| Backgrounds People enjoy great convenience provided by civil nuclear energy;at the same time,we should not ignore its negative effects.With the development of nuclear stations and civil radiological nuclear equipments,they can also bring extreme worry about the diseases induced by radiation exposure and nuclear attacks.The worldwide scientific research shows that patients with acute radiation sickness can get satisfactory therapeutic effects if the radiation dose is under 6Gy,and almost no case of long time survival time is reported if the radiation dose is over 8Gy.Severe bone marrow forms and intestinal forms of acute radiation sickness bring big challenge to scientists.Patients with intestinal forms of acute radiation sickness which means rapid progression and short survival period will get low treatment success rate.The clinical symptoms of these patients are as follows: abdominal pain,vomiting,diarrhea,bloody stool,acute peritonitis and intestinal obstruction.Radiotherapy is a common method for the treatment of tumor.When it kills tumor cells,it also has some effects on surrounding normal tissue and cells.Studies have shown that in patients with pelvic and abdominal tumor radiation therapy,over 75% patients occur different degrees of radiation damage and the incidence of acute radiation enteritis was 25%-75%.Acute radiation sickness means that radiation damage causes tissues and cells damage,so stem cells which can repair damaged tissue provide an important way for the treatment of acute radiation sickness.Stem cell technology is one of the most dynamic areas in life sciences research currently.As we know,stem cells exhibit the capacity for self-renewal to regenerate or differentiate for tissue repair and it is very important for maintaining internal environment stable and recovering damaged tissues.Mesenchymal stem cells(MSCs)originate from the early stage of mesoderm and ectoderm and they were found in bone marrow at first.They also come from adipose and umbilical cord blood.In vivo or vitro,MSCs induced by certain conditions can differentiate into fat,cartilage,bone,nerve,muscle,cardiac muscle,a variety of other types such as endothelial cells.Studies show that the transplantation of stem cells may remarkably affect the radiation enteritis mainly characterized by crypt stem cell damaged.It is reported that bone marrow mesenchymal stem cells(BMSCs)transplanted to the intestinal tissue could repair the damaged tissue by epithelium renewal potential arising from the high level of stem cell proliferation ability.Small intestinal damage is the heaviest when the abdominal radiation damage happens.The small intestinal epithelial cells metabolism is active and they are sensitive to radiation.BMSCs with strong proliferation ability,multi-directional differentiation potential,low immunogenicity are easily isolated from bone marrow,purificated and amplificated in vitro.BMSCs paly an important role in supporting hematopoietic function.They can also promote the stem cells' implantation,self-replicating and immune regulation.Radiation injury can stimulateMSCs to migrate to the damaged tissues and then MSCs promote tissue's repair and reconstruction.Researchers find that some cytokines such as IL-1?IL-6?IL-8?IL-11?TNF-??G-CSF can play a role against radiation injury.The safety of MSCs treatment is good.MSCs are thought to be the most advanced cell therapy tools,with the availability of FDA-approved clinical products.We have also developed clinical research on ABMSCs transplantation in treating inflammatory bowel disease with a satisfactory effect.In this study,we built models of radiation enteropathy with different radiation dose in Beagle dogs.We launched an experimental study on ABMSC in treating radiation enteropathy and used Quantibody Canine Cytokine Array in order to explore its possible mechanism.Objective Developing Beagle dogs model of acute radiation enteropathy with different radiation doses,and to detect the effects of ABMSCs transplantation in treating radiation enteropathy.Quantibody Canine Cytokine Array is used in order to explore the possible mechanism of ABMSCs transplantation in treating radiation enteropathy.Methods Part one: 1.The construction of acute radiation enteritis models of Beagle dogs.Twenty dogs were divided into 4 groups randomly: control group(group A),10 Gy radiation group(group B),12 Gy radiation group(group C),14 Gy radiation group(group D).Group B?C?D Beagle dogs were given single-dose from X ray delivered at dose rates of 250 c Gy/min.2.We investigated clinical features of the dogs before and after radiation,including diarrhea,intestinal bleeding,stool state,abdominal pain,body weight and temperature.The Kaplan-Meier method was used to analyze the survival rate of irradiated dogs.3.The degrees of intestinal tract injury for all animal models after radiation exposure were evaluated from histological features.4.Intestinal absorption test: The dogs were administered 5% D-xylose by oral gavage and the blood D-xylose content was measured in the fixed time within 7 days post-irradiation.The sample treatment was performed by phloroglucinol colorimetric method.5.Intestinal barrier function test: Plasma DAO activity was tested by ELISA in the test groups within 7 days after radiation. 