机械敏感性离子通道Piezo在正畸牙周组织中表达和功能的研究

发布时间：2018-06-21 23:06

本文选题：机械敏感性离子通道 + Piezo　；参考：《第四军医大学》2014年博士论文

【摘要】：研究目的：压电离子通道家族Piezo是一种可以被机械力刺激直接激活的非选择性阳离子通道，在膀胱、肺、结肠以及背根神经节等多个组织及器官中有丰富的表达。在敲除了Piezo的小鼠背根神经细胞中，快反应机械敏感电流被抑制，因此Piezos离子通道被认为在细胞的机械力感受方面有重要的作用。牙周膜成纤维细胞和成骨细胞都是可对机械力做出反应的力效应细胞，而Piezo在牙周组织中的表达和功能目前尚未见报道。本课题拟通过对牙周组织及牙周细胞中Piezo离子通道的表达、定位、追踪及功能的体内外研究，探索该通道在正畸牙周组织及细胞中所扮演的角色。研究方法： 1.采用间接免疫荧光法、RT-PCR对大鼠牙周组织以及牙周膜成纤维细胞（PDLF）、成骨细胞（MC3T3）和牙周膜干细胞（PDLSCs）中的Piezos离子通道进行检测，证实其在牙周组织及细胞中的表达，观察其表达特点及定位，以便进一步探讨Piezos离子通道在牙周组织中所扮演的角色。 2.通过使用正畸螺旋拉簧建立大鼠正畸牙移动模型，观察了正畸牙移动时Piezos离子通道在大鼠牙周组织的表达变化趋势，并同时观察三叉神经节中Piezos离子通道的变化，以了解当正畸力作用于牙周组织时，对三叉神经节中的Piezos离子通道表达的影响，探讨该通道是否可能参与痛觉感受及损伤修复的过程。 3.通过使用体外周期性张应力加载系统，对体外培养的PDLF、MC3T3以及PDLSCs加载周期性伸张和放松的应力，在RNA以及蛋白水平两个方面观察Piezos离子通道的表达，以研究Piezos在应力作用下随时间而变化的趋势。 4.通过使用Piezos离子通道的特异性阻滞剂GsMTx4对Piezos离子通道进行阻断，以研究当周期性张应力作用于PDLF时Piezos离子通道的功能，并同时检测ATP的释放浓度，以进一步了解Piezos在细胞机械反应以及疼痛传导过程中的作用。 5.通过使用大鼠缺血再灌注脑匀浆对PDLSCs进行神经向诱导，观察在此过程中周期性张应力和Piezos阻滞剂GsMTx4的协同作用，以探讨Piezos离子通道感受机械力后在神经损伤修复中的作用。研究结果： 1.间接免疫荧光检测可见Piezo1和Piezo2在牙周成纤维细胞中都有明显的阳性表达，在靠近固有牙槽骨处的成骨细胞中也有该通道蛋白的表达，但是在固有牙槽骨的骨细胞中未见有Piezos蛋白的特异性阳性表达。两种蛋白在牙周膜里的表达分布并不均匀，表达部位主要在胞膜上，部分在胞浆里。 2.在PDLF细胞和MC3T3-E1细胞中，Piezo1和Piezo2在基因转录水平上的表达量均较高，细胞免疫荧光检测可见Piezos在胞核无表达，部分游离分布在胞浆及胞膜中，部分呈点状聚集；而在PDLSCs中，未见有Piezo通道蛋白的特异性表达。 3.大鼠正畸牙移动模型中， Piezo1的表达量表现为先增高后下降的趋势，其中6d、9d组比0d组有显著增高（P0.05）；Piezo2的表达量表现为缓慢增高的趋势，其中9d、12d组比0d组有显著增高（P0.05）。 4.正畸力作用于大鼠牙周组织时，三叉神经节中的Piezo1mRNA表达呈现逐渐上升的趋势，至6d、9d时差异具有统计学意义（P0.05）；而Piezo2的表达量9d前增高不明显，9d时出现显著上调（P0.05）。Western blot检测Piezo1的蛋白表达量缓慢增高至9d组最高；Piezo2的蛋白表达量到6d出现明显增高，并持续增高至12d。 5.通过实时定量PCR和western blot检测，PDLF受到外力牵拉30分钟后Piezos mRNA和蛋白表达即出现明显的上升，Piezo1在4h时到达最高，之后开始降低，但到加力24h组中仍然显著高于对照组（P0.05）；Piezo2在2h时即到达最高，之后开始降低，但到加力24h组中仍然显著高于对照组（P0.05）。MC3T3受到外力牵拉Piezos的蛋白表达变化不明显，仅Piezo1mRNA在24h有明显上升（P0.05），Piezo2mRNA在24h内的变化不明显。PDLSCs受到外力牵拉2h后Piezos mRNA出现显著增高，之后开始下降，，但到加力24h组中仍然高于对照组（P0.05）；不加力的对照组PDLSCs中的几乎无Piezos蛋白表达，加力2h后开始出现，12h、24h组中出现显著的增高（P0.05）。 6.加力24h后PDLF的ATP释放较未加力的对照组有显著的增高（P0.05），在同时使用GsMTx4阻断Piezos离子通道的分组中，ATP的释放有明显的降低（P0.05），但仍然高于对照组的ATP浓度（P0.05）。 7.在PDLSCs进行神经向诱导24h后，通过western blot检测Piezos蛋白表达量有所增加；加入周期性应力作用后，Piezos的上调更为显著，使用GsMTx4对Piezos离子通道进行阻滞后，Piezos的表达变化不明显；NSE在神经向诱导的PDLSCs中有表达，同时使用GsMTx4作用时，表达量高于神经向诱导单独作用；当周期性张应力、神经向诱导和GsMTx4共同作用时，NSE的表达量到达最高。结论： 1. Piezos离子通道在牙周组织及细胞中有丰富的表达，结合该通道已有的生理特性研究基础，推测它可能参与了牙周机械感觉的传导。该通道在正畸牙移动中呈现先上升后下降的表达趋势，可能与神经纤维密度的增加有关，推测它随应力作用时间的推移而在细胞中的累积应与正畸牙周神经末梢的损伤修复有关。 2.当牙周组织中的Piezos离子通道随着正畸力的作用而表达上调的同时，三叉神经节中的Piezos表达也出现相应的趋势，推测位于牙周组织中的Piezos离子通道感受机械刺激，并向三叉神经节传导。 3.在应力作用下Piezos离子通道还可以引起ATP的大量释放，提示Piezos可能参与了正畸牙周疼痛的信号传导过程。 4.对Piezos通道的阻滞可以促进神经向诱导的PDLSCs中神经标志物的表达，推测这是由于阻断了机械力激活的钙离子内流所致，所以在牙周感觉神经末梢上的Piezos离子通道在感受到机械刺激后，可能对神经纤维的生长产生一定影响，从而在正畸牙周神经损伤修复过程中起到一定的作用。
[Abstract]:The purpose of the study is:
