钠通道所介导的FHF2在疼痛中的作用以及钠通道在人前列腺癌中表达特征的研究
发布时间：2019-06-06 10:29
【摘要】：成纤维细胞生长因子同源性因子（Fibroblast growth factor homologousfactors，即FHFs）由于不能分泌到细胞外，故属于成纤维细胞生长因子（Fibroblast growth factor，即FGF）家族中的一类特殊亚家族，共包括四个成员:FHF1-FHF4，其均可在细胞内与电压依赖性钠离子（Nav）通道相互作用并影响通道的表达和功能以及神经元的兴奋性。目前发现FHF4敲除可致小鼠共济失调，选择性敲除小鼠海马神经元上FHF2可导致小鼠学习和记忆能力减退。背根神经节（dorsal root ganglion，即DRG）神经元是痛觉传导的初级神经元，其上有多种钠通道表达，其中Nav1.7选择性表达于无髓鞘的小DRG，可通过强化阈下刺激并设定Nav1.8和Nav1.9激活开放的阈值而影响神经元兴奋性及痛觉信号传导。FHFs作为钠离子通道的一种重要调节蛋白，其对DRG上Nav通道功能调节及痛觉影响目前尚未清楚。本课题首先在DRG神经元上研究不同FHFs亚型的表达特征，进而在过表达系统中确定FHFs主要表达亚型（FHF2)对Nav1.7通道的功能影响，然后用腺病毒介导的shRNA特异性敲除大鼠DRG上的FHF2并观察其对钠通道的影响，最后利用DRG上FHF2条件性敲除小鼠观察FHF2在痛觉中的作用。 电压依赖性钠离子通道不仅在可兴奋细胞中表达，也可表达于多种肿瘤细胞，包括前列腺癌细胞。钠离子通道可影响癌细胞迁移、入侵和代谢。尽管有报道称某些钠通道亚型在大鼠前列腺癌细胞中表达上调，但在正常人前列腺组织（normal human prostate，即NP）、前列腺增生患者（benignprostatic hyperplasia，即BPH）及人前列腺癌（prostatic cancer，即PC）中综合系统比较钠通道表达水平的研究尚未报道。本文拟用实时定量聚合酶链反应（real-time quantitive polymerase chain reaction，即qPCR）研究NP、BPH、PC中钠离子通道α亚基表达水平的差异，从而为前列腺癌的临床诊断及治疗提供依据。 第一部分钠通道所介导的FHF2在疼痛中作用的研究 目的：（1）测定FHFs在大鼠DRG神经元上的表达特征。（2）在异源性表达系统观察FHFs主要表达亚型对Nav1.7通道电流大小及门控特性的影响。（3）在小DRG神经元上观察敲低FHF2对钠通道的影响。（4）利用DRG上FHF2条件性敲除小鼠观察FHF2在痛觉中的作用。 方法：（1）应用全细胞膜片钳技术观察FHF2对钠电流的影响。（2）应用实时定量PCR (quantitative RT-PCR,即qPCR)及免疫荧光染色及激光共聚焦显微镜技术定量和定位分析FHF在DRG中的表达特征。（3）应用热痛仪或Von Frey纤维刺痛针，分别测定DRG中FHF2条件性敲除小鼠的热痛阈和机械痛阈。 结果：（1）FHFs家族中，FHF2亚家族在大鼠DRG中表达量最高，，以FHF2U亚型为主，且主要在小直径DRG中表达。（2）在过表达系统CHO细胞中，FHF2U增大Nav1.7电流密度近一倍，激活曲线无明显变化，失活曲线向右移动，失活减慢。（3）用腺病毒携带shRNA敲除大鼠DRG上FHF2后，小直径DRG上总钠电流为零，通过免疫组化分析，可见DRG细胞膜上钠通道蛋白荧光明显降低。（4）FHF2条件性敲除小鼠与对照组比较，其热痛阈值提高，分别为11.96±0.64s和10.08±0.59s(P0.05)，机械刺痛阈值也提高，分别为4.14±0.23g和3.27±0.24g(P0.05)，均具有统计学差异。 结论：（1）FHF2是大鼠DRG神经元中的主要表达亚型。（2）过表达FHF2U可显著增大Nav1.7电流密度，失活减慢。（3）敲除DRG神经元上FHF2可导致小直径DRG神经元中钠通道呈功能丧失的表型，且钠通道在DRG神经元细胞膜上表达降低。（4）DRG上FHF2条件性敲除小鼠的热痛及机械刺痛阈值显著提高，很可能是因为FHF2缺失导致的钠通道功能/表达受到抑制。 第二部分钠通道在人前列腺癌中表达特征的研究 目的：检测正常人前列腺组织（NP）、人前列腺增生组织（BPH）及人前列腺癌细胞（PC-3、LnCap）中Nav1.1-Nav1.9mRNA的表达水平 方法：（1）应用qPCR技术检测钠通道各个亚型在人NP、BPH、PC-3和LnCap的表达情况。（2）应用全细胞膜片钳技术记录癌细胞钠电流。 结果：（1）正常人前列腺组织中Nav1.5mRNA为主要表达亚型，BPH中以Nav1.2和Nav1.5为主要表达亚型。（2） PC-3和LnCap细胞中则以Nav1.6、Nav1.7mRNA表达占优。（3）比较以上四类细胞中Nav1.5、Nav1.6、Nav1.7mRNA表达水平发现，前列腺癌细胞（PC-3、LnCap）中Nav1.6、Nav1.7mRNA表达水平是NP和BPH的6~27倍，且在PC-3细胞中的表达量明显高于LnCap细胞（P0.05）。（4）在PC-3细胞中可记录到TTX敏感的钠电流，但在LnCap细胞中未记录到。 结论：虽然正常人前列腺组织与前列腺增生组织表达所有钠离子通道亚型，但其表达水平都很低；与之相比，人前列腺癌细胞中Nav1.6和Nav1.7表达明显上调，且在高转移性前列腺癌细胞系PC-3细胞上调最明显。此结果提示高表达的Nav1.6和Nav1.7可作为人类某些前列腺癌的临床诊断标志。
[Abstract]:Fibroblast growth factor homologies (FHFs) belong to a class of special subfamilies in the family of fibroblast growth factor (FGF), including four members: FHF1-FHF4. It can interact with the voltage-dependent sodium ion (Nav) channel in the cell and influence the expression and function of the channel and the excitability of the neurons. It is found that FHF2 knockout can lead to the ataxia of the mouse, and the expression of FHF2 on the hippocampal neurons of the selective knockout mice can lead to the decrease of the learning and memory ability of the mice. The dorsal root ganglion (DRG) neurons are the primary neurons of the hyperalgesia, on which there are a variety of sodium channel expressions, in which Nav1.7 is selectively expressed in a small DRG without a pulp, The activation of the neurons and the conduction of the pain signal can be affected by the threshold stimulation and the setting of the open thresholds for Nav1.8 and Nav1.8. FHFs, as an important regulation protein of sodium ion channel, has not been clear on the regulation of the function of Nav channel and the effect of pain on the DRG. In this paper, we first study the expression of the different FHFs subtypes on the DRG neurons, and then determine the function of the main expression subtype of FHFs (FHF2) on the Nav1.7 channel in the over-expression system, and then use the adenovirus-mediated shRNA to specifically knock the FHF2 on the rat DRG and observe its effect on the sodium channel. Finally, the role of FHF2 in the hyperalgesia was observed by