【作者】 朱晓冬；

【导师】 程焱；

【作者基本信息】 天津医科大学 ， 神经病学， 2010， 博士

【摘要】 研究目的：1.通过立体定向技术将6-OHDA直接注射到前脑内侧束来建立一种简单、有效的PD大鼠模型,为下一步研究奠定基础。2.建立大鼠骨髓间充质干细胞体外分离、扩增方法。3.判断重组腺病毒Ad-GFP、Ad-GFP-NGF、Ad-GFP-NOGGIN对rBMSCs的转染效率、转染对细胞的影响以及目的蛋白的表达情况。4.对比观察化学诱导剂、NGF及Noggin对rBMSCs像神经元样细胞方向分化的诱导作用。5.探讨携带NGF及Noggin的rBMSCs对PD模型大鼠神经功能的修复作用及其机制。研究方法：1.应用6-OHDA立体定向颅内注射制备PD大鼠模型。用阿朴吗啡皮下注射检测造模是否成功。2.取SD大鼠长骨骨髓作为供体来源,经贴壁筛选法进行体外培养并纯化rBMSCs。通过细胞表面标志、成骨细胞诱导和成脂细胞诱导对培养的细胞进行鉴定。3.通过不同浓度的重组腺病毒选择转染最合适的MOI值。将rBMSCs分成5组分别为未转染组、Ad-GFP转染组、Ad-GFP-NGF转染组、Ad-GFP-Noggin转染组和Ad-GFP-NGF、Ad-GFP-Noggin联合转染组,观察转染对rBMSCs生长的影响,通过免疫荧光检测目的蛋白的表达。4.通过化学诱导剂和基因修饰诱导rBMSCs向神经元样细胞方向分化,并检测其神经细胞标志物的表达。5.通过立体定向颅内注射将经过基因修饰的rBMSCs移植入模型大鼠损毁侧纹状体。记录单位时间阿朴吗啡诱发的模型大鼠旋转次数,观察大鼠神经功能恢复情况。通过免疫荧光检测移植细胞在脑内存活、分布以及向神经元、星形胶质细胞方向分化的情况。免疫组化法检测细胞移植后第四周大鼠中脑黑质TH阳性细胞计数。通过高效液相检测大鼠纹状体DA、DOPAC、HVA含量。结果：1.6-OHDA颅内注射制备的帕金森病大鼠模型具有良好的稳定性,该模型的制备为下一步实验奠定了基础。2.贴壁法能够筛选获得纯度较高,活性良好的rBMSCs,具有相关表型,能向成骨细胞和成脂细胞分化。3.重组腺病毒Ad-GFP、Ad-GFP-NGF、Ad-GFP-Noggin在MOI=50pfu/cell条件下对rBMSCs转染效率高,对rBMSCs毒性最小。经NGF、Noggin修饰的重组腺病毒转染后的rBMSCs在发出绿色荧光的同时,能分别表达相应蛋白产物,免疫荧光染色呈红色荧光。联合转染组rBMSCs则同时呈现NGF、Noggin免疫荧光阳性反应。4.rBMSCs在化学诱导剂作用下,5h后出现神经元样形态,免疫荧光染色显示神经元标志物NF(H)呈阳性,星形胶质细胞标志物GFAP呈阴性,但细胞大量死亡。各转染组rBMSCs中Ad-GFP转染组细胞形态无改变,Ad-GFP-NGF转染组、Ad-GFP-Noggin转染组及联合转染组rBMSCs在转染后72h呈现神经元样形态,部分细胞免疫荧光染色显示NF(H)阳性,GFAP阴性,分化后的细胞可长期存活。其中联合转染组NF(H)阳性率最高。5.经立体定向将细胞移植入模型大鼠损毁侧纹状体一周后,大鼠神经功能均有所改善,由阿朴吗啡诱发的旋转次数明显减少,其中联合转染组最为明显。镜下发现,移植细胞自注射部位向周围扩散,分布于纹状体、皮层下、海马及对侧半球。移植后两周,免疫荧光检测部分存活移植细胞NF(H)有阳性表达,另有部分细胞呈GFAP阳性。联合转染组NF(H)和GFAP阳性细胞比例最高。移植后四周,免疫组化检测各组模型黑质TH阳性细胞生存率,基因修饰组有增多的趋势,但各组差异无统计学意义。高效液相检测大鼠纹状体DA、DOPAC、HVA含量损毁侧同健侧之比,各细胞移植组比值均较未移植组和PBS组高,其中Ad-GFP-NGF和Ad-GFP-Noggin联合转染组比值最高。结论：1.6-OHDA立体定向颅内注射法制备PD大鼠模型具有良好的稳定性及可重复性。2.通过贴壁筛选法能有效纯化骨髓,获得高纯度rBMSCs,并且该方法简单实用,对细胞影响小,能满足大多数研究需求。通过流式细胞技术检测细胞表面标志,以及成骨、成脂诱导能够对rBMSCs进行有效鉴定。3.重组腺病毒Ad-GFP、Ad-GFP-NGF和Ad-GFP-Noggin均能安全、有效地转染体外培养的大鼠BMSCs,并稳定表达GFP、NGF和Noggin蛋白。4.体外环境下,化学诱导剂和NGF和Noggin蛋白均能将rBMSCs诱导成为神经元样细胞,但化学诱导剂对细胞存活有明显影响,而后两者不影响细胞存活,并可促进细胞向神经元方向分化。5.立体定向将基因修饰的rBMSCs移植入大鼠损毁侧纹状体,移植细胞可在脑内存活、迁移并分化为具有神经元或星形胶质细胞表型的细胞,通过多种途径有效改善模型大鼠神经功能,促进DA的释放。更多还原

