本文關鍵詞：excADP調控大鼠脊髓星形膠質細胞AMPK活性參與神經病理性疼痛的作用及機制　出處：《第三軍醫(yī)大學》2011年博士論文　論文類型：學位論文

  更多相關文章： 脊髓背角 星形膠質細胞 磷酸腺苷激活蛋白激酶 三磷酸腺苷

【摘要】：膠質細胞活化是維持和促進神經病理性疼痛(Neuropathic pain,NPP)的主要驅動力。既往發(fā)現(xiàn)細胞外ATP在誘發(fā)和維持膠質細胞活化過程中起著重要作用,但其來源與產生機制不明。 前期關于ATP與星形膠質細胞活化之間關系的研究結果提示星形膠質細胞上P2Y類受體激活可能參與了細胞內ATP的合成過程,現(xiàn)需要證明其是否參與細胞外ATP的生成并探索其機制。AMPK是細胞內調控線粒體ATP合成功能的主要蛋白,其上游信號與細胞外ADP(excADP)激活的P2Y類受體下游信號存在著一定的一致性,其是否是聯(lián)系P2Y類受體與線粒體之間的信號蛋白尚需證明。 本課題旨在探討AMPK在體外培養(yǎng)及在體大鼠脊髓背角部位星形膠質細胞中的激活機制及作用規(guī)律。擬以體外培養(yǎng)的大鼠脊髓背角星形膠質細胞和坐骨神經慢性壓迫損傷(CCI)大鼠模型為研究對象,采用形態(tài)學、分子生物學及行為學等實驗技術,探討excADP激活AMPK后促進細胞內ATP合成、轉運、釋放的作用,并將AMPK阻斷劑應用于離體細胞和動物模型觀察其影響星形膠質細胞活化和疼痛進程的效果。研究結果將對了解ATP致痛機制和尋找治療NPP的新靶點提供理論依據。 方法:原代培養(yǎng)的星形膠質細胞取材于SD乳鼠腰段脊髓背角,ADP或ADPβs作為刺激物,熒光素酶法測定細胞內外ATP濃度,JC-1熒光染色觀察線粒體電位變化,免疫組織化學染色和免疫印跡技術觀察AMPK的表達,MTT法測定細胞增殖活性。制作鞘內插管后的CCI大鼠模型,經鞘內導管每日注入AMPK特異性阻斷劑Compound C,分別在坐骨神經結扎后0、3、7、14、21d測定大鼠患趾熱痛閾及機械痛閾,并在第14d應用免疫組織化學法觀察患側腰段脊髓背角部位GFAP的表達變化。結果: 第一部分: 1.經過差速篩選和純化的星形膠質細胞生長狀態(tài)良好。GFAP染色陽性率可達到98%甚至更高,符合本實驗要求。 2.ADP和ADPβs作用后,星形膠質細胞增殖數(shù)呈劑量和時間依賴性增長,高峰值出現(xiàn)在100μM作用24h后;為觀察ADP及ADPβs是否通過G蛋白耦連受體作用,設定ADP和ADPβs在條件培養(yǎng)基濃度為100μM,作用時間設定為24h,觀察不同濃度的GDPβs對細胞增殖數(shù)的影響,結果顯示GDPβs在較小濃度對細胞的增殖效應即出現(xiàn)抑制效應,在100μM濃度后劑量效應逐漸減弱;同樣設定ADP和ADPβs在條件培養(yǎng)基濃度為100μM,作用時間設定為24h,觀察不同濃度的MRS2179預處理阻斷P2Y1后細胞增殖數(shù)的變化時,不同于GDPβs的劑量抑制效應,MRS2179出現(xiàn)細胞增殖的量效促進效應, ADP組在MRS2179 100μM、ADPβs組在200μM后量效增殖效應才逐漸減弱。 3.設定ADP及其模擬劑ADPβs在條件培養(yǎng)基內的濃度均為100μM,作用于細胞的時間為24h,結果表明無論是ADP還是ADPβs均可明顯提高細胞內外的ATP濃度,預先給予G蛋白的非特異性阻斷劑GDPβs可有效阻斷ADP及ADPβs引發(fā)的細胞內外ATP濃度的升高,但預先給予P2Y1受體的特異性阻斷劑MRS2179不僅未能抑制ADP和ADPβs引起的ATP蓄積,反而促使細胞內外的ATP濃度更大幅度增高。 4.設定ADP及ADPβs在條件培養(yǎng)基內的濃度均為100μM,作用于細胞的時間為24h。利用JC-1直接對細胞線粒體內膜進行染色發(fā)現(xiàn),熒光顯微鏡下PBS對照組及MRS2179組細胞內紅色熒光呈散在顆粒狀分布,胞體及突起內均有著色,綠色熒光較弱,呈彌散分布,顆粒感不強。