6.We also recorded endoscopic damage of the animals.7.Endoscopies were performed before and after radiation,and changes of intestinal mucosa were observed from imaging,morphological point of view.They were evaluated according to the appearance of the mucosal damage,vascular congestion,and inflammatory infiltration.According to the score,the intestinal damage was stratified as absent or slight(0),mild(1,2,3,4),moderate(5,6,7,8),or severe(9,10,11,12).Part two: 1.The construction of acute radiation enteritis model of dogs.Thirty-five dogs were divided into 7 groups randomly: control group(group A),10 Gy radiation with saline transplantation group(group B),12 Gy radiation with saline transplantation group(group C),14 Gy radiation with saline transplantation group(group D),10 Gy radiation with MSCs transplantation group(group E),12 Gy radiation with MSCs transplantation group(group F)and 14 Gy radiation with MSCs transplantation group(group G).Group B?C?D?E?F?G Beagle dogs treated by tridimensional conformal radiotherapy(3D-CRT)on abdominal irradiation were given single-dose from X ray delivered at dose rates of 250 c Gy/min.We investigated clinical features,intestinal absorption and barrier function,endoscopic and histologic damage of the animals in different groups.The degrees of intestinal tract injury for all animal models after radiation and after MSCs transplantation treatment were evaluated from histological features.Kaplan-Meier survival analysis was used to observe the survival time of all experimental animals groups.2.The culture of ABMSCs: Percoll density centrifugations were used to dissociate BMSCs from canine tibial plateau.Identification of ABMSCs: The surface markers of CD29,CD44,CD45 and CD90 were used to to identify the cultured BMSCs by immunofluorescence and flow cytometry.The osteogenic differentiations and identifications were carried on BMSCs.MTT assay was used observe the cell viability.3.ABMSCs transplantation: ABMSCs were transplanted into the mesenteric artery using femoral artery puncture and DSA imaging technology.4.Four aspects including diarrhea,intestinal bleeding,stool state,abdominal pain were involved the grading on symptoms of intestinal radiation injury.The four grades were divided according to the severity of intestinal radiation injury.5.Clinical features: The changes of body weight and temperature were observed after irradiation and after MSCs transplantation.We also recorded the diarrhea,intestinal bleeding,stool state,abdominal pain of the animals.6.Endoscopies were performed before and after ABMSCs transplantation,and changes of intestinal mucosa were observed from imaging,morphological point of view,including the appearance of the mucosal damage,vascular congestion and inflammatory infiltration.According to the score,the intestinal damage was stratified as absent or slight(0),mild(1-4),moderate(5-8),or severe(9-12).8.Comparison of survival time of all experimental animals groups was conducted in the observed time points.Part three: 1.Eighteen dogs were divided into 3 groups randomly: control group,normal saline(NS)group and MSCs transplantation group.2.The culture of ABMSCs in MSCs transplantation group: Percoll density centrifugations were used to dissociate BMSCs from canines.Identification of ABMSCs: The surface markers of CD29,CD44,CD45 and CD90 were used to to identify the cultured BMSCs by immunofluorescence and flow cytometry.The osteogenic differentiations and identifications were carried on BMSCs.3.The construction of acute radiation enteritis model of dogs: 18 Beagle dogs treated by tridimensional conformal radiotherapy(3D-CRT)on abdominal irradiation were given single-dose from X ray delivered at dose rates of 250 c Gy/min.12 Gy was used as radiation dose.3.ABMSCs transplantation: ABMSCs were transplanted into the mesenteric artery using femoral artery puncture and DSA imaging technology.4.Endoscopy were performed before and after ABMSCs transplantation,in different groups and the intestinal samples were reserved for Quantibody Canine Cytokine Array 1 and Quantibody Canine Cytokine Array 2 analysis.5.QAC-CYT 1 was used to test GM-CSF?VEGF?IL-2?IL-10?MCP-1?IL-6?RAGE?SCF?IL-8?TNF-?.QAC-CYT 2 was used to test Erythropoietin?FGF-7?HGF?HGFR?IFN-??IL-1 ??IL-12p40?IL-17A?MIP-1??TNF RI.6.Preparation of samples.7.Handling glass slides.8.Completely air dry the glass slide.9.Prepare cytokine standard dilutions.10.Incubation and fluorescence detection.11.Data analysis with QAC-CYT 1 and QAC-CYT 2.12.The cytokines levels changes were evaluated using ELISA.13.Gene ontology analysis was used to explore the possible mechanism and possible gene pathway of ABMSC transplantation in treating radiation enteropathy.Results 1.We built Beagle dogs models of acute radiation enteropathy with different doses successfully.1)Establish an assessment method of combining symptom manifestations,endoscopy and histological changes.Using this method,we found that the severity of clinical symptoms was related to radiation dose.With the radiation dose increasing,the symptoms including vomiting,diarrhea,became earlier,heavier and longer for duration.2)The endoscopic images showed that the intestinal tract damages were correlated with the irradiation doses.Intestinal mucosal damage caused by radiation may exhibit congestion,edema,punctate congestion,hemorrhage,diffuse