發(fā)布時間：2018-04-12 23:37

  本文選題：骨骼肌成肌細胞 + 嘌呤能P2受體　； 參考：《第二軍醫(yī)大學(xué)》2014年博士論文

【摘要】：喉返神經(jīng)損傷導(dǎo)致的聲帶麻痹的治療一直是喉科領(lǐng)域的難點和熱點問題，盡管神經(jīng)修復(fù)可使失神經(jīng)喉肌獲得再生，但喉肌功能仍難以恢復(fù)正常。其重要原因之一是喉肌肌衛(wèi)星細胞（成肌細胞）功能受損。肌衛(wèi)星細胞是骨骼肌成肌細胞，其向受損部位遷移是骨骼肌再生過程的關(guān)鍵環(huán)節(jié)。受損肌纖維釋放的信號分子誘導(dǎo)成肌細胞遷移至受損部位，其通過直接融合生成新生肌纖維或與受損肌纖維融合而完成修復(fù)，此過程涉及復(fù)雜的化學(xué)趨向誘導(dǎo)、細胞骨架重組等程序化的分子細胞事件。研究表明嘌呤能受體P2家族不同成員的時空差異性表達與成肌細胞的功能調(diào)控密切相關(guān)，我們課題組已研究表明在骨骼肌損傷后成肌細胞P2Y6表達上調(diào)并呈特異性的極性分布，提示P2Y6受體可能參與前體細胞和肌管的遷移，但確切機制尚不清楚。本課題擬通過基因修飾、特異性藥物干預(yù)等技術(shù)，闡明P2Y6受體信號通路調(diào)控成肌細胞遷移的作用及信號轉(zhuǎn)導(dǎo)機制。本研究旨在揭示P2Y6受體調(diào)控成肌細胞對失神經(jīng)喉肌再生的作用和確切機制，為促進失神經(jīng)喉肌等骨骼肌的再生和功能恢復(fù)，提供新的治療思路及理論依據(jù)。本研究共分為三個部分。 第一部分P2Y6RNAi慢病毒成肌細胞穩(wěn)定株的構(gòu)建 目的：采用shRNA干擾技術(shù)沉默P2Y6受體表達并構(gòu)建shP2Y6成肌細胞。方法：利用shRNA干擾技術(shù)針對不同的基因位點構(gòu)建一系列的shP2Y6C2C12細胞及空白病毒shCtrl C2C12細胞，并通過qPCR和WB驗證轉(zhuǎn)染效率，并挑選出干擾效率最好的兩株細胞。結(jié)果：共構(gòu)建空白病毒對照株C2C12UETP和8個慢病毒穩(wěn)定株C2C12shB202-C2C12shB209，其中C2C12shB203和C2C12shB206干擾效率最好，分別為（78.1%±6.4%）和（71.8%±2.9%）。結(jié)論：成功構(gòu)建P2Y6RNAi慢病毒成肌細胞穩(wěn)定株，且shB203的干擾效率最好。 第二部分P2Y6受體調(diào)控成肌細胞增殖、分化、遷移的作用 目的：探討P2Y6受體調(diào)控骨骼肌成肌細胞增殖、分化和遷移的確切作用。方法：基因干預(yù)組分為空質(zhì)粒C2C12shCtrl組和慢病毒C2C12shP2Y6-1、shP2Y6-2組。藥物刺激組分為：10μM、50μM、100μMUDP處理組、1μM、5μM、10μMMRS2578處理組和空白對照組。MTS和CCK-8法檢測不同處理組細胞增殖能力的差異；qPCR方法檢測基因干擾組成肌細胞分化指標(biāo)Myogenin、MyoD的mRNA水平的差異；細胞劃痕和Transwell遷移方法檢測不同處理組成肌細胞遷移能力的差異，結(jié)果：與對照組相比，不同處理組間成肌細胞的增殖活性降低無統(tǒng)計學(xué)意義（p0.05）；與對照組相比，基因干擾組成肌細胞在分化不同時間點（0d、1d、2d、3d、5d、6d）的MyoD、Myogenin轉(zhuǎn)錄水平的變化差異無統(tǒng)計學(xué)意義（p0.05）。而基因干擾組和10μMMRS2578處理組的成肌細胞的遷移能力顯著增加，與對照組比較，差異有統(tǒng)計學(xué)意義（p0.05）。結(jié)論：P2Y6受體參與調(diào)控成肌細胞的增殖和分化環(huán)節(jié)中細胞遷移運動。 第三部分P2Y6受體調(diào)控成肌細胞遷移作用的機制研究 目的：用WB檢測基因干擾組β-catenin、N-cadherin、MMP2的蛋白表達；利用基因芯片分析P2Y6受體調(diào)控成肌細胞遷移相關(guān)的信號通路。方法：利用慢病毒C2C12shP2Y6干擾組和空質(zhì)粒C2C12shCtrl組，通過基因芯片分析比較并找出兩組間的基因表達水平差異超過2倍（p0.05）的基因，將這些基因提交IPA分析，，預(yù)測出遷移相關(guān)信號通路。結(jié)果：與對照組相比，基因干預(yù)組β-catenin的蛋白表達明顯升高，差異具有統(tǒng)計學(xué)意義（p0.01）�；蛐酒治龅贸�124個表達升高和21個表達降低的基因，其中42個差異表達最顯著，30個遷移相關(guān)分子中有21個的表達變化預(yù)示遷移能力的增加，分析得出28種相關(guān)信號通路，并預(yù)測P2Y6受體調(diào)控成肌細胞遷移相關(guān)的經(jīng)典HGF和NF-κB信號通路網(wǎng)絡(luò)。結(jié)論：基因芯片分析顯示P2Y6沉默后大量遷移分子的激活預(yù)示著細胞遷移的功能被激活，遷移運動與細胞骨架運動蛋白β-catenin的調(diào)控相關(guān)，IPA分析預(yù)測其調(diào)控成肌細胞遷移的信號通路可能是經(jīng)典HGF和NF-κB通路，確切作用途徑有待進一步證實。 全文結(jié)論： 小鼠骨骼肌發(fā)育和損傷再生過程中，P2Y6受體的特異性時空表達模式提示其可能通過調(diào)控成肌細胞功能參與骨骼肌發(fā)育和再生。功能學(xué)實驗證實了P2Y6受體為參與調(diào)控成肌細胞的增殖和分化環(huán)節(jié)中成肌細胞的遷移能力及細胞骨架調(diào)控直接相關(guān)�；蛐酒C實了P2Y6受體調(diào)控的下游靶基因主要是與細胞運動及骨架調(diào)控相關(guān)的基因，而經(jīng)典HGF和NF-κB通路網(wǎng)絡(luò)可能是P2Y6受體調(diào)控成肌細胞遷移的主要信號轉(zhuǎn)導(dǎo)途徑。
[Abstract]:Treatment of recurrent laryngeal nerve injury caused by vocal cord paralysis is always a difficult and hot problem in the field of throat, although nerve repair can make denervated laryngeal muscle regeneration, but still difficult to restore the normal laryngeal muscle function. One of the important reasons is the laryngeal muscle satellite cells (myoblasts) function of muscle satellite cells are damaged. Skeletal muscle cells, the migration is a key link to the damaged parts of the skeletal muscle regeneration