本文選題：機(jī)械敏感性離子通道 + Piezo　； 參考：《第四軍醫(yī)大學(xué)》2014年博士論文

【摘要】：研究目的： 壓電離子通道家族Piezo是一種可以被機(jī)械力刺激直接激活的非選擇性陽(yáng)離子通道，在膀胱、肺、結(jié)腸以及背根神經(jīng)節(jié)等多個(gè)組織及器官中有豐富的表達(dá)。在敲除了Piezo的小鼠背根神經(jīng)細(xì)胞中，快反應(yīng)機(jī)械敏感電流被抑制，因此Piezos離子通道被認(rèn)為在細(xì)胞的機(jī)械力感受方面有重要的作用。牙周膜成纖維細(xì)胞和成骨細(xì)胞都是可對(duì)機(jī)械力做出反應(yīng)的力效應(yīng)細(xì)胞，而Piezo在牙周組織中的表達(dá)和功能目前尚未見(jiàn)報(bào)道。本課題擬通過(guò)對(duì)牙周組織及牙周細(xì)胞中Piezo離子通道的表達(dá)、定位、追蹤及功能的體內(nèi)外研究，探索該通道在正畸牙周組織及細(xì)胞中所扮演的角色。 研究方法： 1.采用間接免疫熒光法、RT-PCR對(duì)大鼠牙周組織以及牙周膜成纖維細(xì)胞（PDLF）、成骨細(xì)胞（MC3T3）和牙周膜干細(xì)胞（PDLSCs）中的Piezos離子通道進(jìn)行檢測(cè)，證實(shí)其在牙周組織及細(xì)胞中的表達(dá)，觀察其表達(dá)特點(diǎn)及定位，以便進(jìn)一步探討Piezos離子通道在牙周組織中所扮演的角色。 2.通過(guò)使用正畸螺旋拉簧建立大鼠正畸牙移動(dòng)模型，觀察了正畸牙移動(dòng)時(shí)Piezos離子通道在大鼠牙周組織的表達(dá)變化趨勢(shì)，并同時(shí)觀察三叉神經(jīng)節(jié)中Piezos離子通道的變化，以了解當(dāng)正畸力作用于牙周組織時(shí)，對(duì)三叉神經(jīng)節(jié)中的Piezos離子通道表達(dá)的影響，探討該通道是否可能參與痛覺(jué)感受及損傷修復(fù)的過(guò)程。 3.通過(guò)使用體外周期性張應(yīng)力加載系統(tǒng)，對(duì)體外培養(yǎng)的PDLF、MC3T3以及PDLSCs加載周期性伸張和放松的應(yīng)力，在RNA以及蛋白水平兩個(gè)方面觀察Piezos離子通道的表達(dá)，以研究Piezos在應(yīng)力作用下隨時(shí)間而變化的趨勢(shì)。 4.通過(guò)使用Piezos離子通道的特異性阻滯劑GsMTx4對(duì)Piezos離子通道進(jìn)行阻斷，以研究當(dāng)周期性張應(yīng)力作用于PDLF時(shí)Piezos離子通道的功能，并同時(shí)檢測(cè)ATP的釋放濃度，以進(jìn)一步了解Piezos在細(xì)胞機(jī)械反應(yīng)以及疼痛傳導(dǎo)過(guò)程中的作用。 5.通過(guò)使用大鼠缺血再灌注腦勻漿對(duì)PDLSCs進(jìn)行神經(jīng)向誘導(dǎo)，觀察在此過(guò)程中周期性張應(yīng)力和Piezos阻滯劑GsMTx4的協(xié)同作用，以探討Piezos離子通道感受機(jī)械力后在神經(jīng)損傷修復(fù)中的作用。 研究結(jié)果： 1.間接免疫熒光檢測(cè)可見(jiàn)Piezo1和Piezo2在牙周成纖維細(xì)胞中都有明顯的陽(yáng)性表達(dá)，在靠近固有牙槽骨處的成骨細(xì)胞中也有該通道蛋白的表達(dá)，但是在固有牙槽骨的骨細(xì)胞中未見(jiàn)有Piezos蛋白的特異性陽(yáng)性表達(dá)。兩種蛋白在牙周膜里的表達(dá)分布并不均勻，表達(dá)部位主要在胞膜上，部分在胞漿里。 2.在PDLF細(xì)胞和MC3T3-E1細(xì)胞中，Piezo1和Piezo2在基因轉(zhuǎn)錄水平上的表達(dá)量均較高，細(xì)胞免疫熒光檢測(cè)可見(jiàn)Piezos在胞核無(wú)表達(dá)，部分游離分布在胞漿及胞膜中，部分呈點(diǎn)狀聚集；而在PDLSCs中，未見(jiàn)有Piezo通道蛋白的特異性表達(dá)。 3.大鼠正畸牙移動(dòng)模型中， Piezo1的表達(dá)量表現(xiàn)為先增高后下降的趨勢(shì)，其中6d、9d組比0d組有顯著增高（P0.05）；Piezo2的表達(dá)量表現(xiàn)為緩慢增高的趨勢(shì)，其中9d、12d組比0d組有顯著增高（P0.05）。 4.