耳蜗雪旺细胞来源的Wnt1对耳蜗移植干细胞分化的影响及其分子机制研究
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摘要
1目的
神经干细胞（neural stem cells, NSC）的移植为神经系统退行性疾病和损伤的治疗提供了新的思路，但是移植入内耳的神经干细胞向神经元分化的效率很低，这成为移植的外源性神经干细胞替代治疗螺旋神经神经元（spiral ganglion neurons, SGNs）变性的主要障碍。在本研究中，我们验证了耳蜗螺旋神经元损伤后的局部微环境更有利于移植的外源性神经干细胞向神经元方向分化，并初步阐明了其分子机制。
2方法
我们通过向耳蜗圆窗龛局部给予药物ouabain建立大鼠螺旋神经元损伤动物模型。然后将分离、培养的神经干细胞移植进入正常、及螺旋神经元损伤后的耳蜗鼓阶。用免疫荧光染色和实时定量RT-PCR等技术来鉴定螺旋神经元损伤后内耳局部微环境对移植的神经干细胞分化的的影响，并初步阐明其分子机制。最后我们使用transwell共培养系统通过体外实验验证我们的假设。
3结果
通过将外源性干细胞植入螺旋神经元损伤的动物耳蜗内，我们发现，与对照组耳蜗相比，移植的神经干细胞在螺旋神经元损伤耳蜗中更易分化成MAP2阳性的神经元。采用实时定量PCR和免疫荧光法，我们也证明了在螺旋神经元损伤耳蜗内，螺旋神经节内卫星细胞（雪旺细胞的一类，外周神经节内的雪旺细胞称卫星细胞）中Wnt1（Wnt信号通路配体）的表达显着增加。我们进一步证实，神经干细胞表达Wnt信号通路受体和Wnt信号通路胞内关键组件。在以上结果基础上我们提出假设，雪旺细胞所产生Wnt1可能参与了移植神经干细胞向神经元分化的调控。我们使用transwell共培养系统在体外验证这一假设。我们将感染了慢病毒载体高表达Wnt1的耳蜗雪旺氏细胞与神经干细胞共培养，结果显示：与高表达Wnt1的雪旺细胞共培养的神经干细胞，分化为MAP2阳性神经元的比例显着增加，然而这种促进分化的作用可被DKK1（Wnt信号通路抑制剂）抑制。
4结论
这些结果表明，耳蜗雪旺氏细胞来源的Wnt1可激活移植神经干细胞的胞内Wnt信号通路，从而促进其向神经元的分化。详尽的阐明损伤微环境的改变对移植外源性干细胞的影响，有助于我们提出更有效的策略以突破移植神经干细胞向神经元的分化率低这一屏障。
1Objective
While neural stem cell (NSC) transplantation is widely expected to become atherapy for nervous system degenerative diseases and injuries, the low neuronaldifferentiation of NSCs which were transplanted into the inner ear is a majorobstacle for the successful treatment of spiral ganglion neuron (SGN)degeneration. In this study, we validated whether the local microenvironmentinfluences the neuronal differentiation of transplanted NSCs in the inner ear.
2Methods
Ouabain was locally administered to the round window niche to establish theSGN degeneration animal model. NSCs were isolated, cultured and transplantedinto the scala tympani of control or SGN-degenerative cochleae.Immunofluorescence staining and real-time RT-PCR were used to identify theeffect of the inner ear local microenvironment on the neuronal differentiation oftransplanted NSCs. Lentiviral vector infection and transwell co-culture systemwas used in vitro.
3Results
Using a rat SGN degeneration model, we demonstrated that transplantedNSCs were more likely to differentiate into MAP2+neurons in SGN-degenerative cochleae than in control cochleae. Using real-time quantitativePCR and an immunofluorescence assay, we also proved that the expression ofWnt1(a ligand of Wnt signaling) increases significantly in Schwann cells in theSGN-degenerative cochlea. We further verified that NSC cultures expressreceptors and signaling components for Wnts. Based on these expressionpatterns, we hypothesized that Schwann cell-derived Wnt1and Wnt signalingmight be involved in the regulation of the neuronal differentiation oftransplanted NSCs. We verified our hypothesis in vitro using a co-culture system.We transduced a lentiviral vector expressing Wnt1into cochlear Schwann cellcultures and co-cultured them with NSC cultures. Co-culture with Wnt1-expressing Schwann cells resulted in a significant increase in the percentage ofNSCs that differentiated into MAP2+neurons, whereas this differentiationenhancing effect was prevented by Dkk1(an inhibitor of the Wnt signalingpathway).
4Conclusion
These results suggested that Wnt1derived from cochlear Schwann cellsenhanced the neuronal differentiation of transplanted NSCs through Wntsignaling pathway activation. Alterations of the microenvironment deservedetailed investigation because they may help us to conceive effective strategiesto overcome the barrier of the low differentiation rate of transplanted NSCs.
