低剂量辐射对成体神经干细胞增殖分化的刺激作用及其Wnt/β-catenin分子机制的研究
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摘要
研究目的：
帕金森病（Parkinson’s disease, PD）是以黑质多巴胺神经元选择性变性丢失、进行性发展为特征的严重中枢退行性疾病，目前尚缺乏针对神经元死亡的临床治疗手段。近年研究表明，胚胎和成体来源的神经干细胞可能提供新生神经元或神经营养因子，能够从根源上补充丢失的神经元，并保护神经元，为预防和治疗PD提供了新希望。
本课题针对PD的干细胞治疗新方法和干预新靶点的探索研究，通过实验研究中脑和海马来源的成体神经干细胞增殖、细胞分化和神经营养因子特性，探索低剂量电离辐射刺激或激活成体神经干细胞增殖和细胞分化的新方法，分析Wnt/β-catenin信号途径的可能介导机制，以及PD疾病状态下黑质多巴胺神经元的神经营养信号作用等问题，为进一步探索建立PD的细胞治疗方法和靶向干预策略提供实验依据。
研究方法：
1.神经干细胞培养：中脑及海马来源的神经干细胞体外培养。
2.低剂量辐射方法：采用直线加速器X-线电离辐射处理方法。
3. PD动物模型制备：采用MPTP注射制备小鼠亚急性PD模型。
4. BrdU掺入标记技术：BrdU注射标记细胞核显示细胞增殖。
5.流式细胞术：检测中脑及海马神经干细胞生存、坏死和凋亡。
6. Fluoro-Jade C染色方法：组织切片显示黑质神经元变性死亡。
7. Western Blot：定量分析Wnts、BDNF、TrkB等蛋白的表达水平。
8.免疫细胞化学：显示Wnts、BDNF、TrkB等分子表达与变化情况。
9.激光共聚焦显微镜术：观察Wnts、BDNF、TrkB等细胞定位与变化。
主要结果：
第一部分：中脑和海马来源的神经干细胞增殖、细胞分化、神经营养因子产生特性的比较研究
1)免疫细胞化学观察结果显示中脑神经干细胞BrdU/Nestin掺入标记率明显低于海马神经干细胞，说明中脑神经干细胞具有较低增殖率。
2)流式细胞术检测分析表明中脑来源的神经干细胞的细胞存活数量较多、细胞凋亡较少。
3)细胞分化检测发现中脑和海马神经干细胞均可向Tuj-1、Nurr1、TH、GFAP、CNPase阳性细胞分化,中脑神经干细胞分化为TH和Nurr1阳性细胞数量较高，而海马神经干细胞更易分化为GFAP阳性星形胶质细胞。
4)神经营养因子检测证实中脑和海马神经干细胞在增殖和迁移分化中，均表达BDNF、GDNF、CDNF和DJ-1分子。中脑神经干细胞表达BDNF和DJ-1阳性细胞较多，GDNF阳性细胞数较少，而CDNF两者比较无差异。
这些结果证明中脑神经干细胞具有较低的细胞增殖率、较高的多巴胺能神经细胞分化率、BDNF等营养因子合成的细胞特性，提示中脑来源的神经干细胞可能是适合于PD细胞治疗的重要干细胞材料。第二部分：低剂量辐射对神经干细胞增殖、细胞分化的刺激作用、及其
Wnt/β-catennin信号分子机制
1)通过干细胞对照、低剂量辐射（0.3Gy）、高剂量辐射（3.0Gy）组的流式细胞检测，发现低剂量辐射组细胞增殖活跃、细胞存活数目增加、细胞凋亡减少，与对照组、高剂量辐射组之间有显著性差异（P<0.05）。
2)免疫细胞化学和Western blot实验发现低剂量辐射组的神经干细胞表达Tuj-1水平或阳性细胞增加，与对照组和高剂量辐射组比较有显著性差异（P<0.05），但GFAP的表达水平和GFAP阳性细胞数量三组之间无统计学差异。
3)免疫细胞化学和Western blot实验发现低剂量辐射促进神经干细胞Wnt1、Wnt3a、Wnt5a和β-catenin表达水平和阳性细胞数目增加,与对照组、高剂量辐射组比较有显著性差异（P<0.01）。
4) Wnt信号阻断实验发现Wnt信号阻断剂IWR1处理能够显著抑制低剂量辐射对干细胞Wnt1、Wnt3a、Wnt5a和β-catenin表达的刺激效应。Western blot条带灰度分析表明，两组之间有显著性差异（P<0.01)。
5) Wnt信号阻断实验证实给予Wnt信号阻断剂处理能够显著抑制低剂量辐射对干细胞增殖、细胞迁移、细胞分化的刺激效应。相差显微镜下细胞增殖下降、细胞凋亡增加、克隆能力下降、迁移下降、分化能力明显降低，两组之间具有显著差异（P<0.01）。
这些结果表明低剂量辐射能够刺激中脑和海马来源的成体神经干细胞增殖和细胞分化的作用，具有促进干细胞向神经细胞的分化作用。这种低剂量辐射刺激效应可能是通过Wnt/β-catenin信号途径的激活机制而实现的。
第三部分： MPTP动物模型中脑黑质多巴胺神经元上神经营养因子受体Trks的细胞定位和变化特征
1)免疫细胞化学和Western Blot实验证明黑质具有丰富神经营养因子受体TrkB、TrkC表达。其中黑质致密部多巴胺神经元具有TrkB、TrkC阳性免疫染色细胞定位，但缺乏TrkA的阳性表达。
