發(fā)布時(shí)間：2018-05-21 10:59

  本文選題：μ阿片受體 + I1咪唑受體��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年碩士論文

【摘要】：藥物成癮已成為嚴(yán)重的全球性公共衛(wèi)生問(wèn)題和突出的社會(huì)問(wèn)題。根本原因在于藥物成癮的分子生物學(xué)機(jī)制尚未闡明，缺乏理想的藥物靶標(biāo)和醫(yī)學(xué)生物學(xué)干預(yù)手段。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn)胍丁胺自身不成癮，通過(guò)激活I(lǐng)1咪唑啉受體（I1-imidazoline receptor，I1R）增強(qiáng)阿片鎮(zhèn)痛、拮抗阿片耐受，對(duì)阿片軀體和精神依賴(lài)具有較好的預(yù)防與治療作用，對(duì)稽延癥狀有效，具備理想的防復(fù)吸候選藥物特點(diǎn)。進(jìn)一步研究發(fā)現(xiàn)，內(nèi)源性胍丁胺和上述外源性胍丁胺作用類(lèi)似，對(duì)阿片成癮亦具有顯著抑制作用。據(jù)此本課題組提出了“胍丁胺——I1R可能構(gòu)成了又一新的阿片功能調(diào)節(jié)系統(tǒng)”的科學(xué)假說(shuō)。深入研究I1R調(diào)節(jié)阿片功能的相關(guān)分子生物學(xué)機(jī)制，對(duì)促進(jìn)I1R成為新型抗阿片成癮的藥物靶標(biāo)具有重要的理論意義和實(shí)際應(yīng)用價(jià)值，將為揭示阿片成癮的分子機(jī)制提供有益的線(xiàn)索。遺憾的是I1R和α2腎上腺素受體（α2adrenergic receptor, α2AR）幾乎在所有組織與細(xì)胞中同時(shí)表達(dá)，并且作用于I1R的藥物也都能作用于α2AR，無(wú)特異性I1R工具藥物。這些嚴(yán)重阻礙了I1R調(diào)節(jié)阿片功能分子機(jī)制的研究進(jìn)展。 大量研究工作證實(shí)，大鼠腎上腺髓質(zhì)嗜鉻細(xì)胞瘤細(xì)胞（pheochromocytoma cell，PC12）經(jīng)神經(jīng)生長(zhǎng)因子誘導(dǎo)后具有一定神經(jīng)元生物學(xué)特征，表達(dá)有I1R，但不表達(dá)α2AR和μ阿片受體。本研究采用慢病毒感染技術(shù)實(shí)現(xiàn)了在PC12細(xì)胞中穩(wěn)定表達(dá)了大鼠μ阿片受體（ratmu opioid receptor，rMOR），采用流式細(xì)胞分選技術(shù)篩選出高表達(dá)rMOR的PC12細(xì)胞，經(jīng)單克隆細(xì)胞的放大培養(yǎng)后獲得了多個(gè)表達(dá)rMOR的單克隆細(xì)胞系，經(jīng)相關(guān)鑒定后建立具有神經(jīng)元特性、無(wú)α2AR干擾、具有rMOR與野生型I1R共表達(dá)的穩(wěn)定細(xì)胞系，為研究胍丁胺調(diào)節(jié)阿片功能的細(xì)胞生物學(xué)機(jī)制提供了一個(gè)較為理想的細(xì)胞模型。在此模型上，我們觀(guān)察了胍丁胺對(duì)慢性嗎啡處理引起的cAMP超射的抑制作用及抗嗎啡細(xì)胞增殖抑制作用。在此基礎(chǔ)上，利用生化藥理學(xué)技術(shù)在此細(xì)胞模型上研究了胍丁胺調(diào)節(jié)阿片功能的可能分子機(jī)制。 1、PC12細(xì)胞-rMOR穩(wěn)定表達(dá)細(xì)胞系的建立 為了達(dá)到建立PC12細(xì)胞-μ阿片受體穩(wěn)定表達(dá)細(xì)胞系的實(shí)驗(yàn)?zāi)康�，我們首先�?gòu)建了慢病毒表達(dá)載體pBPLV-rMOR-eGFP，包裝獲得慢病毒，對(duì)PC12細(xì)胞進(jìn)行感染，感染72h后細(xì)胞生長(zhǎng)狀態(tài)良好。用二脒基苯基吲哚（4,6-diamidino-2-phenylindole，DAPI）對(duì)此細(xì)胞核進(jìn)行染色后，在熒光顯微鏡下可觀(guān)察90%的PC12細(xì)胞有增強(qiáng)型綠色熒光蛋白（enhanced fluorescent protein，eGFP）表達(dá)，熒光均勻分布于整個(gè)細(xì)胞中，表明含有rMOR的pBPLV-rMOR-eGFP慢病毒載體在PC12細(xì)胞中可以高效表達(dá)，提示此實(shí)驗(yàn)方法可行。 