الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
ype I diabetes mellitus is a disorder characterized by the loss of β-cell mass and function. In most patients with type 1 diabetes, β-cell destruction is caused by an immune response against β-cells, and islet-related autoantibodies are detected in patients’ sera. Almost all pancreatic β-cells are rapidly destroyed, leading to a state of insulin dependency. Streptozotocin (STZ) is a widely used chemical for the induction of experimental diabetes in rodents as pancreatic β-cell is a specific target of STZ. This work was aimed to study the role of UCB-MSCs as a therapeutic modality in type I diabetes mellitus induced by streptozotocin in rats. It was also aimed to compare the efficacy of UCB-MSCs versus CM in restoration of normal endocrine pancreatic parenchyma.
Forty adult male albino rats weighing 150-200 gm were used in this study. They were divided into four main groups ten rats each:
group I (control group):
This group consisted of ten animals. It was further subdivided into two subgroups:
Subgroup ІA:
It included 4 rats that received no treatment.
Subgroup IB:
It included 6 rats that were injected once by 1ml of 0.1 mol/L citrate buffer intraperitoneally. Then, 3 rats were sacrificed after 2 weeks. The other 3 rats were sacrificed after 4 weeks.
group ІІ (Diabetic group):
This group included 10 rats that were injected intraperitoneal by a single dose of 1ml of streptozotocin (STZ) 35 mg/kg body weight, dissolved in 0.1 mol/L citrate buffered solution (pH 4.4). Three days after STZ injection, blood glucose
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was measured and blood glucose >250 mg/dL was considered diabetic and included in the study.
Animals of this group were subdivided equally into 2 subgroups (5 animals in each subgroup):
Subgroup IIA:
Rats were sacrificed 2 weeks after confirmation of being d iabetic.
Subgroup IIB:
Rats were sacrificed 4 weeks after confirmation of being diabetic.
group III (Diabetic + UC-MSCs):
This group included 10 rats. The animals were induced to become diabetic by STZ as in group II. After 3 days from STZ injection, each rat was injected with 1× 106 cells/ml of UC-MSCs into tail vein.
Animals of this group were subdivided equally into 2 subgroups (5 animals in each subgroup):
Subgroup IIIA:
Rats were sacrificed after 2 weeks from injection of UC-MSCs.
Subgroup IIIB:
Rats were sacrificed after 4 weeks from injection of UC-MSCs.
group IV (Diabetic + CM):
This group included 10 rats. Animals of this group were induced to become diabetic by STZ as in group II. After 3 days from STZ injection, the rats received a dose of 0.5 ml of CM that was injected intramuscularly once per week.
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Animals of this group were subdivided equally into 2 subgroups (5 animals in each subgroup):
Subgroup IVA:
Rats were sacrificed after 2 weeks from the first injection of CM.
Subgroup IVB:
Rats were sacrificed after 4 weeks from the first injection of CM.
Before scarification body weights were measured and blood samples were collected for laboratory blood glucose level, serum insulin and C-peptide level then after scarification, pancreatic specimens were obtained, fixed and processed for histological study.
Morphometric studies were done for surface area of the islets of Langerhans, area percentage of positive insulin cells using anti-insulin antibody-stained sections and the mean area percentage of caspase-3 positive cells using an image analyzer. Statistical analysis was done for evaluation of the morphometric data and level of pancreatic serum insulin and C-peptide using SPSS.17 program.
The mean of body weight revealed a significant decrease
(P0.05) in diabetic group II (subgroups IIA and IIB) compared to other subgroups. In UCB-MSCs treated group (subgroup IIIA and IIIB) showed significant increase (P0.05) when compared to subgroups IIA and IIB. In CM treated group (subgroup IVA and IVB) showed significant increase (P0.05) as compared to subgroups IIA and IIB.
The mean of blood glucose level showed a significant increase (P0.05) in diabetic group (subgroups IIA and IIB) to other subgroups. In UCB-MSCs treated group (subgroup IIIA and IIIB) showed significant decrease (P0.05) when compared to subgroups IIA and IIB. In CM treated group (subgroup IVA
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and IVB) showed significant decrease (P0.05) as compared to diabetic group (subgroups IIA and IIB).
Serum insulin and C-peptide showed a significant decrease (P0.05) in subgroups IIA and IIB compared to control group I. This level showed significant increase (P0.05) in subgroup IIIA and subgroup IIIB when compared to subgroups IIA and IIB. Also, the value revealed significant increase (P0.05) in both subgroup IVA and subgroup IVB when compared to subgroups IIA and IIB.
On light microscopic examination of H&E-stained sections of diabetic group II, subgroup IIA showed focal structural changes in islets of Langerhans. The affected islet cells showed variable structural changes. Cells showed vacuolated cytoplasm with small and darkly stained nuclei. Moreover, subgroup IIB revealed more noticeable structural changes than subgroup IIA. These changes extended to include alteration in the structural morphology associated with existence of many empty spaces. Some cells of the islets of Langerhans showed deep acidophilic cytoplasm with flattened eccentric nuclei.
In UCB-MSCs treated group III, subgroup IIIA, showed apparent increase in number of normal sized and shaped cells of the islet of Langerhans with vesicular nuclei. Minimal inflammatory cells infiltration with little capillary congestion. Meanwhile, in subgroup IIIB noticeable areas of nearly restoration of normal morphological structure of Islets as number of Islets cells were apparently increased.
In CM treated group, subgroup IVA, showed focal structural changes of islet of Langerhans associated with capillary congestion. Nuclei of some cells of islet were small and darkly stained. Subgroup IVB showed improvement in morphology, size, and apparent increase in cellular density of islets. Many cells appeared with central vesicular nuclei with pale acidophilic cytoplasm
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In immunohistochemical staining for Anti-insulin-stained sections showed progressive decrease of insulin immunopositively cells in all subgroups of diabetic group II to be maximum in subgroup IIB. In UCB-MSCs treated group, insulin immunopositively cells were increased in both subgroups (IIIA, IIIB) but they were more pronounced in subgroup IIIB. In CM treated group, insulin immunopositively cells were increased in both subgroups (IVA, IVB). These results were confirmed by the statistical study.
Morphometric and statistical study for mean surface area of islets of Langerhans revealed significant decrease (P0.05) in diabetic group (subgroups IIA and IIB) in relation to other subgroups. In UCB-MSCs treated group (subgroups IIIA and IIIB) and CM treated group (subgroups IVA and IVB), there was significant increase (P0.05) as compared to diabetic group (subgroups IIA and IIB).
Morphometric and statistical study for area percentage of positive insulin cells revealed significant decrease (P0.05) in diabetic group (subgroups IIA and IIB) in relation to other subgroups. UCB-MSCs treated group (subgroups IIIA and IIIB) and CM treated group (subgroup IVA and IVB) showed a significant increase (P0.05) as compared to diabetic group (subgroups IIA and IIB).
Caspase-3 immune histochemical stained sections revealed intense positive reaction in many cells in diabetic group (subgroups IIA and IIB). However minimal reaction was noticed in cells in UCB-MSCs treated group (subgroups IIIA and IIIB). Moderate reaction was distinguished in most of cells in CM treated group (subgroups IVA and IVB). Morphometric and statistical study for mean area percentage of caspase-3 positive cells revealed significant elevation (P0.05) in diabetic group (subgroups IIA and IIB) in comparison to other subgroups.