الفهرس 
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Abstract
Summary
Salivary glands are responsible for maintaining the health of the oral cavity and are routinely damaged by therapeutic radiation for head and neck cancer as well as by autoimmune diseases such as Sjogren’s syndrome. Regenerative approaches based on the reactivation of endogenous stem cells or the transplant of exogenous stem cells hold substantial promise in restoring the structure and function of these organs to improve patient quality of life. However, these approaches have been hampered by a lack of knowledge on the identity of salivary stem cell populations and their regulators. Recently, tissue-specific stem cell therapy has attracted public attention as a next-generation therapeutic reagent.
The aim of this work is to assess the regenerative potential of salivary gland-derived stem cells transplantation in gamma irradiated rat submandibular salivary glands.
46 adult male albino rats were used in this study. They were sacrificed by IV administration of anesthetic overdose. Both submandibular SGs were harvested from 5 healthy donor rats and used as a source of stem cells. The collected samples were chopped into a homogenous pulp. Then, mechanically digested tissues centrifuged for tissue collection. Cells were treated with collagenase II enzyme and centrifuged to obtain the cell pellets. Then the cell pellets were subjected to magnetic cell sorting of c-kit+ cells.
Steps proceeded by plating, suspending in media, subculturing and passaging after reaching confluence. characterization and assessment of stemness after isolation by flow cytometry was carried out using CD24 stem cell marker and isolation of c-kit+ cells was confirmed by Fluorescent Analysis Cell Sorting (FACS). Then, labeling c-kit+ stem cells with GFP was carried out.
The cultured cells were divided into 2 groups; 3 day old cells (i), in which the cells were allowed to stay in culture for 3 days before transplantation, and 10 day old cells (ii), in which the cells were allowed to stay in culture for 10 days.
The rats were grouped as follows:
• group I (normal control): (n=5 rats), served as the normal untreated control group.
• group II (irradiation group “IR”): (n=18 rats), were subjected to a whole body gamma radiation with a single dose of 6 Gy.
• group III (irradiation and transplantation group “IR+TR”): (n=18 rats).
• They were subjected to a whole body gamma radiation with a single dose of 6 Gy.
• This group was subdivided into 2 subgroups;
• Subgroup A (n=9 rats), were injected locally into the SMGs with SG derived stem cells at 3 days of stem cells separation and propagation culture.
• Subgroup B (n=9 rats), were injected locally into the SMGs with SG derived stem cells at 10 days of stem cells separation and propagation culture.
The rats in group II (IR) and group III (IR+TR) including subgroups IIIA and group IIIB were subjected to whole body irradiation with a single dose of 6 Gy. Then, the cells were prepared for transplantation and counted using the Neubaue Haemocytometer. 24 hours post-irradiation, transplantation was done under general anesthesia, the rats in group IIIA were locally injected in both SMGs with the cell suspension containing cells of group i, while the rats in group IIIB were injected with the cells of group ii.
After 3 days, 1 week and 2 weeks after the transplantation procedure, 6 rats from group II and group III were sacrificed for SMSGs collection and preparation for histopathological and immunohistochemical analysis.
At 3 days, 1 week and 2 weeks after radiation exposure, 6 rats from each group were sacrificed by anesthetic overdose. The skin was removed, and then the SMSGs were carefully dissected and routinely processed into paraffin blocks. 5 ϻm thick sections were cut, and stained by Harris’s haematoxylin and eosin for histological evaluation of any structural changes in the salivary glands using ordinary light microscope. Then the sections were dewaxed and labeled for PCNA, used for detection of proliferating cells, caspase 3, for detection of apoptotic cells and c-kit, specific as a stem cell marker. The mean values were calculated.
The immunostained sections were examined using ordinary light microscope to assess the prevalence of immunopositivity of PCNA staining, caspase 3 and c-kit in the studied cases. Then, Image Analysis Computer System to assess area percentage of the positive cells using Image J, image analysis software in 4 fields (x200) in each slide and the mean value was calculated and the measured values were expressed as mean ± S.D and the statistical significance of the difference was estimated using ANOVA and Bonferroni’s test for multiple comparison.
The results show that successful isolation of salivary gland stem cells from SMSGs, based on their ability to adhere to plastic plates, was achieved and the cells positively expressed CD24. C-kit+ cells were successfully sorted and positively expressed c-kit. The cells were sub-cultured and expanded up to passage 2.
Three days postdate of transplantation, the acini of group II showed vacuolization, nuclear pleomorphism and hyperchromatism. Few blood vessels and ducts were dilated. The connective tissue stroma contained many dilated blood vessels engorged with RBCs. However, in IIIA and IIIB subgroups, mild disturbance of acinar cell outline and architecture was detected, mild acinar vacuolization, nuclear pleomorphism and hyperchromatism. Also, few dilated ducts and blood vessels.
One week postdate of transplantation, some acini of group II were partly atrophied occupying a smaller area than normal. The acinar nuclei were pleomorphic and hyperchromatic. The duct system was dilated. The ductal cells showed nuclear changes seen as clumped chromatin or nuclear blebbing. Indistinct acinar outline was observed. Severe intracytoplasmic vacuolization was noted in the acini. The connective stroma contained dilated blood vessels with areas of extravasated RBCs. However, the IIIA and IIIB subgroups, revealed slight alteration of the acinar architecture, mild intracytoplasmic acinar vacuolization but progressive in some cases in subgroup IIIB, hyperchromatic variable sized nuclei and slightly dilated duct system.