using the FHF2 conditional knockout mice on the DRG. The voltage-dependent sodium ion channel can be expressed not only in the excitable cells, but also in a variety of tumor cells, including prostate cancer The sodium ion channel can affect the migration, invasion and generation of cancer cells. Thanks. Although some of the sodium channel subtypes have been reported to be up-regulated in rat prostate cancer cells, normal human prostate, i.e., NP, prostate hyperplastic, i.e., BPH, and human prostate cancer are reported. R (i. e. PC) The study of the level of sodium channel expression in the integrated system in PC has not yet been reported. In this paper, real-time quantitative polymerase chain reaction (qPCR) is used to study the difference of the expression level of sodium ion channels and subunits in NP, BPH and PC, so as to provide the basis for the clinical diagnosis and treatment of prostate cancer. The FHF2 mediated by the first partial sodium channel is reported in pain The purpose of this study was to (1) determine the FHFs in the DRG neurons of the rat The expression profile of the expression of the main expression of FHFs on the Nav1.7 channel was observed in the heterologous expression system. Effects of characteristics on small DRG neurons. (3) Low FHF2 on small DRG neurons was observed for sodium Effect of the channel. (4) FHF2 was observed in pain using the FHF2 conditional knockout mouse on the DRG The effect of the method: (1) the application of whole-cell patch clamp technique to observe FHF2 (2) Real-time quantitative PCR (qPCR) and immunofluorescence staining and laser confocal microscopy (FHF) were applied to the quantitative and location analysis of FHF in DR. Expression profiles in G. (3) Thermal pain or Von Frey fiber stinging needles were used to determine the heat of FHF2 conditional knockout mice in the DRG, respectively Results: (1) The expression of FHF2 subfamily in the rat DRG was the highest in the FHFs family, mainly in the FHF2U subtype and mainly in the FHFs family. in that CHO cell of the overexpressing system, the FHF2U increase the current density of the Nav1.7, the activation curve has no obvious change, and the inactivation curve To move to the right, the inactivation was slowed down. (3) After the expression of FHF2 on the DRG of the rat DRG with the adenovirus, the total sodium current on the small diameter DRG was zero, and the sodium on the cell membrane of the DRG was found through the immunohistochemical analysis. (4) The threshold of thermal pain of FHF2-conditioned knockout mice was 11.96-0.64s and 10.08-0.59s (P0.05). The threshold of mechanical stinging was also increased (4.14-0.23g and 3.27-0.24g (P0.05). Conclusion: (1) FHF2 is the rat DRG. Primary expression subtypes in neurons. (2) Overexpression of FHF2U can significantly increase Nav1 . (3) The expression of FHF2 on the DRG neurons can lead to a functional loss of the sodium channel in the small-diameter DRG neurons, and the sodium channel is in the DRG The decrease of the expression of FHF2 on the cell membrane of the neurons. (4) The thermal pain and the mechanical stinging threshold of the FHF2 conditional knockout mice on the DRG were significantly increased, possibly due to the loss of the FHF2 due to the deletion of the FHF2 Channel function/ expression is inhibited. The second part of sodium channel The purpose of the study of the expression of human prostate cancer: to detect the Nav1.8 in the normal human prostate tissue (NP), the human prostate hyperplasia (BPH) and the human prostate cancer cell (PC-3, LnCap). -Na The expression level of v1.9mRNA: (1) The detection of each subtype of sodium channel in human NP, BP by qPCR The expression of H, PC-3 and LnCap. (2) The results are as follows: (1) Nav1.5 mRNA in normal prostate tissue is the main expression subtype, and N in BPH v1.2 and Nav1.5 are the main expression subtypes. (2) In PC-3 and LnCap