congestion,haemorrhage,ulceration and exfoliation from mild to severe(10Gy-14Gy)depending on irradiation dose.3)After irradiation,all of the animals exhibited changes in body temperature with different ranges.There was a declining trend in weight at each detection time point for all dogs.4)Histological changes showed mucosal injury aggravated with irradiation increased.The mild changes appeared in the 8 Gy group,and dogs treated with 10-14 Gy exhibited partially damaged mucosa,glandular dilatation,inflammatory infiltration,vascular congestion and hemorrhage.5)Kaplan-Meier survival analysis found that early toxicity and mortality were dependent upon the total dose.,and high-dose abdominal irradiation was linked to severe intestinal toxicity and increased mortality.During a period of 40 days post-irradiation,the irradiated dogs receiving higher doses survived shorter than those receiving lower doses(P?0.001).2.We isolated,cultured and effectively labled for the dog bone marrow mesenchymal stem cells successfully.Both observation from immunofluorescence staining technology and flow cytometric analysis with special antibody confirmed that cultured BMSCs were successful.BMSCs had the potential to differentiate into osteoblasts in vitro,which had been tested by inducing BMSCs to differentiate into osteoblasts in vitro.3.The intestinal mucosal injury showed a trend of the more serious pathological injury with the greater radiation dose.In group E and group F,the inflammatory infiltration scores after MSCs transplantation were lower than that before MSCs treatment(P=0.034,P=0.003 respectively).We also observed significant lower total histologic scores after MSCs transplantation in group E and group F(P=0.003,P=0.005 respectively).During a period of 40 days post-irradiation,the irradiated dogs receiving higher doses survived shorter than those receiving lower doses(P?0.001).Compared with group C,group F exhibited substantially longer survival time(P=0.049)and improved relief of clinical symptoms.Autologous bone marrow stem cells transplantations were capable of curing acute radiation enteritis in with Beagle dogs a dose of 12 Gy at their abdomens.4.Mechanisms of BMSCs treated acute radiation enteropathy may exhibit the secretion and changes of cytokines.We found the changes in a single antibody array between radiation group and control group: IL-2(Fold change=0),IL-6(Fold change=0),RAGE(Fold change=0),TNF-?(Fold change=0.040),MCP-1(Fold change=0.266),GM-CSF(Fold change=0),VEGF(Fold change=0.134),SCF(Fold change=0.024),IFN-?(Fold change=0),IL-1b(Fold change=4.365),MIP-1?(Fold change=2.109).Between treatment group and control group,we found the changes as follows: IL-2(Fold change=0.130),RAGE(Fold change=0.002),IL-8(Fold change=935.046),TNF-?(Fold change=0.031),MCP-1(Fold change=16.724),GM-CSF(Fold change=0.128),VEGF(Fold change=0.0365),IFN-?(Fold change=0),IL-1?(Fold change=3.091),IL-12p40(Fold change=0.469).Between treatment group and radiation group,the changes were detected as follows: IL-8(Fold change=750.675),MCP-1(Fold change=62.847),VEGF(Fold change=2.727),SCF(Fold change=22.322),Erythropoietin(Fold change=0.119).In multiple antibody array analysis,MCP-1 showed significant difference between MSCs transplantation group and NS group(P=0.039),and ELISA analysis also showed the difference(P=0.012).And gene ontology analysis shows that Cytokine-cytokine receptor interaction,African trypanosomiasi and Malaria maybe the possible gene pathways.Conclusions 1.Beagle dogs models of acute radiation enteropathy have been established successfully,and animal clinical manifestations,endoscopic performance,histological changes and survival time to evaluate radiation intestinal damage at different irradiation doses have been built.With the radiation dose increasing,the symptoms including vomiting,diarrhea,became earlier,heavier and longer for duration.The endoscopic images showed that the intestinal tract damage were correlated with the irradiation doses.Intestinal mucosal damage caused by radiation may exhibit congestion,edema,punctate congestion,hemorrhage,diffuse congestion,haemorrhage,ulceration and exfoliation from mild to severe(10Gy to 14Gy)depending on irradiation dose.2.We isolated,cultured and effectively labled the dog bone marrow mesenchymal stem cells successfully.ABMSCs were transplanted into the mesenteric artery using femoral artery puncture and DSA imaging technology 2 days after radiation.Kaplan-Meier survival analysis found that early toxicity and mortality were dependent upon the total dose.Compared with group C(12Gy radiation with MSCs transplantation group),group F(12Gy radiation with MSCs transplantation group)exhibited substantially longer survival time(P=0.049)and improved symptoms relief?histology changes and intestinal function.3.We found that ABMSCs transplantation could improve animal clinical symptoms and play an important role in repairing radiation damage.Compared with saline treatment group,MCP-1 showed increase in ABMSCs treatment group(P=0.039).ELISA analysis also showed the difference between ABMSCs treatment group and saline treatment group(P=0.012).Gene ontology analysis showed that Cytokine-cytokine receptor interaction,African trypanosomiasi and Malaria might be the possible gene pathways. |
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