在上述預(yù)實(shí)驗(yàn)基礎(chǔ)上，我們應(yīng)用此方法對(duì)PC12細(xì)胞進(jìn)行感染，在病毒感染3d后，采用流式細(xì)胞儀對(duì)高表達(dá)eGFP的PC12細(xì)胞進(jìn)行分選（eGFP高表達(dá)的細(xì)胞占全部有熒光的細(xì)胞比例約為15.7%）。將分選得到的高表達(dá)eGFP細(xì)胞利用有限稀釋法（0.8個(gè)細(xì)胞/100μL）接種于96孔板，逐級(jí)擴(kuò)大培養(yǎng)獲得在PC12細(xì)胞中穩(wěn)定表達(dá)rMOR的單克隆細(xì)胞系。 用RT-PCR對(duì)有熒光表達(dá)的PC12單克隆細(xì)胞系進(jìn)行mRNA檢測(cè)，結(jié)果發(fā)現(xiàn)感染后有熒光表達(dá)的PC12細(xì)胞在1.2kd附近有明顯條帶，這與rMOR基因理論分子量（1198bp）的位置相同，表明rMOR有表達(dá)，而未感染病毒的PC12細(xì)胞未見(jiàn)此特異性條帶出現(xiàn)。從而在mRNA水平證明了rMOR在PC12細(xì)胞中的表達(dá)。在此基礎(chǔ)上，利用阿片受體放射性配體[3H]-二丙諾啡為工具，采用放射配體受體結(jié)合實(shí)驗(yàn)技術(shù)從擴(kuò)大培養(yǎng)獲得的34株表達(dá)eGFP的PC12單克隆細(xì)胞系中成功篩選出了15株μ阿片受體表達(dá)水平較高的細(xì)胞系。進(jìn)一步選定第12號(hào)克隆進(jìn)行進(jìn)一步研究工作。利用飽和實(shí)驗(yàn)對(duì)rMOR親合力和表達(dá)水平進(jìn)行研究。結(jié)果發(fā)現(xiàn)rMOR與[3H]-二丙諾啡結(jié)合的Kd值為0.51±0.07nM，與文獻(xiàn)報(bào)道相似，Bmax為1.58±0.15pmol/mg （n=3）。 那么，在此表達(dá)系統(tǒng)中表達(dá)的rMOR是否具有細(xì)胞信號(hào)傳遞功能呢？為了回答此問(wèn)題，我們用10μM嗎啡分別對(duì)PC12細(xì)胞和PC12-rMOR細(xì)胞進(jìn)行10min刺激，采用蛋白免疫印跡的方法檢測(cè)p-ERK與ERK的比值，以確定嗎啡對(duì)rMOR的激活情況，以此檢測(cè)PC12細(xì)胞中表達(dá)的rMOR激活后對(duì)細(xì)胞信號(hào)的傳遞功能。結(jié)果發(fā)現(xiàn)，與PC12細(xì)胞相比，PC12-rMOR細(xì)胞的ERK磷酸化水平顯著升高（P0.001，n=3）。上述結(jié)果表明在PC12細(xì)胞中表達(dá)的rMOR蛋白具有與野生型μ阿片受體相同的生物學(xué)特征。 2、胍丁胺在PC12-rMOR細(xì)胞上抗嗎啡依賴(lài)和嗎啡耐受形成的作用 表達(dá)有阿片受體的細(xì)胞系經(jīng)嗎啡慢性處理后，在納洛酮催促下會(huì)出現(xiàn)cAMP顯著升高，稱(chēng)此為cAMP超射。被學(xué)界公認(rèn)為阿片依賴(lài)的細(xì)胞生物學(xué)指標(biāo)。在本實(shí)驗(yàn)中，用嗎啡（1~100μM）慢性處理PC12-rMOR細(xì)胞24小時(shí)后，納洛酮（10μM）催促15min可顯著引起胞內(nèi)cAMP濃度升高。與未經(jīng)嗎啡處理的對(duì)照組相比，20μM的嗎啡可引起胞內(nèi)cAMP含量升高255±25.2%（p 0.01，n＝5），而同樣的方式處理未表達(dá)rMOR的PC12細(xì)胞時(shí)，對(duì)細(xì)胞內(nèi)cAMP含量無(wú)影響。胍丁胺（10~100μM）伴隨嗎啡（20μM）慢性慢性處理后，胍丁胺（10μM～100μM）能夠濃度依賴(lài)性抑制納洛酮（10μM）催促引起的cAMP超射，胍丁胺濃度為100μM時(shí)能夠?qū)AMP超射水平抑制65.2%，與嗎啡處理組相比具有顯著性差異（p 0.01， n=5）。 大量文獻(xiàn)報(bào)道，嗎啡耐受的形成與嗎啡長(zhǎng)期作用于機(jī)體導(dǎo)致中樞神經(jīng)系統(tǒng)神經(jīng)元發(fā)生凋亡有關(guān)。而本課題組前期研究發(fā)現(xiàn)胍丁胺對(duì)阿片耐受具有拮抗作用，其作用機(jī)制尚不十分清楚。因此，我們?cè)赑C12-rMOR細(xì)胞上采用CCK-8細(xì)胞活力檢測(cè)技術(shù)評(píng)價(jià)嗎啡和胍丁胺慢性處理96小時(shí)后對(duì)活細(xì)胞數(shù)量的影響以及胍丁胺是否能抑制嗎啡對(duì)活細(xì)胞數(shù)量的影響。