Temporal induction of Lhx8 by optogenetic control system for efficient bone regeneration.
Temporal induction of Lhx8 by optogenetic control system for efficient bone regeneration.
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DOI：
10.1186/s13287-021-02412-8
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发表时间：
2021-06-10
影响因子：
7.5
通讯作者：
Zhou C
中科院分区：
文献类型：
--
作者：
Huang D;Li R;Ren J;Luo H;Wang W;Zhou C
必需基因的时空调控对于在再生过程中以精确的方式控制细胞的生长和分化至关重要。近年来，光遗传学被认为是一种有效的靶基因调控技术，有望成为再生医学的重要工具。在这项研究中，我们使用光遗传学控制系统来精确调节Lhx 8的表达，以促进有效的骨再生。采用实时定量PCR和Western blotting检测Lhx 8和成骨标志基因的表达。碱性磷酸酶染色和茜素红染色检测碱性磷酸酶活性和钙结节。构建了一个定制的光遗传学表达系统来调控Lhx 8，其表达在蓝光中被激活，而在黑暗中不被激活。我们还使用了一个关键的颅骨缺损模型，在体内骨再生的分析。此外，显微计算机断层扫描（micro-CT），三维重建，定量骨测量，组织学和免疫组织化学分析，以探讨体内新骨的形成。在BMSCs向成骨细胞分化过程中，Lhx 8的表达水平先升高后降低。Lhx 8促进BMSC的早期增殖，但抑制随后的成骨分化。在分化的早期阶段通过蓝光刺激BMSC中Lhx 8的光遗传学激活导致细胞增殖的显著增加，从而允许足够数量的分化的BMSC进入后期成骨分化阶段。对临界颅骨缺损模型的分析显示，在移植的BMSC中Lhx 8的脉冲光遗传学激活超过5天的时间导致体内骨生成的显著增加。Lhx 8在平衡BMSCs的增殖和成骨分化中起关键作用。在BMSCs分化的早期阶段，光遗传学激活Lhx 8的表达导致更好的成骨，这将是一个有前途的策略，精确的骨再生。在线版本包含补充材料，可通过10.1186/s13287-021-02412-8获得。
The spatiotemporal regulation of essential genes is crucial for controlling the growth and differentiation of cells in a precise manner during regeneration. Recently, optogenetics was considered as a potent technology for sophisticated regulation of target genes, which might be a promising tool for regenerative medicine. In this study, we used an optogenetic control system to precisely regulate the expression of Lhx8 to promote efficient bone regeneration. Quantitative real-time PCR and western blotting were used to detect the expression of Lhx8 and osteogenic marker genes. Alkaline phosphatase staining and alizarin red staining were used to detect alkaline phosphatase activity and calcium nodules. A customized optogenetic expression system was constructed to regulate Lhx8, of which the expression was activated in blue light but not in dark. We also used a critical calvarial defect model for the analysis of bone regeneration in vivo. Moreover, micro-computed tomography (micro-CT), three-dimensional reconstruction, quantitative bone measurement, and histological and immunohistochemistry analysis were performed to investigate the formation of new bone in vivo. During the osteogenic differentiation of BMSCs, the expression levels of Lhx8 increased initially but then decreased thereafter. Lhx8 promoted the early proliferation of BMSCs but inhibited subsequent osteogenic differentiation. The optogenetic activation of Lhx8 in BMSCs in the early stages of differentiation by blue light stimulation led to a significant increase in cell proliferation, thus allowing a sufficient number of differentiating BMSCs to enter the later osteogenic differentiation stage. Analysis of the critical calvarial defect model revealed that the pulsed optogenetic activation of Lhx8 in transplanted BMSCs over a 5-day period led to a significant increase in the generation of bone in vivo. Lhx8 plays a critical role in balancing proliferation and osteogenic differentiation in BMSCs. The optogenetic activation of Lhx8 expression at early stage of BMSCs differentiation led to better osteogenesis, which would be a promising strategy for precise bone regeneration. The online version contains supplementary material available at 10.1186/s13287-021-02412-8.
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