The piezoelectric ion channel family Piezo is a non selective cation channel that can be activated directly by mechanical stimulation. It has a rich expression in many tissues and organs such as bladder, lung, colon and dorsal root ganglion. In the dorsal root nerve cells knocking Piezo mice, the fast reverse mechanical sensitive current is suppressed, so Piezos ion passes. The channel is considered to play an important role in the mechanosperception of cells. Periodontal ligament fibroblasts and osteoblasts are force effector cells that respond to mechanical forces. The expression and function of Piezo in periodontal tissues are not yet reported. This topic is intended to express the expression of Piezo ion channels in periodontal and periodontal cells. Localization, tracking and functional in vivo and in vitro studies to explore the role of the channel in orthodontic periodontal tissues and cells.
Research methods:
1. by indirect immunofluorescence, RT-PCR was used to detect the periodontal tissue and periodontal ligament fibroblasts (PDLF), osteoblasts (MC3T3) and periodontal ligament stem cells (PDLSCs), to detect the expression of Piezos in the periodontal tissues and cells, to observe their expression characteristics and location, so as to further explore the Piezos ion channel in the rat. The role of the periodontium.
2. a orthodontic orthodontic tooth movement model was established by using orthodontic spiral spring. The changes of the expression of Piezos ion channel in the periodontal tissue of the rat were observed and the changes of the Piezos ion channel in the trigeminal ganglia were observed to understand the Piezos ion in the trigeminal ganglia when the orthodontic force acted on the periodontal tissue. The effect of channel expression on whether the channel may participate in the process of pain perception and injury repair is discussed.
3. by using in vitro cyclic tensile stress loading system, the expression of Piezos ion channel was observed in two aspects of PDLF, MC3T3 and PDLSCs in vitro, and the expression of Piezos ion channel was observed in the two aspects of RNA and protein level, so as to study the trend of change with time under stress.
4. the Piezos ion channel was blocked by the specific blocker GsMTx4 using the Piezos ion channel to study the function of the Piezos ion channel when the periodic tensile stress acted on PDLF, and to detect the release concentration of ATP at the same time, so as to further understand the role of Piezos in the process of cell mechanical reaction and the conduction of pain.
5. through the use of rat cerebral ischemia-reperfusion brain homogenate to induce the neural direction of PDLSCs, the synergistic effect of periodic Zhang Yingli and Piezos blocker, GsMTx4, was observed to explore the role of Piezos ion channel in the repair of nerve injury after the mechanical force of the ion channel.