結(jié)果發(fā)現(xiàn)，嗎啡（160μM～2.5mM）能濃度依賴(lài)性減少活細(xì)胞的數(shù)量，當(dāng)嗎啡濃度達(dá)640μM時(shí)對(duì)減少活細(xì)胞數(shù)量的減少與鹽水對(duì)照比較具有統(tǒng)計(jì)學(xué)差異（p 0.05，n＝3）。當(dāng)嗎啡濃度達(dá)到750μM時(shí)對(duì)PC12-rMOR活細(xì)胞數(shù)量減少作用具有顯著性差異（p 0.01，n＝3），當(dāng)達(dá)到mM水平細(xì)胞出現(xiàn)死亡。胍丁胺（2.5μM～160μM）能濃度依賴(lài)性促進(jìn)活細(xì)胞數(shù)量的增加，當(dāng)胍丁胺濃度達(dá)2.5μM時(shí)與鹽水對(duì)照比較具有統(tǒng)計(jì)學(xué)差異（p 0.05，n＝3），160μM具有顯著性差異（p 0.01，n＝3），濃度達(dá)到640μM時(shí)其效應(yīng)消失，但未檢測(cè)到對(duì)細(xì)胞的毒性。在此細(xì)胞模型上，用不同濃度胍丁胺（2.5μM~160μM）與750μM嗎啡共同處理PC12-rMOR細(xì)胞96小時(shí)，胍丁胺2.5μM就能顯著地抑制嗎啡對(duì)細(xì)胞增殖的阻礙作用，隨著濃度增加，抑制效應(yīng)也隨之加強(qiáng)，當(dāng)胍丁胺濃度達(dá)160μM時(shí)能顯著地抑制嗎啡對(duì)活細(xì)胞數(shù)量減少作用（p 0.01，n＝3）。 3、胍丁胺調(diào)節(jié)阿片功能的可能分子機(jī)制 在上述細(xì)胞模型上利用蛋白免疫印跡方法研究發(fā)現(xiàn)：1）分別用20μM嗎啡和100μM胍丁胺慢性處理PC12-rMOR細(xì)胞72小時(shí)均能顯著激活p-ERK（p 0.01，n=3），胍丁胺伴隨嗎啡預(yù)處理PC12-rMOR細(xì)胞72小時(shí)后與單純嗎啡處理組相比，p-ERK不但沒(méi)進(jìn)一步加強(qiáng)，反而呈現(xiàn)降低趨勢(shì)；2）20μM嗎啡慢性處理PC12-rMOR細(xì)胞72小時(shí)后，，CREB和p-CREB顯著下降（p 0.01，n=3），100μM胍丁胺處理使PC12-rMOR細(xì)胞72小時(shí)使CREB和p-CREB顯著升高，胍丁胺伴隨嗎啡處理能顯著抑制嗎啡所致的CREB和p-CREB降低（p 0.05，n=3）；3）20μM嗎啡慢性處理72小時(shí)后IκB水平顯著升高，100μM胍丁胺處理72小時(shí)后IκB顯著降低（p 0.05，n=3），胍丁胺伴隨嗎啡給藥能顯著抑制嗎啡慢性作用引起的IκB升高（p 0.01，n=3）；20μM嗎啡慢性處理72小時(shí)后NF-κB表達(dá)水平顯著降低（p 0.05，n=3），100μM胍丁胺處理72小時(shí)NF-κB水平顯著升高（p 0.05，n=3），胍丁胺伴隨嗎啡處理能顯著拮抗嗎啡引起的NF-κB降低（p 0.05，n=3）。 研究結(jié)論： 1.成功實(shí)現(xiàn)了rMOR在PC12細(xì)胞中的穩(wěn)定表達(dá)，并具有與野生型μ阿片受體相同的受體功能特征，為體外研究胍丁胺調(diào)節(jié)阿片功能的分子機(jī)制提供了理想的研究模型； 2.胍丁胺（10μM～100μM）在PC12-rMOR細(xì)胞模型上能濃度依賴(lài)性抑制慢性嗎啡處理、納洛酮催促引起的cAMP超射，即在該細(xì)胞模型上胍丁胺能較好地抑制嗎啡依賴(lài)的形成； 3.胍丁胺在PC12-rMOR細(xì)胞模型上能濃度依賴(lài)性抑制嗎啡慢性處理所致細(xì)胞凋亡，可能是其拮抗嗎啡耐受形成的機(jī)制之一； 4.胍丁胺調(diào)節(jié)阿片功能分子機(jī)制可能與抑制嗎啡所致p-ERK升高和拮抗CREB、p-CREB、NF-κB的降低有關(guān)。
[Abstract]:In this paper , we have found that the mechanism of I1R ' s molecular biological mechanism has not been clarified yet , and there is a lack of ideal drug targets and medical biological intervention methods . It is very important to study the mechanism of I1R to modulate opioid addiction .