process. The damaged muscle fibers induced release of signal molecules into muscle cells migrate to the site of injury, by direct fusion of angiogenesis and muscle fiber or damaged muscle fiber fusion and complete repair, this process involves complex chemotaxis induced. Molecular and cellular events cytoskeletal reorganization and other procedures. The results indicate that the expression of purinergic regulation of temporal and spatial differences of different members of P2 family receptors and muscle cell function is closely related to our research group The study showed that in the skeletal muscle myoblasts and P2Y6 expression was polar distribution specificity, suggesting that migration of P2Y6 receptors may be involved in precursor cells and myotubes, but the exact mechanism is not clear. This paper through genetic modification of specific drug intervention techniques, clarify the regulation of P2Y6 receptor signaling pathway into effect and the signal transduction mechanism of muscle cell migration. The purpose of this study is to reveal the P2Y6 receptor expression in myoblasts on denervated laryngeal muscle regeneration and the exact mechanism, in order to promote the recovery of denervated skeletal muscle and laryngeal muscle function, and provide a new treatment ideas and theoretical basis. This research is divided into three parts.
Construction of a stable strain of P2Y6RNAi lentivirus myoblast
Objective: the expression of shRNA silencing P2Y6 receptor and construct shP2Y6 myoblasts. Methods: using shRNA interference technology in different locus to construct a series of shP2Y6C2C12 cells and blank virus shCtrl C2C12 cells, and the transfection efficiency of qPCR and WB verification, and pick out the interference efficiency of two cell lines. The best results: Construction Control line C2C12UETP and 8 lentiviral stable strains C2C12shB202-C2C12shB209, C2C12shB203 and C2C12shB206 interference efficiency best, respectively (78.1% + 6.4%) and (71.8% + 2.9%). Conclusion: the successful construction of lentivirus P2Y6RNAi myoblasts stable strains, and the best jamming efficiency of shB203.
The effect of the second part of P2Y6 receptor on the proliferation, differentiation and migration of myocytes
Objective: To investigate the P2Y6 receptor expression in skeletal myoblast proliferation, differentiation and migration of the exact role. Methods: the gene intervention group were divided into C2C12shCtrl group and empty plasmid lentivirus C2C12shP2Y6-1, shP2Y6-2 group. Drug stimulation group was divided into: 10 M, 50 M, 100 MUDP group, 1 M, 5 M, 10 MMRS2578 treatment group and blank control was detected the proliferation ability of.MTS group and CCK-8 method difference; qPCR method for detection of gene interference index of differentiation of muscle cells Myogenin, the difference of MyoD mRNA level; differences in detection of cell scratch assay and Transwell migration method of different treatment composition of muscle cell migration results: Compared with the control group, among different treatment groups, the proliferation of skeletal muscle cells decreased activity was not statistically