PC12细胞—大鼠μ阿片受体稳定表达系的建立与胍丁胺调节阿片功能分子机制研究

发布时间：2018-05-21 10:59

本文选题：μ阿片受体 + I1咪唑受体　；参考：《中国人民解放军军事医学科学院》2014年硕士论文

【摘要】：药物成瘾已成为严重的全球性公共卫生问题和突出的社会问题。根本原因在于药物成瘾的分子生物学机制尚未阐明，缺乏理想的药物靶标和医学生物学干预手段。本实验室前期研究发现胍丁胺自身不成瘾，通过激活I1咪唑啉受体（I1-imidazoline receptor，I1R）增强阿片镇痛、拮抗阿片耐受，对阿片躯体和精神依赖具有较好的预防与治疗作用，对稽延症状有效，具备理想的防复吸候选药物特点。进一步研究发现，内源性胍丁胺和上述外源性胍丁胺作用类似，对阿片成瘾亦具有显著抑制作用。据此本课题组提出了“胍丁胺——I1R可能构成了又一新的阿片功能调节系统”的科学假说。深入研究I1R调节阿片功能的相关分子生物学机制，对促进I1R成为新型抗阿片成瘾的药物靶标具有重要的理论意义和实际应用价值，将为揭示阿片成瘾的分子机制提供有益的线索。遗憾的是I1R和α2肾上腺素受体（α2adrenergic receptor, α2AR）几乎在所有组织与细胞中同时表达，并且作用于I1R的药物也都能作用于α2AR，无特异性I1R工具药物。这些严重阻碍了I1R调节阿片功能分子机制的研究进展。大量研究工作证实，大鼠肾上腺髓质嗜铬细胞瘤细胞（pheochromocytoma cell，PC12）经神经生长因子诱导后具有一定神经元生物学特征，表达有I1R，但不表达α2AR和μ阿片受体。本研究采用慢病毒感染技术实现了在PC12细胞中稳定表达了大鼠μ阿片受体（ratmu opioid receptor，rMOR），采用流式细胞分选技术筛选出高表达rMOR的PC12细胞，经单克隆细胞的放大培养后获得了多个表达rMOR的单克隆细胞系，经相关鉴定后建立具有神经元特性、无α2AR干扰、具有rMOR与野生型I1R共表达的稳定细胞系，为研究胍丁胺调节阿片功能的细胞生物学机制提供了一个较为理想的细胞模型。在此模型上，我们观察了胍丁胺对慢性吗啡处理引起的cAMP超射的抑制作用及抗吗啡细胞增殖抑制作用。在此基础上，利用生化药理学技术在此细胞模型上研究了胍丁胺调节阿片功能的可能分子机制。 1、PC12细胞-rMOR稳定表达细胞系的建立为了达到建立PC12细胞-μ阿片受体稳定表达细胞系的实验目的，我们首先构建了慢病毒表达载体pBPLV-rMOR-eGFP，包装获得慢病毒，对PC12细胞进行感染，感染72h后细胞生长状态良好。用二脒基苯基吲哚（4,6-diamidino-2-phenylindole，DAPI）对此细胞核进行染色后，在荧光显微镜下可观察90%的PC12细胞有增强型绿色荧光蛋白（enhanced fluorescent protein，eGFP）表达，荧光均匀分布于整个细胞中，表明含有rMOR的pBPLV-rMOR-eGFP慢病毒载体在PC12细胞中可以高效表达，提示此实验方法可行。在上述预实验基础上，我们应用此方法对PC12细胞进行感染，在病毒感染3d后，采用流式细胞仪对高表达eGFP的PC12细胞进行分选（eGFP高表达的细胞占全部有荧光的细胞比例约为15.7%）。将分选得到的高表达eGFP细胞利用有限稀释法（0.8个细胞/100μL）接种于96孔板，逐级扩大培养获得在PC12细胞中稳定表达rMOR的单克隆细胞系。用RT-PCR对有荧光表达的PC12单克隆细胞系进行mRNA检测，结果发现感染后有荧光表达的PC12细胞在1.2kd附近有明显条带，这与rMOR基因理论分子量（1198bp）的位置相同，表明rMOR有表达，而未感染病毒的PC12细胞未见此特异性条带出现。从而在mRNA水平证明了rMOR在PC12细胞中的表达。在此基础上，利用阿片受体放射性配体[3H]-二丙诺啡为工具，采用放射配体受体结合实验技术从扩大培养获得的34株表达eGFP的PC12单克隆细胞系中成功筛选出了15株μ阿片受体表达水平较高的细胞系。进一步选定第12号克隆进行进一步研究工作。利用饱和实验对rMOR亲合力和表达水平进行研究。结果发现rMOR与[3H]-二丙诺啡结合的Kd值为0.51±0.07nM，与文献报道相似，Bmax为1.58±0.15pmol/mg （n=3）。那么，在此表达系统中表达的rMOR是否具有细胞信号传递功能呢？为了回答此问题，我们用10μM吗啡分别对PC12细胞和PC12-rMOR细胞进行10min刺激，采用蛋白免疫印迹的方法检测p-ERK与ERK的比值，以确定吗啡对rMOR的激活情况，以此检测PC12细胞中表达的rMOR激活后对细胞信号的传递功能。结果发现，与PC12细胞相比，PC12-rMOR细胞的ERK磷酸化水平显著升高（P0.001，n=3）。上述结果表明在PC12细胞中表达的rMOR蛋白具有与野生型μ阿片受体相同的生物学特征。 2、胍丁胺在PC12-rMOR细胞上抗吗啡依赖和吗啡耐受形成的作用表达有阿片受体的细胞系经吗啡慢性处理后，在纳洛酮催促下会出现cAMP显著升高，称此为cAMP超射。