本文選題：紫杉醇 + 神經(jīng)毒性��； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：紫杉醇(paclitaxel, PT)是臨床上常用的抗腫瘤藥物,具有很好的抗腫瘤治療效果,但PT的神經(jīng)毒性作用或PT誘發(fā)的周圍神經(jīng)病變可使病人出現(xiàn)嚴(yán)重的神經(jīng)病理性疼痛癥狀,而且這種疼痛癥狀用常規(guī)的鎮(zhèn)痛藥物難以緩解,嚴(yán)重影響進一步抗腫瘤治療。到目前為止,PT的神經(jīng)毒性作用或PT誘發(fā)的周圍神經(jīng)病變的發(fā)病機制還未完全被闡明。PT誘發(fā)的神經(jīng)病理性疼痛癥狀與背根神經(jīng)節(jié)(dorsal root ganglion, DRG)初級傳入神經(jīng)元的毒性有關(guān)。因此,研究PT對DRG神經(jīng)元表型變化的影響對于闡明PT的神經(jīng)毒性作用機制具有一定的指導(dǎo)意義,探討緩解PT的神經(jīng)毒性作用有助于開發(fā)PT誘發(fā)的周圍神經(jīng)病變新的治療手段。本研究利用體外培養(yǎng)的DRG神經(jīng)元和PT誘發(fā)的大鼠周圍神經(jīng)病變模型研究胰島素樣生長因子-1(insulin-like growth factor-1, IGF-1)緩解PT誘發(fā)的神經(jīng)毒性及其機制。 第一部分 IGF-1對具有紫杉醇毒性的培養(yǎng)的背根神經(jīng)節(jié)神經(jīng)元的保護作用 根據(jù)神經(jīng)生化免疫反應(yīng)的特殊標(biāo)記可將DRG神經(jīng)元分為神經(jīng)肽免疫反應(yīng)(neuropeptide-immunoreactive, NP-IR)和神經(jīng)絲免疫反應(yīng)(neurofilament-IR, NF-IR)神經(jīng)元兩種主要表型。降鈣素基因相關(guān)肽(calcitonin gene-related peptide, CGRP)是一種在DRG神經(jīng)元內(nèi)表達并發(fā)揮多種生物學(xué)功能的感覺神經(jīng)肽,CGRP-IR神經(jīng)元可代表NP-IR神經(jīng)元。神經(jīng)絲(neurofilament, NF)是神經(jīng)元特異性中間絲,其中神經(jīng)絲蛋白-200(neurofilament-200,NF-200)對正常神經(jīng)元的維持發(fā)揮重要作用,NF-200-IR神經(jīng)元可代表NF-IR神經(jīng)元。PT是否能夠影響DRG神經(jīng)元的神經(jīng)化學(xué)表型目前仍不清楚。 IGF-1是一種促生長的肽類激素,通過激活其酪氨酸激酶受體IGF-1R促進神經(jīng)元的存活。IGF-1及其受體IGF-1R均在DRG神經(jīng)元有表達,IGF-1在促進DRG神經(jīng)元軸突的生長及調(diào)控感覺神經(jīng)肽的表達方面發(fā)揮重要作用。IGF-1是否通過細胞外信號調(diào)節(jié)蛋白激酶1/2(extracellular signal-regulated protein kinasel/2, ERK1/2)和磷脂酰肌醇3-激酶(phosphatidylinositol3-kinase, PI3K)／絲氨酸-蘇氨酸激酶(serine-threonine kinase, Akt)信號通路調(diào)節(jié)PT誘發(fā)的DRG神經(jīng)元的表型變化尚需進一步探討。 為了檢測PT神經(jīng)毒性作用的劑量依賴關(guān)系,將原代培養(yǎng)48h的DRG神經(jīng)元分為以下各組：(1) PT0.01μmol/L;(2) PT0.1μmol/L;(3) PT1μmol/L;(4)對照組：DRG神經(jīng)元僅在培養(yǎng)液內(nèi)孵育作為對照。DRG神經(jīng)元在以上條件下繼續(xù)培養(yǎng)24h后,在倒置相差顯微鏡下觀察DRG神經(jīng)元活細胞生長狀態(tài),用微管相關(guān)蛋白-2(microtubule-associated protein2, MAP2)進行免疫熒光標(biāo)記后測量單個神經(jīng)元突起的長度。結(jié)果顯示,DRG神經(jīng)元突起長度的縮短與PT的濃度具有劑量依賴性。 為了檢測IGF-1對具有PT毒性的DRG神經(jīng)元的保護作用,將原代培養(yǎng)48h的DRG神經(jīng)元分為以下各組：(1) PT(1μmol/L);(2) PT(1μmol/L)+IGF-1(20nmol/L);(3)ERKl/2抑制劑PD98059(10μmol/L)+PT (1μmol/L)+IGF-1(20nmol/L);(4) PI3K抑制劑LY294002(10μmol/L)+PT (1μmol/L)+IGF-1(20nmol/L);(5)對照組：DRG神經(jīng)元僅在培養(yǎng)液內(nèi)孵育作為對照。