2)MPTP动物模型观察发现MPTP处理引起黑质多巴胺神经元凋亡和TrkB、TrkC阳性细胞的丢失。Western Blot实验以β-Actin作内参（42KD），进一步验证了黑质TrkB和TrkC（145KD大小）蛋白表达水平下降。
3)通过比较MPTP模型黑质内TrkB和TrkC阳性细胞数目，发现TrkB和TrkC阳性神经元均发生丢失，但黑质致密部TrkB阳性神经元仍具有较多存活，其细胞丢失明显少于TrkC阳性细胞。
这些结果表明黑质较多TrkB阳性神经元能够在MPTP注射损伤后存活，表明BDNF-TrkB信号对于PD状态下多巴胺神经元保护具有重要作用。
初步结论与意义：
本课题通过体外、体内实验研究，获得以下结果或初步结论：
1)中脑来源的神经干细胞具有较低增殖率、向多巴胺神经元高分化能力、BDNF等营养因子产生特性，表明其在PD的细胞治疗中具有优良的应用潜力；
2)低剂量电离辐射具有刺激中脑和海马来源神经干细胞增殖和细胞分化的作用，其生物刺激效应与Wnt/β-catenin信号途径激活明显相关；
3)动物模型证实黑质内较多TrkB神经元能够在MPTP注射损伤后存活，表明BDNF-TrkB信号通路对于PD状态下多巴胺神经元保护具有重要意义。
本研究结果初步提供了低剂量辐射刺激或激活成体神经干细胞增殖和细胞分化的新方法，发现Wnt/β-catenin信号途径介导其生物刺激效应的可能机制，为进一步探索帕金森病的干细胞治疗和靶向干预新策略提供了重要实验依据。
PURPOSE OF STUDY
Parkinson’s disease (PD) is one common and severe neurological diseasethat characterizes with a selective degeneration of dopamine (DA) neurons inthe substantia nigra and disease progression, and at present it is still lack ofclinical cure method targeting on arresting neuronal progressive death.Growing evidences have shown that embryonic and adult neural stem cellswhich can potentially produce new neurons and neurotrophic factors in vivo andin vitro. Therefore, the cell replacement strategy by transplantation of neuralstem cells and inducement of dopaminergic neurons derived neural stem cells tosupply lost and decreased neurons is highly recommended for the preventionand treatment and of PD.
In this project, by focusing on new intervention method or manipulatingmolecular target for cell therapy against PD， we have performed theseexperiments, i.e. comparison of midbrain and hippocampus-derived adult neuralstem cells in proliferation, cell survival, differentiation and neurotrophicproperties; stimulatory effects of low-dose radiation on proliferation anddifferentiation of adult neural stem cells and Wnt/β-catenin signalingmechanism, and dynamic changes of neurotrophic Trks signaling in the dopaminergic neurons of substance nigra in MPTP-induced PD model. Weexpect to find a new intervention method activating adult neural stem cells forcell therapy against PD in human beings.
MATERIAL AND METHODS
1．Cell culture of neural stem cells: The midbrain and hippocampus-derivedneural stem cells were prepared and cultured.
