【摘要】：成纖維細胞生長因子同源性因子（Fibroblast growth factor homologousfactors，即FHFs）由于不能分泌到細胞外，故屬于成纖維細胞生長因子（Fibroblast growth factor，即FGF）家族中的一類特殊亞家族，共包括四個成員:FHF1-FHF4，其均可在細胞內(nèi)與電壓依賴性鈉離子（Nav）通道相互作用并影響通道的表達和功能以及神經(jīng)元的興奮性。目前發(fā)現(xiàn)FHF4敲除可致小鼠共濟失調(diào)，選擇性敲除小鼠海馬神經(jīng)元上FHF2可導致小鼠學習和記憶能力減退。背根神經(jīng)節(jié)（dorsal root ganglion，即DRG）神經(jīng)元是痛覺傳導的初級神經(jīng)元，其上有多種鈉通道表達，其中Nav1.7選擇性表達于無髓鞘的小DRG，可通過強化閾下刺激并設定Nav1.8和Nav1.9激活開放的閾值而影響神經(jīng)元興奮性及痛覺信號傳導。FHFs作為鈉離子通道的一種重要調(diào)節(jié)蛋白，其對DRG上Nav通道功能調(diào)節(jié)及痛覺影響目前尚未清楚。本課題首先在DRG神經(jīng)元上研究不同F(xiàn)HFs亞型的表達特征，進而在過表達系統(tǒng)中確定FHFs主要表達亞型（FHF2)對Nav1.7通道的功能影響，然后用腺病毒介導的shRNA特異性敲除大鼠DRG上的FHF2并觀察其對鈉通道的影響，最后利用DRG上FHF2條件性敲除小鼠觀察FHF2在痛覺中的作用。 電壓依賴性鈉離子通道不僅在可興奮細胞中表達，也可表達于多種腫瘤細胞，包括前列腺癌細胞。鈉離子通道可影響癌細胞遷移、入侵和代謝。盡管有報道稱某些鈉通道亞型在大鼠前列腺癌細胞中表達上調(diào)，但在正常人前列腺組織（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）中綜合系統(tǒng)比較鈉通道表達水平的研究尚未報道。本文擬用實時定量聚合酶鏈反應（real-time quantitive polymerase chain reaction，即qPCR）研究NP、BPH、PC中鈉離子通道α亞基表達水平的差異，從而為前列腺癌的臨床診斷及治療提供依據(jù)。 第一部分鈉通道所介導的FHF2在疼痛中作用的研究 目的：（1）測定FHFs在大鼠DRG神經(jīng)元上的表達特征。（2）在異源性表達系統(tǒng)觀察FHFs主要表達亞型對Nav1.7通道電流大小及門控特性的影響。（3）在小DRG神經(jīng)元上觀察敲低FHF2對鈉通道的影響。（4）利用DRG上FHF2條件性敲除小鼠觀察FHF2在痛覺中的作用。 方法：（1）應用全細胞膜片鉗技術觀察FHF2對鈉電流的影響。（2）應用實時定量PCR (quantitative RT-PCR,即qPCR)及免疫熒光染色及激光共聚焦顯微鏡技術定量和定位分析FHF在DRG中的表達特征。（3）應用熱痛儀或Von Frey纖維刺痛針，分別測定DRG中FHF2條件性敲除小鼠的熱痛閾和機械痛閾。 結果：（1）FHFs家族中，F(xiàn)HF2亞家族在大鼠DRG中表達量最高，，以FHF2U亞型為主，且主要在小直徑DRG中表達。（2）在過表達系統(tǒng)CHO細胞中，F(xiàn)HF2U增大Nav1.7電流密度近一倍，激活曲線無明顯變化，失活曲線向右移動，失活減慢。（3）用腺病毒攜帶shRNA敲除大鼠DRG上FHF2后，小直徑DRG上總鈉電流為零，通過免疫組化分析，可見DRG細胞膜上鈉通道蛋白熒光明顯降低。（4）FHF2條件性敲除小鼠與對照組比較，其熱痛閾值提高，分別為11.96±0.64s和10.08±0.59s(P0.05)，機械刺痛閾值也提高，分別為4.14±0.23g和3.27±0.24g(P0.05)，均具有統(tǒng)計學差異。 結論：（1）FHF2是大鼠DRG神經(jīng)元中的主要表達亞型。（2）過表達FHF2U可顯著增大Nav1.7電流密度，失活減慢。（3）敲除DRG神經(jīng)元上FHF2可導致小直徑DRG神經(jīng)元中鈉通道呈功能喪失的表型，且鈉通道在DRG神經(jīng)元細胞膜上表達降低。（4）DRG上FHF2條件性敲除小鼠的熱痛及機械刺痛閾值顯著提高，很可能是因為FHF2缺失導致的鈉通道功能/表達受到抑制。 第二部分鈉通道在人前列腺癌中表達特征的研究 目的：檢測正常人前列腺組織（NP）、人前列腺增生組織（BPH）及人前列腺癌細胞（PC-3、LnCap）中Nav1.1-Nav1.9mRNA的表達水平 方法：（1）應用qPCR技術檢測鈉通道各個亞型在人NP、BPH、PC-3和LnCap的表達情況。（2）應用全細胞膜片鉗技術記錄癌細胞鈉電流。 結果：（1）正常人前列腺組織中Nav1.5mRNA為主要表達亞型，BPH中以Nav1.2和Nav1.5為主要表達亞型。（2） PC-3和LnCap細胞中則以Nav1.6、Nav1.7mRNA表達占優(yōu)。（3）比較以上四類細胞中Nav1.5、Nav1.6、Nav1.7mRNA表達水平發(fā)現(xiàn)，前列腺癌細胞（PC-3、LnCap）中Nav1.6、Nav1.7mRNA表達水平是NP和BPH的6~27倍，且在PC-3細胞中的表達量明顯高于LnCap細胞（P0.05）。（4）在PC-3細胞中可記錄到TTX敏感的鈉電流，但在LnCap細胞中未記錄到。 結論：雖然正常人前列腺組織與前列腺增生組織表達所有鈉離子通道亞型，但其表達水平都很低；與之相比，人前列腺癌細胞中Nav1.6和Nav1.7表達明顯上調(diào)，且在高轉移性前列腺癌細胞系PC-3細胞上調(diào)最明顯。此結果提示高表達的Nav1.6和Nav1.7可作為人類某些前列腺癌的臨床診斷標志。
[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
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.25

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