cells, Na (3) The expression level of Nav1.5, Nav1.6, Nav1.7 mRNA in the above four types of cells was found to be 6-27 times that of NP and BPH, and the expression of Nav1.5, Nav1.7 and Nav1.7 mRNA in prostate cancer cells (PC-3, LnCap) was 6-27 times that of NP and BPH, and the expression in PC-3 cells The amount is significantly higher than that of LnCap cells (P0.05). (4) TTX-sensitive cells can be recorded in PC-3 cells The sodium current, but not recorded in the LnCap cell, was not recorded in the LnCap cell. Conclusion: Although the normal prostate tissue and the prostate hyperplasia tissue express all of the sodium ion channel subtypes, the level of expression is very low; in contrast, the expression of Nav1.6 and Nav1.7 in human prostate cancer cells is significantly up-regulated and is high The up-regulation of PC-3 cells in metastatic prostate cancer cell lines is the most obvious. This result suggests a high expression of Nav1.6 and Nav
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
本文编号：2494289
[Abstract]:Fibroblast growth factor homologies (FHFs) belong to a class of special subfamilies in the family of fibroblast growth factor (FGF), including four members: FHF1-FHF4. It can interact with the voltage-dependent sodium ion (Nav) channel in the cell and influence the expression and function of the channel and the excitability of the neurons. It is found that FHF2 knockout can lead to the ataxia of the mouse, and the expression of FHF2 on the hippocampal neurons of the selective knockout mice can lead to the decrease of the learning and memory ability of the mice. The dorsal root ganglion (DRG) neurons are the primary neurons of the hyperalgesia, on which there are a variety of sodium channel expressions, in which Nav1.7 is selectively expressed in a small DRG without a pulp, The activation of the neurons and the conduction of the pain signal can be affected by the threshold stimulation and the setting of the open thresholds for Nav1.8 and Nav1.8. FHFs, as an important regulation protein of sodium ion channel, has not been clear on the regulation of the function of Nav channel and the effect of pain on the DRG. In this paper, we first study the expression of the different FHFs subtypes on the DRG neurons, and then determine the function of the main expression subtype of FHFs (FHF2) on the Nav1.7 channel in the over-expression system, and then use the adenovirus-mediated shRNA to specifically knock the FHF2 on the rat DRG and observe its effect on the sodium channel. Finally, the role of FHF2 in the hyperalgesia was observed by using the FHF2 conditional knockout mice on the DRG. The voltage-dependent sodium ion channel can be expressed not only in the excitable cells, but also in a variety of tumor cells, including prostate cancer The sodium ion channel can affect the migration, invasion and generation of cancer cells. Thanks. Although some of the sodium channel subtypes have been reported to be up-regulated in rat prostate cancer cells, normal human prostate, i.e., NP, prostate hyperplastic, i.e., BPH, and human prostate cancer are reported. R (i. e. PC) The study of the level of sodium channel expression in the integrated system in PC has not yet been reported. In this paper, real-time quantitative polymerase chain reaction (qPCR) is used to study the difference of the expression level of sodium ion channels and subunits in NP, BPH and PC, so as to provide the basis for the clinical diagnosis and treatment of prostate cancer. The FHF2 mediated by the first partial sodium channel is reported in pain The purpose of this study was to (1) determine the FHFs in the DRG neurons of the rat The expression profile of the expression of the main expression of FHFs on the Nav1.7 channel was observed in the heterologous expression system. Effects of characteristics on small DRG neurons. (3) Low FHF2 on small DRG neurons was observed for sodium Effect