神经干细胞（neural stem cells, NSC）的移植为神经系统退行性疾病和损伤的治疗提供了新的思路，但是移植入内耳的神经干细胞向神经元分化的效率很低，这成为移植的外源性神经干细胞替代治疗螺旋神经神经元（spiral ganglion neurons, SGNs）变性的主要障碍。在本研究中，我们验证了耳蜗螺旋神经元损伤后的局部微环境更有利于移植的外源性神经干细胞向神经元方向分化，并初步阐明了其分子机制。
2方法
我们通过向耳蜗圆窗龛局部给予药物ouabain建立大鼠螺旋神经元损伤动物模型。然后将分离、培养的神经干细胞移植进入正常、及螺旋神经元损伤后的耳蜗鼓阶。用免疫荧光染色和实时定量RT-PCR等技术来鉴定螺旋神经元损伤后内耳局部微环境对移植的神经干细胞分化的的影响，并初步阐明其分子机制。最后我们使用transwell共培养系统通过体外实验验证我们的假设。
3结果
通过将外源性干细胞植入螺旋神经元损伤的动物耳蜗内，我们发现，与对照组耳蜗相比，移植的神经干细胞在螺旋神经元损伤耳蜗中更易分化成MAP2阳性的神经元。采用实时定量PCR和免疫荧光法，我们也证明了在螺旋神经元损伤耳蜗内，螺旋神经节内卫星细胞（雪旺细胞的一类，外周神经节内的雪旺细胞称卫星细胞）中Wnt1（Wnt信号通路配体）的表达显着增加。我们进一步证实，神经干细胞表达Wnt信号通路受体和Wnt信号通路胞内关键组件。在以上结果基础上我们提出假设，雪旺细胞所产生Wnt1可能参与了移植神经干细胞向神经元分化的调控。我们使用transwell共培养系统在体外验证这一假设。我们将感染了慢病毒载体高表达Wnt1的耳蜗雪旺氏细胞与神经干细胞共培养，结果显示：与高表达Wnt1的雪旺细胞共培养的神经干细胞，分化为MAP2阳性神经元的比例显着增加，然而这种促进分化的作用可被DKK1（Wnt信号通路抑制剂）抑制。
4结论
这些结果表明，耳蜗雪旺氏细胞来源的Wnt1可激活移植神经干细胞的胞内Wnt信号通路，从而促进其向神经元的分化。详尽的阐明损伤微环境的改变对移植外源性干细胞的影响，有助于我们提出更有效的策略以突破移植神经干细胞向神经元的分化率低这一屏障。
1Objective
While neural stem cell (NSC) transplantation is widely expected to become atherapy for nervous system degenerative diseases and injuries, the low neuronaldifferentiation of NSCs which were transplanted into the inner ear is a majorobstacle for the successful treatment of spiral ganglion neuron (SGN)degeneration. In this study, we validated whether the local microenvironmentinfluences the neuronal differentiation of transplanted NSCs in the inner ear.
2Methods
Ouabain was locally administered to the round window niche to establish theSGN degeneration animal model. NSCs were isolated, cultured and transplantedinto the scala tympani of control or SGN-degenerative cochleae.Immunofluorescence staining and real-time RT-PCR were used to identify theeffect of the inner ear local microenvironment on the neuronal differentiation oftransplanted NSCs. Lentiviral vector infection and transwell co-culture systemwas used in vitro.
3Results
Using a rat SGN degeneration model, we demonstrated that transplantedNSCs were more likely to differentiate into MAP2+neurons in SGN-degenerative cochleae than in control cochleae. Using real-time quantitativePCR and an immunofluorescence assay, we also proved that the expression ofWnt1(a ligand of Wnt signaling) increases significantly in Schwann cells in theSGN-degenerative cochlea. We further verified that NSC cultures expressreceptors and signaling components for Wnts. Based on these expressionpatterns, we hypothesized that Schwann cell-derived Wnt1and Wnt signalingmight be involved in the regulation of the neuronal differentiation oftransplanted NSCs. We verified our hypothesis in vitro using a co-culture system.We transduced a lentiviral vector expressing Wnt1into cochlear Schwann cellcultures and co-cultured them with NSC cultures. Co-culture with Wnt1-expressing Schwann cells resulted in a significant increase in the percentage ofNSCs that differentiated into MAP2+neurons, whereas this differentiationenhancing effect was prevented by Dkk1(an inhibitor of the Wnt signalingpathway).
4Conclusion
These results suggested that Wnt1derived from cochlear Schwann cellsenhanced the neuronal differentiation of transplanted NSCs through Wntsignaling pathway activation. Alterations of the microenvironment deservedetailed investigation because they may help us to conceive effective strategiesto overcome the barrier of the low differentiation rate of transplanted NSCs.
引文
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2. ALTSCHULER RA, O'SHEA KS, MILLER JM. Stem cell transplantation forauditory nerve replacement. Hear Res2008;242:110-116.
3. COLEMAN.B., HARDMAN.J.,.A. C. fate of embryonic stem cellstransplanted into the deafened mammalian cochlea. Cell Transplant2006;15:369–380.
4. PARKER MA. neural stem cells injected into the sound-damaged cochleamigrate throughout the cochlea and express markers of haircells,suppprting cells, and spiral ganglion cells. Hear Res2007;232:29-43.
5. MATSUOKA AJ. In vivo and in vitro characterization of bonemarrow-derived stem cells in the cochlea. laryngoscope2006;116:1363-1367.
6. KONDO T. Sonic hedgehog and retinoic acid synergistically promotesensory fate specification from bone marrow-derived pluripotent stem cells.PNAS2005;102:4789-4799.
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