DRG神經(jīng)元在以上條件下繼續(xù)培養(yǎng)24h后,在倒置相差顯微鏡下觀察DRG神經(jīng)元活細胞生長狀態(tài),用MAP2進行免疫熒光標(biāo)記后測量單個神經(jīng)元突起的長度,用WST-1檢測DRG神經(jīng)元細胞活力,用Hoechst33342染色法檢測DRG神經(jīng)元細胞凋亡,用MAP2和CGRP或NF-200免疫熒光雙標(biāo)記檢測DRG神經(jīng)元表型的變化,用real-time PCR分析技術(shù)檢測CGRP mRNA和NF-200mRNA的表達變化,用Western blot分析技術(shù)檢測CGRP和NF-200蛋白的表達。結(jié)果顯示,PT可影響DRG神經(jīng)元的生長狀態(tài),使DRG神經(jīng)元突起的長度變短,細胞活力減低,細胞凋亡率升高,CGRP-IR神經(jīng)元和NF-200-IR神經(jīng)元表型的比例、CGRP和NF-200蛋白及其mRNA的水平下降；IGF-1可使具有PT毒性的DRG神經(jīng)元的突起延長,細胞活力增加,細胞凋亡率降低,IGF-1可挽救PT誘發(fā)的CGRP-IR神經(jīng)元表型的比例下降,但對NF-200-IR神經(jīng)元表型的變化沒有影響；IGF-1對DRG神經(jīng)元生長狀態(tài)的改善和對DRG神經(jīng)元特定表型的影響作用是通過激活ERK1/2和PI3K/Akt細胞信號轉(zhuǎn)導(dǎo)通路而實現(xiàn)的。本研究的結(jié)果表明,IGF-1可通過抑制具有PT毒性的DRG神經(jīng)元凋亡而增加神經(jīng)元細胞活力和促進神經(jīng)元突起的生長,IGF-1主要是對支配皮膚的DRG神經(jīng)元產(chǎn)生保護作用,PT對DRG神經(jīng)元不同亞群的神經(jīng)毒性作用以及IGF-1對具有PT毒性的特定神經(jīng)元表型神經(jīng)營養(yǎng)作用,為進一步研究IGF-1緩解PT誘發(fā)的神經(jīng)毒性提供了實驗依據(jù)。 第二部分 IGF-1緩解紫杉醇誘發(fā)的神經(jīng)病理性疼痛的實驗研究 由于PT的神經(jīng)毒性可誘發(fā)周圍神經(jīng)病變,這種周圍神經(jīng)病變的神經(jīng)病理性疼痛癥狀用常規(guī)的鎮(zhèn)痛藥物難以緩解。因此,通過神經(jīng)營養(yǎng)的角度改善神經(jīng)元的功能狀態(tài),是緩解這種周圍神經(jīng)病變所致的神經(jīng)病理性疼痛癥狀的一個具有研究前景和應(yīng)用前景的領(lǐng)域。IGF-1對不同神經(jīng)損傷的改善作用和其特有的神經(jīng)營養(yǎng)作用,以及IGF-1通過激活特有的細胞信號轉(zhuǎn)導(dǎo)通路而發(fā)揮生物學(xué)效應(yīng)的特性,提供了通過干預(yù)新的潛在性治療靶點緩解PT誘發(fā)的神經(jīng)病理性疼痛的可行性。因此,本研究利用腹膜腔注射(intraperitoneally injection, i.p.)PT建立PT誘發(fā)的神經(jīng)病理性疼痛大鼠動物模型,分別鞘內(nèi)注射IGF-1、ERK1/2抑制劑PD98059+IGF-1、 PI3K抑制劑LY294002+IGF-1,檢測各組大鼠觸覺痛覺閾值和熱痛覺閾值的變化和L4-6DRG神經(jīng)元CGRP和NF-200及其mRNA的表達變化。結(jié)果顯示,PT可使大鼠觸覺痛覺閾值和熱痛覺閾值降低,鞘內(nèi)注射IGF-1可使大鼠觸覺痛覺閾值和熱痛覺閾值升高,并使其提前恢復(fù)至正常水平,預(yù)先鞘內(nèi)注射ERK1/2抑制劑PD98059或PI3K抑制劑LY294002可阻斷IGF-1的效應(yīng)。PT可使大鼠L4-6DRG神經(jīng)元CGRP及其mRNA的表達上調(diào),而NF-200及其mRNA的表達下調(diào)。而鞘內(nèi)注射IGF-1后,DRG神經(jīng)元CGRP及其mRNA的表達趨于正常,而NF-200及其mRNA的表達則不受IGF-1的影響。IGF-1所致的DRG神經(jīng)元CGRP及其mRNA的表達變化效應(yīng)可被預(yù)先鞘內(nèi)注射ERK1/2抑制劑PD98059或PI3K抑制劑LY294002所阻斷。這些結(jié)果表明,IGF-1可通過ERK1/2和PI3K/Akt細胞信號轉(zhuǎn)導(dǎo)通路緩解PT誘發(fā)的神經(jīng)病理性疼痛,這可能是由于IGF-1改善了不同亞群的DRG神經(jīng)元抑制了促炎癥反應(yīng)神經(jīng)肽的表達而實現(xiàn)的。IGF-1的這些作用為進一步開發(fā)PT誘發(fā)的神經(jīng)病理性疼痛新的治療策略提供了實驗依據(jù)。
[Abstract]:The neurotoxic effects of PT on DRG neurons and the pathogenesis of PT - induced peripheral neuropathy have not been fully elucidated .

