【作者】 吴雷；

【导师】 方加胜；

【作者基本信息】 中南大学 ， 神经外科， 2009， 博士

【摘要】 脑出血(Intracerebral hemorrhage,ICH)是指原发的非外伤性脑实质内出血,占全部卒中患者的21%～48%,发病后1个月内死亡率30%～50%,超过30%的存活者遗留功能障碍。目前针对ICH的治疗方法并不多,外科治疗中采取的单纯血肿碎吸治疗、血肿微创清除、神经内窥镜治疗及开颅血肿清除等方法,目的都是尽量减少脑血肿的占位效应和降低血肿降解产物造成的影响,但疗效和预后无法令人满意。干细胞的发现为治疗中枢神经系统损伤带来了新的曙光,有研究报道胚胎干细胞、神经干细胞、骨髓基质干细胞等移植有助于神经功能恢复,然而,伦理道德及取材等多方面的限制使干细胞移植治疗中枢神经系统损伤进展缓慢。近年来,脂肪基质干细胞(adipose-derivedstem cells,ADSCs)逐渐走入各国科研工作者的视野,由于其完全不存在上述限制,使其获得了广泛的应用前景。本实验,通过观察大鼠脑出血模型移植ADSCs治疗后不同指标的变化,以期为ADSCs的临床应用提供理论基础。共分为3章。第一章大鼠脂肪基质干细胞的生物学特性研究目的:分离脂肪基质干细胞,体外传代培养,观察,诱导其分化,并进行鉴定,为实验研究奠定基础。方法:从SD大鼠腹股沟脂肪垫分离得到脂肪基质干细胞,用含血清培养基对其培养、扩增、传代。将得到的脂肪基质干细胞置于神经干细胞培养基及成脂肪诱导培养基中,观察细胞变化,并利用免疫荧光及免疫组化技术对分化后细胞鉴定,同时对细胞进行冻存复苏等实验。结果:从大鼠腹股沟脂肪组织可获取大量ADSCs,电镜下细胞呈低分化状态,体外诱导后细胞可表达神经细胞标记物(Nestin,GFAP,NSE)及脂肪细胞标记物(油红O),冻存复苏后细胞仍有活性。结论:脂肪基质干细胞具有自我更新及多向分化潜能,可经神经干细胞向神经细胞分化,并可向脂肪细胞分化。体外长期培养,冻存复苏后仍保持干细胞特性。第二章ADSCs移植对脑出血模型运动功能的影响目的:探讨脂肪基质干细胞移植对大鼠脑出血模型运动功能的影响及其可能原因。方法:体外培养扩增的ADSCs标记后于造模成功后24小时立体定注入移植组大鼠右侧脑室(设立PBS移植组、模型组进行对照)。移植后3、7、14、21、28 d通过Longa评分观察大鼠运动功能情况。各组大鼠按时捕杀取脑制作切片,利用免疫荧光观察ADSCs存活、分布情况,利免疫组化观察各组出血灶周围凋亡细胞情况及血管生成情况。结果:移植的细胞自侧脑室及针道逐渐向损伤灶迁移;移植组在7、14、21、28 d有更好地神经功能评分;在各时间点移植组凋亡细胞明显少于对照组,7 d后血管密度高于对照组。结论:脂肪基质干细胞移植可通过抑制病灶周围细胞凋亡并促进血管生成进而改善脑出血后大鼠运动功能。第三章ADSCs移植改善中枢神经系统功能的分子生物学机制探讨目的:在分子生物水平探讨ADSCs移植改善脑出血大鼠模型运动功能的部分原因。方法:用ELISA方法检测ADSCs细胞分泌BDNF,VEGF情况,RT-PCR及Western—blot检测移植组与对照组脑组织BDNF,VEGF mRNA及蛋白表达情况。结果:ADSCs在体外培养条件下自身即可分泌BDNF及VEGF,移植后各时间点脑组织BDNF,VEGF在mRNA及蛋白层面与对照组相比均上调。结论:ADSCs移植后可通过BDNF,VEGF等可溶性因子发挥营养支持,保护,减轻损伤反应,增强血管发生,促进修复的作用。更多还原

