excADP调控大鼠脊髓星形胶质细胞AMPK活性参与神经病理性疼痛的作用及机制

发布时间：2018-01-16 14:14

本文关键词：excADP调控大鼠脊髓星形胶质细胞AMPK活性参与神经病理性疼痛的作用及机制　出处：《第三军医大学》2011年博士论文　论文类型：学位论文

【摘要】：胶质细胞活化是维持和促进神经病理性疼痛(Neuropathic pain,NPP)的主要驱动力。既往发现细胞外ATP在诱发和维持胶质细胞活化过程中起着重要作用,但其来源与产生机制不明。前期关于ATP与星形胶质细胞活化之间关系的研究结果提示星形胶质细胞上P2Y类受体激活可能参与了细胞内ATP的合成过程,现需要证明其是否参与细胞外ATP的生成并探索其机制。AMPK是细胞内调控线粒体ATP合成功能的主要蛋白,其上游信号与细胞外ADP(excADP)激活的P2Y类受体下游信号存在着一定的一致性,其是否是联系P2Y类受体与线粒体之间的信号蛋白尚需证明。本课题旨在探讨AMPK在体外培养及在体大鼠脊髓背角部位星形胶质细胞中的激活机制及作用规律。拟以体外培养的大鼠脊髓背角星形胶质细胞和坐骨神经慢性压迫损伤(CCI)大鼠模型为研究对象,采用形态学、分子生物学及行为学等实验技术,探讨excADP激活AMPK后促进细胞内ATP合成、转运、释放的作用,并将AMPK阻断剂应用于离体细胞和动物模型观察其影响星形胶质细胞活化和疼痛进程的效果。研究结果将对了解ATP致痛机制和寻找治疗NPP的新靶点提供理论依据。方法:原代培养的星形胶质细胞取材于SD乳鼠腰段脊髓背角,ADP或ADPβs作为刺激物,荧光素酶法测定细胞内外ATP浓度,JC-1荧光染色观察线粒体电位变化,免疫组织化学染色和免疫印迹技术观察AMPK的表达,MTT法测定细胞增殖活性。制作鞘内插管后的CCI大鼠模型,经鞘内导管每日注入AMPK特异性阻断剂Compound C,分别在坐骨神经结扎后0、3、7、14、21d测定大鼠患趾热痛阈及机械痛阈,并在第14d应用免疫组织化学法观察患侧腰段脊髓背角部位GFAP的表达变化。结果: 第一部分: 1.经过差速筛选和纯化的星形胶质细胞生长状态良好。GFAP染色阳性率可达到98%甚至更高,符合本实验要求。 2.ADP和ADPβs作用后,星形胶质细胞增殖数呈剂量和时间依赖性增长,高峰值出现在100μM作用24h后;为观察ADP及ADPβs是否通过G蛋白耦连受体作用,设定ADP和ADPβs在条件培养基浓度为100μM,作用时间设定为24h,观察不同浓度的GDPβs对细胞增殖数的影响,结果显示GDPβs在较小浓度对细胞的增殖效应即出现抑制效应,在100μM浓度后剂量效应逐渐减弱;同样设定ADP和ADPβs在条件培养基浓度为100μM,作用时间设定为24h,观察不同浓度的MRS2179预处理阻断P2Y1后细胞增殖数的变化时,不同于GDPβs的剂量抑制效应,MRS2179出现细胞增殖的量效促进效应, ADP组在MRS2179 100μM、ADPβs组在200μM后量效增殖效应才逐渐减弱。 3.设定ADP及其模拟剂ADPβs在条件培养基内的浓度均为100μM,作用于细胞的时间为24h,结果表明无论是ADP还是ADPβs均可明显提高细胞内外的ATP浓度,预先给予G蛋白的非特异性阻断剂GDPβs可有效阻断ADP及ADPβs引发的细胞内外ATP浓度的升高,但预先给予P2Y1受体的特异性阻断剂MRS2179不仅未能抑制ADP和ADPβs引起的ATP蓄积,反而促使细胞内外的ATP浓度更大幅度增高。 4.设定ADP及ADPβs在条件培养基内的浓度均为100μM,作用于细胞的时间为24h。利用JC-1直接对细胞线粒体内膜进行染色发现,荧光显微镜下PBS对照组及MRS2179组细胞内红色荧光呈散在颗粒状分布,胞体及突起内均有着色,绿色荧光较弱,呈弥散分布,颗粒感不强。与对照组相比,100μM ADP及ADPβs作用24h后细胞内红色颗粒状荧光增强、增多,在100μM MRS2179预处理后再加入ADP或ADPβs,红光荧光强度进一步增强,尤其是在细胞核周围红色颗粒状荧光密集分布,绿色荧光相应减弱,红色:绿色荧光比值明显增大。