| 【Background】With the increase of head and neck malignant tumor, surgery is a primarytreatment to tumors, but they often need to receive radiotherapy. Due to the relative closeposition with malignant tumor, the adjacent normal salivary gland tissue is often damagedby radiation, Which leading to the normal salivary gland function is impaired and salivasecretion function disorder, resulting in complications such as xerostomia, mucosalchronic inflammation and secondary rampant caries. Clinical treatment technology islimited to symptomatic treatment for this condition currently. Tissue engineeringtechnology, especially the in-depth research of stem cell therapy has provided a feasibleway to the salivary gland regeneration.【Objective】The aim of this program is to research the adipose-derived stem cells treatment in theradiation-damaged salivary glands tissue of mice for salivary gland regeneration, observewhether ASCs differentiate into acinar cells in vivo leading to regeneration of the salivary gland and salivary secretion reconstruction. The research will provide experimental basisand reference for ASCs complex treatment for salivary gland regeneration, provideimportant theoretical and experimental evidence in treatment for salivary glands damageand salivary gland dysfunction.【Methods】1. Establishment of mice animal model with the radiation-damaged salivary glands tissue:Six weeks old female C57mice were taken with local irradiation for the submandibulargland after anesthetized, which were divided into5groups, ten mice in each group.5groups were respectively given with single12Gy,15Gy,18Gy,21Gy and24Gy linearaccelerator irradiation, blank control group was set, the same with10mice, a total of60mice. Mice were fed in SPF conditions, survival and diet of mice was observed, weightand blood routine was tested, the saliva flow and salivary amylase of mice was measuredbefore radiation,3months and6months later after radiation. The mice submandibulargland of each group were made into sections for cell morphology observation includingHE staining, PAS staining, transmission electron microscope observation and cellapoptosis detection, in order to observe damage of salivary glands. The best radiation dosefor making model and the best time to conduct stem cells for therapeutic intervention wasverified. The mice animal model with the radiation-damaged salivary glands tissue wasestablished by the best radiation dose.2. Isolation, culture and identification of mice adipose-derived stem cells: Cells wereisolated from the bilateral inguinal fat pads from2weeks C57mice by collagenasedigestion, expanded and routine cultured. The growth curve were drawn by MTTcolorimetry. The surface markers of cells were analyzed by flow cytometry. Cells werecultrued in osteogenic or adipogenic medium respectively in vitro and verified.3. The experiments of ASCs fluorescently labeled in vitro and traced in vivo: OrdinaryC57mice were labled by DiI and DEPI fluorescent dye in vitro. ASCs of GFP transgenicC57mice were cultured and only labled by DEPI fluorescent dye in vitro. All observedunder the fluorescence microscope. ASCs labeled by DiI fluorescent dye in vitro andASCs of transgenic mice were respectively injected into the C57mice with localized radiative damaged salivary through caudal vein and gland local injection, the injectedmice were killed after24h later, the submandibular gland were made into frozen sections,stained by DEPI fluorescent dye and observed under the fluorescence microscope.4. The study of ASCs treatment in the radiation-damaged salivary glands tissue of micethrough caudal vein and gland local injection: Mice were divided into five groups. A:normal control group; B: saline+caudal vein injection group; C: ASCs+caudal veininjection group; D: saline+gland local injection group; E: ASCs+gland local injectiongroup. Morphology of acinar cells and function of salivary gland were observed, data weretaken Statistical analysis.The mice submandibular gland of each group were made intosections for cell morphology observation including HE staining, PAS staining,transmission electron microscope observation and cell apoptosis detection3month laterafter injection. The apoptosis index of cells were observed to judge the salivary glandregeneration.5. Statistical Methods: Data were taken statistical analysis with SPSS19.0statisticalsoftware. T-test taken between the two groups and Single-factor variance test betweengroups, P<0.05was considered statistically significant.