骨髓间充质干细胞对放射性肠损伤修复作用的实验研究
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摘要
放射性肠损伤是肿瘤放射治疗和核战争以及核事故时常见的放射性组织损伤,对放射性肠损伤的治疗目前常采用的是对症支持疗法,效果不理想。骨髓间充质干细胞(mesenchymal stem cells, MSCs)是骨髓内非造血实质干细胞,在不同的诱导条件下MSCs具有跨胚层多向分化潜能,.本研究利用骨髓间充质干细胞在体外可以诱导分化为肠上皮细胞的特点,通过静脉注入MSCs于放射性肠损伤大鼠体内,观察肠组织的病理变化,进一步探究MSCs在体内环境下对放射性肠损伤的修复作用。
研究结果表明,将标记的MSCs静脉注入放射性肠损伤模型动物体内,经冰冻切片,共聚焦显微镜观察标记细胞位置,发现2 d、7 d时MSCs在损伤的肠壁内聚集并逐渐增多,30 d治疗组肠粘膜细胞分裂相增多、间质纤维增生减少,证实MSCs对放射性肠损伤具有修复作用,同时也验证了本研究制作的放射性肠损伤动物模型适合医学动物实验研究。
Radiaction intestinal damage is seen frequently in the nuclear accident and the nuclear explosion of wartime, and is one of the most common complications in radiotherapy course of abdominal tumor. The apoptosis and proliferation inhibition occur significantly in the intestinal crypt stem cells after the abdomen is irradiated with a certain dose, which lead further to the damages of the structure of mucosa, the dysfunction of intestinal barrier, intestinal infection and the development of chronic diseases. At present, the treatment of radioactive intestinal injury mainly is still the support of the symptoms, but curative effect is not good. Thus, it is of great significance to explore effective measures for the promotion of intestinal radiation damage repair. Bone marrow mesenchymal stem cells (MSCs) are present in the bone marrow of non-hematopoietic stem cells, which have the differentiation potential. In appropriate conditions, MSCs can be divided into bone, cartilage, tendon, fat and other organizations, also differentiate into intestinal epithelial cells, liver cells, alveolar cells, pancreas cells, skin and nerve cells and so on. MSCs can be used in transplantations between different species and different individuals for the characteristic of MSC low immunogenicity.
At the intestinal radiation damage, the intestine oneself loses the ability of repair because the intestine crypt stem cells were seriously damaged. It is the focus of this study whether the exogenous MSCs home to damaging intestine through the transplantation and have the repair function of intestinal injury. In this study, based on the multi-polarization theory of MSCs, we used the rat bone marrow mesenchymal stem cells which were cultured in vitro, purified, and injected into the animal model bodys of the intestinal radiation damage, which explore the repair effect of MSCs on the radioactive intestinal injury and its mechanisms.
1 Culture of rat MSCs in vitro
In this study, we used the adherent culture methods to get a large number of MSCs. The MSCs were detected by the flow cytometry, in which the percentage of G0-G1 cells were 76.65% of the total number of the cells. And the unlimited proliferation of MSCs was further illuminated.
2 Induction and regeneration of MSCs in vitro
The purified MSCs were added into the osteoblasts solution, which inductively cultured for two weeks. Then, the induced product of the cells was identified with the alkaline phosphatase staining method. The results showed that rat bone marrow mesenchymal stem cells cultured in vitro can be induced to osteoblasts which were positive by specific staining. Therefore, the MSCs are proved to have the differentiation potentia.
3 establishment of animal model of radioactive intestinal injury
The Wistar female rats, 240– 260 g, were irradiated with 5 Gy (0.287 Gy/min for 17.42 min) for 5 times, radiation dose rate is 0.287/MIN. The interval of irradiation at two times was 72 h. The exposed area was 8.5 cm×8.5 cm. The source-skin distance was 60 cm.
4 Observation of MSC ecesis position in vivo
The MSCs labeled with DAPI were injected into animal model. The animals were executed by luxation 2 and 7 d after injection, respectively. The ileum tissues were taken out to make into frozen section which was observed with confocal microscopy. There were circular fluorescent images in the frozen section in the first 48 h after injection of MSCs labeled with fluorescent. The circular fluorescent image was the lumina tissue of micro-circulatory system confirmed by pathology. The result shows that, because the MSCs enter into the blood vessel endothelium of the injuried tissue of microcirculation system, the MSCs firstly repaired the small vessels and improved the supply of the local blood and oxygen, and the state of damaged cell nutrition. Finaly, the cells and tissues were repaired.
Some experiments showed that bone marrow mesenchymal stem cells (BMSCs) can differentiate into intestinal epithelial cells by using inducing factors in vitro. In this study we found that bone marrow-derived mesenchymal stem cells can differentiate and replace injuried and necrotic cells at the proper circumstance with various kinds of biological factors and the emergency request of body.