與對照組相比,100μM ADP及ADPβs作用24h后細胞內紅色顆粒狀熒光增強、增多,在100μM MRS2179預處理后再加入ADP或ADPβs,紅光熒光強度進一步增強,尤其是在細胞核周圍紅色顆粒狀熒光密集分布,綠色熒光相應減弱,紅色:綠色熒光比值明顯增大。 第二部分: 1.正常體外培養(yǎng)的大鼠腰段脊髓背角部位星形膠質細胞上存在AMPK蛋白的表達,陽性區(qū)域多集中在細胞核周圍; 2.100μM ADP或ADPβs作用24h后,細胞內的AMPK表達強度增加,AMPK陽性表達部位擴大到細胞突起,部分區(qū)域細胞出現(xiàn)不規(guī)則生長,胞體變大,突起延長。MRS2179+ADP組和MRS2179+ADPβs組細胞與單純ADP或ADPβs組細胞相比,細胞胞體肥大,突起粗壯,細胞間間隔不明顯,AMPK著色熒光進一步增強,分布到細胞的各個部位,Western Blot檢測到AMPK蛋白含量相應增加; 3.預先使用AMPK的特異性阻斷劑Compound C直接阻斷AMPK的信號通路,熒光素酶法測定ATP濃度,MTT法進行細胞計數(shù),JC-1染色觀察線粒體內膜電位。結果發(fā)現(xiàn)使用20μM Compound C預處理后,無論是否合用MRS2179預先阻斷P2Y1,與對照組相比細胞內外的ATP濃度、線粒體內膜電位及細胞數(shù)量均無明顯的升高; 4.ADP、ADPβs及Compound C作用于星形膠質細胞24h后,細胞培養(yǎng)基內的谷氨酸(Glu)濃度未發(fā)生具有顯著統(tǒng)計學意義的變化。 第三部分: 1.經腰骶部進行留置鞘內導管的大鼠四肢活動正常,死亡率、致殘率較低,導管固定較好,位置比較容易驗證,可以用于CCI大鼠痛覺行為學觀察; 2.大鼠的痛覺行為學測定表明,腰段蛛網膜下腔每日注入10μl濃度為200μM的Compound C后,熱痛閾在坐骨神經結扎后的第7、14、21d,機械痛閾在坐骨神經結扎后的第7、14d的閾值均有顯著的提高(p0.05),而生理鹽水鞘內注射則對CCI大鼠熱痛閾及機械痛閾無顯著的抑制作用(p0.05); 3.采用免疫組織化學(DAB顯色)方法觀察對照組及CCI損傷術后3d、7d、14d、21d各時間點大鼠脊髓背角星型膠質細胞活化反應(照片6及圖2)。從圖中可以看出,CCI術后3d損傷側脊髓背角GFAP染色開始加強,數(shù)量增多,14 d最明顯。提示CCI3d、7 d、14d、21d損傷側脊髓背角星型膠質細胞均發(fā)生活化,且CCI 14 d星型膠質細胞激活程度最高; 4.通過對CCI術后14d大鼠患側脊髓背角部位星形膠質細胞GFAP染色觀察發(fā)現(xiàn),鞘內不注藥或鞘內注射生理鹽水的CCI大鼠脊髓背角部位GFAP染色較對照組明顯加深,被GFAP著色的星形膠質細胞胞體較大,突起較粗,單位面積內OD值也有顯著的增加(p0.05),而鞘內注射Compound C的CCI大鼠脊髓背角染色加深不明顯,細胞數(shù)量也無明顯增加。 結論: 1.通過測定培養(yǎng)細胞內外ATP濃度和線粒體內膜電位,證明excADP可以促進星形膠質細胞內ATP的合成和釋放,而P2Y1受體激活后對ATP的過度合成和釋放起抑制作用。 2.AMPK可能參與了excADP引起的細胞內外ATP蓄積過程,AMPK蛋白的表達受P2Y1受體活性的影響。 3.特異性阻斷星形膠質細胞內的AMPK可以抑制excADP引起的細胞內外ATP蓄積和細胞活化。 4.通過鞘內注入AMPK特異性阻斷劑可以顯著抑制CCI大鼠患側脊髓背角的星形膠質細胞活化和大鼠的痛覺敏感化程度。 綜上所述,脊髓背角部位的星形膠質細胞內的AMPK蛋白參與了excADP引發(fā)的細胞活化和慢性疼痛形成過程。當細胞內的AMPK在excADP刺激下表達增強后,星形膠質細胞合成和釋放ATP增加,這可能是ADP參與脊髓平面痛覺中樞敏感化的機制之一。因此AMPK有望成為新的痛覺治療作用位點。
[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.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R363

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