發(fā)布時(shí)間：2018-05-24 18:36

  本文選題：腰椎間盤突出 + 自體髓核移植��； 參考：《蘇州大學(xué)》2015年碩士論文

【摘要】：目的1.初步闡明自體髓核(nucleus pulposus,NP)移植誘導(dǎo)的成年大鼠下肢痛覺過敏中背根神經(jīng)節(jié)(DRG)神經(jīng)元上電壓門控性鈉通道(VGSCs)的表達(dá)和功能的變化特征。2.探討胱硫醚-?-合成酶(CBS)在下肢痛覺過敏中的作用及其對(duì)電壓門控性鈉通道表達(dá)和功能調(diào)控的影響。方法1.選擇成年SD雄性大鼠隨機(jī)分為LDH組和Sham組,LDH組在左側(cè)L5、L6背根神經(jīng)節(jié)處以自體尾椎髓核移植來建立大鼠腰椎間盤突出模型,Sham組僅暴露L5、L6神經(jīng)根,其他處理與LDH組相同。測(cè)量?jī)山M大鼠術(shù)前及術(shù)后7天,14天,21天的左后肢機(jī)械刺激縮足反射閾值(PWT)和熱刺激縮足反射閾值(PWL)的變化。鞘內(nèi)注射CBS拮抗劑羧甲基羥胺半鹽酸(AOAA),同樣運(yùn)用以上PWT、PWL測(cè)量方法評(píng)測(cè)大鼠的下肢疼痛反射閾值,從而檢測(cè)CBS在LDH模型中的作用。2.運(yùn)用逆行標(biāo)記的熒光素(Di I)來標(biāo)記支配后肢的L5-L6背根神經(jīng)節(jié)(DRG)中、小神經(jīng)元,運(yùn)用全細(xì)胞膜片鉗方法研究LDH大鼠L5-L6 DRG神經(jīng)元興奮性及電壓門控性鈉通道電流的變化特性。3.運(yùn)用Western Blotting方法檢測(cè)大鼠L5-L6 DRG神經(jīng)元中Na V1.7,Na V1.8以及CBS蛋白表達(dá)情況。4.運(yùn)用免疫組織化學(xué)檢測(cè)LDH大鼠L5-L6 DRG神經(jīng)元中CBS和Na V1.7,Na V1.8蛋白共表達(dá)情況。結(jié)果1.自體髓核移植明顯增加大鼠下肢痛覺過敏,機(jī)械刺激縮足反射閾值(PWT)在術(shù)后1周至3周顯著低于Sham組,1周時(shí)達(dá)到疼痛閾值的最低值,熱刺激縮足反射閾值(PWL)在術(shù)后1周時(shí)顯著降低,2周時(shí)基本恢復(fù)到原基線水平,并且PWT和PWL的顯著降低與CBS的表達(dá)上調(diào)相關(guān)聯(lián);鞘內(nèi)注射CBS的拮抗劑AOAA后,可以顯著翻轉(zhuǎn)LDH大鼠疼痛閾值,并呈現(xiàn)時(shí)間和劑量依賴性。2.全細(xì)胞膜片鉗記錄顯示,與Sham組相比,LDH組L5-L6 DRG神經(jīng)元興奮性顯著增加;進(jìn)一步檢測(cè)顯示自體髓核移植后電壓門控性鈉通道的電流密度顯著增加。3.數(shù)據(jù)分析后顯示,與Sham組相比,LDH組的失活曲線顯著左移,激活曲線沒有顯著變化。另外,自體髓核移植處理后,電壓門控性鈉通道的失活時(shí)間顯著增長,激活時(shí)間沒有顯著變化。4.運(yùn)用免疫組化的實(shí)驗(yàn)方法檢測(cè)后發(fā)現(xiàn)CBS與Na V1.7,Na V1.8在L5-L6神經(jīng)元中能夠共表達(dá)。自體髓核移植大鼠L5-L6 DRG神經(jīng)元中Na V1.7和Na V1.8受體蛋白表達(dá)顯著增加,與移植無關(guān)的T10-T12 DRG神經(jīng)元Na V1.7和Na V1.8的蛋白表達(dá)水平?jīng)]有顯著差異。5.連續(xù)鞘內(nèi)注射AOAA可以顯著翻轉(zhuǎn)LDH組大鼠L5-L6相關(guān)DRG神經(jīng)元的興奮性,還可以顯著降低電壓門控性鈉通道的電流密度,并使電壓門控性鈉通道的失活曲線右移。另外AOAA還可以顯著降低LDH組大鼠L5-L6相關(guān)DRG神經(jīng)元中Na V1.7和Na V1.8的高表達(dá)。結(jié)論以上實(shí)驗(yàn)結(jié)果表明硫化氫合酶CBS可能是通過電壓門控性鈉通道的激活和Na V1.7和Na V1.8蛋白的高表達(dá),增加電壓門控性鈉通道電流密度,導(dǎo)致自體髓核移植處相關(guān)DRG神經(jīng)元興奮性的增加進(jìn)而參與外周痛覺信號(hào)的調(diào)制,最終導(dǎo)致大鼠下肢痛覺過敏。本研究在一定程度上揭示了LDH病人疼痛產(chǎn)生的部分分子機(jī)制,可能會(huì)為LDH的治療提供一些有效的治療方案。-?-
[Abstract]:Objective 1. to preliminarily elucidate the expression and function of voltage gated sodium channel (VGSCs) on the dorsal root ganglion (DRG) neurons of the lower extremities induced by autologous nucleus pulposus (NP) transplantation in adult rats. The role of.2. to explore the role of cystthioether - synthetase (CBS) in the hyperalgesia of the lower extremities and its voltage gated sodium channel Method 1. the adult SD male rats were randomly divided into LDH group and Sham group. The LDH group was in the left L5 and L6 dorsal root ganglion with autologous tail marrow nucleus transplantation to establish the rat lumbar disc herniation model. The Sham group only exposed L5, L6 nerve root, and the other treatments were the same as LDH group. The two groups of rats were measured before and 7 days, 14 after the operation. The changes of the left hind limb mechanical stimulation of the contraction foot reflex threshold (PWT) and the thermal stimulation contraction reflex threshold (PWL). The intrathecal injection of CBS antagonist carboxymethyl hydroxylamine semi hydrochloric acid (AOAA) is also used to evaluate the pain reflex threshold of the lower extremities of the rats by the above PWT and PWL methods, and to detect the function of CBS in LDH model by.2. to use retrograde labeling. Di I to mark the L5-L6 dorsal root ganglion (DRG) in the hind limbs (DRG), small neurons, using