5 Pathological changes after MSCs injected into rats
The nuclear fission in both model and treatment groups all increased, and it in the 30 d treatment group increased significantly as compared with that in the model group. The inflammatory cells seepage in both groups all decreased, and started to restore after 15 d. There were the degeneration, necrosis and defluxion of epithelial cells in both groups, more severe in the 3 d model group. The mucosa in the treatment group was thicker than that in the corresponding model group. The interstitial fibrosis proliferated obviously in the 30 d model group. The intestinal necrosis is lighter in the 7 d treatment group than that in the model group in the treatment group, especially in ileum.
研究结果表明,将标记的MSCs静脉注入放射性肠损伤模型动物体内,经冰冻切片,共聚焦显微镜观察标记细胞位置,发现2 d、7 d时MSCs在损伤的肠壁内聚集并逐渐增多,30 d治疗组肠粘膜细胞分裂相增多、间质纤维增生减少,证实MSCs对放射性肠损伤具有修复作用,同时也验证了本研究制作的放射性肠损伤动物模型适合医学动物实验研究。
Radiaction intestinal damage is seen frequently in the nuclear accident and the nuclear explosion of wartime, and is one of the most common complications in radiotherapy course of abdominal tumor. The apoptosis and proliferation inhibition occur significantly in the intestinal crypt stem cells after the abdomen is irradiated with a certain dose, which lead further to the damages of the structure of mucosa, the dysfunction of intestinal barrier, intestinal infection and the development of chronic diseases. At present, the treatment of radioactive intestinal injury mainly is still the support of the symptoms, but curative effect is not good. Thus, it is of great significance to explore effective measures for the promotion of intestinal radiation damage repair. Bone marrow mesenchymal stem cells (MSCs) are present in the bone marrow of non-hematopoietic stem cells, which have the differentiation potential. In appropriate conditions, MSCs can be divided into bone, cartilage, tendon, fat and other organizations, also differentiate into intestinal epithelial cells, liver cells, alveolar cells, pancreas cells, skin and nerve cells and so on. MSCs can be used in transplantations between different species and different individuals for the characteristic of MSC low immunogenicity.
At the intestinal radiation damage, the intestine oneself loses the ability of repair because the intestine crypt stem cells were seriously damaged. It is the focus of this study whether the exogenous MSCs home to damaging intestine through the transplantation and have the repair function of intestinal injury. In this study, based on the multi-polarization theory of MSCs, we used the rat bone marrow mesenchymal stem cells which were cultured in vitro, purified, and injected into the animal model bodys of the intestinal radiation damage, which explore the repair effect of MSCs on the radioactive intestinal injury and its mechanisms.
1 Culture of rat MSCs in vitro
In this study, we used the adherent culture methods to get a large number of MSCs. The MSCs were detected by the flow cytometry, in which the percentage of G0-G1 cells were 76.65% of the total number of the cells. And the unlimited proliferation of MSCs was further illuminated.
2 Induction and regeneration of MSCs in vitro
The purified MSCs were added into the osteoblasts solution, which inductively cultured for two weeks. Then, the induced product of the cells was identified with the alkaline phosphatase staining method. The results showed that rat bone marrow mesenchymal stem cells cultured in vitro can be induced to osteoblasts which were positive by specific staining. Therefore, the MSCs are proved to have the differentiation potentia.
3 establishment of animal model of radioactive intestinal injury
The Wistar female rats, 240– 260 g, were irradiated with 5 Gy (0.287 Gy/min for 17.42 min) for 5 times, radiation dose rate is 0.287/MIN. The interval of irradiation at two times was 72 h. The exposed area was 8.5 cm×8.5 cm. The source-skin distance was 60 cm.
4 Observation of MSC ecesis position in vivo
The MSCs labeled with DAPI were injected into animal model. The animals were executed by luxation 2 and 7 d after injection, respectively. The ileum tissues were taken out to make into frozen section which was observed with confocal microscopy. There were circular fluorescent images in the frozen section in the first 48 h after injection of MSCs labeled with fluorescent. The circular fluorescent image was the lumina tissue of micro-circulatory system confirmed by pathology. The result shows that, because the MSCs enter into the blood vessel endothelium of the injuried tissue of microcirculation system, the MSCs firstly repaired the small vessels and improved the supply of the local blood and oxygen, and the state of damaged cell nutrition. Finaly, the cells and tissues were repaired.
Some experiments showed that bone marrow mesenchymal stem cells (BMSCs) can differentiate into intestinal epithelial cells by using inducing factors in vitro. In this study we found that bone marrow-derived mesenchymal stem cells can differentiate and replace injuried and necrotic cells at the proper circumstance with various kinds of biological factors and the emergency request of body.
5 Pathological changes after MSCs injected into rats
The nuclear fission in both model and treatment groups all increased, and it in the 30 d treatment group increased significantly as compared with that in the model group. The inflammatory cells seepage in both groups all decreased, and started to restore after 15 d. There were the degeneration, necrosis and defluxion of epithelial cells in both groups, more severe in the 3 d model group. The mucosa in the treatment group was thicker than that in the corresponding model group. The interstitial fibrosis proliferated obviously in the 30 d model group. The intestinal necrosis is lighter in the 7 d treatment group than that in the model group in the treatment group, especially in ileum.
引文
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