發(fā)布時(shí)間：2018-06-22 20:40

  本文選題：Pannexin3 + 半通道��； 參考：《武漢大學(xué)》2014年博士論文

【摘要】：第一部分：Pannexin3在牙髓組織及成牙本質(zhì)細(xì)胞中的表達(dá)及分布 目的：明確Pannexin3基因及蛋白在牙髓組織及成牙本質(zhì)細(xì)胞中的表達(dá)及分布情況。 方法：收集15-20歲的健康正畸牙或者智齒,提取牙髓組織mRNA,采用PCR技術(shù)檢測(cè)牙髓內(nèi)Pannexin3基因的相對(duì)含量；應(yīng)用組織免疫熒光方法觀察牙髓組織中Pannexin3蛋白表達(dá)及分布情況。采用組織塊法培養(yǎng)牙髓干細(xì)胞,誘導(dǎo)分化為成牙本質(zhì)細(xì)胞,利用PCR、Western blot、細(xì)胞免疫熒光技術(shù)從基因及蛋白水平鑒定成牙本質(zhì)細(xì)胞中Pannexin3的表達(dá)及分布。 結(jié)果：組織塊法體外培養(yǎng)牙髓細(xì)胞,經(jīng)礦化誘導(dǎo)7天后細(xì)胞分化為成牙本質(zhì)細(xì)胞。PCR、Western blot檢測(cè)結(jié)果顯示牙髓組織及成牙本質(zhì)細(xì)胞中有Pannexin3基因及蛋白表達(dá),免疫熒光結(jié)果發(fā)現(xiàn)Pannexin3蛋白主要分布于人牙髓組織的成牙本質(zhì)細(xì)胞層的胞體及細(xì)胞突起部分和成牙本質(zhì)細(xì)胞胞漿近胞核及細(xì)胞膜等位置。 結(jié)論：Pannexin3主要分布在人牙髓組織以及成牙本質(zhì)細(xì)胞胞漿及胞膜上。 第二部分：Pannexin3參與成牙本質(zhì)細(xì)胞釋放ATP 目的：研究Pannexin3縫隙連接蛋白在外界冷熱刺激下對(duì)成牙本質(zhì)細(xì)胞釋放ATP的影響。 方法：構(gòu)建Pannexin3過(guò)表達(dá)慢病毒轉(zhuǎn)染載體PLL3.7\CMV-PANNEXIN3\CMV-RFP,通過(guò)病毒感染獲取能夠高表達(dá)Pannexin3的成牙本質(zhì)細(xì)胞；合成siRNA、構(gòu)建shRNA分別干擾成牙本質(zhì)細(xì)胞Pannexin3基因表達(dá)。采用PCR、Western blot技術(shù)對(duì)過(guò)表達(dá)及干擾效果進(jìn)行評(píng)價(jià)。將細(xì)胞分為對(duì)照組、過(guò)表達(dá)組、siRNA干擾組、shRNA干擾組及甘珀酸(CHX)功能阻斷組共5組,對(duì)各組細(xì)胞分別進(jìn)行冷熱刺激。使用奎納克林進(jìn)行染色觀察細(xì)胞ATP釋放情況,通過(guò)Image Pro-plus計(jì)算熒光變化量,采用生物熒光法對(duì)釋放到細(xì)胞外的ATP含量進(jìn)行測(cè)定。 結(jié)果：PCR、Western blot及熒光照片結(jié)果證明Pannexin3過(guò)表達(dá)、siRNA、shRNA能有效感染成牙本質(zhì)細(xì)胞,實(shí)現(xiàn)細(xì)胞Pannexin3基因的高表達(dá)和干擾效果。冷熱刺激后成牙本質(zhì)細(xì)胞內(nèi)的ATP迅速釋放到細(xì)胞外,Pannexin3過(guò)表達(dá)細(xì)胞釋放速度明顯高于對(duì)照組,使用siRNA、shRNA干擾或者CHX阻斷Pannexin3通道都能抑制冷熱刺激引起的細(xì)胞內(nèi)ATP釋放到胞外。 結(jié)論：Pannexin3能在成牙本質(zhì)細(xì)胞形成半通道,冷熱刺激時(shí)細(xì)胞通過(guò)Pannexin3半通道釋放ATP到細(xì)胞外。 第三部分：ATP參與牙髓感覺(jué)神經(jīng)疼痛刺激傳遞 目的：了解ATP在牙髓感覺(jué)神經(jīng)疼痛受體激活、Ca離子流動(dòng)以及疼痛刺激過(guò)程中的作用。 方法：取小鼠三叉神經(jīng)節(jié),采用免疫熒光方法檢測(cè)小鼠三叉神經(jīng)節(jié)中P2X3受體表達(dá)情況,采用酶消化法分離培養(yǎng)三叉神經(jīng)元細(xì)胞,通過(guò)形態(tài)觀察及外周蛋白免疫熒光方法進(jìn)行細(xì)胞鑒定。將神經(jīng)元細(xì)胞分為ATP刺激組、α βme-ATP刺激組、A-317491抑制組共三組,采用膜片鉗技術(shù)對(duì)各組神經(jīng)元細(xì)胞在接受刺激后細(xì)胞內(nèi)Ca離子流動(dòng)情況進(jìn)行分析。以SD大鼠為實(shí)驗(yàn)對(duì)象,磨除大鼠磨牙釉質(zhì)及部分牙本質(zhì),制備牙本質(zhì)敏感模型,按注射藥物不同分為：PBS、ATP、α, βme-ATP及A-317491四組,分別添加冷熱刺激,記錄刺激后5分鐘內(nèi)各組動(dòng)物抬爪或者舔舐的次數(shù)。 結(jié)果：組織免疫熒光顯示P2X3受體集中分布在三叉神經(jīng)節(jié)的邊緣,散在分布于中間部分。采用酶消化法獲得三叉神經(jīng)元細(xì)胞,細(xì)胞呈現(xiàn)為網(wǎng)狀、線(xiàn)狀排列。ATP、 α, βme-ATP刺激三叉神經(jīng)元細(xì)胞后出現(xiàn)Ca離子內(nèi)流,A-317491能抑制Ca離子流動(dòng)。冷熱刺激后牙本質(zhì)敏感大鼠出現(xiàn)抬爪或者舔舐動(dòng)作,ATP、α, βme-ATP能夠顯著增加動(dòng)物對(duì)刺激的反應(yīng)程度及頻度,而A-317491則明顯降低了動(dòng)物的反應(yīng)行為。 結(jié)論：ATP激活三叉神經(jīng)節(jié)上的P2X3受體,使Ca離子內(nèi)流,從而導(dǎo)致動(dòng)物產(chǎn)生疼痛感覺(jué)。