In this study , we observed the inhibitory effect of guanidinamine on chronic morphine treatment and the inhibition of morphine - induced cell proliferation .

1 . Establishment of a stable expression cell line of PC12 cells - rMOR

In order to achieve the experimental purpose of establishing a PC12 cell - 渭o(jì)pioid receptor stable expression cell line , we first constructed the slow virus expression vector pBPLV - rMOR - eGFP , which was used to obtain the lentivirus , which was infected with PC12 cells . After 72 hours of infection , the cells grew well . The fluorescence microscope showed that 90 % of PC12 cells were expressed with enhanced fluorescent protein ( eGFP ) , and the fluorescence was distributed homogeneously throughout the cell , suggesting that the pBPLV - rMOR - eGFP vector containing rMOR could be expressed efficiently in PC12 cells , suggesting that the experimental method is feasible .

The PC12 cells were infected by this method . After 3 days of viral infection , the PC12 cells expressing eGFP were sorted by flow cytometry ( the percentage of cells with high expression of eGFP was about 15.7 % ) . The highly expressed eGFP cells obtained were seeded on 96 well plates by a finite dilution method ( 0.8 cells / 100 渭L ) , and the monoclonal cell line stably expressing rMOR in PC12 cells was obtained by step - by - step expansion .

The expression of rMOR in PC12 cells was demonstrated by using radioligand receptor binding assay . The results showed that the Kd value of rMOR was 0.51 鹵 0.07 nM , similar to that reported in the literature , and Bmax was 1 . 58 鹵 0 . 15 pmol / mg ( n = 3 ) .

In order to answer this question , we stimulated PC12 cells and PC12 - rMOR cells with 10 渭M morphine for 10 min . The ratio of p - ERK and ERK was detected by Western blot to determine the activation of rMOR in PC12 cells . The results showed that the ERK phosphorylation level of PC12 cells increased significantly compared with PC12 cells ( P0.001 , n = 3 ) . The above results indicate that the rMOR protein expressed in PC12 cells has the same biological characteristics as the wild - type 渭 opioid receptor .

Effects of guanamine on morphine dependence and morphine tolerance formation in PC12 - rMOR cells

In this experiment , the concentration - dependent inhibition of naloxone ( 10 - 100 渭M ) in PC12 cells treated with morphine ( 1 - 100 渭M ) increased 255 鹵 25 . 2 % ( p 0.01 , n = 5 ) .