whole cell patch clamp method to study the excitatory and voltage-gated sodium channel current change characteristics of L5-L6 DRG neurons in LDH rats.3. using Western Blotting method to detect the L5-L6 DRG neurons in rats. .4. immunohistochemical staining was used to detect the co expression of CBS and Na V1.7, Na V1.8 protein in the L5-L6 DRG neurons of LDH rats. Results 1. autologous nucleus pulposus transplantation significantly increased the hyperalgesia of the lower limbs of the rats. The threshold of mechanical stimulation contraction reflex (PWT) was significantly lower than that in the Sham group from 1 to 3 weeks after the operation, and the minimum value of the pain threshold was reached at 1 weeks, and the heat stimulation was achieved. The threshold of the contraction reflex (PWL) decreased significantly at 1 weeks after the operation and was basically restored to the original baseline level at 2 weeks, and the significant reduction of PWT and PWL was associated with the up regulation of CBS expression. After the intrathecal CBS antagonist AOAA, the pain threshold of LDH rats could be reversed significantly, and the time and dose dependent.2. whole cell patch clamp recording showed, and Sha. Compared with group M, the excitability of L5-L6 DRG neurons in group LDH increased significantly. Further detection showed that the current density of voltage gated sodium channel after autotransplantation of autologous nucleus pulposus was significantly increased after.3. data analysis. Compared with the Sham group, the inactivation curve in the LDH group was significantly left, and the activation curve was not significantly changed. In addition, after autologous nucleus pulposus transplantation, the voltage was changed. The inactivation time of the gated sodium channel increased significantly and the activation time did not change significantly..4. was detected by CBS and Na V1.7, and Na V1.8 was co expressed in L5-L6 neurons. The Na V1.7 and Na receptor receptor protein expression in L5-L6 DRG neurons in the autologous nucleus pulposus transplanted rats increased significantly. There is no significant difference in the protein expression level of Na V1.7 and Na V1.8 in the T12 DRG neurons..5. continuous intrathecal AOAA can significantly overturn the excitatory of L5-L6 related DRG neurons in the LDH group, and can significantly reduce the current density of the voltage gated sodium channel and move the inactivation curve of the voltgated sodium channel to the right. To reduce the high expression of Na V1.7 and Na V1.8 in the L5-L6 related DRG neurons in the LDH group. Conclusion the above experimental results suggest that the hydrogen sulfide synthase CBS may be activated by voltage gated sodium channels and the high expression of Na V1.7 and Na V1.8 protein, increasing the current density of voltage gated sodium channels, leading to the associated nerve related to the transplantation of autologous nucleus pulposus. The increase of element excitability then participates in the modulation of peripheral pain signals and eventually leads to the hyperalgesia of the lower extremities. This study reveals some of the molecular mechanisms of pain in LDH patients, and may provide some effective treatment for the treatment of LDH. - -
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R681.5

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本文編號(hào)：1930123