[Abstract]:Part one: the expression and distribution of Pannexin3 in dental pulp and odontoblasts.
Objective: to clarify the expression and distribution of Pannexin3 gene and protein in dental pulp and odontoblasts.
Methods: a 15-20 year old healthy orthodontic tooth or wisdom tooth was collected to extract the mRNA of the pulp tissue. The relative content of Pannexin3 gene in the pulp was detected by PCR technique. The expression and distribution of Pannexin3 protein in the pulp tissue were observed by tissue immunofluorescence. The tissue block method was used to cultivate the pulp stem cells and induce the differentiation into the dentin formation. The expression and distribution of Pannexin3 in odontoblasts were identified by PCR, Western blot and cellular immunofluorescence.
Results: the dental pulp cells were cultured in vitro by tissue mass method. After 7 days of mineralization, the cells differentiated into odontoblast.PCR. The results of Western blot detection showed that there were Pannexin3 gene and protein expression in dental pulp and odontoblast cells. The results of immunofluorescence showed that the main Pannexin3 protein was distributed in the odontoblast layer of human dental pulp tissue. The location of cell bodies and cell protrusions and odontoblasts, cytoplasm, nuclei and cell membrane.
Conclusion: Pannexin3 is mainly distributed in human dental pulp and in the cytoplasm and membrane of odontoblasts.
The second part: Pannexin3 is involved in odontoblast release from ATP.
Objective: To study the effect of Pannexin3 connexin on the release of ATP from odontoblasts in cold and heat stimuli.
Methods: the Pannexin3 overexpressed lentivirus transfection vector PLL3.7CMV-PANNEXIN3CMV-RFP was constructed to obtain the odontoblast cells with high expression of Pannexin3 through viral infection, and siRNA was synthesized and shRNA interfered with the expression of Pannexin3 gene in odontoblast cells. PCR, Western blot technique was used to evaluate the overexpression and interference effect. The cells were divided into the control group, the overexpression group, the siRNA interference group, the shRNA interference group and the CHX function block group in 5 groups. The cells of each group were treated with cold and hot stimulation. The ATP release of the cells was observed by the coloring of krindin, the changes of fluorescence were calculated by Image Pro-plus, and the content of ATP released to the cell by bioluminescence was used. Test.