Two weeks postdate of transplantation, incomplete restoration of normal acinar structure was observed in IR group. There was mild intracytoplasmic vacuolization and variability of nuclear size. The duct system was slightly dilated, whereas blood vessels appeared normal. In both IIIA and IIIB subgroups, the glands were almost normal with restoration of the histological appearance and structure of acini, slightly dilated duct system and normal vascular component.
The positive staining for GFP might indicate the homing and/or differentiation of the transplanted stem cells in irradiated SG tissue which shows that transplanted cells can regenerate radiation damaged salivary gland and form new ducts that may contribute to regeneration.
PCNA positive immunoreaction was observed as brown stain in the nuclei of the acinar cells. The SMSGs of group II (14.56 ±0.36) presented high numbers of proliferating cells 3 days postdate of transplantation. Also, in the other subgroups IIIA (4.98 ±1.44) and IIIB (6.14 ±2.03), the number of proliferating cells increased reaching maximum level 3 days post-irradiation. A subsequent decrease in the proliferation rate was noted up to 7 days postdate of transplantation in group II (2.38 ±0.73). The proliferation rate of subgroups IIIA (4.42 ±3.75) and IIIB (3.83 ±1.58) followed the same pattern of group II. After 2 weeks, all groups declined in the proliferation rate with the irradiation group II (3.93 ±1.15) being higher in numbers.
Regarding caspase 3 activity, the rate of apoptosis was increasing till reaching peak activity at 1 week postdate of transplantation, then declined at 2 weeks reaching levels more than those reached in the transplantation group. While the other subgroups IIIA and IIIB, the rate of apoptosis peaked at 3 days post transplantation then declined at 1 week. After 2 weeks, in the 2 groups, the rate of apoptosis declined more with the irradiation group II being higher in numbers.
The current study showed positive expression of c-kit exclusively in and around some ducts in all the studied groups. In the control samples, the excretory and striated duct cells showed the expression of c-kit. In the irradiation group II, c-kit immunoexpression was observed to be confined to the ductal compartment. At 1 week postdate of transplantation, the immunoexpression declined than that expressed at 3 days postdate of transplantation.
In the transplantation group III, a larger number of ducts showed positive expression of c-kit, which peaked at 1 week post transplantation. These results are indicative of potential long-term recovery.
The statistical analysis revealed significant increase in cell proliferation (PCNA) in both groups II and III compared to the control group I at 3 days where the difference was statistically highly significant (P ˂0.05), followed by increase in the mean values of subgroups IIIA and IIIB 1 week post transplantation compared to groups I and II, where there was a significant difference between groups I, IIIA and between II, IIIA but insignificant in groups II and IIIB compared to the control group I (P ˃0.05). Then followed by a decline in the mean values 2 weeks post transplantation, where it almost reached the control values in subgroup IIIB producing insignificant difference compared to group I, however still high in group II.
Regarding caspase 3 results, the statistical analysis revealed highly significant increase in the mean values of groups II and subgroups IIIA and IIIB 3 days post transplantation compared to the control group I (P ˂0.05), however, there was no statistically significant difference between groups II, IIIA and IIIB (P ˃0.05). Followed by decline in the mean values in subgroups IIIA and IIIB 1 week post transplantation, producing highly significant difference compared to group II (P ˂0.05), however, there was no statistically significant difference between groups IIIA and IIIB (P ˃0.05).
At 2 weeks post transplantation, group II showed the statistically significantly highest mean caspase 3 expression, followed by group IIIB, IIIA and group I. The statistical difference between the groups was highly significant (HS) (P ˂0.05). The difference was statistically highly significant in groups II and IIIB compared to control group I, in subgroups IIIA and IIIB compared to group II and between subgroups IIIA and IIIB (P ˂0.05). However, there was no statistically significant difference between groups I and IIIA, where the mean value in this subgroup almost reached the control values (P ˃0.05).
Concerning c-kit expression, at 3 days post transplantation, subgroup IIIB showed the statistically significantly highest mean c-kit expression, followed by group II, subgroup IIIA and group I. The statistical difference between the groups was insignificant (NS) (P ˃ 0.05). At 1 week post transplantation, subgroup IIIB showed the statistically significantly highest mean c-kit expression, followed by subgroup IIIA, group II and control group I. The statistical difference between the groups was significant (S) (P ˂0.05). The difference was statistically significant in subgroup IIIB compared to control group I, and in subgroup IIIB compared to group II (P ˂0.05), however, there was no statistically significant difference between control group I, group II, and subgroup IIIA and between subgroups IIIA and IIIB (P ˃0.05).
At 2 weeks post transplantation, group II showed the statistically significantly highest mean c-kit expression, followed by subgroups IIIA, IIIB and control group I. The statistical difference between the groups was insignificant (NS) (P ˃0.05).
Finally, it can be concluded that transplantation of these c-kit+ SMSGSCs could result in amelioration of the severely reduced quality of life of surviving cancer patients.