2．Low dose ionizing radiation treatment: The midbrain and hippocampusNSCs were treated by X-ray ionizing radiation on which effected by linearaccelerator.
3．Preparation of PD animal model: PD mouse model was prepared bysystemic MPTP injection in C57/BL mice.
4．BrdU incorporation experiment: BrdU was incorporated into nucleus toshow cell division or proliferation state.
5．Flowcytometry: Cell survival, necrosis and apoptosis of cultured neural stemcells were examined by flowcytometry analysis.
6．Fluoro-Jade C staining: The neuronal degeneration in the substantia nigra ofMPTP model was visualized by Fluoro-Jade C staining.
7．Western blot analysis: Western blot analysis were used to confirm expressionlevels of Wnts、BDNF and TrkB proteins.
8．Immunocytochemistry and double labeling: Cell expression and changes ofWnts、BDNF and TrkB were performed by immunocytochemistry.
9．Laser scanning confocal microscopy: Cellular localization pattern andchanges of Wnts、BDNF and TrkB were observed by laser scanning confocalmicroscopy.
MAJOR RESULTS
1． Comparison study of midbrain and hippocampus-derived neural stemcells in proliferation, survival, differentiation and neurotrophicproperties
①Double immunofluorescence and laser scanning confocal microscopyconfirmed that nestin and BrdU was co-expressed in the cultured neuralstem cells. The midbrain-derived NSCs showed less or slowerproliferation than that of hippocampus-derived NSCs.
②The midbrain-derived NSCs showed higher rate of survival and lowerrate of apoptosis in comparison with that of hippocampus NSCs byflowcytometry.
③Differentiation into Tuj-1, TH, Nurr-1, and GFAP-positive cells weredetected in cell culture. Quantitative analysis indicated thatmidbrain-derived NSCs showed Tuj-1+commitment rate similar to thatof hippocampus NSCs, but higher rate of Nurr-1+and TH+cells thanthat of hippocampus NSCs. In contrast, however, hippocampus NSCsexhibited higher rate of GFAP+cell differentiation than that of midbrainNSCs.
④Immunocytochemistry and Western blot showed expression of BDNF,GDNF, CDNF and DJ-1both midbrain NSCs and hippocampus NSCs.The midbrain NSCs and migrating cells exhibited strong BDNF andDJ-1staining, similar CDNF staining, and weaker GFAP staining incomparison with that of hippocampus NSCs.
This result has indicated that midbrain neural stem cells show uniqueproperties in low proliferation, high neuronal commitment, and generation ofneurotrophic factors such as BDNF with therapeutic potential for PD.
2. Stimulatory effects of low-dose radiation on cell proliferation,differentiation of adult neural stem cells, and Wnt/β-catenin signalmediating mechanism
①Flowcytometry was used to examine stimulating effects on proliferationof neural stem cells. Increasing of cell survival, proliferation anddecreasing of cell apoptosis were detected in low-dose radiation(0.3Gy) group, by comparing to that of control and high-dose radiation（3.0Gy）group（P<0.05vs control or high-dose group）
②Immunocytochemistry showed that expression of Tuj-1or Tuj-1-positive cells was increased in low-dose radiation(0.3Gy) group, incomparison with that control and high-dose radiation（3.0Gy）group（P<0.05vs control or high-dose group）.
③Western blot further indicated that expression of Wnt1,Wnt3a,Wnt5aand β-catenin were up-regulated in low-dose radiation (0.3Gy) group,in comparison with that of control and high dose radiation（3.0Gy）group（P<0.05vs control or high-dose group).
④Wnt signaling block experiment showed that IWR1significantly inhibitstimulating effect on expression of Wnt1,Wnt3a,Wnt5a and β-cateninproteins after low-dose radiation by Western blot analysis.
⑤Immunocytochemistry and confocal microscopy further confirmed thatIWR1could inhibit stimulating effect on proliferation, migration anddifferentiation of cultured neural stem cells after low-dose radiation.Under phase contrast microscopy, increasing of cell apoptosis,decreasing of proliferation of neural stem cells, and abilities ofneurospheres formations and migration and differentiation were clearly observed in0.3Gy+IWR1group.
