BDNF及其前体蛋白在炎性痛中的调控作用研究

发布时间：2018-09-15 06:18

【摘要】：第一部分炎性痛致大鼠脊髓后角及背根节ProBDNF及其受体的表达变化 目的：探讨完全弗氏佐剂(Complete Freund's Adjuvant, CFA)致炎性疼痛后大鼠脊髓后角和背根节(Dorsal root ganglion, DRG)内ProBDNF及其受体P75NTR和Sortilin的表达变化及意义。 方法：大鼠随机分为正常对照组和实验组,实验组大鼠左侧足底皮下注射CFA和生理盐水混合溶剂100μl,建立炎性疼痛模型,实验组分为注射CFA后1d、7d和14d组。在建模前和建模后lh、6h及1d、3d、7d和14d采用Von Frey纤维检测大鼠50%机械缩足阈值(paw withdrawal threshold, PWT)的变化；采用免疫组织化学及western blot方法观察不同时间点ProBDNF及P75NTR、Sortilin在脊髓后角及DRG的表达变化。 结果： 1.足底注射CFA1h后50%PWT即下降,并在6h至1d左右达到最低值,3d后逐渐上调,至14d仍低于基础值。 2.足底注射CFA1d后,注射侧脊髓后角ProBDNF表达较对侧明显上调,这种上调持续至注射CFA后7d,CFA注射14d后,脊髓后角ProBDNF表达逐渐下降,但仍高于正常组,两侧表达差异不明显。脊髓的western blot结果显示,ProBDNF表达的明显增高出现在炎性痛的后期即CFA7d时,且注射侧表达明显强于对侧。DRG的免疫组化和western blot结果表明,注射CFA后1d即可导致注射侧DRG内ProBDNF的表达上调,注射侧表达强于对侧。与注射CFA1d比,注射CFA后7d时ProBDNF的表达进一步上调,且DRG内表达ProBDNF小直径神经元数量逐渐减少,中、大直径神经元数量明显增多。至注射CFA后14d,DRG内ProBDNF的表达下调。 3.脊髓内P75NTR的免疫组化和western blot结果表明,足底注射CFA1d时,P75NTR在脊髓后角的表达达高峰,两侧表达差异不明显；至CFA7d,脊髓内P75NTR的表达仍较强。注射CFA14d时P75NTR的表达较7d明显下降。DRG内P75NTR的免疫组化和Western blot结果显示,在炎性痛早期即CFA1d时,P75NTR的表达即明显增高,且两侧表达差异无显著性；持续至CFA7d时,P75NTR的表达仍明显增高,与CFA1d组比差异不明显,至注射CFA14d时P75NTR的表达下降。 4. Sortilin在正常脊髓后角浅层仅有较弱阳性产物表达,注射CFA后不同时间点脊髓内Sortilin的表达与正常对照组比差异不明显。DRG内Sortilin的表达量在炎性痛后不同时间点也无显著变化,但注射侧DRG内表达Sortilin的小直径神经元数量减少,中直径神经元数量增多,这与proBDNF的表达模式类似。 结论： 足底注射CFA能诱导大鼠产生为期2周以上的炎性痛病程；炎性痛时脊髓后角和DRG内ProBDNF及P75NTR的表达上调可能参与了外周痛觉信号的传导和中枢敏化的形成。DRG内表达Sortilin的细胞类型变换可能与proBDNF及BDNF的运输及释放有关。 第二部分炎性痛致大鼠脊髓及背根节内BDNF基因不同外显子的表达变化 目的：探讨完全弗氏佐剂(Complete Freund's Adjuvant, CFA)致炎性疼痛后大鼠脊髓和DRG内BDNF总的mRNA及包含不同BDNF外显子1mRNA的表达变化及意义,从而揭示炎性痛不同时期、不同部位BDNF基因表达调节的差异性机制。 方法：大鼠随机分为正常组和实验组,实验组大鼠左侧足底皮下注射CFA和生理盐水混合溶剂100ul,建立炎性疼痛模型,实验组包括注射CFA后1d、7d和14d组。采用普通PCR以及Realtime-PCR方法检测BDNF总的mRNA及BDNF不同外显子(exon Ⅰ, exon ⅡA, exon ⅡB, exon ⅡC, exon Ⅲ, exon Ⅳ和exon ⅨA)在脊髓和DRG内的表达变化。 结果： 1.