【作者】 张磊；

【导师】 王建枝； 刘丽江；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 中风是全球成年人致死性疾病的第三位，并长期位于成年人致残性疾病的首位。脑出血(ICH)约占西方人群中所有中风性疾病的10-15％，在亚洲人群中比例更高，至少达到30％。生长因子是一类参与调节神经细胞生存、增殖、分化发育的多肽。很多生长因子在脑创伤或中风后给药都显示出神经保护和神经营养作用。粒细胞集落刺激因子(G-CSF)是生长因子类细胞因子的一员，分子量约20KD。粒细胞集落刺激因子现已广泛的应用于临床，包括肿瘤化疗所致的白细胞减少症、骨髓的重构、造血干细胞的募集等。近年来研究发现，一些中风模型的造模小鼠给予粒细胞集落刺激因子后可诱导受损脑组织周围的神经发生，最终促进小鼠神经功能的恢复。但粒细胞集落刺激因子在脑出血过程中是否扮演相同角色尚未见报道。本研究试图探讨粒细胞集落刺激因子在脑出血中是否具有神经保护作用。我们选择健康wistar雄性大鼠40只，分为阴性对照组(20只)和G-CSF治疗组(20只)，全部进行脑出血模型造模。我们采用胶原酶Ⅶ定位注射大鼠尾壳核诱导大鼠脑出血的方法复制脑出血模型。造模24小时后G-CSF治疗组进行腹腔注射G-CSF(15μg／kg)，连续治疗5天，对照组给予生理盐水。我们每周通过肢体放置实验检测大鼠神经功能恢复情况，连续观察4周后发现，给药组可以明显促进脑出血大鼠神经功能的恢复。为了探讨G-CSF促进脑出血大鼠神经功能恢复的机制，我们用免疫组化的方法检测了脑片的三个指标，分别为神经干细胞标志nestin，造血干细胞标志CD34和星形胶质细胞标志GFAP，还通过Hoechest33258荧光显色的方法检测了脑出血大脑半球的细胞凋亡情况。最后，我们测量了出血坏死区病灶的面积。实验结果显示：1．G-CSF可促进出血灶周边组织神经干细胞的表达。2．G-CSF可动员骨髓造血干细胞入脑参与神经修复。3．G-CSF可增强脑出血后星形胶质细胞的增殖。4．G-CSF有抗凋亡的作用。5．两组大鼠的病灶面积未见明显差异。综上所述，我们的研究证实粒细胞集落刺激因子可促进脑出血大鼠神经功能的恢复，其机制可能与粒细胞集落刺激因子促进神经干细胞表达、动员造血干细胞入脑、刺激星形胶质细胞增殖以及其抗凋亡作用均有关系，但其具体机制仍需进一步深入研究揭示。更多还原

【Abstract】 Stroke is the third leading cause of death and a leading cause of long-term disability inadults worldwide.Intracerebral hemorrhage (ICH) represents 10-15% of all strokes in theWestern population and at least 30% in Asian countries.Growth factors are polypeptideswhich regulate survival,proliferation,maturation of developing neuronal cells.Manyfactors display endogenous neuroprotective and neurotrophic effects when they areexogenously administrated after brain trauma or stroke.Granulocyte colony-stimulating factor (G-CSF),is a member of the cytokine family ofgrowth factors.G-CSF has been used extensively in the treatment of chemotherapy-inducedneutropenia,as well as for bone marrow reconstitution and stem cell mobilization.Morerecently,it has been shown that administration of G-CSF in stroke mouse models couldinduce neurogenesis near the damaged area of these mice,leading to neurological andfunctional recovery.Until now,the role of G-CSF on ICH is not fully understood.In the present study,we studied the potential neuroprotective effect of G-CSF in a ratmodel of ICH.For the induction of intracerebral hemorrhage and G-CSF treatment,maleWistar rats were grouped as ICH-only control group (n = 20) and G-CSF-treated ICH group(n = 20).Experimental ICH was induced by the stereotaxic administration of collagenasetypeⅦinto caudate nucleus.The recombinant human G-CSF (15μg/kg),dissolved innormal saline,was administered through intraperitoneal,at 24 h after the ICH induction anddaily afterwards for 5 days.The same volume of normal saline was adminstrated to theICH-only control group.The limb placement tests were examined weekly for 4 weeks afterintracerebral hemorrhage was induced.We found that intraperitoneal injection of G-CSF for5 days could efficiently attenuate the ICH-induced neuronal behavioral impairment.To study the possible mechanisms underlying the behavioral improvement by G-CSF,we measured the expression of nestin,CD34 and GFAP by immunohistochemistry.We alsoobserved apoptosis by the immunofluorescence staining of Hoechst33258 and measured the sizes of necrosis area.The results are shown as follows:1.G-CSF may stimulate the expression of neural stem cells in the peri-necrosis area ofthe hemorrhagic hemisphere in the rats.2.G-CSF may stimulate HSCs to migrate into the brain regions through bloodcirculation after ICH induction.3.G-CSF can promote proliferation of astrocytes.4.G-CSF has anti-apoptotic effect after ICH.5.No differences were found in the sizes of necrosis area between G-CSF-treatedgroup and control.In summary,we have found in the present study that administration of G-CSF canimprove the ICH-induced neurobehavioral impairments in rats.The mechanism mayinvolve proliferation of neural stem cells;the mobilization of HSCs to damaged areas of thebrain;the increased astrocytes;and the anti-apoptotic effect of G-CSF.更多还原