These results have indicated that low-dose radiation could stimulate cellproliferation, migration and differentiation of adult neural stem cells mostpossibly by triggering Wnt/β-catenin signaling pathway.
3. Localization and changes of neurotrophic factor receptor TrkB and TrkCin dopaminergic neuron of substantia nigra in MPTP-mouse model
①Double immunofluorescence and Western Blot analysis confirmed thatexpression of TrkB or TrkC were abundant in the substantia nigra,especially TrkB and TrkC-positive neurons were numerously observedin the substantia nigra pars compacta except of TrkA.
②Western Blot analysis verified that down-regulation of TrkB and TrkCproteins expression were found in dopaminergic neurons of substancenigra of MPTP mouse model.
③Though TrkB and TrkC positive cells were decreased in nigral neuronsin MPTP model, TrkB-positive survival neurons remained more incontrast to TrkC positive cells after MPTP insult. It showed that lostof TrkB positive cells is less than that of TrkC positive cells afterMPTP-insulted C57/BL mice.
This result indicated that TrkB-positive dopaminergic neurons might beless sensitive to MPTP insult in the substantia nigra, suggesting thatBDNF-TrkB may crucially function in protection of dopaminergic neuronsunder PD state.
SUMMARY AND CONCLUSIONS
In summary, we have obtained these important results in this study：
1．Midbrain-derived neural stem cells showed unique biological propertiesin low proliferation, high dopaminergic neurons commitment, andneurotrophic factor such as BDNF generation with therapeutic potentialfor PD.
2．Low-dose radiation could stimulate cell proliferation, cell differentiationof midbrain-and hippocampus-derived neural stem cells, and theseeffects were most possibly mediated by triggering Wnt/β-cateninsignaling pathway.
3．PD animal study indicated that TrkB-positive dopaminergic neuronswere less sensitive to MPTP insult in the substantia nigra of adult mice,suggesting that BDNF-TrkB might crucially act in neuroprotection ofdopaminergic neurons under PD state.
In this study, we have studied and found new method that low-doseradiation can effective stimulate proliferation and differentiation of adult neuralstem cells possibly by Wnt/β-catenin signaling activation mechanism. Data ofthis study has thus provided important evidence for further investigation ofpractical cell therapy and molecular targeting strategy in treatment of PD.
帕金森病（Parkinson’s disease, PD）是以黑质多巴胺神经元选择性变性丢失、进行性发展为特征的严重中枢退行性疾病，目前尚缺乏针对神经元死亡的临床治疗手段。近年研究表明，胚胎和成体来源的神经干细胞可能提供新生神经元或神经营养因子，能够从根源上补充丢失的神经元，并保护神经元，为预防和治疗PD提供了新希望。
本课题针对PD的干细胞治疗新方法和干预新靶点的探索研究，通过实验研究中脑和海马来源的成体神经干细胞增殖、细胞分化和神经营养因子特性，探索低剂量电离辐射刺激或激活成体神经干细胞增殖和细胞分化的新方法，分析Wnt/β-catenin信号途径的可能介导机制，以及PD疾病状态下黑质多巴胺神经元的神经营养信号作用等问题，为进一步探索建立PD的细胞治疗方法和靶向干预策略提供实验依据。
研究方法：
1.神经干细胞培养：中脑及海马来源的神经干细胞体外培养。
2.低剂量辐射方法：采用直线加速器X-线电离辐射处理方法。
3. PD动物模型制备：采用MPTP注射制备小鼠亚急性PD模型。