significant (P0.05); compared with the control group, the muscle cells in the differentiation of gene interference at different time points (0d, 1D, 2D, 3D, 5D, 6D, MyoD, Myogenin) There were no significant differences in the transcriptional level (P0.05). The gene interference group and 10 MMRS2578 group of myoblast migration increased significantly, compared with the control group, the difference was statistically significant (P0.05). Conclusion: the P2Y6 receptor is involved in the regulation of cell migration into the link of the proliferation and differentiation of muscle cells in motion.
The mechanism of the third part of P2Y6 receptor to regulate the migration of myoblast
Objective: to group beta -catenin, WB was used to detect N-cadherin gene interference, the expression of MMP2 protein by gene chip analysis; P2Y6 receptor regulates myoblast migration related signaling pathways. Methods: using lentiviral C2C12shP2Y6 interference group and empty plasmid group C2C12shCtrl by gene chip analysis was found between the two groups of gene expression over 2 times (P0.05) genes, these genes will be submitted to IPA analysis, predict the migration related signaling pathways. Results: compared with control group, intervention group -catenin beta gene protein expression was significantly increased, the difference was statistically significant (P0.01). The gene chip analysis of 124 and 21 increased expression of decreased expression of genes. One of the 42 most significant differential expression, increased expression of 21 indicates the migration of 30 migration related molecules, analyzed 28 kinds of signaling pathways, and predicted by P2Y6 Body control myoblast migration related to classical HGF and NF- kappa B signaling network. Conclusion: the gene chip analysis showed that P2Y6 gene silencing molecules indicates that activation of the great migration of cell migration function is activated, the regulation of migration and cytoskeleton movement protein beta -catenin, IPA signal pathway analysis prediction of myoblast migration the regulation may be the classic HGF and NF- B pathway, the exact mechanism needs to be further confirmed.
The full text conclusion:
Mouse skeletal muscle development and injury regeneration process, expression pattern suggests that it may be through the regulation of myoblast functions involved in the development and regeneration of skeletal muscle specific temporal P2Y6 receptor. Functional experiments confirmed that the P2Y6 receptor is involved in the regulation of proliferation and differentiation of muscle cells to link the directly related to migration and cytoskeletal regulation of myoblast the gene chip confirmed the downstream target gene of P2Y6 receptor regulation is mainly associated with cell motility and skeleton regulated genes HGF and NF-, and the classical B pathway network may be P2Y6 receptor signal transduction regulation into muscle cell migration pathway.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R767.4

【參考文獻】

相關(guān)期刊論文 前1條

1 ;MAPK signal pathways in the regulation of cell proliferation in mammalian cells[J];Cell Research;2002年01期

本文編號：1741964