Nurr1基因修饰胚胎中脑神经干细胞移植治疗帕金森病

乔奕胜; 陈孝祥; 徐蛟天; 王威; 张超; 宋晓斌; 杨智勇; 邓兴力

doi:10.12259/j.issn.2095-610X.S20210919

Nurr1基因修饰胚胎中脑神经干细胞移植治疗帕金森病

doi: 10.12259/j.issn.2095-610X.S20210919

乔奕胜^{1, 2},
陈孝祥¹,
徐蛟天¹,
王威¹,
张超¹,
宋晓斌¹,
杨智勇¹,
邓兴力^1, ,

1.
昆明医科大学第一附属医院神经外一科，云南昆明　650032
2.
云南省滇东北区域中心医院神经医学中心，云南昭通　547000

基金项目: 国家自然科学基金资助项目（81360197）；云南省科技厅-昆明医科大学应用基础研究联合专项计划基金资助项目（2017FB130）

详细信息

作者简介:
乔奕胜（1993年～），男，云南安宁人，在读硕士研究生，主要从事神经外科临床工作

通讯作者:
邓兴力，E-mail：dxlkmmu@163.com

中图分类号: R742.5
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出版历程
- 收稿日期: 2021-07-11

Transplantation of Nurr1 Gene-modified Embryo Midbrain Neural Stem Cells for Treatment of Parkinson’s Rats

1.
Dept. of Neurosurgery，The First Affiliated Hospital to Kunming Medical University，Kunming Yunnan 650032
2.
Neurology Center of Northeast Yunnan Regional Central Hospital，Zhao tong Yunnan 547000，China

摘要

摘要: 目的探讨移植Nurr1基因修饰胚胎中脑神经干细胞对帕金森大鼠的治疗效果。方法（1）建立SD大鼠帕金森病模型；（2）原代培养SD大鼠胚胎中脑神经干细胞，并于神经干细胞过表达Nurr1；（3）移植治疗分组：假手术组（A组），移植神经干细胞组(B组)，移植Nurr1修饰神经干细胞组（C组）；（4）在移植后3周、6周、9周和12周分别对各组大鼠进行行为学测试，观察各组症状改善情况；（5）移植12周后，分别检测各组中Nurr1、TH蛋白表达情况和Nurr1荧光情况。结果（1）原代培养大鼠胚胎中脑神经干细胞，并于神经干细胞成功过表达Nurr1基因。（2）免疫荧光、Western Blot证实过表达Nurr1可以促进神经干细胞向多巴胺能神经元转化（P < 0.05）。（3）移植Nurr1修饰神经干细胞可以有效的改善帕金森大鼠的症状（P < 0.05），Western Blot证实小胶质细胞可显著促进移植后的神经干细胞在分化为多巴胺能神经元(P < 0.05)。（4）移植后Nurr1能促进神经干细胞向多巴胺能神经元转化，提高移植神经干细胞的存活，达到更好治疗效果。结论 Nurr1基因可以促进神经干细胞向多巴胺能神经元转化；移植修饰Nurr1神经干细胞可以有效治疗帕金森大鼠。
- Nurr1 /
- 胚胎中脑神经干细胞 /
- 帕金森病
Abstract: Objective To investigate the therapeutic effect of transplantedNurr1 gene-modified Embryo Midbrain neural stem cells on Parkinson’ s rats. Methods 1. Establishment of Parkinson’ s disease model in SD rats.2. Primary culture Embryo Midbrain neural stem cells （NCSs） and Nurr1 gene-modified neural stem cells.3. Transplantation treatment group: sham operation group （sham group）, Transplanted neural stem cell group （NCS group）, transplanted Nurr1 gene-modified neural stem cells group （NNCS group）. 4. At 3 weeks, 6 weeks, 9 weeks, and 12 weeks after the combined transplantation, the behavioral tests of rats in each group were performed to observe the improvement of symptoms in each group. 5. After 12 weeks of transplantation, the expression of Nurr1, TH protein and Nurr1 fluorescence were detected in each group. Results 1. Primary cultured neural stem cells, and successfully overexpressed Nurr1 in neural stem cells. 2. Immunofluorescence and Western Blot confirmed that overexpression of Nurr1 could promote the transformation of neural stem cells into dopaminergic neurons （P < 0.05）. 3. Transplantation of NNCS can effectively improve the symptoms of Parkinson’ s rats （P < 0.05）. 4. After transplantation, Nurr1 can promote the transformation of neural stem cells into dopaminergic neurons, greatly improve the survival of transplanted neural stem cells and achieve better therapeutic effect. Conclusion Nurr1 can advance the transformation of neural stem cells into dopaminergic neurons. Transplantation of overexpression of Nurr1 neural stem cells can effectively treat Parkinson’ s rats.
- Nurr1 /
- Neural stem cells /
- Parkinson Disease

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图 1 神经干细胞的培养及鉴定

A：神经干细胞原代培养7 d；B、C：巢蛋白（Nestin）鉴定阳性。

Figure 1. Culture and Identification of Neural Stem Cells

下载: 全尺寸图片幻灯片

图 2 过表达Nurr1和对神经干细胞分化的影响

A：转染72 h后荧光显微镜检测Nurr1呈绿色；B、C：转染7 d后RT-PCR检测Nurr1的mRNA表达；D：分化培养7 d后检测TH荧光呈红色；E、F、G：分化7 d后western检测Nurr1和TH表达。与对照组比较，^*** P < 0.001。

Figure 2. Overexpression of Nurr1 and its effect on differentiation of neural stem cells

下载: 全尺寸图片幻灯片

图 3 细胞移植后分析阿扑吗啡诱导的旋转评分

各组分别在移植后3、6、9、12周，利用阿扑吗啡诱导检测转圈行为与C组比较，^*P < 0.05；，^**P < 0.001。

Figure 3. Apomorphine-induced rotation scores were analyzed after cell transplantation.