第二部分: 1.正常体外培养的大鼠腰段脊髓背角部位星形胶质细胞上存在AMPK蛋白的表达,阳性区域多集中在细胞核周围; 2.100μM ADP或ADPβs作用24h后,细胞内的AMPK表达强度增加,AMPK阳性表达部位扩大到细胞突起,部分区域细胞出现不规则生长,胞体变大,突起延长。MRS2179+ADP组和MRS2179+ADPβs组细胞与单纯ADP或ADPβs组细胞相比,细胞胞体肥大,突起粗壮,细胞间间隔不明显,AMPK着色荧光进一步增强,分布到细胞的各个部位,Western Blot检测到AMPK蛋白含量相应增加; 3.预先使用AMPK的特异性阻断剂Compound C直接阻断AMPK的信号通路,荧光素酶法测定ATP浓度,MTT法进行细胞计数,JC-1染色观察线粒体内膜电位。结果发现使用20μM Compound C预处理后,无论是否合用MRS2179预先阻断P2Y1,与对照组相比细胞内外的ATP浓度、线粒体内膜电位及细胞数量均无明显的升高; 4.ADP、ADPβs及Compound C作用于星形胶质细胞24h后,细胞培养基内的谷氨酸(Glu)浓度未发生具有显著统计学意义的变化。第三部分: 1.经腰骶部进行留置鞘内导管的大鼠四肢活动正常,死亡率、致残率较低,导管固定较好,位置比较容易验证,可以用于CCI大鼠痛觉行为学观察; 2.大鼠的痛觉行为学测定表明,腰段蛛网膜下腔每日注入10μl浓度为200μM的Compound C后,热痛阈在坐骨神经结扎后的第7、14、21d,机械痛阈在坐骨神经结扎后的第7、14d的阈值均有显著的提高(p0.05),而生理盐水鞘内注射则对CCI大鼠热痛阈及机械痛阈无显著的抑制作用(p0.05); 3.采用免疫组织化学(DAB显色)方法观察对照组及CCI损伤术后3d、7d、14d、21d各时间点大鼠脊髓背角星型胶质细胞活化反应(照片6及图2)。从图中可以看出,CCI术后3d损伤侧脊髓背角GFAP染色开始加强,数量增多,14 d最明显。提示CCI3d、7 d、14d、21d损伤侧脊髓背角星型胶质细胞均发生活化,且CCI 14 d星型胶质细胞激活程度最高; 4.通过对CCI术后14d大鼠患侧脊髓背角部位星形胶质细胞GFAP染色观察发现,鞘内不注药或鞘内注射生理盐水的CCI大鼠脊髓背角部位GFAP染色较对照组明显加深,被GFAP着色的星形胶质细胞胞体较大,突起较粗,单位面积内OD值也有显著的增加(p0.05),而鞘内注射Compound C的CCI大鼠脊髓背角染色加深不明显,细胞数量也无明显增加。结论: 1.通过测定培养细胞内外ATP浓度和线粒体内膜电位,证明excADP可以促进星形胶质细胞内ATP的合成和释放,而P2Y1受体激活后对ATP的过度合成和释放起抑制作用。 2.AMPK可能参与了excADP引起的细胞内外ATP蓄积过程,AMPK蛋白的表达受P2Y1受体活性的影响。 3.特异性阻断星形胶质细胞内的AMPK可以抑制excADP引起的细胞内外ATP蓄积和细胞活化。 4.通过鞘内注入AMPK特异性阻断剂可以显著抑制CCI大鼠患侧脊髓背角的星形胶质细胞活化和大鼠的痛觉敏感化程度。综上所述,脊髓背角部位的星形胶质细胞内的AMPK蛋白参与了excADP引发的细胞活化和慢性疼痛形成过程。当细胞内的AMPK在excADP刺激下表达增强后,星形胶质细胞合成和释放ATP增加,这可能是ADP参与脊髓平面痛觉中枢敏感化的机制之一。因此AMPK有望成为新的痛觉治疗作用位点。
[Abstract]:Activation of glial cells is the main driving force to maintain and promote Neuropathic pain (NPP). It has been discovered that extracellular ATP plays an important role in inducing and maintaining glial activation, but its origin and mechanism is unknown.
