【作者】 张轶丹；

【导师】 赵建军；

【作者基本信息】 长春中医药大学 ， 中医内科学， 2013， 博士

【摘要】 目的：通过研究大鼠脑出血后神经干细胞的反应过程及在这一过程中相关因子的变化,探讨破血化瘀、填精补髓法对神经干细胞增殖分化的影响;并以脑源性神经营养因子(BDNF)、碱性成纤维细胞生长因子(bFGF)和内源性干细胞因子(SCF)为切入点,通过探讨该法对脑源性神经营养因子(BDNF)、碱性成纤维细胞生长因子(bFGF)和内源性干细胞因子(SCF)的调控机理,研究其对脑出血周围组织损伤修复的机制和对NSC增殖、分化的影响。据此进一步确定破血化瘀、填精补髓法的治疗作用,拓宽了现代医学理论基础,为出血性中风治疗提供一条行之有效的方法,为临床的广泛运用提供科学依据；从NSC入手研究中医药治疗出血性脑卒中,阐明脑出血后NSC增殖、分化的机制,用中医药疗法加以诱导和加强,发挥NSC的自我修复潜力,以促进出血性脑卒中神经功能恢复,重建受损的神经网络。将脑病发病病机关键学说--髓虚毒损进一步完善,并丰富脑髓理论。方法：本研究选择Wistar大鼠,采用Bederson方法造成实验性脑出血模型,在造模后1、3、7d即症状的急性期分别取材,观察实验性脑出血大鼠血肿、水肿和神经功能缺损等整体动物行为学指标在破血化瘀、填精补髓治法的影响下的变化；实验观察实验性脑出血大鼠内源性NSC增殖分化在破血化瘀、填精补髓治法的影响下的变化；并进一步观察破血化瘀、填精补髓治法对诱导NSC增殖分化的的神经营养因子BDNF、碱性成纤维细胞bFGF、内源性干细胞因子SCP蛋白表达的影响。进一步设计实验选择观察表达相对明显的蛋白其相应受体的蛋白的表达情况及该信号转导通路的mRNA的表达情况。结果：(1)在给药第3天和7天分别观察对照药、实验方剂药及模型组大鼠神经功能缺损评分,与模型组大鼠比较,各组大鼠症状明显改善,神经功能缺损评分明显降低,并具有统计学意义(P<0.05或P<0.01)。(2)在给药第三天、第七天,对照药组,实验方剂低、中,高剂量组与模型组大鼠比较脑出血面积明显降低,具有显著统计学意义(P<0.05,P<0.001,P<0.001)。(3)在给药第三天后,对照药组、实验方剂中、高剂量组与模型组大鼠比较脑含水量明显降低,有显著性差异(P<0.05,P<0.01)。(4)给药第三天,实验方剂中剂量组Nestin阳性细胞光密度明显增高,与模型组比有显著的统计学意义(P<0.05)。受试药高剂量组Nestin阳性细胞总而积及光密度明显增高,与模型组比较具有显著的统计学意义(P<0.05)。第七天,对照组、实验方剂低剂量组Nestin阳性细胞总面积明显增高,与模型组比较具有显著的统计学意义(P<0.05),实验方剂中、高剂量组Nestin阳性细胞总面积及光密度明显增高,与模型组比有显著的统计学意义(P<0.01,P<0.01)。(5)给药第一天,与模型组比较,实验方剂高剂量组BrdU阳性细胞明显增高,与模型组比有显著的统计学意义(P<0.05)。第三天,实验方剂高、低剂量组BrdU阳性细胞明显增高,与模型组比有显著的统计学意义(P<0.05,P<0.05)。第七天,实验方剂高、中剂量组BrdU阳性细胞明显增高,与模型组比有显著的统计学意义(P<0.01,P<0.05)。(6)随着时间的变化,神经干细胞的分化与实验方剂药物浓度明显关联。在给药第三天,第七天,实验方剂高剂量组和中剂量组BrdU/GFAP、BrdU/NSE双标阳性细胞明显增高,与模型组比较具有显著的统计学意义(P<0.05, P<0.05, P<0.001)。(7)对照组、实验方剂低、中、高剂量组大鼠BDNF阳性细胞数目在术后第一、三、七天,随时间变化有显著的变化,与第一天比,第三、七天BDNF阳性细胞数目明显增加,有显著的统计学意义(P<0.05,P<0.01)。(8)在对照组及实验方剂中剂量组大鼠bFGF阳性细胞数在术后第一、三、七天,随时间变化有显著的变化,与第一天比较,第三、七天bFGF阳性细胞数目明显增加,有显著的统计学意义(P<0.01,P<0.01)。实验方剂低、高剂量组大鼠bFGF阳性细胞数数在术后第一、三、七天,随时间变化有显著的变化,与第一天比,第七天bFGF阳性细胞数目明显增加,具有显著的统计学意义(P<0.05)。(9)对照组、实验方剂中、高剂量组大鼠SCF阳性细胞数目在术后第一、三、七天,随时间变化有显著的变化,与第一天比,第七天SCF阳性细胞数目明显增加,有显著的统计学意义(P<0.05)。(10)与模型组大鼠比较,实验方剂各组大鼠的BDNFmRNA表达量明显增加,有统计学意义。结论：通过本实验研究,证明了无论是在改善脑出血大鼠神经功能缺损,还是在促进血肿吸收、降低脑含水量方面,应用“破血化瘀,填精补髓”法方剂都有明显的作用；成功的诱导内源性NSC增殖、分化及参与病损的修复；从蛋白水平来研究其作用机制,并证明其可能为促进脑出血灶血肿周围损伤组织中脑源性神经营养因子BDNF,碱性成纤维细胞bFGF,内源性干细胞因子SCF升高,从而促进内源性NSC增殖、分化及参与病损的修复；并从基因水平来研究其作用机制,并证明其可能为促进脑出血灶血肿周围损伤组织中SCFmRNA表达有关。更多还原

