| Objective: Cerebral infarction is common and severe disease which usually results in death and disability. No approach has been demonstrated to have benefits on most patients with cerebral infarction except thrombolytic therapy at early stage and rehabilitation. How to improve the neurological function of patients suffering from cerebral infarction is a key point which needs to be explored. Cell substitute therapy is thought to be a prospective means in settling this problem. Plasticity of HSCs gives us a possibility for clinical application of HSCs. The aim of this study was to verify whether HSCs transplanted can be alive and differentiate into neural stem cell,neural cell and vascular endothelial cell and rebuild neurovascular circulation in boundary zone around infarcton core and effects of this therapy by evaluating the survival,migration and differentiation of HSCs,NSS and infarction volume after transplantation. The influences of NGF on the survival,migration and differentiation of HSCs was aslo assessed. The effects were appraised between HSCs transplant- ation and HSCs mobilization to explore more effectively HSCs therapeutic strategy for cerebral infarction.Methods 1 The bone marrow stem cells were mobilizated into the peripheral blood by hyodermic of recombinant human granulocyte colonystimulating factor (rhG-CSF). Stem cells obtained by Ficoll gradient separation were detected by immunohistochemistry for CD34 which is specific surface antigen of HSCs. MCAO models were done according to reforming Longa's method and all adult male rats were randomly divided into 5 groups: group A (HSCs group), group B(HSCs and NGF group), group C (NGF group), group D (the control group), group E (HSCs mobilization group). 7 days after MCAO, we injected 1×106 HSCs labeled with (bromodeoxyuridine) Brdu as a tracer into the region where was: 1.2mm behind bregma, 3mm right side of midline, 4mm below duramater in group A,1×106 HSCs labeled with Brdu along with NGF 200ng in group B; NGF 200ng in group C, PBS in group D. Rats of group E were injected subcutaneously with rhG-CSF (20ug/kg) for the first time, later 10ug/kg/day for five consecutive days. NSS tests were performed to assess the improvement of neurological functions at 1,2,3,4 weeks. Rats were killed and taken off the brain at the point of 1,4 weeks after transplantation. Immunohisto- chemisty was used to observe the distribution of CD34,Nestin,VEGF,vWF cells in the infarct area and investigate the survival,migration and differentiation of HSCs transplanted by detecting Brdu/CD34,Brdu/Nestin,Brdu/Nse,Brdu/vWF. Infarction volume was measured by TTC staining at the point of 4 weeks. 2 Statistical Methods: Quantitative data analyzed by T-test analysis and analysis of variance. Results were regarded as significance when P<0.05, SAS 6.12 Statistical analysis software was used to deal with the data.Results1 5.24-7.96×106 cells can be obtained from every rat, in which 91.5% of cells were HSCs.2 The labled rate of HSCs with Brdu was 98%.3 Before transplantation, the living rate of HSCs was 100% on excluding test.4 The recovery of neurological function of rats after transplan- tationAt 1 week the neurological deficit of rats in group E improved significantly compared with the other four groups (P<0.05) in which there were no significant differences; At 2 weeks neurological function of rats in group A,B,C,E enhanced significantly compared with group D (P<0.05). The differences between NSS before transplantation and 2 weeks after transplantation in group E was the hightest. NSS did not remarkably descend in group A,B,C; At 3 weeks the improvement of neurological function in group A,B was better than group D, but no significant difference was found between group A and B. The differences in group C,D,E did not increase remarkably; At 4 weeks NSS were obviously lower in group A,B,C,E than group D. The recovery of neurological function of rats in group B was the best, and group A was the second (P<0.05), there was not remarkable contrast between group C and group E.5 Immunohistochemisty showed5.1 At the point of 1 week5.1.1 CD34-positive cells were found in all group. In group A,B around position of injection there are more positive cells than those in group D (P<0.05); There were more CD34-positive cells in group B than in group A; There were only a few CD34- positive cells in group C,D,E, in which there were no significant differences.5.1.2 In all groups there were Nestin-positive cells in the marginal zone of the infarction and injection. There were more positive cells in group A,B,C than those in group D(P<0.05); A significant increase in numbers of positive cells in group B was detected compared with group A (P<0.05); There were fewer positive cells of group C than group A; No difference was found between group D and group E.5.1.3 There were statistically significant for vWF-positive cells among all groups. Positive cells of group A,B,C,E were more than those of group D (P<0.05); There were much more vWF cells in group B than those in