Preliminary study on the relationship between ATP and astrocyte activation results suggest that P2Y receptor activation of astrocytes may be involved in the synthesis process of intracellular ATP, we need to prove whether it is involved in the formation of ATP cells and explore the mechanism of.AMPK protein is the main intracellular regulation of mitochondrial ATP synthesis function, its upstream the signal and extracellular ADP (excADP) P2Y receptor downstream signaling activation has a certain consistency, whether it is the connection between P2Y receptor signaling proteins and mitochondria still need to be proved.
The purpose of this study is to investigate AMPK in vitro and in vivo rat spinal cord dorsal horn parts of astrocytes in the activation mechanism and the role of law. The rat spinal dorsal horn astrocytes and sciatic nerve chronic constriction injury (CCI) in cultured rat model by morphology as the research object, molecular biology and behavior study on experimental technology, excADP activated AMPK cells promote ATP synthesis, transport, release, and AMPK blocking agent used in vitro and animal model to observe the effect of astrocyte activation and pain process effect. The research results will provide theoretical basis for the new target for understanding ATP induced pain and mechanism for the treatment of NPP.
Methods: primary cultured astrocytes derived from SD rat lumbar spinal dorsal horn, ADP or ADP beta s as a stimulant, determination of the intracellular ATP concentration of luciferase method, observe the changes of mitochondrial potential JC-1 staining, immunohistochemical staining and Western blot to detect the expression of AMPK, determination of cell proliferation activity by MTT the CCI model in rats. After intrathecal intubation, after intrathecal catheter daily injection of a specific inhibitor of AMPK Compound C, respectively after sciatic nerve ligation in rats suffering from toe 0,3,7,14,21d determination of thermal and mechanical pain threshold, and the 14d should observe the expression changes of patients side lumbar GFAP spinal dorsal horn parts with immune the results of histochemical method:
Part one:
1. the positive rate of.GFAP staining of astrocytes after differential screening and purification can reach 98% or even higher, which is in line with the requirements of this experiment.
The role of 2.ADP and ADP beta s, astrocyte proliferation in a dose and time dependent growth, the peak value occurred at 100 M after 24h; to observe ADP and ADP beta s through G protein coupled receptors, ADP and ADP set the beta s in conditioned medium concentration is 100 M time is set to 24h, observe the effects of GDP beta s with different concentration on the cell proliferation, the results show that GDP beta s in lower concentration on cell proliferation effect that inhibitory effect gradually weakened at 100 M concentration after dose effect; also set the ADP and ADP beta s in conditioned medium concentration was 100 M, the action time is set to 24h, observe the changes of the number of blocked P2Y1 cell proliferation after MRS2179 pretreatment of different concentrations, the inhibitory effect is different from the GDP beta s dose, dose effect MRS2179 cell proliferation promoting effect of ADP in MRS2179 group, 100 M, ADP beta s group at 200 M after the dose effect proliferation effect Gradually diminished.
The 3. set of ADP concentration and its simulation agent ADP beta s medium in the condition was 100 M, the effect on the cell time for 24h, the results show that both the ADP or ADP beta s can significantly increase the concentration of ATP inside and outside cells, nonspecific pretreatment with G protein beta blockers GDP s effectively inhibited the increase of ADP ADP and beta s triggered intracellular ATP concentration, but pretreatment with P2Y1 receptor antagonist of MRS2179 not only failed to inhibit ADP and ADP beta s induced ATP accumulation, ATP concentration makes cells inside and outside of the more substantial increase.
4. set ADP and ADP beta s in the condition of culture medium concentration was 100 M, the effect on the cell time is 24h. using JC-1 directly on the cell mitochondrial staining, fluorescence microscope PBS control group and MRS2179 group cells with red fluorescence were scattered in the granular distribution, were immunostained cell bodies and processes. The green fluorescence is weak, diffuse distribution, grain is not strong. Compared with the control group, 100 M ADP and ADP s 24h beta cells after red granular fluorescence enhancement, increased in 100 M after pretreatment with MRS2179 before adding ADP or ADP beta s, red fluorescence intensity was further enhanced, especially in the nucleus around the red fluorescence dense granular distribution of green fluorescence decreased, red: green fluorescence ratio increased significantly.