4. BrdU掺入标记技术：BrdU注射标记细胞核显示细胞增殖。
5.流式细胞术：检测中脑及海马神经干细胞生存、坏死和凋亡。
6. Fluoro-Jade C染色方法：组织切片显示黑质神经元变性死亡。
7. Western Blot：定量分析Wnts、BDNF、TrkB等蛋白的表达水平。
8.免疫细胞化学：显示Wnts、BDNF、TrkB等分子表达与变化情况。
9.激光共聚焦显微镜术：观察Wnts、BDNF、TrkB等细胞定位与变化。
主要结果：
第一部分：中脑和海马来源的神经干细胞增殖、细胞分化、神经营养因子产生特性的比较研究
1)免疫细胞化学观察结果显示中脑神经干细胞BrdU/Nestin掺入标记率明显低于海马神经干细胞，说明中脑神经干细胞具有较低增殖率。
2)流式细胞术检测分析表明中脑来源的神经干细胞的细胞存活数量较多、细胞凋亡较少。
3)细胞分化检测发现中脑和海马神经干细胞均可向Tuj-1、Nurr1、TH、GFAP、CNPase阳性细胞分化,中脑神经干细胞分化为TH和Nurr1阳性细胞数量较高，而海马神经干细胞更易分化为GFAP阳性星形胶质细胞。
4)神经营养因子检测证实中脑和海马神经干细胞在增殖和迁移分化中，均表达BDNF、GDNF、CDNF和DJ-1分子。中脑神经干细胞表达BDNF和DJ-1阳性细胞较多，GDNF阳性细胞数较少，而CDNF两者比较无差异。
这些结果证明中脑神经干细胞具有较低的细胞增殖率、较高的多巴胺能神经细胞分化率、BDNF等营养因子合成的细胞特性，提示中脑来源的神经干细胞可能是适合于PD细胞治疗的重要干细胞材料。第二部分：低剂量辐射对神经干细胞增殖、细胞分化的刺激作用、及其
Wnt/β-catennin信号分子机制
1)通过干细胞对照、低剂量辐射（0.3Gy）、高剂量辐射（3.0Gy）组的流式细胞检测，发现低剂量辐射组细胞增殖活跃、细胞存活数目增加、细胞凋亡减少，与对照组、高剂量辐射组之间有显著性差异（P<0.05）。
2)免疫细胞化学和Western blot实验发现低剂量辐射组的神经干细胞表达Tuj-1水平或阳性细胞增加，与对照组和高剂量辐射组比较有显著性差异（P<0.05），但GFAP的表达水平和GFAP阳性细胞数量三组之间无统计学差异。
3)免疫细胞化学和Western blot实验发现低剂量辐射促进神经干细胞Wnt1、Wnt3a、Wnt5a和β-catenin表达水平和阳性细胞数目增加,与对照组、高剂量辐射组比较有显著性差异（P<0.01）。
4) Wnt信号阻断实验发现Wnt信号阻断剂IWR1处理能够显著抑制低剂量辐射对干细胞Wnt1、Wnt3a、Wnt5a和β-catenin表达的刺激效应。Western blot条带灰度分析表明，两组之间有显著性差异（P<0.01)。
5) Wnt信号阻断实验证实给予Wnt信号阻断剂处理能够显著抑制低剂量辐射对干细胞增殖、细胞迁移、细胞分化的刺激效应。相差显微镜下细胞增殖下降、细胞凋亡增加、克隆能力下降、迁移下降、分化能力明显降低，两组之间具有显著差异（P<0.01）。
这些结果表明低剂量辐射能够刺激中脑和海马来源的成体神经干细胞增殖和细胞分化的作用，具有促进干细胞向神经细胞的分化作用。这种低剂量辐射刺激效应可能是通过Wnt/β-catenin信号途径的激活机制而实现的。
第三部分： MPTP动物模型中脑黑质多巴胺神经元上神经营养因子受体Trks的细胞定位和变化特征
1)免疫细胞化学和Western Blot实验证明黑质具有丰富神经营养因子受体TrkB、TrkC表达。其中黑质致密部多巴胺神经元具有TrkB、TrkC阳性免疫染色细胞定位，但缺乏TrkA的阳性表达。
2)MPTP动物模型观察发现MPTP处理引起黑质多巴胺神经元凋亡和TrkB、TrkC阳性细胞的丢失。Western Blot实验以β-Actin作内参（42KD），进一步验证了黑质TrkB和TrkC（145KD大小）蛋白表达水平下降。
3)通过比较MPTP模型黑质内TrkB和TrkC阳性细胞数目，发现TrkB和TrkC阳性神经元均发生丢失，但黑质致密部TrkB阳性神经元仍具有较多存活，其细胞丢失明显少于TrkC阳性细胞。
这些结果表明黑质较多TrkB阳性神经元能够在MPTP注射损伤后存活，表明BDNF-TrkB信号对于PD状态下多巴胺神经元保护具有重要作用。
初步结论与意义：
本课题通过体外、体内实验研究，获得以下结果或初步结论：
1)中脑来源的神经干细胞具有较低增殖率、向多巴胺神经元高分化能力、BDNF等营养因子产生特性，表明其在PD的细胞治疗中具有优良的应用潜力；
2)低剂量电离辐射具有刺激中脑和海马来源神经干细胞增殖和细胞分化的作用，其生物刺激效应与Wnt/β-catenin信号途径激活明显相关；
3)动物模型证实黑质内较多TrkB神经元能够在MPTP注射损伤后存活，表明BDNF-TrkB信号通路对于PD状态下多巴胺神经元保护具有重要意义。
本研究结果初步提供了低剂量辐射刺激或激活成体神经干细胞增殖和细胞分化的新方法，发现Wnt/β-catenin信号途径介导其生物刺激效应的可能机制，为进一步探索帕金森病的干细胞治疗和靶向干预新策略提供了重要实验依据。
PURPOSE OF STUDY
Parkinson’s disease (PD) is one common and severe neurological diseasethat characterizes with a selective degeneration of dopamine (DA) neurons inthe substantia nigra and disease progression, and at present it is still lack ofclinical cure method targeting on arresting neuronal progressive death.Growing evidences have shown that embryonic and adult neural stem cellswhich can potentially produce new neurons and neurotrophic factors in vivo andin vitro. Therefore, the cell replacement strategy by transplantation of neuralstem cells and inducement of dopaminergic neurons derived neural stem cells tosupply lost and decreased neurons is highly recommended for the preventionand treatment and of PD.