The results of the study:
1. the indirect immunofluorescence showed that Piezo1 and Piezo2 had obvious positive expression in the periodontal fibroblasts. The expression of the channel protein was also expressed in the osteoblasts near the alveolar bone, but no specific positive expression of Piezos protein was found in the bone cells of the inherent alveolar bone. The expression of the two proteins in the periodontal ligament was expressed. The distribution is not uniform. The location of the expression is mainly on the cell membrane and partly in the cytoplasm.
2. in PDLF and MC3T3-E1 cells, the expression of Piezo1 and Piezo2 at the gene transcription level was higher. The cell immunofluorescence detection showed that Piezos was not expressed in the nucleus and partial dissociation was distributed in the cytoplasm and the cytoplasm, and partial aggregation was found, but there was no specific expression of Piezo channel protein in PDLSCs.
The expression of Piezo1 expression in the orthodontic tooth movement model of 3. rats showed a tendency to increase first and then decrease, of which 6D, 9D group was significantly higher than that of group 0d (P0.05), and the expression of Piezo2 showed a trend of slow increase, of which 9D, 12D group was significantly higher than that of the 0d group (P0.05).
4. the expression of Piezo1mRNA in the trigeminal ganglia increased gradually when the orthodontic force acted on the periodontal tissue of the rat, and the difference of the 9D time difference was statistically significant (P0.05), while the expression of Piezo2 was not obvious before 9D, and the expression of P0.05.Western blot was up to the highest in 9D group when 9D was significantly up (P0.05). The protein expression of Piezo2 increased significantly to 6D and increased to 12D.
5. by real-time quantitative PCR and Western blot detection, the expression of Piezos mRNA and protein expression rose obviously after 30 minutes of pulling out by external force. Piezo1 at 4H reached the highest, and then began to decrease, but it was still significantly higher than the control group (P0.05) in the addition of 24h group (P0.05); Piezo2 in 2H, then began to decrease, but then to add force. It was still significantly higher than the control group (P0.05) (P0.05) that the protein expression of.MC3T3 was not significantly changed by external force traction, but only Piezo1mRNA was significantly increased in 24h (P0.05). The change of Piezo2mRNA in 24h was not obvious. The Piezos mRNA appeared significantly higher after the external traction 2h, and then began to decline, but it was still higher than the control group. Group B (P0.05); there was almost no Piezos protein expression in PDLSCs of the non intensified control group, and 2H began to appear after loading 2h, and there was a significant increase in 12h and 24h group (P0.05).
After 6. addition of 24h, the release of ATP in PDLF was significantly higher than that in the control group (P0.05). The release of ATP was significantly reduced in the group of GsMTx4 blocking Piezos ion channels at the same time (P0.05), but still higher than the ATP concentration of the control group (P0.05).
7. after 24h was induced in PDLSCs, the expression of Piezos protein was increased by Western blot. After adding periodic stress, the up regulation of Piezos was more significant. The expression of Piezos was not obvious after the use of GsMTx4 to block the Piezos ion channel; NSE was expressed in the PDLSCs of the neural induction, and GsMT was used at the same time. When X4 acted, the expression level was higher than that of the nerve induction. When the tension stress was induced, the expression of NSE reached the maximum when the nerve was induced to interact with GsMTx4.
Conclusion:
The 1. Piezos ion channel is rich in the periodontal tissue and cell, which is based on the physiological characteristics of the channel. It is presumed that it may participate in the conduction of the periodontal mechanical sensation. The channel appears in the orthodontic tooth movement and then decreases. It may be related to the increase of the density of the nerve fibers. The accumulation of action time and accumulation in cells should be related to the repair of nerve endings in orthodontic periodontal tissue.
2. when the Piezos ion channel in the periodontal tissue is up-regulated with the effect of orthodontic force, the expression of Piezos in the trigeminal ganglia also has a corresponding trend. It is speculated that the Piezos ion channel located in the periodontal tissue is induced by mechanical stimulation and conduction to the trigeminal ganglion.
3. under stress, Piezos ion channels can also cause a large amount of ATP release, suggesting that Piezos may participate in the signal transduction process of orthodontic periodontal pain.
4. block of Piezos channel can promote the expression of nerve markers in PDLSCs induced by nerve. It is presumed that this is due to the blocking of the internal flow of calcium ions activated by mechanical force, so the Piezos ion channel on the sensory nerve endings of the periodontium may have a certain effect on the growth of nerve fibers after feeling the mechanical stimulation. It plays a certain role in the process of orthodontic periodontal nerve repair.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R783.5

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