【Abstract】 Purpose:This paper explores the effect that the method of stasis-break and enriching essence and marrow proliferate and differentiation NSCs by researching rat’s reaction process of NSCs after hematencephalon and changes of correlation factor in this process; takes BDNF, BFGF, SCF as cut-in point,researches the mechanism and impact that this methord acts on repairing texture around hematencephalon part and proliferating and polarising NSCs by disscussing regulation and control mechanism for BDNF, BFGF, SCF. In view of above conclusion, confirm the therapeutical effect on the methord of stasis-break and enriching essence and marrow further, broaden its modern medical theories foundation about curing hemorrhagic stroke,provide scientific evidence for clinical wide spread application; With the view to induce NSCs’proliferation and polarization and take part in curing lesion by traditional Chinese medicine, explores a new interposing regulatory mechanism for reconstructing neurological function, provides new thought and curing means for traditional Chinese medicine deep research on this field, further deepens biological mechanism of action on Promoting neurological function damage repair by traditional Chinese medicineMethod:The research makes a model of experimental hematencephalon by Bederson on Wistar rat, draws a materials from the first, the third and the seventh day respectively during acute stage of semeiotic, observe the effect that the method of stasis-break and enriching essence and marrow changes experimental hematencephalon rats’hematoncus, oedema, neurologic impairment and other indexes of the whole ethology. Experiments observe how the method of stasis-break and enriching essence and marrow affects endogenous NSC proliferation and polarization of the experimental hematencephalon rat and further observe how the method of stasis-break and enriching essence and marrow affects BDNF, BFGF and SCF.Result:(1) Control drugs, experimental prescription drugs and model group rats neurological deficit scores, compared with the model group rats, the rats symptoms significantly improved neurological deficit scores were observed in the administration of3and7days reduce and statistically significant (P<0.05or P<0.01, respectively). (2) In the administration of the third day, the seventh day, the drug-control group, experimental prescription low, medium and high dose group and the model group comparison cerebral hemorrhage area was significantly lower, with a statistically significant (P<0.05, P <0.001,P<0.001).(3) On the third day after the administration, the drug control group, experimental recipe, the high-dose group compared with the model group rats brain water content was significantly lower, with a significant difference (P<0.05, P<0.01).(4) Administration of the third day of Traditional middle dose group Nestin positive cells in the optical density was significantly increased compared with the model group statistically significant (P<0.05). The drug to be tested high-dose group nestin-positive cells of the total area and the optical density was significantly higher, significant statistical significance (P <0.05) compared with the model group. The seventh day, Nestin positive cells in the total area of the control group, the experimental prescription low dose group was significantly higher compared with the model group with significant statistical significance (P<0.05) and high dose groups of Traditional Nestin positive cells in a total area of light The density was significantly increased compared with the model group statistically significant (P<0.01, P <0.01).(5) The first day of the administration, compared with model group, the experimental prescription high dose group of BrdU-positive cells was significantly higher compared with the model group statistically significant (P<0.05). The third day, the experimental prescription, BrdU-positive cells in the low dose group was significantly higher compared with the model group statistically significant (P<0.05, P<0.05). The seventh day of Traditional middle dose group of BrdU-positive cells was significantly higher compared with the model group statistically significant (P<0.01, P<0.05).(6)The neural stem cell differentiation and experimental prescription drug concentrations were significantly associated with the change of time. In the administration of the third day, the seventh day, the the experimental prescription high dose group and middle dose group BrdU/GFAP, BrdU/NSE double-labeled positive cells was significantly higher compared with the model group has significant statistically significant (P<0.05, P<0.05, P<0.001).(7) The control group, the experimental prescription low, medium and high dose group were BDNF positive cells after the first three or seven days, over time a significant change, with the first day than the third, seven days BDNF The number of positive cells increased significantly, there is a significant statistical significance (P<0.05, P<0.01).(8) In the control group and the experimental prescription dose group bFGF positive cells after the first three, seven days, over time there are significant changes, compared with the first day of the third, seven-day bFGF positive cells increased significantly, there is a significant statistical significance (P<0.01, P<0.01). Of Traditional high-dose rats bFGF positive cells after surgery first, third, seven days a significant change over time, than the first day, the seventh day of bFGF positive cells increased significantly, with significant statistically significant (P<0.05).(9) And the control group, the experimental prescription, high-dose group the number of positive cells in rat SCF after the first three or seven days, over time a significant change, with the first day of the seventh day the number of positive cells in SCF increased significantly, there is a significant statistical significance (P<0.05).(10)Compared with the model group rats, the rats of the experimental prescription BDNFmRNA expression was significantly increased, with statistical significance.Conclusion:According to the experiments research,proves that the method of stasis-break and enriching essence and marrow can obviously improve hematencephalon rat’s neurologic impairment, promote hematoma absorption, reduce BWC;success in inducing proliferation and differentiation of endogenous NSC, participate in lesion impairment;proves that its mechanism of action may increase BDNF, BFGF and SCF around hematencephalon part from protein level, thereby, promotes proliferation and differentiation of endogenous NSC, participate in lesion impairment and proves that its mechanism of action increase SCF,mRNA and C-kitmRNA receptor around hematencephalon partfrom gene level.更多还原