足底注射CFA1d后,注射侧脊髓内BDNF总的mRNA的表达明显升高,注射侧强于对侧；上调持续至注射CFA后7d左右达高峰,对侧表达也有增高,但注射侧表达仍强于对侧；至14d时,BDNF mRNA表达下调,但仍强于正常对照组。注射侧DRG内BDNF mRNA的表达在注射CFA1d后明显升高,注射侧强于对侧；上调持续至注射CFA后7d；至14d时,BDNF mRNA表达下调,与正常对照组相比差异没有显著性。 2.足底注射CFA1d时,exon Ⅰ在注射侧脊髓的表达明显上调,与对侧相比有明显差异；exon ⅡA和exon ⅨA在对侧脊髓表达有上调。至注射CFA7d时,注射侧exon ⅡA、exon ⅡB和exon ⅨA的表达较1d时明显增高,并明显高于对侧。注射CFA14d时,脊髓两侧exon ⅡB的表达仍高于正常组,对侧脊髓内exon ⅡA和exon ⅨA的表达有上调。 3.足底注射CFA1d时,注射侧DRG内BDNF基因exon Ⅰ、exon ⅡA、exon ⅡB和exon Ⅳ的表达明显上调,与对侧相比有明显差异。至注射CFA7d时,exon Ⅰ的表达较CFA1d时进一步增高,且明显高于对侧,其他外显子的表达无明显变化。至注射CFA后14d时,各外显子的表达与正常对照组比差异均不明显。 结论： 在炎性痛的不同时期、不同部位BDNF外显子出现差异性的表达变化,BDNF外显子的变化与总的BDNF mRNA的变化趋势基本相符。 第三部分炎性痛致大鼠脊髓后角乙酰化组蛋白3的表达变化及吗啡的干预影响 目的：探讨完全弗氏佐剂(Complete Freund's Adjuvant, CFA)致大鼠炎性疼痛后脊髓后角乙酰化组蛋白3(Acetylated Histone3,ACH3)水平的表达变化及意义,进一步说明组蛋白乙酰化在炎性疼痛产生和发展过程中发挥的作用以及吗啡干预对其的影响。 方法：大鼠左侧足底皮下注射CFA和生理盐水混合溶剂100ul,建立炎性疼痛模型。大鼠随机分为正常组和实验组(注射CFA后1d、7d组),后部分实验增加CFA1d+吗啡干预组。采用免疫组织化学和免疫荧光化学方法观察不同组脊髓后角ACH3的表达变化以及ACH3与神经元、星形胶质细胞和小胶质细胞的双标情况；采用Von Frey纤维检测吗啡对炎性痛早期大鼠50%PWT的变化,并观察吗啡干预对炎性痛早期脊髓后角ACH3及BDNF表达的影响。 结果： 1.免疫组织化学结果显示,注射CFA后1d,脊髓后角ACH3的表达明显下调,与正常大鼠相比差异显著；至注射CFA后7d,ACH3表达水平逐渐恢复。免疫荧光双标结果表明,正常大鼠脊髓后角ACH3的表达主要在神经元,部分在胶质细胞。注射CFA1d后,ACH3的下调主要发生在神经元；而至CFA7d时表达ACH3的神经元数量明显增加,且表达ACH3的星形胶质细胞和小胶质细胞数量也增加。 2.腹腔注射吗啡(10mg/kg)可明显增高大鼠CFA1d时的50%PWT。吗啡可抑制炎性痛早期脊髓后角ACH3的下调,其作用在于增加了表达ACH3的神经元及胶质细胞的数量,尤其是星形胶质细胞和小胶质细胞。 3.正常大鼠脊髓后角可见较弱BDNF表达,注射CFA1d后注射侧脊髓后角BDNF的表达明显上调,且注射侧表达强于对侧；吗啡干预对脊髓后角BDNF的表达无明显影响。 结论： CFA诱导的炎性痛可导致脊髓后角短暂的ACH3下调,这种下调可被吗啡逆转。在炎性痛的发生发展过程中,ACH3的表达变化有细胞类型的转换。吗啡干预对炎性痛早期脊髓后角BDNF的表达无明显影响。
[Abstract]:Part one expression of ProBDNF and its receptor in spinal dorsal horn and dorsal root ganglion in rats induced by inflammatory pain
AIM: To investigate the expression of ProBDNF and its receptors P75NTR and Sortin in dorsal root ganglion (DRG) and spinal dorsal horn of rats with inflammatory pain induced by Complete Freund's adjuvant (CFA).
