The Study Of Treating The Radioactive Damage Of Salivary Glands In Mice By Injecting ADSCs Composite PRF/EPCs

[Background]: In clinic, surgery and radiotherapy is also used for head and necktumor, however, they both can lead to the irreversible damage of salivary glands; andsome autoimmune diseases can also lead to reduce the function of salivary gland or evenvanish. Salivary hypofunction and consequent xerostomia are the most common and themost significant complications, in which salivary gland hypofunction caused byradiotherapy is the majority.Nearly64%of long-term survivors of head and neck cancer after radiotherapy experience moderate to severe xerostomia[1]. Salivary hypofunction may lead to a seriesof sequelae, as well as health problems, such as xerostomia, difficult swallowing, loss oftaste, dental caries and oral candidiasis. In the end, these complications may lead tonutritional insufficiency and weight loss, lower quality of life. Besides, salivaryhypofunction by radiotherapy not only affects the patient’s quality of life,[2]but also somepatients had to terminate radiotherapy, which lead to tumors cannot continue to effectivelycontrol[3-5]. But at present therapy is only a symptomatic treatment, which can only relievesymptoms, and can’t cure these diseases fundamentally.[Objective]: This experiment is trying to cure the mice of radioactive saliva glandsdamage by injecting ADSCsl/PRF/EPCs to seek a more safe, convenient and effectiveway to promote restoration of the damaged salivary glands. It may provide a new newthought and method for salivary gland tissue engineering.[Methods]:1) Experiment in vitro:To cultivate ADSCs by digestion method andcultivate EPCs cells by induced ADSCs, and identify two kinds of these cells byimmunofluorescence, cell phenotypic characterization, HE dyeing method; To study thebiological characteristics of PRF of different shapes by gross observation, scanningelectron microscopy (SEM); Simultaneously, expand the scale culture of cells by RCCSsystem by gross observation, DIL cell staining, HE staining method and scanning electronmicroscopy to observe the influence of RCCS on cells;2) Experiment in vivo: The preparation model of radioactive damaged salivary glandsin mice by the18Gy local irradiation; Mice were randomly divided into5groups, andgave local injections. To observe the different changes of the submandibular gland tissuesin different groups in mice by weight changes, saliva flow rate, gross observation,histological section, method of transmission electron microscopy.[Results]:(一) Experiment in vitro:1) Mice ADSCs are fusiform, expressing thestem cell phenotype, with a concomitant adipogenesis and osteogenic differentiationpotential;2) EPCs are typical epithelioid structure, can express CD31, with some lumensample organization form;3) Both gel PRF and drying PRF can serve as a kind of verygood natural scaffold materials, can act as a carrier for cell growth, can promote cell proliferation and differentiation;4) Compared with the traditional method, the simulatedmicrogravity rotating bioreactor system can promote cell proliferation and differentiationmore effectively; The doubling time is (3.62795+0.25936)by the traditional method,doubling time is (2.98975+0.10133) days by rotating culture system, the doubling time isfaster (P <0.05);(二) Experiment in vivo:1) The mice after radiation lose weight, and reducesaliva production; After3months, HE dyeing show that the structure of submandibulargland acinar cells are damaged, almost all cells are liquefied; And there are a large numberof mononuclear stroma cell infiltration. Transmission electron microscopy (SEM) showthat structure of the normal cells damaged, such as cell edema, cell degeneration, nucleussecretion are statistically significant.2) The weight of other four inject groups arerespectively27.14±1.57g、29.45±1.65g、36.78±2.78g、39.86±2.42g， compare to20.78±1.27g in the PBS group, P<0.05; and the saliva flow rate are respectively52.75±8.03μl/min、66.5±5.37μl/min、78.37±8.41μl/min、87.25±8.94μl/min, compare to PBSgroup35.37±7.98μl/min，（P <0.05）; The delay time of saliva are respectively80±3.92s、81±5.57s、76±4.36s、72±3.22s, compare to PBS group92±4.59s,(P <0.05); HEdyeing of ADSCs/PRF/EPCs group show that most of the cellular structure arecomplete,there are no obvious cavity structure,even there are similar new acini and newblood vessels sample structure generate; Salivary gland biopsy tem observation show thatthe cells in local injections of ADSCs/EPCs/PRF group are basically complete, cellmembrane and nucleus are clear, a lot of normal endoplasmic reticulum and mitochondriacan be seen in cytoplasm; Apoptosis rate was10.6±1.6%. Compared with the PBS group,the amount of saliva in mice have a certain degree of recovery, all indicators havesignificant statistical significance (P <0.05); ADSCs compound with PRF/EPCs groupmay have a protection to promote the repair and regeneration of acinar cells.[Conclusion]:(1) Experiment in vitro:1. To establish the ADSCs cell line of micesuccessfully;2.To master the basic skills how to successfully induce the EPCs to ADSCsin mice in vitro;3.It is an original found that the feasibility of PRF act as a naturalbiological scaffold materials and carrier;4.To make full use of the characteristics of RCCS, and culture cells by it. The result shows pycnosis, mitochondrial swelling andendoplasmic reticulum expansion, even the cytoplasm of normal component disapeared;that the unique system can promote cell proliferation more effectively, which provides arich source of seed cells for the next step of research in vivo;(2) Experiment in vivo:1. The research established an animal models of the salivaryglands radioactive injury successfully of the project animal models in mice;2. Theexperiment had made an in-depth study to explore for the biological characteristics ofsalivary gland radioactive injury in mice by the local injections of ADSCs/PRF/EPCs;This compound are rich, convenient, safe and effective, especially there is little injury topatients. The experiments also show that the composite injection group have promoted thedamaged salivary gland tissue vascularized, acinar cell repaired, and also stimulatesalivary flow rate in mice effectively, improve the restoration of damaged mice salivaryglands. This idea has a broad prospect for tissue engineering research of salivary glandregeneration and clinical prevention; even it can provide the experimental basis for thetreatment of salivary gland injury research.