layers of
purified islet were 38.01 ± 12.47%, 30.33 ± 12.33 and 2.15 ± 1.17%, respectively.
Majority of β-cells were centralized whilst the other two cell types were placed in the
peripheral regions. A similar pattern of abundance of β-cell, α-cell and δ-cell population
was determined by flow cytometry analysis (37.52 ± 9.74%, 31.72 ± 5.67%, and 2.73 ±
2.73% respectively). Flow cytometry findings of the endocrine cell population within
caprine islets were consistent with the microscopic investigation results. The
morphological study of caprine islet revealed arrangement of the different islet cell
types, which can lead to better understanding of different cell type interactions in
caprine islets. The cytoarchitectural study of caprine islet can be occasioned to the
comparison of similarity and dissimilarity of caprine islet cytoarchitectural features with
other species islets and their physiological structures. However, anatomical study of
caprine islet was conducted to in vitro and in vivo function assessment.
Two agents of antioxidant and secretagogue were considered to enhance viability and
functionality of caprine islets. Tocopherol as an antioxidant agent could offer antiapoptosis
reaction and secretagogue agent, 3-isobutyl-1-methylxanthine (IBMX),
enhance insulin secretion of caprine islets in the culture. The impact of supplementing
antioxidant (tocopherol) and secretagogue factors (IBMX) on caprine islet viability
during a short period maintenance of caprine islet culture was also assessed. The treated
caprine islets with mentioned agents showed stability of islet morphology in cell
culture, viability and functionality due to microscopy observation, FDA/PI staining and
insulin secretion while stimulated by a high and low glucose stimulation, respectively
(Day 1; 0.24 ± 0.09 μg/L, 0.13 ± 0.02 μg/L, Day 3; 0.19 ± 0.07 μg/L, 0.07 ± 0.02 μg/L,
Day 5; 0.22 ± 0.05 μg/L, 0.11 ± 0.02 μg/L). These results can be promoted via
molecular and gene expression studies on caprine islet.
The insulin promoter gene (PDX1) as one of the most important genes in pancreas and
islet was studied in caprine islets. Because it plays essential roles in pancreas and islet
expansion, pancreas growth, islet formation within the pancreas and insulin secretion
from β-cells. The PDX-1 and its protein expression were simultaneously assessed in the
supplemented caprine islet culture. The results showed PDX1 gene up-regulation during
five days of tocopherol and 3-isobutyl-1-methylxanthine supplemented caprine islet
culture compared with the control group, serum-free media, with the relative
quantification (RQ) value, day 1; 7.85 ± 1.20, day 3; 1.84 ± 0.14, and day 5; 6.80 ± 2.08
fold. Enhancement of PDX-1 expression in caprine islet results to produce more
effective islets for in vivo study and xenotransplantation approach.
The final aim of this study was to assess caprine islet functionality after
xenotransplantation into a diabetic murine model. The optimal islets (size range between 50-250 μm) of viable purified caprine islets were transplanted into the recipient
group (immunosuppressed diabetic mice) and compared with control groups of nondiabetic
mice, un-grafted diabetic mice without immunosupresive drug injection,
immunosuppressed mice with sham graft and islets grafted mice without
immunosuppressive drug injection. Glucose tolerance test, blood glucose monitoring
and microscopic examination of transplanted graft collectively indicated a reversion of
diabetic status in STZ induced immunosuppressed mice. Non-fasting blood glucose
level, 8.04 ± 0.44 mM/L, decreased from 23.3 ± 5.4 mM/L, meanwhile serum insulin
level increase from 0.01 ± 0.001 μg/L to 0.56 ± 0.17 μg/ L and recipient mice body
weights increased from 23.64 ± 0.31g to 25.85 ± 0.34 g (p < 0.05).
In conclusion, the combination of tocopherol and IBMX was capable to impair the rate
of apoptosis, improve the viability of caprine islets for short period culture and enhance
the duodenal homeobox gene and protein expression. It might be considered a potential
treatment to improve islet viability in vitro before islet transplantation. As the first
attempt of using purified caprine islets, results indicate that the grafted islets were
capable to retrieval diabetes in immunosuppressed STZ-injected mice. |
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