| Objective:To observe the effect of the mobilization of bone marrow stem cells through the changes of CD34 + cells in the peripheral blood and brain tissue; To reveal the protective effect of Naomaitong associated with bone marrow stem cell mobilization agent on brain nerve cell damage after focal cerebral ischemia, through the observation of the nerve function, brain water content , cerebral infarction area, the dynamic changes of brain tissue pathology in rats after cerebral ischemia injury ; To explore the protective effect and the mechanism of action of Naomaitong associated with bone marrow stem cell mobilization agent on brain damage after focal cerebral ischemia, through the observation of the changes of neuronal apoptosis and related apoptosis-regulating gene expression.Method:In this study, SD rats were divided into sham-operated group, model group, BMSCs mobilized group (mobilization), Naomaitong group, the Naomaitong associated with BMSCs mobilization group (associated group) according to the random number table, then were divided into 2d,3d,7d,14d group. Cerebral ischemia animal model was duplicated in accordance with improved Longa's focal middle cerebral artery occlusion (MCAO) model; sham group, model group, the mobilization group before making model four days were intragastric administration with physiological saline one time each day, to add one time before making model ,then were intragastric administration once daily after making model until rats were killed (intragastric administration volume of 1ml?100g-1?d-1); Naomaitong group and the associated group were intragastric administration with Naomaitong four days before making model, plus intragastric administration before making model one time, intragastric administration once daily after making model until rats were killed (40.5mg?100g-1?d-1, 40.5mg? ml-1). The associated group and mobilization group rats were injected G-CSF three days before making model, were injected two days after making model once daily. To observe the changes of WBC in peripheral blood, CD34+ in blood and brain tissue. To observe the changes of weight and the survival of rats, to judge the nerve function with a comprehensive evaluation of rat nerve function score, to calculate brain tissue water content and cerebral infarction area of rats.The pathological changes of brain tissue were observed under light microscope, the changes of cell ultrastructure were observated under transmission electron microscopy, to observe nerve cell apoptosis by TUNEL and the changes of Bcl-2,Bax, Fas , FasL, Caspase-3 expression under immunohistochemistry chemical.Results:①Compared with the sham-operated group , WBC and CD34+ cells in peripheral blood of the model 2d, 3d group were higher, and peak amplitude was in 3d group, and then gradually declined.Compared with the model group, the WBC, CD34 + cells of the mobilization and the associated group 2d, 3d were higher and the most significantly in 3d, the Naomaitong group was no significantly change. The WBC count of associated group 2d, 3d were significantly higher than the mobilization group, the WBC in the peripheral blood of the associated group of 7d maintained a high level, the CD34+ cells in the blood of the associated group 3d, 7d increased more significantly than the mobilization group.②There were no expression of CD34+ cells in the sham group. Compared with the sham-operated group,the expression of CD34+ cells in the brain tissue of the model rats were higher, reached its peak at the 7d, then gradually reduced. Compared with the model group, the expression of the CD34+ cells of the mobilization groups and the associated group were significantly higher, and the most significantly was the 7d.There were no significantly changes of CD34+ cells express in Naomaitong group.Compared with the Naomaitong group , the expression of CD34+ cells in the brain tissue of the mobilization group were significantly higher.The expression of CD34+ cells in the brain tissue of the associated group were increased more significantly than the Naomaitong group and the mobilization group ,and the most significantly was the 7d.③Compared with the sham-operated group , the mortality rate of the model rats were significantly higher, the weight loss at 2d, 3d, 7d was higher, the weight growth rate at 14d was lower, the nerve function score was lower, the brain water content was higher, the area of cerebral infarction was higher. There had no obvious pathological damage in the sham group, the model group at different time points nerve cells had different degrees of damage and structural damage ,the most serious at 7d group. Compared with the model group, the weight loss of the Naomaitong group 2d, 7d and the mobilization group 3d and the associated group 2d, 3d, 7d reduced, the weight growth rate of 14d group were higher, nerve function score was significantly higher, the water content of brain tissue were significantly lower, the cerebral infarction significantly lower, the damage of brain tissue pathology reduced. And the associated group effected more significantly than the the Naomaitong group and mobilization group, especially at the 14dth. With the time of ischemia the nerve function damage in rats at the 2dth were most serious, then gradually resumed with time, the brain tissue and water content of cerebral infarction at the 7dth were the most serious, then reduced at the 14dth, the nerve cell injury were the most serious at the 7dth, reduced at the 14dth.The amount of the nerve cell degeneration and necrosis decreased significantly, interstitial edema was lighter, especially at the 14dth of the associated group.④Compared with the sham-operated group,the nerve cells apoptosis increased in the model group. The nerve cells apoptosis in the Naomaitong group 2d, 14d, mobilization group 2d, 3d and the associated group 2d, 3d and 14d nerve cells apoptosis were significantly less than the model group,the Naomaitong 14d group nerve cells apoptosis were significantly less than the mobilization group. Compared with the associated group ,the nerve cells apoptosis increased significantly in the mobilization group 2d and the Naomaitong group 2d,3d. The nerve cells apoptosis most serious at the 2dth in the model group, Naomaitong group, mobilization group and the associated group.⑤Compared with the sham-operated group, Fas, FasL expressed more strengthen in the model group. Compared with the model group , Fas, FasL expressed more weakenly in the Naomaitong group 7d,14d, mobilization 2d group, the associated group, Fas, FasL expression significantly higher in the mobilization group 2d than the Naomaitong group. Fas, FasL expressed significantly lower in the model, the Naomaitong group, the associated group 7d, 14d group and the mobilization group 14d than at the 2dth, the 14d group than in their 3d group Fas, FasL expression were lower.⑥Compared with the sham-operated group, Bcl-2, Bax expressed higher than the model group, compared with the sham-operated group,the Naomaitong group 14d, the associated group 3d, 7d, 14d group Bcl-2, Bax expressed higher, Bax expressed lower in the Naomaitong group 7d, mobilization 3d ,7d group, the associated group. Bcl-2 expressed higher in the Naomaitong group 14d than in the mobilization group. Bax expressed lower at the 14dth than in all of its 2d, 3d group.⑦Compared with the sham-operated group, Caspase-3 expressed higher in the model group,Caspase-3 expressed lower in the Naomaitong group 7d,14d and the associated group than the model group, the model group,mobilization group and the Naomaitong group 7d, 14d and the associated group 3d, 7d, 14d Caspase-3 expressed lower than at 2d, Caspase-3 expressed lower in the Naomaitong group 7d, 14d group than the 3d, Caspase-3 expressed lower in the model, mobilization group 14d than at 3d.Conclusion:①G-CSF could mobilize the bone marrow stem cells, promote CD34+ significantly increased in the blood and brain tissue,Naomaitong could improve the micro-environment of BMSCs mobilization. Their combined effect was more significant, and the role of a longer time.②The mortality and weight loss rate increased, weight growth rate reduced, neurological defects, cerebral edema, infarction lesions formation and nerve cells damaged after cerebral ischemia , most serious at 7d. The protective effect was obvious along with the days passed by Naomaitong ,BMSCs mobilization and the associated of both of them protected, and enhance the role of a trend extended over time, the most significantly was associated 14d.③A lot of nerve cells apoptosis after cerebral ischemia, Caspase-3, Fas, FasL expressed increased, Bcl-2, Bax expressed increased, especially Bax increased. Naomaitong, BMSCs mobilization and the associated of both of them could reduce the number of nerve cells apoptosis, increased the Bcl-2 expression which could suppress apoptosis, reduced Fas, FasL, Bax Caspase-3 expression which could promote apoptosis.The effection was more significantly after their joint application.
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