【Abstract】 Intracerebral hemorrhage is defined as spontaneous haemorrhage in brain tissue not caused by injury of central nervous system,which covers about 21%～48%of stroke.30%～50%patients die of it in one month and neural function disturbances are remained in over 30% survivors.By now therapeutic measures are not abundant,surgical therapy includes aspiration of haematoma,neural endoscope measures, clearance of haematoma by operation and so on.The aims of all the therapeutic measures are to decrease effects of the occupancy and degradation products of haematoma.While the effectivenesses are not satisfactory.The appearance of stem cell bring hopes to patients with central nervous system injury.Recently,there are many reports about amendment of neural functions by stem cells transplantation,such as neural stem cells,embryonic stem cells,bone marrow stromal cells and so on.However,the restrictions about ethics and collection of cells made the study develop slowly and hard to be used clinically.Recent years, ADSCs(adipose-derived stem cells) have become hot spots of stem cells research.Above-mentioned restrictions not existing make ADSCs have broader perspective.So we transplanted ADSCs into rats model of intracerebral hemorrhage and observed the changes of some indexs to offer theary foundation for clinical application. PartⅠThe study on biological character of ADSCsObjective:To isolate,cultiviate,induce differentiation and identify ADSCs,observe the biological character in vitro,thus to build a foundation for applied research.Methods:Cells isolated from rats adipose of groin were cultured in medium which contained blood serum. When bespread the culture flask,cells were collected and inoculate into the mediums of neural stem cells and adipose cells.The changes of cells were observe and the differentiated cells were evaluated by immunohistochemistry and immunofluorescence.At the same time,the test of reserve ADSCs under the low-temperature condition and others were carried out.Results:The adpose tissue of rats groin contains lots of ADSCs.The cells are poorly differentiated under electron microscope. The appearance of ADSCs changed when culivated in the the mediums of neural stem cells and adipose cells medium.The differentiated cells present Nestine,GFAP,NSE and oil red O positive.The anabiosised reserved ADSCs still have activity.Conclusions:ADSCs have the ability to proliferation and self- refreshment,can differentiate into neural cells through NSCs and adipose cells.ADSCs can be cultivate long term in vitro,and it’s character of stem cells still remain after being anabiosised. PartⅡEffects of adipose-derived stem cells transplantation on motor function in intracerebral hemorrhage modelObjective:To explore the effects of adipose-derived stem cells (ADSCs) transplantation on motor function in rats with intracerebral hemorrhage and the reasons.Methods:ADSCs were cultured, proliferated and labelled in vitro.After the rats of ADSCs-transplanted group underwent intracerebral hemorrhage for 24 hours,ADSCs were stereotaxically transplanted into the right ventricular(PBS-transplanted group and model group were designed to be contrasted).The motor function were evaluated at 3,7,14,21,28 d after transplantation.Every group was enthanized on time and brain sections were made.Some sections were used in immunohistochemistry to observe the distribution of transplanted cells,and others were used in immunohistochemistry for apoptosis and angiogenesis detection.Results:The transplanted cells migrated to vicinities of hemorrhage focus gradually from ventricular and the hole of needle.The ADSCs-transplanted group has better motor function at 7,14,21,28 d,fewer apoptotic cells at 3,7,14,21,28 d and more angiogenesis at 7,14,21,28 d after transplantation.Conclusions: ADSCs transplantation can improve the motor function by reducing apoptotic cells and increaseing angiogenesis in intracerebral hemorrhage rats. PartⅢTo explore the reasons of ADSCs transplantation improving CNS function on the level of molecular organismObjective:To explore some reasons of ADSCs transplantation improving the motor function of intracerebral hemorrhage rats on the level of molecular organism.Methods:ELISA was used to detect whether ADSCs can excrete BDNF and VEGF.The changes of BDNF and VEGF mRNA and protein in brain tissue of two groups were detected by RT-PCR and Western-blot.Results:ADSCs can excrete BDNF and VEGF in vitro.BDNF,VEGF mRNA and protein express higher in the brain tissue of ADSCs-transplanted group at 3,7, 14,21,28 d after transplantation.Conclusions:ADSCs can protect, nuish neural cells and can increase angiogenesis,reduce damage by some dissoluble cytokine such as VEGF and BDNF.更多还原