Results: PCR, Western blot and fluorescence photos showed that Pannexin3 was overexpressed, siRNA, shRNA could effectively infect dentin cells and realize the high expression and interference effect of the cell Pannexin3 gene. After cold and hot stimulation, the ATP in dentin cells quickly released to the cells, and the release rate of Pannexin3 overexpression cells was significantly higher than that of the control group. Blocking Pannexin3 channels with siRNA, shRNA interference or CHX can inhibit the release of intracellular ATP from cold and heat stimuli to the extracellular domain.
Conclusion: Pannexin3 can form a half channel in odontoblasts. When cold and heat stimulates, the cells release the ATP through the Pannexin3 half channel.
The third part: ATP participates in the transmission of pulp sensory nerve pain.
Objective: To investigate the role of ATP in receptor activation, Ca ion flow and pain stimulation in pulp sensory nerve pain.
Methods: the mouse trigeminal ganglia was taken and the expression of P2X3 receptor in the trigeminal ganglia of mice was detected by immunofluorescence. The trigeminal neuron cells were isolated and cultured by enzyme digestion. The cells were identified by morphological observation and peripheral protein immunofluorescence. The neuron cells were divided into ATP stimulation group, alpha beta me-ATP stimulation group, A-317491 A total of three groups of inhibition groups were used to analyze the flow of Ca ions in the cells of each group after receiving stimulation by patch clamp technique. Taking SD rats as the experimental object, the dentin sensitive model was prepared by grinding the enamel and partial dentin of the rat molars, which were divided into four groups: PBS, ATP, alpha, beta me-ATP and A-317491, respectively. Heat and cold stimulation were used to record the number of claw or licking in each group within 5 minutes after stimulation.
Results: the tissue immunofluorescence showed that the P2X3 receptor was concentrated on the edge of the trigeminal ganglion and scattered in the middle part. The trigeminal neuron cells were obtained by enzyme digestion. The cells were reticular and linear arranged in.ATP. Alpha, beta me-ATP stimulated the Ca ion internal flow after the trigeminal neuron cells, and A-317491 could inhibit the flow of Ca ions. ATP, alpha, and beta me-ATP could significantly increase the degree and frequency of response to stimulation, while A-317491 significantly reduced the response behavior of animals.
Conclusion: ATP activates the P2X3 receptor on the trigeminal ganglion, which causes Ca influx, causing the animals to feel pain.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R780.2