The second part:
1. the expression of AMPK protein was found on astrocytes in the dorsal horn of lumbar spinal cord in normal rats, and the positive region was mostly around the nucleus.
2.100 M ADP or ADP s beta 24h, intracellular AMPK expression increased, AMPK positive cells were expanded to cell processes, part of cells appeared irregular growth, larger cell body, neurite extension of.MRS2179+ADP group and MRS2179+ADP beta cells in s group with pure ADP or ADP beta cells in s group compared to cells the cell body hypertrophy, cell protrusions stout, interval is not obvious, AMPK color fluorescence enhanced, distributed to various parts of the cell, Western Blot detected AMPK protein content increased;
Specificity of 3. pretreatment with the AMPK antagonist Compound C directly blocking AMPK signaling pathway, ATP concentration was measured by luciferase method, MTT method was used to observe cell count, mitochondrial membrane potential JC-1 staining. The results showed that the use of 20 M Compound C after pretreatment with MRS2179, regardless of whether blocking P2Y1, compared with the control group, the concentration of ATP cells the number of mitochondrial membrane potential and cell were not significantly increased;
4.ADP, ADP s and Compound C beta in astrocytes after 24h cell culture medium of glutamic acid (Glu) concentration did not change significantly.
The third part:
1. the activities of the limbs in the lumbar sacral region were normal, the mortality rate was low, the disability rate was low, the catheter fixation was good, the location was relatively easy to verify, and it could be used for the observation of pain behavior in CCI rats.
The pain behavior of 2. rats determination showed that lumbar subarachnoid injection daily 10 L concentration of 200 M Compound C, 7,14,21d in the thermal pain threshold after sciatic nerve ligation, both in the mechanical pain threshold after sciatic nerve ligation of the 7,14d threshold significantly increased (P0.05), and saline intrathecal injection of CCI on the pain threshold of rats thermal and mechanical threshold without significant inhibitory effect (P0.05);
3. by immunohistochemical method (DAB staining) were observed in control group and CCI injury 3D, 7d, 14d, 21d at different time points in rat spinal dorsal horn astrocytes activation (Photo 6 and Figure 2). It can be seen from the figure, after CCI 3D GFAP in dorsal horn of spinal cord injury with the increase in the number of strong staining, and 14 d. The most obvious tip CCI3d, 7 d, 14d, 21d damage in spinal cord dorsal horn astrocytes were activated, and CCI 14 d of astrocyte activation of the highest degree;
4. by CCI 14d after spinal dorsal horn of rats parts of astrocytes were observed by GFAP staining found that intrathecal GFAP parts of the spinal dorsal horn of CCI rats without injection or intrathecal injection of saline was significantly increased compared with the control group, GFAP staining of astrocytes in the larger cell body, coarse protrusions the unit area, OD value was significantly increased (P0.05), and intrathecal injection of Compound C CCI rat spinal cord dorsal horn deep staining is not obvious, the number of cells is not significantly increased.
Conclusion:
1., by measuring intracellular and extracellular ATP concentration and mitochondrial intimal potential, it is proved that excADP can promote the synthesis and release of ATP in astrocytes, while P2Y1 receptor activation inhibits the over synthesis and release of ATP.
2.AMPK may be involved in the accumulation of intracellular and extracellular ATP induced by excADP, and the expression of AMPK protein is affected by the activity of P2Y1 receptor.
3. the specific blocking of AMPK in astrocytes can inhibit the accumulation of ATP and cell activation by excADP.
4. intrathecal injection of AMPK specific blockers can significantly inhibit the activation of astrocytes in the dorsal horn of the injured side of CCI rats and the degree of pain sensitivity in rats.
In summary, parts of the spinal dorsal horn astrocytes within the AMPK protein involved in excADP induced cell activation and chronic pain formation. When the enhanced expression of AMPK in cells under excADP stimulation, increase the synthesis and release of ATP in astrocytes, which may be one of the mechanisms of ADP in spinal cord pain central sensitization. Therefore, AMPK is expected to become the new site of pain treatment.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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