【Results】1. The mice animal model with the radiation-damaged salivary glands tissue wasestablished：The results showed that with the local radiation dose increases, systemiceffects and submandibular gland damage in mice were more obvious. Body weight anddetermination of blood cells of mice in the18Gy local irradiation group were generallynormal, but submandibular gland were seriously injured. Saliva flow and saliva amylasecontent decreased by66.7%and64%respectively. HE staining and PAS staining showedthe number of acini significantly reduced. Transmission electron microscopy observedSerous cells degenerated. The apoptotic index was24±4.1%. compared to3monthslater after18Gy local irradiation, salivary glands tissue injury further intensified, salivaryflow and salivary amylase content of mice continued to decline, HE staining and PASstaining showed acinar further reduce, transmission electron microscope showed theSerous cells degeneration, chromatin and nuclear pyknosis6months later after18Gy irradiation. Apoptotic index was51.9±3.9%, compared with AI of3months later after18Gy local irradiation were statistically significant (P <0.05).2. The adipose-derived stem cells of mice were cultured successfully：ASCs presentedfusiform shape and vortex-like arrangement.The growth curve was like S shape.Theresults of Flow cytometry showed that ASCs expressed CD29, CD44and CD90, but notexpressed CD31, CD4and CD45. Alizarin red staining and Oil Red O staining showedpositive reaction.3. The phenomenon of ASCs homing in the radiation-damaged salivary glands tissue ofmice after injection in vivo were observed：ASCs were labeled by DiI the DEPIfluorescent dyes successfully in vitro. ASCs labeled by DiI and ASCs of transgenic micewere found in the salivary gland tissue with localized radiative damaged of C57micethrough Caudal vein injection; ASCs with DiI staining also found through glands localinjection.4.The functional and morphological improvement of C57mice Salivary gland tissue withlocalized radiative damaged were observed after injection of ASCs in vivo.①Saliva flow and salivary amylase content of mice in group C and group E than in Band group D, but lower than group A. Data of group C and group E compared with othergroups were statistically significant（P＜0.05）, but group C and group E compared witheach other were no statistically significant. Data of group B and group D compared witheach other were also no statistically significant（P＞0.05）.②HE staining and PAS staining results show that acinar tissue morphology of miceSalivary gland tissue of group C and group E significantly improved compared to group Band groupD, but relatively poor compared to group A.③C57mice Salivary gland tissue ultrastructure observation under transmission electronmicroscopy (sem): Serous cell organelles of submandibular gland were structural integrityin group A. Serous cell degeneration and necrosis, nuclear pyknosis, zymogen granulesdecrease obviously in Group B and group E. Acini damage lighter, zymogen granules isrich, cellular edema is lighter, the serous cell structure is basically complete in group Cand group F. ④Apoptosis detection: Apoptosis index (AI) was0+4.4%in group A,49.5±5.6%ingroup B,31.7±3.6%in group C,52.1±4.9%in group D and27.6±6.4%in group E. AIof Group B, group C, group D and group E were statistically significant compared withgroup A respectively (P <0.05). AI of group C compared with group E, group B comparedwith group D were no significant difference (P>0.05). AI of Group C and Group Ecompared with group B and group D respectively were statistically significant (P <0.05).【Conclusion】1. The mice submandibular gland was damaged obviously in18Gy local irradiation group,but the whole body is mild affected. So18Gy is the best radiological dose for modeling.The mice animal model with the radiation-damaged salivary glands tissue was establishedsuccessfully. Acinar cells had a continuing losses over an extended period after localirradiation,3months after Submandibular gland local irradiation is the best time toconduct stem cells for therapeutic intervention.2. ASCs are excellent seed cells in tissue engineering, Due to abundant content, easierobtaining, fast growth, Strong differentiation ability, easier Isolation, culture, digestion andexpansion.3. ASCs homing in the radiation-damaged salivary glands tissue after injection in vivo,which confirmed that stem cells can specificity homing in damaged salivary gland tissue.4. ASCs were probably transformed into the acinar cells in vivo after caudal vein injectionor gland local injection, leading to restoration and reconstruction partly of damagedsalivary gland function and morphology. |
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