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 馬斌;劉玉柱;鎖云華;;激光聯(lián)合脫敏藥治療牙本質(zhì)過(guò)敏的療效觀察[J];人民軍醫(yī);2006年11期

2 楊洪俠;王華薇;倫杰;;調(diào)鉭、脫敏、充填法治療重癥牙本質(zhì)過(guò)敏的臨床體會(huì)[J];口腔醫(yī)學(xué);2007年06期

3 李江;陳衛(wèi);;酚醛樹(shù)脂治療牙本質(zhì)過(guò)敏的療效觀察[J];中國(guó)當(dāng)代醫(yī)藥;2010年35期

4 李光清;張學(xué)君;;極固寧治療牙本質(zhì)過(guò)敏的體會(huì)[J];中國(guó)醫(yī)藥指南;2013年11期

5 ;應(yīng)用直流電解除牙體z┒粗票甘鋇難辣局使鬧J];安醫(yī)學(xué)報(bào);1960年04期

6 李清泉;;硝酸鉀飽和液治療牙本質(zhì)過(guò)敏[J];人民軍醫(yī);1985年05期

7 宋鳳至，，于樹(shù)珍;牙本質(zhì)過(guò)敏的病因及治療體會(huì)[J];廣東牙病防治;1996年02期

8 孫衛(wèi)斌;如何測(cè)評(píng)牙本質(zhì)過(guò)敏的癥狀（二）[J];口腔醫(yī)學(xué);1996年02期

9 孫衛(wèi)斌;如何測(cè)評(píng)牙本質(zhì)過(guò)敏的癥狀(四)[J];口腔醫(yī)學(xué);1997年01期

10 史亞青;;咀嚼茶葉防治牙本質(zhì)過(guò)敏[J];健康天地;2007年07期

相關(guān)會(huì)議論文 前7條

1 趙碧霞;;中西藥結(jié)合治療生理磨損性牙本質(zhì)過(guò)敏[A];FDI、CSA臨床口腔進(jìn)展學(xué)術(shù)會(huì)議論文匯編[C];1999年

2 尹忠梅;李萍;邵明英;;綜合治療磨損所致牙本質(zhì)過(guò)敏的療效觀察[A];全國(guó)第四次牙體牙髓病學(xué)術(shù)會(huì)議論文匯編[C];1995年

3 王躍才;;脫敏王治療重癥牙本質(zhì)過(guò)敏臨床效果觀察[A];玉溪市醫(yī)學(xué)會(huì)第八屆口腔學(xué)術(shù)年會(huì)論文集[C];2007年

4 李雅萍;趙信義;李石保;;脫敏材料對(duì)牙本質(zhì)通透性的影響[A];第八屆全國(guó)口腔材料學(xué)術(shù)交流會(huì)暨中華口腔醫(yī)學(xué)會(huì)口腔材料專(zhuān)業(yè)委員會(huì)2013年會(huì)論文集[C];2013年

5 徐逸敏;馬曉蓬;劉國(guó)勤;劉筠;;醫(yī)用膠治療牙本質(zhì)過(guò)敏的臨床觀察[A];FDI、CSA臨床口腔進(jìn)展學(xué)術(shù)會(huì)議論文匯編[C];1999年

6 李亞華;楊春英;盧愛(ài)工;李秀英;;100例新型脫敏含漱液和自制托盤(pán)載藥聯(lián)合治療牙本質(zhì)過(guò)敏的療效隨訪(fǎng)及護(hù)理分析[A];全國(guó)口腔護(hù)理學(xué)術(shù)交流暨專(zhuān)題講座會(huì)議論文匯編[C];2004年

7 黃云亮;;3M Adper Prompt自酸蝕黏結(jié)劑治療牙本質(zhì)過(guò)敏的療效觀察[A];2004年上海市口腔醫(yī)學(xué)學(xué)術(shù)年會(huì)論文匯編[C];2004年