the first portion

Protective effects of IGF - 1 on cultured dorsal root ganglia neurons with paclitaxel toxicity

Neurofilament - related peptide ( CGRP ) plays an important role in the maintenance of normal neurons . Neurofilament - 200 ( NF - 200 ) plays an important role in the maintenance of normal neurons .

IGF - 1 is a kind of growth - promoting peptide hormone . IGF - 1 and its receptor IGF - 1R play an important role in promoting the expression of IGF - 1 and its receptor IGF - 1R in DRG neurons .

In order to detect the dose - dependent relationship of PT neurotoxicity , the DRG neurons cultured for 48 h were divided into the following groups : ( 1 ) PT0 . 01 渭mol / L ; ( 2 ) PT0 . 1 渭mol / L ; ( 3 ) PT0 . 1 渭mol / L ; ( 4 ) control group : DRG neurons were incubated in culture medium for 24 h .

In order to detect the protective effects of IGF - 1 on DRG neurons with PT toxicity , the DRG neurons cultured for 48 h were divided into the following groups : ( 1 ) PT ( 1 渭mol / L ) ; ( 2 ) PT ( 1 渭mol / L ) + IGF - 1 ( 20 nmol / L ) ; ( 4 ) the expression of CGRP mRNA and NF - 200 mRNA was detected by using the method of Western blot analysis .
IGF - 1 can prolong the number of DRG neurons with PT toxicity , increase the cell viability , decrease the apoptosis rate , and the ratio of IGF - 1 to the PT - induced CGRP - IR neuronal phenotype is decreased , but there is no effect on the changes of the phenotype of NF - 200 - IR neurons ;
IGF - 1 plays a role in improving the growth status of DRG neurons and the effect on the specific phenotype of DRG neurons . The results of this study suggest that IGF - 1 can increase neuronal cell viability and promote neuronal outgrowth by inhibiting the apoptosis of DRG neurons with PT toxicity . IGF - 1 is primarily a protective effect on DRG neurons innervated skin , and the effects of PT on the neurotoxic effects of different subgroups of DRG neurons and the neurotrophism of IGF - 1 on specific neuronal phenotype with PT toxicity provide experimental basis for further study of IGF - 1 response to PT - induced neurotoxicity .

the second part

An experimental study of IGF - 1 in alleviating neuropathic pain induced by paclitaxel

鐢變簬PT鐨勭緇忔瘨鎬у彲璇卞彂鍛ㄥ洿紲炵粡鐥呭彉,榪欑鍛ㄥ洿紲炵粡鐥呭彉鐨勭緇忕梾鐞嗘，

本文編號：2006103