It is found that morphine ( 160 渭M ~ 2.5 mM ) can reduce the number of viable cells after 96 hours of chronic treatment with morphine and guanamine . The results show that morphine ( 160 渭M ~ 2.5 mM ) can reduce the number of viable cells in a dose - dependent manner . There was significant difference in the number of viable cells ( p 0.01 , n = 3 ) in PC12 - rMOR cells when the concentration of morphine reached 750 渭M , and when the concentration of guanamine was 2.5 渭M , the effect of morphine on cell proliferation was significantly different ( p 0.01 , n = 3 ) .

3 . Possible molecular mechanisms of the regulation of opioid function by guanamine

It was found that : 1 ) p - ERK ( p 0.01 , n = 3 ) was significantly activated in PC12 - rMOR cells treated with 20 渭M morphine and 100 渭 M guanamine for 72 hours .
2 ) After 72 hours of treatment of PC12 - rMOR cells with 20 渭M morphine , CREB and p - CREB significantly decreased ( p 0.01 , n = 3 ) , and 100 渭M guanamine treatment resulted in a significant increase of CREB and p - CREB in PC12 - rMOR cells 72 hours , while the concomitant morphine treatment significantly inhibited the decrease in CREB and p - CREB induced by morphine ( p 0.05 , n = 3 ) ;
3 ) The level of I魏B was significantly increased after 72 h treatment with 20 渭M morphine , and the level of I魏B was significantly decreased ( p 0.05 , n = 3 ) after 72 h treatment with 100 渭g of guanamine , and the increase of I魏B ( p 0.01 , n = 3 ) induced by morphine was significantly inhibited by the administration of guanamine with morphine .
The level of NF - 魏B was significantly decreased ( p 0.05 , n = 3 ) and the level of NF - 魏B was significantly increased ( p 0.05 , n = 3 ) after 72 h treatment with 20 渭M morphine ( p 0.05 , n = 3 ) .

Conclusions of the study :

1 . The stable expression of rMOR in PC12 cells was successfully achieved , and the same receptor functional characteristics as wild type 渭 opioid receptors were found .


2 . Guanitidine ( 10 渭M ~ 100 渭M ) inhibited the formation of morphine dependence on PC12 - rMOR cell model with a concentration - dependent inhibition of chronic morphine treatment , naloxone - induced cAMP overshot , that is , guanidinamine in the cell model .


3 . The concentration - dependent inhibition of guanamine on PC12 - rMOR cell model can inhibit the cell apoptosis induced by chronic morphine treatment , which may be one of the mechanisms to inhibit the formation of morphine tolerance ;


4 . The mechanism of regulating opioid receptors by guanamine may be related to the inhibition of morphine - induced increase in p - ERK and antagonism of CREB , p - CREB , NF - 魏B .
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R965

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 吳寧,蘇瑞斌,徐波,李錦,秦伯益;大鼠μ阿片受體及人咪唑啉-1受體在CHO細(xì)胞中的共同穩(wěn)定表達(dá)[J];軍事醫(yī)學(xué)科學(xué)院院刊;2004年04期

2 周連芳;朱永平;;Changes of CREB in rat hippocampus, prefrontal cortex and nucleus accumbens during three phases of morphine induced conditioned place preference in rats[J];Journal of Zhejiang University Science;2006年02期

3 蘇瑞斌,李錦,高凱,裴鋼,秦伯益;咪唑克生對(duì)嗎啡鎮(zhèn)痛、耐受和身體依賴(lài)的影響<英文>[J];Acta Pharmacologica Sinica;2000年11期

4 蘇瑞斌,李錦,秦伯益;雙向阿片功能調(diào)節(jié)劑:胍丁胺(英文)[J];Acta Pharmacologica Sinica;2003年07期

5 李錦，李昕，裴剛，秦伯益;Analgesic effect of agmatine and its enhancement on morphine analgesia in mice and rats[J];Acta Pharmacologica Sinica;1999年01期

6 李錦,李昕,裴鋼,秦伯益;Effects of agmatine on tolerance to and substance dependence on morphine in mice[J];Acta Pharmacologica Sinica;1999年03期

7 王秀麗,蘇瑞斌,楊紅菊,吳寧,米衛(wèi)東,李錦;胍丁胺對(duì)神經(jīng)病理性痛大鼠嗎啡鎮(zhèn)痛作用及耐受的影響[J];中華麻醉學(xué)雜志;2005年08期

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