相關(guān)重要報(bào)紙文章 前10條

1 ;老年人牙本質(zhì)過(guò)敏的防治[N];人民政協(xié)報(bào);2000年

2 王燕;牙本質(zhì)過(guò)敏可致牙髓炎[N];衛(wèi)生與生活報(bào);2008年

3 健康時(shí)報(bào)記者 劉橋斌;牙齒怕冷怕酸也是病[N];健康時(shí)報(bào);2008年

4 本報(bào)記者 李健;三成國(guó)人口腔患“感冒”[N];中國(guó)消費(fèi)者報(bào);2008年

5 于峰;牙齒酸痛怎么辦？[N];大眾衛(wèi)生報(bào);2006年

6 山東省濟(jì)南市口腔醫(yī)院副主任醫(yī)師 王輝東;核桃仁可緩解倒牙[N];健康時(shí)報(bào);2009年

7 中南大學(xué)湘雅醫(yī)院 熊爭(zhēng)鳴;倒牙是病莫忽視[N];大眾衛(wèi)生報(bào);2003年

8 程鵬;碘化銀治療老年性牙本質(zhì)過(guò)敏癥[N];農(nóng)村醫(yī)藥報(bào)（漢）;2006年

9 馬艷玲;磨牙患者警惕心病[N];醫(yī)藥養(yǎng)生保健報(bào);2008年

10 楊海川;謹(jǐn)防牙體的磨耗[N];中國(guó)中醫(yī)藥報(bào);2002年

相關(guān)博士學(xué)位論文 前4條

1 付東杰;Pannexin3在牙髓中的表達(dá)及在牙本質(zhì)敏感中的作用[D];武漢大學(xué);2014年

2 李箐;氟/鉀納米羥基磷灰石涂膜的合成及其對(duì)牙本質(zhì)的作用[D];四川大學(xué);2006年

3 詹振林;齲齒修復(fù)與粘結(jié)性能的光學(xué)監(jiān)測(cè)與評(píng)估[D];福建師范大學(xué);2013年

4 唐成芳;原花青素對(duì)牙本質(zhì)及粘接界面耐脫礦和再礦化能力影響的實(shí)驗(yàn)研究[D];第四軍醫(yī)大學(xué);2013年

相關(guān)碩士學(xué)位論文 前10條

1 李晨曦;不同修復(fù)材料對(duì)牙釉質(zhì)及牙本質(zhì)磨損性能的研究[D];華北理工大學(xué);2016年

2 余少華;Er:YAG激光對(duì)牙本質(zhì)過(guò)敏癥的應(yīng)用研究[D];第三軍醫(yī)大學(xué);2016年

3 羅妍彥;半導(dǎo)體激光照射時(shí)間對(duì)牙髓及牙本質(zhì)的影響研究[D];大連醫(yī)科大學(xué);2007年

4 郭敬茹;冷光美白劑對(duì)牙本質(zhì)顯微硬度影響的實(shí)驗(yàn)研究[D];遼寧醫(yī)學(xué)院;2011年

5 李雅萍;牙本質(zhì)通透性的測(cè)定及其應(yīng)用研究[D];第四軍醫(yī)大學(xué);2013年

6 王大銘;勁潤(rùn)牙本質(zhì)保護(hù)膜對(duì)不同粘結(jié)劑與牙本質(zhì)間剪切強(qiáng)度的影響[D];山東大學(xué);2014年

7 王瓊;多肽調(diào)控羥基磷灰石礦化及其在牙本質(zhì)過(guò)敏治療中的應(yīng)用[D];清華大學(xué);2012年

8 馬勇;牙本質(zhì)過(guò)敏癥的不同治療方法對(duì)牙本質(zhì)影響的電鏡觀察[D];河北醫(yī)科大學(xué);2003年

9 林琪;Er，Cr：YSGG激光消融對(duì)牙本質(zhì)結(jié)構(gòu)及成分影響的研究[D];福建醫(yī)科大學(xué);2010年

10 余曉芬;牙本質(zhì)脫敏劑/粘結(jié)劑治療牙本質(zhì)過(guò)敏的臨床療效觀察[D];浙江大學(xué);2009年

本文編號(hào)：2054164