Pannexin3在牙髓中的表达及在牙本质敏感中的作用

发布时间：2018-06-22 20:40

本文选题：Pannexin3 + 半通道　；参考：《武汉大学》2014年博士论文

【摘要】：第一部分：Pannexin3在牙髓组织及成牙本质细胞中的表达及分布目的：明确Pannexin3基因及蛋白在牙髓组织及成牙本质细胞中的表达及分布情况。方法：收集15-20岁的健康正畸牙或者智齿,提取牙髓组织mRNA,采用PCR技术检测牙髓内Pannexin3基因的相对含量；应用组织免疫荧光方法观察牙髓组织中Pannexin3蛋白表达及分布情况。采用组织块法培养牙髓干细胞,诱导分化为成牙本质细胞,利用PCR、Western blot、细胞免疫荧光技术从基因及蛋白水平鉴定成牙本质细胞中Pannexin3的表达及分布。结果：组织块法体外培养牙髓细胞,经矿化诱导7天后细胞分化为成牙本质细胞。PCR、Western blot检测结果显示牙髓组织及成牙本质细胞中有Pannexin3基因及蛋白表达,免疫荧光结果发现Pannexin3蛋白主要分布于人牙髓组织的成牙本质细胞层的胞体及细胞突起部分和成牙本质细胞胞浆近胞核及细胞膜等位置。结论：Pannexin3主要分布在人牙髓组织以及成牙本质细胞胞浆及胞膜上。第二部分：Pannexin3参与成牙本质细胞释放ATP 目的：研究Pannexin3缝隙连接蛋白在外界冷热刺激下对成牙本质细胞释放ATP的影响。方法：构建Pannexin3过表达慢病毒转染载体PLL3.7\CMV-PANNEXIN3\CMV-RFP,通过病毒感染获取能够高表达Pannexin3的成牙本质细胞；合成siRNA、构建shRNA分别干扰成牙本质细胞Pannexin3基因表达。采用PCR、Western blot技术对过表达及干扰效果进行评价。将细胞分为对照组、过表达组、siRNA干扰组、shRNA干扰组及甘珀酸(CHX)功能阻断组共5组,对各组细胞分别进行冷热刺激。使用奎纳克林进行染色观察细胞ATP释放情况,通过Image Pro-plus计算荧光变化量,采用生物荧光法对释放到细胞外的ATP含量进行测定。结果：PCR、Western blot及荧光照片结果证明Pannexin3过表达、siRNA、shRNA能有效感染成牙本质细胞,实现细胞Pannexin3基因的高表达和干扰效果。冷热刺激后成牙本质细胞内的ATP迅速释放到细胞外,Pannexin3过表达细胞释放速度明显高于对照组,使用siRNA、shRNA干扰或者CHX阻断Pannexin3通道都能抑制冷热刺激引起的细胞内ATP释放到胞外。结论：Pannexin3能在成牙本质细胞形成半通道,冷热刺激时细胞通过Pannexin3半通道释放ATP到细胞外。第三部分：ATP参与牙髓感觉神经疼痛刺激传递目的：了解ATP在牙髓感觉神经疼痛受体激活、Ca离子流动以及疼痛刺激过程中的作用。方法：取小鼠三叉神经节,采用免疫荧光方法检测小鼠三叉神经节中P2X3受体表达情况,采用酶消化法分离培养三叉神经元细胞,通过形态观察及外周蛋白免疫荧光方法进行细胞鉴定。将神经元细胞分为ATP刺激组、α βme-ATP刺激组、A-317491抑制组共三组,采用膜片钳技术对各组神经元细胞在接受刺激后细胞内Ca离子流动情况进行分析。以SD大鼠为实验对象,磨除大鼠磨牙釉质及部分牙本质,制备牙本质敏感模型,按注射药物不同分为：PBS、ATP、α, βme-ATP及A-317491四组,分别添加冷热刺激,记录刺激后5分钟内各组动物抬爪或者舔舐的次数。结果：组织免疫荧光显示P2X3受体集中分布在三叉神经节的边缘,散在分布于中间部分。采用酶消化法获得三叉神经元细胞,细胞呈现为网状、线状排列。ATP、 α, βme-ATP刺激三叉神经元细胞后出现Ca离子内流,A-317491能抑制Ca离子流动。冷热刺激后牙本质敏感大鼠出现抬爪或者舔舐动作,ATP、α, βme-ATP能够显著增加动物对刺激的反应程度及频度,而A-317491则明显降低了动物的反应行为。结论：ATP激活三叉神经节上的P2X3受体,使Ca离子内流,从而导致动物产生疼痛感觉。
[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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R780.2

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