下载: 全尺寸图片幻灯片

图 4 移植Nurr1后神经干细胞向多巴胺能神经元转化的情况

A：各组Nurr1和TH蛋白的表达水平；B、C：C组与对照组及B相比，Nurr1和TH表达上调；D、E：：移植治疗12周后。与NNCS组比较，^* P < 0.05，^**P < 0.01。

Figure 4. Transformation of Neural Stem Cells into dopaminergic neurons after Nurr1 transplantation

下载: 全尺寸图片幻灯片

参考文献(18)

[1]	Balestrino R,Schapira A H V. Parkinson disease[J]. Eur J Neurol,2020,27(1):27-42. doi: 10.1111/ene.14108
[2]	陈彪. 左旋多巴在帕金森病治疗中的地位及进展[J]. 中国临床神经科学,2017,25(05):546-550. doi: 10.3969/j.issn.1008-0678.2017.05.012
[3]	Zhu B,Caldwell M,Song B. Development of stem cell-based therapies for Parkinson's disease[J]. Int J Neurosci,2016,126(11):955-962. doi: 10.3109/00207454.2016.1148034
[4]	Zhang Q,Chen W,Tan S,et al. Stem Cells for Modeling and Therapy of Parkinson's Disease[J]. Hum Gene Ther,2017,28(1):85-98. doi: 10.1089/hum.2016.116
[5]	刘罡,吴毅,贾杰,等. 神经干细胞体外分化抗原表达的研究[J]. 中国运动医学杂志,2009,28(01):55-59.
[6]	Li K,Li J,Zheng J,Qin S. Reactive Astrocytes in Neurodegenerative Diseases[J]. Aging Dis,2019,10(3):664-675. doi: 10.14336/AD.2018.0720
[7]	Barker R A,Drouin Ooellet J,Parmar M. Cell-based therapies for Parkinson disease-past insights and future potential[J]. Nat Rev Neurol,2015,11(9):492-503. doi: 10.1038/nrneurol.2015.123
[8]	徐蛟天,陈孝祥,王威,等. Nurr1对小胶质细胞联合神经干细胞共培养促进神经干细胞向多巴胺神经元分化作用研究[J]. 国际神经病学神经外科学杂志,2018,45(1):52-57.
[9]	Liu W,Gao Y,Chang N. Nurr1 overexpression exerts neuroprotective and anti-inflammatory roles via down-regulating CCL2 expression in both in vivo and in vitro Parkinson's disease models[J]. Biochem Biophys Res Commun,2017,482(4):1312-1319. doi: 10.1016/j.bbrc.2016.12.034
[10]	Kadkhodaei B,Ito T,Joodmardi E,et al. Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons[J]. J Neurosci,2009,29(50):15923-15932. doi: 10.1523/JNEUROSCI.3910-09.2009
[11]	Radad K,Moldzio R,AL-Shraim M,et al. Recent Advances on the Role of Neurogenesis in the Adult Brain:Therapeutic Potential in Parkinson's and Alzheimer's Diseases[J]. CNS Neurol Disord Drug Targets,2017,16(7):740-748.
[12]	O'sullivan S S,Johnson M,Williams D R,et al. The effect of drug treatment on neurogenesis in Parkinson's disease[J]. Mov Disord,2011,26(1):45-50. doi: 10.1002/mds.23340
[13]	Madhavan L,Daley B F,Paumier K L,et al. Transplantation of subventricular zone neural precursors induces an endogenous precursor cell response in a rat model of Parkinson's disease[J]. J Comp Neurol,2009,515(1):102-115. doi: 10.1002/cne.22033
[14]	Dong J,Liu X,Wang Y,et al. Nurr1(Cd11bcre)conditional knockout mice display inflammatory injury to nigrostriatal dopaminergic neurons[J]. Glia,2020,68(10):2057-2069. doi: 10.1002/glia.23826
[15]	Wang X,Zhuang W,Fu W,et al. The lentiviral-mediated Nurr1 genetic engineering mesenchymal stem cells protect dopaminergic neurons in a rat model of Parkinson's disease[J]. Am J Transl Res,2018,10(6):1583-1599.
[16]	Smith G A,Rocha E M,Rooney T,et al. A Nurr1 agonist causes neuroprotection in a Parkinson's disease lesion model primed with the toll-like receptor 3 dsRNA inflammatory stimulant poly(I:C)[J]. PLoS One,2015,10(3):e0121072. doi: 10.1371/journal.pone.0121072
[17]	Ahmed A,Isaksen T J,Yamashita T. Protocol for mouse adult neural stem cell isolation and culture[J]. STAR Protoc,2021,2(2):100522. doi: 10.1016/j.xpro.2021.100522
[18]	Raina A,Mahajani S,Bahr M,et al. Neuronal Trans-differentiation by TraNCSription Factors Ascl1 and Nurr1:Induction of a Dopaminergic Neurotransmitter Phenotype in Cortical GABAergic Neurons[J]. Mol Neurobiol,2020,57(1):249-260. doi: 10.1007/s12035-019-01701-x

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