group A; More positive cells can be found in group A than those in group E (P<0.05); There was no real distinction between group C and E.5.1.4 The expression of VEGF in group A,B,C was significantly higher than group D in the marginal zone of the infarction (P<0.05). The level of VEGF expression in group A, B was higher than that group C, but there was no difference between group A and B,the same as D and E (P>0.05).5.1.5 vWF cells increased with the level of VEGF expression improving 1 week after tranplantation. There was the linear correlation between vWF and VEGF.5.2 At the point of 4 weeks5.2.1 No CD34-positive cells were found in all groups. There were significant differences compared with those at 1 week.5.2.2 There were more Nestin cells in group A,B,C than group D; there were more Nestin cells in group A,B than group C, but there were hardly any differences between group A and B; There was no difference between group D and E. There was a decline in the numbers of Nestin cells at 4 weeks compared with those at 1 week (P<0.01).5.2.3 There were much more vWF-positive cells in group A,B,C,E than group D. A=B>C=E>D. At 4 weeks the numbers of vWF-positive cells were more than those at 1 week except group D (P<0.01).5.2.4 There was statistically significant in expression of VEGF among groups. The level of VEGF of group A,B,C was highter than that of group D (P<0.05); It was higher in group B than group A; there was higher in group A than group C; There was no difference between group D and group E. The level of VEGF expression at 4 weeks declined remarkably compared with that at 1 week. The expression of VEGF of group A and C significantly decreaced compared with that at 1 week (P<0.01); No change was observed in group B,D,E.6 The survival and differentiation of HSCs6.1 At the point of 1 week6.1.1 There were some Brdu/CD34-positive cells in group A and B in the injecting points: 6.77±1.24,10.10±1.71, There were more positive cells in group B than those in group A (P<0.01).6.1.2 Brdu/Nestin-positive cells were observed in the marginal zone of the infarction. The numbers of group B (13.17±2.61) were more than those of group A (9.57±1.95), there was statistical significance (P<0.01).6.1.3 A few Brdu/Nse-positive cells were found in the marginal zone of the infarction in group A,B: 8.27±3.58,12.00±1.85, there was statistically significant (P=0.0002).6.1.4 Immunohistochemistry doublestaining for Brdu/vWF showed Brdu/vWF positive cells in group B (2.47±0.78) were more than those of group A (1.03±0.29) (P<0.05).6.2 At the point of 4 week6.2.1 No Brdu/CD34 immunpositive cells were observed in group A and B 4 weeks after transplantation. There was significant difference compared with those at 1 week.6.2.2 Brdu/Nestin-positive cells were found in the marginal zone of the infarction, the numbers of group A were 5.67±1.63, the numbers of group B were 5.47±0.60, there was no significant difference. In contrast at 4 weeks the numbers of Brdu/Nestin-positive cells in group A,B cut down remarkably compared with those at 1 week (P<0.01). 6.2.3 No significant difference was found between group A (17.43±3.78) and group B (22.10±4.37) in Brdu/Nse-positive cells. The quantities of Brdu/Nse-positive cells at 4 weeks were statistically higher than those at 1 week in group A,B (P<0.01).6.2.4 The numbers of Brdu/vWF cells of group A (3.37±1.34) were not fewer than that of group B (4.20±0.68). A significant increase in numbers of Brdu/vWF cells compared with those at 1 week (P<0.01) in group A and B.6.3 There was the linear correlation between Brdu/Nse-positive cells and the differences between NSS before transplantation and 4 weeks after transplantation in group A,B. This showed the more Brdu/Nse-positive cells, the more improvement of neurological functions.6.4 At 1,4 weeks Nestin cells were much more than Brdu/vWF cells in group A,B (P<0.001).7 At the point of 4 weeks after transplantation the percent of infarction volume in the whole volume of the cerebrum was 8.77%,11.15%,20.46%,23.86%,19.89%,P=0.001, there was statistically significant. A=B>C=E>D.Conclusion1 HSCs transplanted in the boundary zone around infarction core of rats can survive and differentiate into neural stem cells,neuron cells.2 HSCs transplanted in the boundary zone around infarction core of rats can differentiate into endothelial cells and increase the level VEGF expression,further promote angiogenesis. 3 HSCs transplantation can improve neurological function recovery of rats with focal cerebral infarction and lessen the size of infarct.4 NGF has the effect on promoting the survival,differentiation of HSCs transplantated in the brain.5 Intracerebral administration of NGF can induce multiplication of endogenesis neural stem cells and improve the neurological functions in rats.6 Endogenesis mobilization of HSCs with rhG-CSF can improve neurological functions, but it would not be better than intracerebral HSCs transplantation in improving the neurological functions in rats. |
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