In situ transplantation of autologous bone marrow stem cells mobilized by Granulocyte colony-stimulating factor for treatment of ischemic cerebral infarction in rats / 中国组织工程研究

ABSTRACT

BACKGROUND:Granulocyte colony-stimulating factor (G-CSF) can strongly mobilize bone marrow hematopoietic stem cells (HSCs). It has been proved that G-CSF has the ability to mobilize both HSCs and mesenchymal stem cells (MSCs).OBJECTIVE:To investigate the therapeutic effect of G-CSF in mobilizing autologous bone marrow stem cells entering cerebral infarction zone on ischemic cerebral infarction in rats.DESIGN:A randomized grouping design, animal experiment.SETYING: Center for Stem Cell Biology and Tissue Engineering of Sun Yat-sen University.MATERIALS: This experiment was carried out at the Animal Experimental Department of Sun Yat-sen University (North District) and Center for Stem Cell Biology and Tissue Engineering of Sun Yat-sen University from September 2004 to January 2005. Totally 200 male Wistar rats were chosen and randomly divided into autologous bone marrow stem cells transplantation group and control group, with 100 rats in each group.METHODS:Rats of two groups were made cerebral infarction models by line occlusion. Transplantation group introduced intraperitoneal injection of 60 μg/kg G-CSF one hour after operation. The control group introduced intraperitoneal injection of saline of the same dosage at the same time. ①All rats were weighed before operation and 24 hours, 48 hours, one week after operation to evaluate body mass loss rate. They were also given neurological grading. Grading criteria: Grade 0 is normal. Grade Ⅰ is that the right forelimb bends. Grade Ⅱ is that the right forelimb grasped weakly when the tail is lifted. Grade Ⅲ is that the rat has no directivity in automatic action and circumrotates to right when the tail is lifted. Grade Ⅳ is that the rat circumrotates to right in automatic action. ②15 rats in each group were selected. 24 hours, 48 hours, one week after operation, we opened the skulls, took out the brain and used 2,3,5-Triphenyltetrazoluim Chloride (TTC) staining to measure infarction volume, hematoxylin-eosin(HE) staining to observe the pathological change , and immunohistochemistry to detect the infiltration of CD34+ cells.MAIN OUTCOME MEASURES:Body mass loss rate, neurological grade,infarction volume, pathological change and infiltration of CD34+ cells.RESULTS: Totally 180 of 200 rats were successfully made cerebral infarction model. 48 rats died in seven days after operation. As a result, 132 rat models were alive and 120 rats were randomly selected for data analysis. ①Measurement of body mass and neurological grading: There was no significant difference in body mass loss rate between two groups 24 hours and 48 hours after operation (P ＜ 0.05);one week after operation, body mass loss rate was significantly lower in transplantation group [(10.5±8.2)%]than in control group [(17.8±7.1)%] (P ＜ 0.05). There was no significant difference in neurology grade between two groups. ②Infarction volume:Infarction volume and the percent of infarction volume in the whole brain in control group were all higher than those in the transplantation group,with significant difference [ (251.69±52.77) mm3 vs(145.72±28.05)mm3,(17.00±2.69)% vs (9.90±1.62)% ,P ＜ 0.01]. ③Pathological change: 24 hours after operation, the brain tissue of two groups got classical pathological change of cerebral ischemia infarction. There were some mono-nucleus cells infiltrating in transplantation group while none in control group. 48 hours after operation, most nerve cells disappeared and the glial cells were degenerated. There were many mono-nucleus cells infiltrating in transplantation group while a few in control group. One week after operation, tissues in the infarction zone were liquescent with many monocaryons and lymphocytes infiltrating around them in control group. In transplantation group, part of the infarction zone was plerosised through proliferation of newly born capillaries and glial cells and inflammatory cells were not evident. ④Immunohistochemistry: CD34+ mono-nucleus cells were detected in the ischemic territory in transplantation group 24 hours after operation while none in the brain of other side and control group. There were CD34+ mono-nucleus cells and pyramidate cells with mutations in transplantation group 48 hours after operation while none in the brain of other side and control group.CONCLUSION:The stem cell transplantation in situ therapy, which employs self-marrow stem cells mobilized by G-CSF can relieve the ischemic degree and reduce the infarction volume.