In this project, by focusing on new intervention method or manipulatingmolecular target for cell therapy against PD， we have performed theseexperiments, i.e. comparison of midbrain and hippocampus-derived adult neuralstem cells in proliferation, cell survival, differentiation and neurotrophicproperties; stimulatory effects of low-dose radiation on proliferation anddifferentiation of adult neural stem cells and Wnt/β-catenin signalingmechanism, and dynamic changes of neurotrophic Trks signaling in the dopaminergic neurons of substance nigra in MPTP-induced PD model. Weexpect to find a new intervention method activating adult neural stem cells forcell therapy against PD in human beings.
MATERIAL AND METHODS
1．Cell culture of neural stem cells: The midbrain and hippocampus-derivedneural stem cells were prepared and cultured.
2．Low dose ionizing radiation treatment: The midbrain and hippocampusNSCs were treated by X-ray ionizing radiation on which effected by linearaccelerator.
3．Preparation of PD animal model: PD mouse model was prepared bysystemic MPTP injection in C57/BL mice.
4．BrdU incorporation experiment: BrdU was incorporated into nucleus toshow cell division or proliferation state.
5．Flowcytometry: Cell survival, necrosis and apoptosis of cultured neural stemcells were examined by flowcytometry analysis.
6．Fluoro-Jade C staining: The neuronal degeneration in the substantia nigra ofMPTP model was visualized by Fluoro-Jade C staining.
7．Western blot analysis: Western blot analysis were used to confirm expressionlevels of Wnts、BDNF and TrkB proteins.
8．Immunocytochemistry and double labeling: Cell expression and changes ofWnts、BDNF and TrkB were performed by immunocytochemistry.
9．Laser scanning confocal microscopy: Cellular localization pattern andchanges of Wnts、BDNF and TrkB were observed by laser scanning confocalmicroscopy.
MAJOR RESULTS
1． Comparison study of midbrain and hippocampus-derived neural stemcells in proliferation, survival, differentiation and neurotrophicproperties
①Double immunofluorescence and laser scanning confocal microscopyconfirmed that nestin and BrdU was co-expressed in the cultured neuralstem cells. The midbrain-derived NSCs showed less or slowerproliferation than that of hippocampus-derived NSCs.
②The midbrain-derived NSCs showed higher rate of survival and lowerrate of apoptosis in comparison with that of hippocampus NSCs byflowcytometry.
③Differentiation into Tuj-1, TH, Nurr-1, and GFAP-positive cells weredetected in cell culture. Quantitative analysis indicated thatmidbrain-derived NSCs showed Tuj-1+commitment rate similar to thatof hippocampus NSCs, but higher rate of Nurr-1+and TH+cells thanthat of hippocampus NSCs. In contrast, however, hippocampus NSCsexhibited higher rate of GFAP+cell differentiation than that of midbrainNSCs.