正畸力作用于大鼠牙周組織時(shí)，三叉神經(jīng)節(jié)中的Piezo1mRNA表達(dá)呈現(xiàn)逐漸上升的趨勢(shì)，至6d、9d時(shí)差異具有統(tǒng)計(jì)學(xué)意義（P0.05）；而Piezo2的表達(dá)量9d前增高不明顯，9d時(shí)出現(xiàn)顯著上調(diào)（P0.05）。Western blot檢測(cè)Piezo1的蛋白表達(dá)量緩慢增高至9d組最高；Piezo2的蛋白表達(dá)量到6d出現(xiàn)明顯增高，并持續(xù)增高至12d。 5.通過(guò)實(shí)時(shí)定量PCR和western blot檢測(cè)，PDLF受到外力牽拉30分鐘后Piezos mRNA和蛋白表達(dá)即出現(xiàn)明顯的上升，Piezo1在4h時(shí)到達(dá)最高，之后開(kāi)始降低，但到加力24h組中仍然顯著高于對(duì)照組（P0.05）；Piezo2在2h時(shí)即到達(dá)最高，之后開(kāi)始降低，但到加力24h組中仍然顯著高于對(duì)照組（P0.05）。MC3T3受到外力牽拉Piezos的蛋白表達(dá)變化不明顯，僅Piezo1mRNA在24h有明顯上升（P0.05），Piezo2mRNA在24h內(nèi)的變化不明顯。PDLSCs受到外力牽拉2h后Piezos mRNA出現(xiàn)顯著增高，之后開(kāi)始下降，，但到加力24h組中仍然高于對(duì)照組（P0.05）；不加力的對(duì)照組PDLSCs中的幾乎無(wú)Piezos蛋白表達(dá)，加力2h后開(kāi)始出現(xiàn)，12h、24h組中出現(xiàn)顯著的增高（P0.05）。 6.加力24h后PDLF的ATP釋放較未加力的對(duì)照組有顯著的增高（P0.05），在同時(shí)使用GsMTx4阻斷Piezos離子通道的分組中，ATP的釋放有明顯的降低（P0.05），但仍然高于對(duì)照組的ATP濃度（P0.05）。 7.在PDLSCs進(jìn)行神經(jīng)向誘導(dǎo)24h后，通過(guò)western blot檢測(cè)Piezos蛋白表達(dá)量有所增加；加入周期性應(yīng)力作用后，Piezos的上調(diào)更為顯著，使用GsMTx4對(duì)Piezos離子通道進(jìn)行阻滯后，Piezos的表達(dá)變化不明顯；NSE在神經(jīng)向誘導(dǎo)的PDLSCs中有表達(dá)，同時(shí)使用GsMTx4作用時(shí)，表達(dá)量高于神經(jīng)向誘導(dǎo)單獨(dú)作用；當(dāng)周期性張應(yīng)力、神經(jīng)向誘導(dǎo)和GsMTx4共同作用時(shí)，NSE的表達(dá)量到達(dá)最高。 結(jié)論： 1. Piezos離子通道在牙周組織及細(xì)胞中有豐富的表達(dá)，結(jié)合該通道已有的生理特性研究基礎(chǔ)，推測(cè)它可能參與了牙周機(jī)械感覺(jué)的傳導(dǎo)。該通道在正畸牙移動(dòng)中呈現(xiàn)先上升后下降的表達(dá)趨勢(shì)，可能與神經(jīng)纖維密度的增加有關(guān)，推測(cè)它隨應(yīng)力作用時(shí)間的推移而在細(xì)胞中的累積應(yīng)與正畸牙周神經(jīng)末梢的損傷修復(fù)有關(guān)。 2.當(dāng)牙周組織中的Piezos離子通道隨著正畸力的作用而表達(dá)上調(diào)的同時(shí)，三叉神經(jīng)節(jié)中的Piezos表達(dá)也出現(xiàn)相應(yīng)的趨勢(shì)，推測(cè)位于牙周組織中的Piezos離子通道感受機(jī)械刺激，并向三叉神經(jīng)節(jié)傳導(dǎo)。 3.在應(yīng)力作用下Piezos離子通道還可以引起ATP的大量釋放，提示Piezos可能參與了正畸牙周疼痛的信號(hào)傳導(dǎo)過(guò)程。 4.對(duì)Piezos通道的阻滯可以促進(jìn)神經(jīng)向誘導(dǎo)的PDLSCs中神經(jīng)標(biāo)志物的表達(dá)，推測(cè)這是由于阻斷了機(jī)械力激活的鈣離子內(nèi)流所致，所以在牙周感覺(jué)神經(jīng)末梢上的Piezos離子通道在感受到機(jī)械刺激后，可能對(duì)神經(jīng)纖維的生長(zhǎng)產(chǎn)生一定影響，從而在正畸牙周神經(jīng)損傷修復(fù)過(guò)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R783.5

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