【Abstract】 Objective:1. Establish a simple and effective rat model of Parkinson’disease by direct injection of 6-hydroxydopamine(6-OHDA) in the middle forebrain bundle(MFB) with stereotactic technique, and offer an experimental basement for the next research.2. Establish a method to isolate and culture the rat bone mesenchymal stem cells(rBMSCs).3. Study the transfect efficiency of three kinds of recombinant adenovirus including Ad-GFP, Ad-GFP-NGF and Ad-GFP-Noggin. The influence of transfection and the expression of NGF and Noggin were observed.4. The effects of chemical supplement, NGF and/or Noggin in inducing rBMSCs into neuron-like cells were discussed.5. To explore the effects and mechanisms on recovery of neurological function observed in rats of PD model grafted by rBMSCs modified with NGF and/or Noggin.Methods:1. Establish a rat model of PD by direct injection of 6-OHDA in MFB. To check if the establishment of PD rat model was successive by hypodermical injection of apomorphine.2. rBMSCs were isolated from rat bone marrow and purified through adherence ability. The cells were identified as rBMSCs by their phenotypical properties and their ability of differentiating into osteocyte and adipocyte.3. The proper MOI was chosen through recombinant adenovirus different density. rBMSCs were divided into 5 groups:rBMSCs, rBMSCs transfected with Ad-GFP, rBMSCs transfected with Ad-GFP-NGF, rBMSCs tarnsfected with Ad-GFP-Noggin and rBMSCs transfected with Ad-GFP-NGF combined with Ad-GFP-Noggin. Detect the influence of transfection to rBMSCs and the expression of NGF and Noggin through immunofluorescence.4. Induce rBMSCs differentiate into neuron-like cell and observe their expression of neuron specific marking proteins NF(H).5. rBMSCs modified by different genes were injected into the lesion side of striatum of PD model rats by stereotactic apparatus. Rotation test evoked by apomorphine was used to study the recovery of neurological deficits of rats. The distribution of survival transplanted cells was observed, and their diffentiation into neurons and astrocytes was detected through immunofluorescence. Four weeks after implantation, the number of TH positive cells in nigra was detected through immunohistochemical assessment. The contents of DA(dopamine)、DOPAC(dihydroxyphenylacetic) and HVA(homovanillic acid) in striatum of model rats were detected by high performance liquid chromatography-electric-chemical discharge(HPLC-ECD).Results:1. The PD model established by injecting 6-OHDA into rat’s MFB is a stable PD model. The establishment of the model had offered a basement for further experiment.2. The cells pure by their adherence ability had basic phenotypical properties of rBMSCs, and could differentiate into osteocyte and adipocyte.3. When MOI=50, Ad-GFP Ad-GFP-NGF and Ad-GFP-Noggin could transfect rBMSCs with high efficiency and low toxicity. rBMSCs transfected with recombinant adenovirus had green fluorescence autonomically at the same time it could express corresponding protein and send out red fluorescence through immunofluorescence. rBMSCs transfected with both Ad-GFP-NGF and Ad-GFP-Noggin had positive reaction to both NGF or Noggin at the same time.4. Neuron-like appearance could be seen after rBMSCs had been cultured in chemical supplement for 5h. NF(H),but astrocytic marking protein GFAP, was positive in them. Most of cells died in this supplement. The shape of rBMSCs transfected with Ad-GFP was not change. In the three groups transfected with Ad-NGF、Ad-Noggin and the two virus, the cells got the shape of neuron 72h later, some of them were positive to NF(H) and negative to GFAP. These cells grew in good state. The ratio of NF(H)+cells in combinating transfected group was the highest.5. One week after injection of the modified rBMSCs into lesion side striatum of rat model, the neurological function of rats was improved, the apomorphine-induced rotation was reduced significantly, the decrement of the combined group was remarkable. Observed by microscope, survival rBMSCs could be seen in striatum, subcortex, hippocampus which around the injective position and even the opposite side hemisphere. Two weeks after implantation, some of the survival cells were positive to NF(H) and some others were positive to GFAP. The rats transplanted with rBMSCs modified with two genes had more survival cells and higher ratio of NF(H)+ or GFAP+ cells than other groups. Four weeks after implantation, the survival rate of TH positive cells in nigra was found an increased tendency in the combined group. But the difference of each group was not statistically significant. The contents of DA, DOPAC and HVA in lesion side and uninjured side were detected by HPLC, their ratio of each group was compared. The ratio of groups that implanted with rBMSCs was higher than the group with PBS and group with no implantation. The ratio of group that implanted with rBMSCs which modified with two genes was the highest in all groups.Conclusion:1. The PD rat model established by intracal injection of 6-OHDA has good stability and repeatability.2. rBMSCs could be isolated and purified from rat bone marrow through their adherence ability and subculture. This method was simple, and would not influence the activity of cells. rBMSCs could be identified effectively by phenotypical properties and multipotency.3. Ad-GFP, Ad-GFP-NGF and Ad-GFP-Noggin could transfect rBMSCs safely. The transfected cells could express GFP, NGF and Noggin effectively.4. Chemical supplement, NGF and Noggin could induce rBMSCs into neuron-like cells in vitro. But chemical supplement interrupted the survival of cells, while NGF and Noggin could improve their differentiation.5. rBMSCs modified with NGF and Noggin could be implanted into the lesion side striatum of rat model. The transplanted rBMSCs could survive and migrate in the brain of model rats. Some of them could differentiate into cells with phenotype of neuron or astrocyte. The transplanted rBMSCs could improve the recovery of neurological function and the delivery of DA in rat model by several effective ways.更多还原