| Background:Type 1 diabetes mellitus is characterized by the permanent destruction of pancreatic β cells. Symptomatic individuals typically have lost more than 80% of their β cell population, resulting in essentially no insulin production and an inability to regulate plasma glucose levels properly. Stem cell transplantation for diabetes therapy may offer a promising possibility that deserves to be explored. Stem cell transplantation has some advantages, such as the easy operation, short operation time, and low invasiveness for patients relative compared with whole organ transplantation. Up to date, the mesenchymal stem cells (MSCs) has been actively investigated their therapeutic values for different kinds of diseases such as diabetes, injury, gastric cancer, etc. The beneficial of MSCs, which include multi-lineage differentiation and immunosuppressive capability, suggesting a role of MSCs therapy for tissue regeneration. However, the distribution and therapeutic effects of transplanted MSCs in diabetes patients are not clarified well. Quantum dots(QDs) is one kind of nanomaterial which have abroad application prospects in molecular imaging. In our previous study, we have syntheticed CdSe/ZnS QDs with near-infrared region. Herein, we evaluated their biosafety, and used CdSe/ZnS QDs to label MSCs, investigating their in vivo biodistribution in rat models with type 1 diabetes, our study lay foundation for further MSCs clinical application for diabetes therapy in near future.Methods:1.Culture of rat MSCsRats were anesthetized by ketamin-xylazin and then placed in ethanol(70%[v/v]). After dissecting out the femur and tibia, bone marrow was aspirated with 22 gauge needle. The cells were in cubatedin a humidified 5% CO2 incubator at 37℃ and the culture medium was changed every 2-3days.2. Identification of rat MSCs2.1 Morphological identification The morphology of MSCs was visualized by the presence of highly refractive intracellular lipid droplets in phase contrast microscopy.2.2. Identification of MSC characteristionMSCs were incubated with an appropriate concentration of CD29, CD31, CD45 and CD90. Quantitative fluorescence analysis was carried out using FACS caliber cytometer.2.3.Functional characterizationOsteogenic differentiation:MSCs were cultured with osteogenic medium for 2 weeks.Mineralization of the extracellular matrix was visualized by staining of the cultures with Alizarin Red S.Adipogenic differentiation:MSCs were cultured with osteogenic medium for 2 weeks.Adipogenic differentiation was visualized by the presence of highly refractive intracellular lipid droplets in phase contrast microscopy, the lipid droplets were stained with Oil Red O.3.Cell viability assayOur lab early has synthesized good biocompatibility quantum dots for targeted imaging. The effect of QDs on MSCs was evaluated by using Cell Counting Kit-8 assay. The absorbance of the sample at 450μL/mL was measured against a background control using the Multiskan microplate reader according to the protocol of the CCK8 assay kit.4. Viarify the distribution of QDs in MSCsAfter incubated with QDs, fix the cells with paraformaldehyde for 15min at room temperature. DAPI fluorescence stain was performed. Observe the cells with the fluorescence microscope.5. Transduct QDs into MSCsMSCs were cultured with different concentrations of QDs transduction medium at 37 ℃ for 6h. And then, the flask were washed three times with saline and covered with new saline. The cells were counted and prepared for quantum dot distribution test and cell transplantation.6. Ex vivo fluorescence imaging of MSCs labeled with QDs in rats with Type 1 diabetes For the ex vivo fluorescence imaging analyses, rats were sacrificed by cervical in 7 days afer MSCs transplantation, five major organs (heart, lungs, pancreas, spleen and liver) were harvested and immediately subjected to fluorescence imaging using the Bruker In-Vivo F PRO system. Results: 1.Identification of rat MSCs ①Morphological identification By the time they were 6-8 days old, cell colonies with different sizes increased in number. As growth continued, adjacent colonies interconnected with each other and a monolayer confluence was obtained after 12-15 days of incubation. In later passages, MSCs exhibited large, flattened or fibroblast-like morphology. ②Identification of MSC characteristionFluorescence-activated cell sorting analysis for surface antigen profiling of MSCs showed that more than 95% of these cells were positive for CD29, CD90, but negative for CD31, CD45.③Functional characterizationOsteogenic differentiation was induced. Alizarin red staining of the extra-cellular calcium in differentiated cells indicated osteogenic differentiation of MSCs into osteoblasts.Adipogenic differentiation was staining with Oil Red O. Lipid droplets were detectable even after 3 days, but a period of 2 weeks was necessary to accomplish maximal lipid accumulation.2. Determination of cytotoxicitySignificant cytotoxicity was observed in MSCs transduced with 32μL/mL or 50μL /mL of QDs, however,>80% of the cells were still alive. In addition, no remarkable cytotoxicity was observed with less than 16.0μL/mL of QDs. 3. Localization of quantum dots inside mesenchymal stem cells After incubated with 15μL/mL quantum dots, the cells were stained with DAPI. Merging image shows that quantum dots is present in the cytoplasm. Within cultured time, we did not observe that QDs were exited out of cells, QDs coexisted with cells well. 4. Ex vivo fluorescence imaging and semiquantitative analysis The strong fluorescence derived from QDs was observed from only the liver in the normal rats transplanted MSCs. On the other hand, bright fluorescence from not only the liver but also the pancreas, could be observed in rats transplanted with MSCs in diabetic rats. The RFI analysis showed that the accumulation rate of MSCs in the liver of rats transplanted with MSCs in normal control was about 41%, while that in rats transplanted with MSCs in diabetic rats was about 32% and that in the pancreas was about 32%.Conclusion: We investigated the behavior and organ-specific accumulation of transplanted ASCs labeled with QDs in a rats model of type 1 diabetes.Using the Bruker In-Vivo F PRO system,accumulation of MSCs in the liver of normal rats, while the ratio in rats transplanted with MSCs in diabetic rats was same to that in the pancreas.Our findings indicate that QDs fluorescence labeling can be useful for showingthe distribution of transplanted MSCs. |
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