of the channel. (4) FHF2 was observed in pain using the FHF2 conditional knockout mouse on the DRG The effect of the method: (1) the application of whole-cell patch clamp technique to observe FHF2 (2) Real-time quantitative PCR (qPCR) and immunofluorescence staining and laser confocal microscopy (FHF) were applied to the quantitative and location analysis of FHF in DR. Expression profiles in G. (3) Thermal pain or Von Frey fiber stinging needles were used to determine the heat of FHF2 conditional knockout mice in the DRG, respectively Results: (1) The expression of FHF2 subfamily in the rat DRG was the highest in the FHFs family, mainly in the FHF2U subtype and mainly in the FHFs family. in that CHO cell of the overexpressing system, the FHF2U increase the current density of the Nav1.7, the activation curve has no obvious change, and the inactivation curve To move to the right, the inactivation was slowed down. (3) After the expression of FHF2 on the DRG of the rat DRG with the adenovirus, the total sodium current on the small diameter DRG was zero, and the sodium on the cell membrane of the DRG was found through the immunohistochemical analysis. (4) The threshold of thermal pain of FHF2-conditioned knockout mice was 11.96-0.64s and 10.08-0.59s (P0.05). The threshold of mechanical stinging was also increased (4.14-0.23g and 3.27-0.24g (P0.05). Conclusion: (1) FHF2 is the rat DRG. Primary expression subtypes in neurons. (2) Overexpression of FHF2U can significantly increase Nav1 . (3) The expression of FHF2 on the DRG neurons can lead to a functional loss of the sodium channel in the small-diameter DRG neurons, and the sodium channel is in the DRG The decrease of the expression of FHF2 on the cell membrane of the neurons. (4) The thermal pain and the mechanical stinging threshold of the FHF2 conditional knockout mice on the DRG were significantly increased, possibly due to the loss of the FHF2 due to the deletion of the FHF2 Channel function/ expression is inhibited. The second part of sodium channel The purpose of the study of the expression of human prostate cancer: to detect the Nav1.8 in the normal human prostate tissue (NP), the human prostate hyperplasia (BPH) and the human prostate cancer cell (PC-3, LnCap). -Na The expression level of v1.9mRNA: (1) The detection of each subtype of sodium channel in human NP, BP by qPCR The expression of H, PC-3 and LnCap. (2) The results are as follows: (1) Nav1.5 mRNA in normal prostate tissue is the main expression subtype, and N in BPH v1.2 and Nav1.5 are the main expression subtypes. (2) In PC-3 and LnCap cells, Na (3) The expression level of Nav1.5, Nav1.6, Nav1.7 mRNA in the above four types of cells was found to be 6-27 times that of NP and BPH, and the expression of Nav1.5, Nav1.7 and Nav1.7 mRNA in prostate cancer cells (PC-3, LnCap) was 6-27 times that of NP and BPH, and the expression in PC-3 cells The amount is significantly higher than that of LnCap cells (P0.05). (4) TTX-sensitive cells can be recorded in PC-3 cells The sodium current, but not recorded in the LnCap cell, was not recorded in the LnCap cell. Conclusion: Although the normal prostate tissue and the prostate hyperplasia tissue express all of the sodium ion channel subtypes, the level of expression is very low; in contrast, the expression of Nav1.6 and Nav1.7 in human prostate cancer cells is significantly up-regulated and is high The up-regulation of PC-3 cells in metastatic prostate cancer cell lines is the most obvious. This result suggests a high expression of Nav1.6 and Nav
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
【参考文献】
相关期刊论文 前3条
1 ;Roles of intracellular fibroblast growth factors in neural development and functions[J];Science China(Life Sciences);2012年12期
2 渠兴乾;程志军;沈伯雄;;电压门控型钠离子通道Na_v1.7的研究进展[J];四川医学;2009年04期
3 杨晨颖;王克威;;电压门控钠通道Na_V1.7与痛信号的产生[J];生理科学进展;2007年04期
本文编号：2494289
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