| Objective: Using stem cell to treat cerebral ischemia in experimental study has achieved a lot recent years, and it has been demonstrated that using recombinant human granulocyte colony stimulating (rhG-CSF)can mobilize the stem cells to peripheral blood , locating in the area adjacent to cerebral infarction core and differentiating into nerve cells. But the previous experiment study was aimed to evaluate the efficacy by giving rhG-CSF as soon as cerebral infarction model was made, there are little experimental study on admini- stration rhG-CSF some time after infarction,howere,it is necessary to know the effect of different time particular late,as the majory of cases may be present very late. In this study, the MCAO model of rats were given subcutaneous injection of rhG-CSF in different time windows after the model finished,observing the rat neural function recovery at different time, to explore the best treatment time; and useding 5-bromo-deoxy-uracil (Brdu) to mark bone marrow stem cells which in a proliferative state after mobilization, observing the migration and differentiation of stem cells were mobilized,and to explore of the possible mechanism that rhG-CSF improve cerebral infarction prognosis by the cell count, infarction volume measurement and testing apoptosis related factors.Methods:1 Select 280-320g healthy male SD rats, MCAO(middle cerebral artery occlusion, MCAO) models were done according to reforming Longa's method and all rats were randomly divided into 3 groups: 24 hours, 72 hours, 7 days according to the administration time, each group was randomly divided into experimental and control group. Each group was of 24 rats. Each experimental group were given cervical subcutaneous injection of recombinant human granulocyte colony stimulating factor (rhG-CSF),the first day was 20ug/kg, later 10ug/kg/day for five consecutive days, while giving the experimental rats Brdu 50mg/kg/day by intraperitoneal injection, until applied to the day killed the rat,to mark the nascent bone marrow stem cell mobilized by rhG-CSF. MCAO model of control groups were given cervical subcutaneous injection of the same dose saline,Brdu 50mg/kg/day intraperitoneal injection, continuous applied to the day killed the rat. After administration 5 days,14 days and 21 days, respectively, to take out the rat brain.The rat brain tissue at 5 days was aimed to study the endogenous mobilization of bone marrow stem cells through the CD34 antigen by immunohistochemistry. The bcl-2/bax antigen staining to understand neural cell apoptosis. At 14 days and 21 days, the Brdu/GFAP, Brdu/Nestin, Brdu/Nse double staining immuno-histochemistry to understand of the survival, migration, differentiation of the cells, The bcl-2/bax antigen staining to understand neural cell apoptosis.Infarction volume was measured by TTC staining after administration 21 days.After administration 5 days,14 days and 21days,through the neurolo- gical deficit score (Neurological Severity Scores, NSS) to understand the improve neurological function in rats.2 Statistical Analysis: SAS V8 software was used to deal with the data,all results were indicated by mean±standard deviation ( x±s).Quantitative data analyzed by t-test analysis and analysis of variance. Mortality rate use multiple samples chi-square test. According to a=0.05 as the test standard, the data for statistical analysis.Results:1 mortality rate:1.1 the mortality after the model were finished:there were no significant difference between group A(20%) and group a(38.5%),groupB(38.5%) and groupb(42.6%),groupC(40%) and groupc(41.5%)(p>0.05); There were no significant difference among group a,group b and group c(p>0.05);There were no significant difference among group A,group B and group C(p>0.05).1.2 the mortality after administration:there were no significant difference between group A (4%)and group a(8%)(P>0.05), group B, group b, group C and group c were 0.there were no significant difference among group A,group B and group C(p>0.05). There were no significant difference among group a,group b and group c(p>0.05)2 neurological deficit scores:2.1 at 5 days,14days and 21days2.1.1 groupA 0.05).2.2 there were no significant difference in group A or group B or group C at 14 days and 21days(p>0.05).3 infarct volume:3.1 group A>group a,group B>group b, group C>group c,there were signifi- cant difference between group A and group a,group B and group b,group C and group c(p<0.05).3.2 There were no significant difference among group a,group b and group c(p>0.05).3.3 There were significant difference among group A,group B and group C(p<0.05). But there were no significant difference between group A and group B(p>0.05).4 Immunohistochemical results:4.1 CD34+ positive cells:at 5 days,14days and 21days.4.1.1 group A>group a, group B>group b, group C>group c,there were signifi- cant difference between group A and group a,group B and group b,group C and group c(p<0.05).4.1.2 There were only a little CD34+ positive cell,and no significant difference among group a,group b and group c(p>0.05).4.1.3 There were many CD34+ positive cells mainly around the infarction area,there were no significant difference among group A group B and group C(p>0.05). 4.2 Bcl-2: at 5 days,14days and 21days.4.2.1 group A>group a,group B>group b, group C>group c,There were signifi- cant difference between group A and group a,group B and group b,group C and group c(p<0.05).4.2.2 There were only a little Bcl-2 positive cells,and no significant differenc- es among group a,group b and group c(p>0.05).4.2.3 There were many Bcl-2 positive cells mainly around the infarction area,there were no significant difference among group A,group B and group C(p>0.05).4.3 bax: at 5 days,14days and 21days.4.3.1 group A0.05)4.3.3 There were a little bax positive cells mainly around the infarction area,there were no significant difference among group A,group B and group C(p>0.05).4.4 GFAP/Brdu:at 14days and 21days.4.4.1 group A0.05)4.4.3 There were a little GFAP/Brdu positive cells mainly around the infarction area,there were no significant difference among group A,group B and group C(p>0.05).4.5 NSE/Brdu:at 14days and 21days.4.5.1 group A>group a,group B>group b, group C>group c,there were significant difference between group A and group a,group B and group b,group C and group c(p<0.05).4.5.2 There were a little NSE/Brdu positive cells mainly around the infarction area, there no significant difference among group a,group b and group c. (p>0.05)4.5.3 There were many NSE/Brdu positive cells around the infarction area, but also hippocampus and the other hemisphere,there no significant difference among group A,group B and group C. (p>0.05)。4.6 Nestin/Brdu:at 14days and 21days.4.6.1 group A>group a,group B>group b, group C>group c,there were significant difference between group A and group a,group B and group b,group C and group c(p<0.05).4.6.2 There were a little Nestin/Brdu positive cells mainly around the infarction area, there no significant difference among group a,group b and group c. (p>0.05)4.6.3 There were many Nestin/Brdu positive cells around the infarction area,but also hippocampus and the other hemisphere,there no significant difference among group A,group B and group C. (p>0.05).Conclusion:1 Using rhG-CSF after cerebral infarction can mobilize bone marrow stem cells and aggregate around the infacrtion area,and differentiation into neural stem cells and neuron cells.2 Using rhG-CSF after cerebral infarction can increase the experission of the anti-apoptotic factor, depress the experission of factor that promote apoptosis.3 Using rhG-CSF after cerebral infarction within 72 hour can decrease the infarction volume.4 Using rhG-CSF after cerebral infarction can improve the neurological deficit scores,there are no difference among administration at 24h,72h and 7days.
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