④Immunocytochemistry and Western blot showed expression of BDNF,GDNF, CDNF and DJ-1both midbrain NSCs and hippocampus NSCs.The midbrain NSCs and migrating cells exhibited strong BDNF andDJ-1staining, similar CDNF staining, and weaker GFAP staining incomparison with that of hippocampus NSCs.
This result has indicated that midbrain neural stem cells show uniqueproperties in low proliferation, high neuronal commitment, and generation ofneurotrophic factors such as BDNF with therapeutic potential for PD.
2. Stimulatory effects of low-dose radiation on cell proliferation,differentiation of adult neural stem cells, and Wnt/β-catenin signalmediating mechanism
①Flowcytometry was used to examine stimulating effects on proliferationof neural stem cells. Increasing of cell survival, proliferation anddecreasing of cell apoptosis were detected in low-dose radiation(0.3Gy) group, by comparing to that of control and high-dose radiation（3.0Gy）group（P<0.05vs control or high-dose group）
②Immunocytochemistry showed that expression of Tuj-1or Tuj-1-positive cells was increased in low-dose radiation(0.3Gy) group, incomparison with that control and high-dose radiation（3.0Gy）group（P<0.05vs control or high-dose group）.
③Western blot further indicated that expression of Wnt1,Wnt3a,Wnt5aand β-catenin were up-regulated in low-dose radiation (0.3Gy) group,in comparison with that of control and high dose radiation（3.0Gy）group（P<0.05vs control or high-dose group).
④Wnt signaling block experiment showed that IWR1significantly inhibitstimulating effect on expression of Wnt1,Wnt3a,Wnt5a and β-cateninproteins after low-dose radiation by Western blot analysis.
⑤Immunocytochemistry and confocal microscopy further confirmed thatIWR1could inhibit stimulating effect on proliferation, migration anddifferentiation of cultured neural stem cells after low-dose radiation.Under phase contrast microscopy, increasing of cell apoptosis,decreasing of proliferation of neural stem cells, and abilities ofneurospheres formations and migration and differentiation were clearly observed in0.3Gy+IWR1group.
These results have indicated that low-dose radiation could stimulate cellproliferation, migration and differentiation of adult neural stem cells mostpossibly by triggering Wnt/β-catenin signaling pathway.
3. Localization and changes of neurotrophic factor receptor TrkB and TrkCin dopaminergic neuron of substantia nigra in MPTP-mouse model
①Double immunofluorescence and Western Blot analysis confirmed thatexpression of TrkB or TrkC were abundant in the substantia nigra,especially TrkB and TrkC-positive neurons were numerously observedin the substantia nigra pars compacta except of TrkA.
②Western Blot analysis verified that down-regulation of TrkB and TrkCproteins expression were found in dopaminergic neurons of substancenigra of MPTP mouse model.
③Though TrkB and TrkC positive cells were decreased in nigral neuronsin MPTP model, TrkB-positive survival neurons remained more incontrast to TrkC positive cells after MPTP insult. It showed that lostof TrkB positive cells is less than that of TrkC positive cells afterMPTP-insulted C57/BL mice.
This result indicated that TrkB-positive dopaminergic neurons might beless sensitive to MPTP insult in the substantia nigra, suggesting thatBDNF-TrkB may crucially function in protection of dopaminergic neuronsunder PD state.
SUMMARY AND CONCLUSIONS
In summary, we have obtained these important results in this study：
1．Midbrain-derived neural stem cells showed unique biological propertiesin low proliferation, high dopaminergic neurons commitment, andneurotrophic factor such as BDNF generation with therapeutic potentialfor PD.
2．Low-dose radiation could stimulate cell proliferation, cell differentiationof midbrain-and hippocampus-derived neural stem cells, and theseeffects were most possibly mediated by triggering Wnt/β-cateninsignaling pathway.
3．PD animal study indicated that TrkB-positive dopaminergic neuronswere less sensitive to MPTP insult in the substantia nigra of adult mice,suggesting that BDNF-TrkB might crucially act in neuroprotection ofdopaminergic neurons under PD state.
In this study, we have studied and found new method that low-doseradiation can effective stimulate proliferation and differentiation of adult neuralstem cells possibly by Wnt/β-catenin signaling activation mechanism. Data ofthis study has thus provided important evidence for further investigation ofpractical cell therapy and molecular targeting strategy in treatment of PD.
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