| Radiation-induced injury often occurs in nuclear explosions, nuclear leakage, nuclear terrorism and the clinical radiotherapy course. The tissues with rapid proliferating and metabolism cells, such as the hematopoietic tissue and intestinal tissue is the most sensitive to radiation. Abdominal tumor patients received a certain dose of radiation whose intestinal stem cells in the crypts and villus will be damaged. Small intestinal structure damaged resulting in the intestinal integrity of physical and immune barriers damaged. Pathogenic organisms and toxic substances in intestinal tract entered into the blood and fluid circulation and resulted in systemic morbid changes, even multiple organs infections and failure and eventual death. Currently, there are no effective treatments for radiation-induced intestinal injury, which is now just treated based on symptoms. Cytokines combination treatment has achieved some effectiveness. However, kinds and combination manner of cytokines have yet to be explored and further optimized. Therefore, developing new therapeutics for radiation-induced intestinal injury is necessary and very important.Mesenchymal stem cells (MSCs) are a class of adult stem cells, distinguished from hematopoietic stem cells (HSCs) and isolate initially from the bone marrow with strong self-renewal and differentiation potential. MSCs can be induced to differentiate into the source of the mesoderm of osteoblast, cartilage and adipocyte or even other embryonic germ layers in appropriate induced-environment. Because of the low immunogenicity, MSCs are considered the most ideal tissue engineering cell. A large number of clinical trials have proved that MSCs transplantation therapy is predominant and feasibility.The mechanisms that MSCs transplanted into the body to play a therapeutic role, there have been several different points (1) MSCs homed to the damaged tissue and differentiated into the corresponding cells and played a role in tissue repair, (2) homed to the damaged tissue to fuse with the corresponding cell, (3) secreted large amounts of growth factors, chemokines and played a therapeutic role through paracrine / endocrine. MSCs secreted large amounts of cytokines into the extracellular in culture process, which had been confirmed by many experiments. Accordingly, we designed this experiment. Firstly we founded a model of radiation-induced intestine injury and then isolated and cultured umbilical cord source MSCs, collected and deal with MSCs hypoxic conditioned medium, observed the therapeutic effects to radiation-induced intestinal damage.1. mouse model of radiation-induced intestinal injury Balb/C mice were divided into five groups (10 mice each group), anesthetized, fixed in plastic box. Head, tail and limbs of mice were shielded with lead bricks. Then mice were received 60Coγray with the doses of 7.0Gy, 8.5Gy, 10.0yG, 11.5Gy, 13.0Gy five doses to abdomen. Survival rate of mice in each group were observed for 30 days after irradiation. According to survival, pathological section and the activities of irradiated mice, we chose 10Gy radiation dose as the follow-up experiments.2. isolation, purification, identification of induction of umbilical cord mesenchymal stem cells Sterile collection of newborn umbilical cord tissue was removed vein and artery. Umbilical cord was cut into small pieces about 2cm3, implanted into culture dish. About five days later, adherent shuttle-type cells could be seen in edges of tissue pieces. Medium was changed to remove floating cells. Tissue pieces were taken away about 10 days after implantation. Digestion and passage were performed until 80% confluence of cells. Cells would be further purification during passage. Cells presented a typical long-spindle. The 3-5 generation of cells were used for osteogenic and adipogenic induction. Alizarin red staining was performed for osteogenic induction 14 days later. Calcium subsection was stained significantly. Adipogenic induced for 8 days and oil red O staining was performed. Lipid droplets were found in microscope. According to cell morphology and potential of differentiation, we determined the cells we isolated were MSCs.3. preparation of MSCs hypoxia conditioned mediumMSCs of 3-5 passage were implanted in culture flasks, incubated in hypoxia incubator for 6 hours。 Then cultivation supernatant of MSCs was collected. Cytokines levels of MSCs hypoxia or non-hypoxic condition medium were tested by Elisa assay. Medium was collected and separated using ultrafiltration units with 3kDa molecular weight cut-off. The high molecular weight fraction was obtained for subsequent experiments in vitro and vivo.4. MSCs and MSCs secreted-high molecular weight fraction protect irradiated-induced intestine injury of Mice40 mice divided into four groups (10 per group) according to weight, which were MSCs, one time MSCs secreted molecule, repeated MSCs secreted molecules and control group. 40 mice were received a dose of 10Gy60Coγirradiation in abdomen. Respective groups were given appropriate treatment within 1h of irradiation. Mouse diarrhea during the acute period was observed. Survival and weight change of mice were recorded within 30 days. Peripheral blood was measured. The results showed that repeated injected MSCs secreted molecules group can significantly reduce diarrhea. Body weight and peripheral blood of mice also had some improvement. Death did not appear within 30 days.5. Pathology of the small intestineMice divided into irradiated-control group, MSCs group, repeated injected MSCs secreted molecules groups (40 mice per group) were received 8.5Gy60Coγray local irradiation in abdomen. Mice of each group were killed at 1, 3, 7, 11, 21, 30, 60 days after irradiation (5 mice each time point) to take out the jejunum. Formalin-fixed small intestine, HE stained. Result showed repeated injected MSCs secreted molecules group significantly increased intestinal epithelial thickness. The parameter of villus height and integrity were more excellent than that of control group. |
PDF Full Text Request