Effect Of Bone Marrow Stem Cells On Repairment Of Intestinal Radiation Damage

The mesenchymal stem cells (MSCs) obtained from bone marrow are nonhematopoietic stromal cells that have high self-renewal potential and are capable of differentiating along multiple mesenchymal lineages. They are capable of differentiating into, and contribute to the regeneration of, mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose. They are ease to expand and can be expand manyfold in culture while retaining their great proliferation and differentiation potential. Besides, MSCs can home to tissues, particularly when injured or inflamed .Accordingly, MSCs is a promising source of stem cells for tissue repair.Radiation-induced tissue injuries can be caused by radiation therapy of malignant tumors, nuclear accidents or radiological terrorism. The tissues with rapid proliferating cells, such as the use of hematopoietic tissue or alimentary tract, are particularly radiation sensitive. Until now, effective treatments for radiation injuries of the gastrointestinal tract need to be developed and basic research is required. Radiation induces sever damages to alimentary tract, making the loss of its physical and immunity barrier shield function. Toxic substances and bacterium migrate into blood circulation or organs and lead to infections. Eventually, it results in multiple organ failure and death. So, radiation-induced alimentary tract damage appears to be a determinant feature in patient mortality.MSCs are being investigated for their potential to regenerate radiation-induced injured tissues by a process referred to as plasticity or transdifferentiation. MSC therapy is a promising approach. Researches have been reported that MSCs can migrate and distribute to various tissues. But, there is little information on the fate and potential therapeutic efficacy of the reinfusion of MSCs following irradiation. This study is to address this question. Firstly, we made a mice model with radiation-induced gastrointestinal tract injuries. Then, expanded MSCs from bone marrow were transplanted by vein. We investigated the effect of the MSCs.Objective:To investigate the effect of bone marrow MSCs on the repairment of intestinal radiation damage.Methods:1. In the research, the objects were SPF C57 BL/6 mice at age of 6-8 weeks. Female mice were used to make radiation-induced alimentary tract damage and received transplantion. The male mice were donors to supply bone marrow MSCs.2. MSCs separated from bone marrow of male C57 BL /6 mice were cultured in vitro. MSC were determined by induced differentiation to the adipocyte and osteoblast.3. Female C57 BL/6 mice as recipients received ⁶⁰Coγ-ray lethal total body irradiation and were divided into three groups randomly. Groupâ… were control group. Groupâ…¡were transplanted bone marrow mononuclear cells. Groupâ…¢were transplanted Bone marrow stem cells and mononuclear cells. Observe the survival rate in 4 weeks and pathological section of the mice; the sex-determining gene Sry was detected by PCR in recipients.4. The calculation was performed using SPSS 11.5 software package. The data were represented as the mean±standard deviation or percentage. Comparisons in experiments were performed using one-way analysis of variance (ANOVA). Multiple Comparisons were performed using Lest-significant Difference (LSD). Survival curve was made by Kaplan-Meier assay. Differences with P≤0.05 were considered to be significant. Results:1. After total body lethally irradiation by 13.5Gy⁶⁰Coγ-ray, mice shed apparently, bent, suffering from weight loss, significant inactivity and weakened response to stimulation. Take intestinal tissue for pathology slice, under general observations, apparent intestinal wall thinning, mesenteric congestion, gore, and mucosal congestion can be seen. After HE staining, large area of intestine epithelial cell necrosis and collapse, nuclear swelling, extensive epithelial necrosis and stripping, villous atrophy and shorten, a large number of inflammatory cells invasion, no new Waterloo gland cells can be seen under light microscope ,it is in line with intestinal injury standard.2. After bone marrow cells cultured for 48 h, the suspended cells were removed for first time. Under inverted microscope we can cells adhesion to the wall; cells were in round shape. After cultured for 7-8 days, the cells changed into a spindle shape, shaping to cell clone with different cells in a swirl shape. After cultured for 10-11 days, the cells fuse into a single layer with a clear direction. When cells fused as much as 80 percent, we carry out transfer of culture. After the passage, the cells grown rapidly, impurities cells were cleared further. In vitro adipogenesis, fat drops began to appear in cells after 72 hours, mainly around the nucleus. With the induction time extension, fat drops gradually gathering to lipid bubble. After oil red 0 staining for cells, we can see fat drops shown dark orange-red. In the osteogenesis induction culture, we can see the bone nodule-like structure significantly higher than the surrounding surface of cells around 25th day. Alizarin red staining showed obvious red nodules of calcium.3. In Groupâ… ,10 mice all died in 7days after irradiation. Average survival time was 4.20 (4.20±0.79) d, and survival rate was 0/10. In Groupâ…¡, 10 mice died in 2 weeks after transplant except 2 mice were died in 7 days. Average survival time was 11.00 (11.00±2.31) d, and survival rate was 0/10. In groupâ…¢, 8 mice survival time exceed 28 days (considered as long-time surviving), and the other 2 mice died in 28 days. Average life time was 27.20 (27.20±1.75) d, survival rate was 8/10. Apply one-way ANOVA to compare each group survival time ,there was statistical significance(F=464.232, P<0.001). Using LSD to compare survival time of groupâ…¢, groupâ…¡, groupâ… , there were statistical significance(P < 0.001). Pathological section displayed that, the intestinal tract mucosa of mice in groupâ…¡were damaged seriously and there were no obviously repair compared with groupâ… , but intestinal tract mucosa of mice in groupâ…¢were nearly normal. The intestinal radiation damage had been definitely repaired. The Sry gene was detected in recipient mice of groupâ…¢, which confirmed that bone marrow MSCs participate in the repair of male mice intestinal tract damage.Conclusion:1. We can made reliable animal model by giving C57 BL/6 mice ⁶⁰Coγ-ray lethal total body irradiation.2. We can obtain fairly pure bone marrow MSCs by idensity gradient centrifugation and adherent culture.3. Bone marrow stem cells and bone marrow mononuclear cells have effect on repair of intestinal radiation damage; it proved the possibility of using bone marrow MSCs to repair intestinal radiation damage.