被学界公认为阿片依赖的细胞生物学指标。在本实验中，用吗啡（1~100μM）慢性处理PC12-rMOR细胞24小时后，纳洛酮（10μM）催促15min可显著引起胞内cAMP浓度升高。与未经吗啡处理的对照组相比，20μM的吗啡可引起胞内cAMP含量升高255±25.2%（p 0.01，n＝5），而同样的方式处理未表达rMOR的PC12细胞时，对细胞内cAMP含量无影响。胍丁胺（10~100μM）伴随吗啡（20μM）慢性慢性处理后，胍丁胺（10μM～100μM）能够浓度依赖性抑制纳洛酮（10μM）催促引起的cAMP超射，胍丁胺浓度为100μM时能够将cAMP超射水平抑制65.2%，与吗啡处理组相比具有显著性差异（p 0.01， n=5）。大量文献报道，吗啡耐受的形成与吗啡长期作用于机体导致中枢神经系统神经元发生凋亡有关。而本课题组前期研究发现胍丁胺对阿片耐受具有拮抗作用，其作用机制尚不十分清楚。因此，我们在PC12-rMOR细胞上采用CCK-8细胞活力检测技术评价吗啡和胍丁胺慢性处理96小时后对活细胞数量的影响以及胍丁胺是否能抑制吗啡对活细胞数量的影响。结果发现，吗啡（160μM～2.5mM）能浓度依赖性减少活细胞的数量，当吗啡浓度达640μM时对减少活细胞数量的减少与盐水对照比较具有统计学差异（p 0.05，n＝3）。当吗啡浓度达到750μM时对PC12-rMOR活细胞数量减少作用具有显著性差异（p 0.01，n＝3），当达到mM水平细胞出现死亡。胍丁胺（2.5μM～160μM）能浓度依赖性促进活细胞数量的增加，当胍丁胺浓度达2.5μM时与盐水对照比较具有统计学差异（p 0.05，n＝3），160μM具有显著性差异（p 0.01，n＝3），浓度达到640μM时其效应消失，但未检测到对细胞的毒性。在此细胞模型上，用不同浓度胍丁胺（2.5μM~160μM）与750μM吗啡共同处理PC12-rMOR细胞96小时，胍丁胺2.5μM就能显著地抑制吗啡对细胞增殖的阻碍作用，随着浓度增加，抑制效应也随之加强，当胍丁胺浓度达160μM时能显著地抑制吗啡对活细胞数量减少作用（p 0.01，n＝3）。 3、胍丁胺调节阿片功能的可能分子机制在上述细胞模型上利用蛋白免疫印迹方法研究发现：1）分别用20μM吗啡和100μM胍丁胺慢性处理PC12-rMOR细胞72小时均能显著激活p-ERK（p 0.01，n=3），胍丁胺伴随吗啡预处理PC12-rMOR细胞72小时后与单纯吗啡处理组相比，p-ERK不但没进一步加强，反而呈现降低趋势；2）20μM吗啡慢性处理PC12-rMOR细胞72小时后，，CREB和p-CREB显著下降（p 0.01，n=3），100μM胍丁胺处理使PC12-rMOR细胞72小时使CREB和p-CREB显著升高，胍丁胺伴随吗啡处理能显著抑制吗啡所致的CREB和p-CREB降低（p 0.05，n=3）；3）20μM吗啡慢性处理72小时后IκB水平显著升高，100μM胍丁胺处理72小时后IκB显著降低（p 0.05，n=3），胍丁胺伴随吗啡给药能显著抑制吗啡慢性作用引起的IκB升高（p 0.01，n=3）；20μM吗啡慢性处理72小时后NF-κB表达水平显著降低（p 0.05，n=3），100μM胍丁胺处理72小时NF-κB水平显著升高（p 0.05，n=3），胍丁胺伴随吗啡处理能显著拮抗吗啡引起的NF-κB降低（p 0.05，n=3）。研究结论： 1.成功实现了rMOR在PC12细胞中的稳定表达，并具有与野生型μ阿片受体相同的受体功能特征，为体外研究胍丁胺调节阿片功能的分子机制提供了理想的研究模型； 2.胍丁胺（10μM～100μM）在PC12-rMOR细胞模型上能浓度依赖性抑制慢性吗啡处理、纳洛酮催促引起的cAMP超射，即在该细胞模型上胍丁胺能较好地抑制吗啡依赖的形成； 3.胍丁胺在PC12-rMOR细胞模型上能浓度依赖性抑制吗啡慢性处理所致细胞凋亡，可能是其拮抗吗啡耐受形成的机制之一； 4.胍丁胺调节阿片功能分子机制可能与抑制吗啡所致p-ERK升高和拮抗CREB、p-CREB、NF-κB的降低有关。
[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opioid 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 卤 0.07 nM , similar to that reported in the literature , and Bmax was 1 . 58 卤 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 卤 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 .
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

【参考文献】