The Experimental Study Of Stem Cells On Prevention Of Irradiation Injury Of Salivary Gland In Mice

【Background】Saliva is secreted by salivary gland, which is necessary for human to live. It helps todigest food, sterilize, clean and protect mouth. However, for the patients with head andneck malignant tumors, most of them need to receive radiotherapy. Salivary will beinjured inevitable as it lies in the head and neck, so it’s secretion function will bedecreased or lost. This will bring about great hardship to patients. At present the methodsonly keep salivary gland off injury. These methods try to decrease the scope of salivaryirradiation. However, the injury of parts of gland is inevitable for the gland malignanttumor. There have been not fundamental therapeutic measures to solve the irradiationinjury. It puzzles many scientists and doctors about how to prevent irradiation injuryfundamentally. Salivary tissue engineering develops gradually as the increasing of the study of tissue engineering. The cells are important for the tissue engineering. Howeversalivary cells are mature terminally differentiated cells, which cannot proliferate infinitely,and it is difficult to extract form the patients with salivary irradiation injury. As themulti-directional differentiation potential of the stem cells is explored, whether the stemcells from other origins can be used to prevent salivary gland irradiation injury? So thisexperiment mainly study whether adipose stem cells（ASCs）and bone mesenchymal stemcell（sBMSCs）can prevent salivary gland irradiation injury, discussing their mechanism ofaction, provides a new way for preventing dry mouth in clinic.【Objective】To investigate the feasibility of stem cells to prevent salivary gland irradiationinjury, and provide experimental evidence for preventing dry mouth in clinic.【Methods】1. ASCs and BMSCs were isolated and cultured from male mice, the characteristics oftwo kinds of cells would be identified by the photos of living cells,HE staining,adipogenic induction, osteogenic induction, MTT assay and Cell surface CD molecularidentification by flow cytometry.2. Found an ideal salivary gland radiation damage model. The female mice were devidedinto6group, and every group included ten mice. Submandibular glands of every groupmice were locally irradiated respectively in the head and neck region with a singledose of0Gy,12Gy,15Gy,18Gy,21Gy,24Gy by a linear accelerator. Weight weremeasured by balance scale within1month after X-irradiation(IR), blood analysis weredone in IR+1week, IR+4week．Saliva were collected in IR+8weeks，IR+12weeks，IR+16weeks, tissue slices were stained by HE and analyzed morphologically at thesame time. Ultrastructure of tissues were observed by transmission electronmicroscope, cell apoptosis was detected by TUNEL.3. The third experiment includes4groups, control group, IR+NS, IR+ASCs, IR+BMSCs. Two kinds of stem cells were injected into the mice by caudal vein, whichwere just irradiated locally18Gy twice every week, totally last for8weeks. The cell concentration is2×105/ml, the volume is0.2ml. The index was measured8weeks afterinjection cells.4. The salivary gland function of every group was measured, which includes weight ofmice, weight of gland, saliva flow rate.5. The salivary gland morphological of every group was observed, which includes HEstaining, PAS staining，tissue ultrastructure were observed by transmission electronmicroscope, cell apoptosis were detected by TUNEL to infer damage of tissue.6. Statistical analysis: using the SPSS19.0software, using One-way classificationANOVA with paired t test for statistical analysis between above-mentioned data ofevery group, compare statistical comparisons between every group.【results】1. ASCs Showed clostridia form, strong proliferation ability, Flow cytometry showedthe cells could express the cell surface specific antigen of ASCs, CD29、CD44andCD90，without CD31、CD34or CD45. The cells could differentiated actively intoadipogenesis and osteogenesis. So we can concluded we cultivated ASC successfully.2. We could cultivated BMSC successfully by Method of whole bone marrow, Thecellular morphology shows different shapes, such as clostridial form, polygon, trigon,irregular and so on. Flow cytometry showed the cells could express CD29、CD44andCD90，without CD31、CD34or CD45. have stable performance, could differentiatedactively into adipogenesis and osteogenesis, MTT shows the cells grows fast, but itcannot hold a candle to ASCs.3. The weight of mice in irradiated group decreased slightly, the blood routine showedthe decreasing of every component in irradiation group than control group, salivaflow rate of irradiation groups are lower than which in control group, as the timeextension, the more irradiation dose, the lower it is. There are obvious differencebetween irradiation group and control group.4. Tissue slice HE showed the submandibular tissue in irradiation group damaged moreseriously than that in control group. the amount of acinar cell decreased, the structureof cells disorder. The detection of apoptosis showed that the tissue of irradiation groups3months after irradiation had many apoptotic cells, the AI of18Gy irradiationgroup was21±2.5%, there was no apoptotic cells in the control group.5. Eight weeks after injection cells into irradiated mice, the weight of mice, weight ofglands and saliva flow rates in IR+ASCs and IR+BMSCs groups were increasedthan IR+NS group, the index in the IR+ASCs group are more than that of IR+BMSCs. there were obvious differences among four groups.6. Eight weeks after injection cells into irradiated mice, HE staining shows the amount ofacinus cells in irradiation groups are lower than control group, there were statisticsdifference between them. The amount of acinus cells in IR+cells groups increasedmore than IR+NS group, There were statistics differences between them.7. PAS staining shows that the tissue slice in irradiation groups was stained lighter thancontrol group. However, the tissue slice in IR+injection cells groups were staineddarker than IR+NS. This indicated that glycogen secreted by acinus cells of irradiatedgroup decreased obviously, comparing to control group, the glycogen that acinuscells of irradiation+injection cells groups secreted increased more than that of IR+NS.8. The ultrastructure observed by transmission electron microscope displayed that thecells of control group had normal morphology, intact organelles. However the cells inIR+NS group damaged most seriously, cellular swelling, cell structure in a mess,lysosome increased obviously. The cellular structure in irradiation+injection cellsgroups became normal, some organelles degenerated, some new small vessels could befould.9. Cell apoptosis: the AI of control approximate0，the apoptosis in irradiation+NS wasmost obvious, the AI was38.6±2.3%，the AI of IR+ASCs group was23±1.8%，the AIof IR+BMSCs was28±2.1%。【Conclusion】1. we cultured ASCs and BMSCs from the female mice successfully, which havemultipotential differentiation, we proved that ASCs of mice are prior to BMSCs inmultiplication capacity and cell viability after cell passage. 2. Salivary gland Radiation damage is dose dependent. Two months later Afterirradiation,18Gy exposure group tissue slice with a lot of inflammatory cellinfiltration, compared with the control, saliva flow close to60%, is the ideal salivarygland radiation damage model.3. Injection stem cells after irradiation could reduce salivary irradiation injury, but itcould not come to normal level. ASCs is more effective than BMSCs in preventingsalivary gland irradiation injury, there were obvious statistics difference between twogroups.