Methods: Rats were randomly divided into normal control group and experimental group. Inflammatory pain model was established by subcutaneous injection of CFA and saline mixed solvent 100 ml into left plantar of rats in experimental group. The experimental group was divided into 1, 7 and 14 days after injection of CFA. The expression of ProBDNF, P75NTR and Sortilin in the posterior horn of spinal cord and DRG were observed by immunohistochemistry and Western blot.
Result:
1. After injection of CFA1, 50% PWT decreased and reached the lowest level from 6 hours to 1 day. It gradually increased after 3 days and was still below the baseline level at 14 days.
2. The expression of ProBDNF in the dorsal horn of the spinal cord was up-regulated significantly after injection of CFA1. The up-regulation lasted until 7 days after injection of CFA and 14 days after injection of CFA. The expression of ProBDNF in the dorsal horn of the spinal cord decreased gradually, but was still higher than that in the normal group, and there was no significant difference between the two sides. The results of immunohistochemistry and Western blot showed that the expression of ProBDNF was up-regulated and the expression of ProBDNF was up-regulated in the injection-side DRG at day 1 after injection of CFA. The number of meridians decreased gradually and the number of large diameter neurons increased significantly. The expression of ProBDNF in DRG was down-regulated 14 days after CFA injection.
3. The immunohistochemical and Western blot results of P75NTR in spinal cord showed that the expression of P75NTR in the dorsal horn of spinal cord reached a peak on the day of CFA1 injection, but there was no significant difference between the two sides. At the day of CFA7, the expression of P75NTR in spinal cord was still strong. The expression of P75NTR was significantly increased in the early stage of inflammatory pain, that is, CFA1d, and there was no significant difference between the two sides. The expression of P75NTR was still significantly increased until the 7th day of CFA, but not significantly different from CFA1d, and the expression of P75NTR was decreased at the 14th day of CFA1 injection.
4. The expression of Sortilin in the superficial layer of the normal spinal dorsal horn was weakly positive. The expression of Sortilin in the spinal cord at different time points after injection of CFA was not significantly different from that in the normal control group. The number of neurons in the middle diameter increased, which is similar to the expression pattern of proBDNF.
Conclusion:
The expression of ProBDNF and P75NTR in the dorsal horn of spinal cord and DRG may be involved in the transmission of peripheral pain signals and the formation of central sensitization.
The second part is the expression of BDNF exons in spinal cord and dorsal root ganglion in rats with inflammatory pain.
AIM: To investigate the changes and significance of total BDNF mRNA and BDNF exon 1 mRNA in spinal cord and DRG in rats with inflammatory pain induced by Complete Freund's Adjuvant (CFA), so as to reveal the differential mechanism of BDNF gene expression regulation in different parts of spinal cord and DRG in different stages of inflammatory pain.
METHODS: Rats were randomly divided into normal group and experimental group. Inflammatory pain model was established by subcutaneous injection of CFA and saline mixed solvent 100ul into the left plantar of rats in experimental group. The experimental group included 1, 7 and 14 days after injection of CFA. Expression changes of II B, exon II C, exon III, exon IV and exon IX A in spinal cord and DRG.
Result:
1. After CFA 1 injection, the expression of BDNF mRNA in the spinal cord of the injected side increased significantly, and the expression of BDNF mRNA in the injected side was stronger than that in the contralateral side. The expression of BDNF mRNA was down-regulated 7 days after injection of CFA, and there was no significant difference between the two groups.
2. The expression of exon I was significantly up-regulated in the spinal cord of the injection side and significantly different from that of the contralateral side when CFA 1 was injected into the sole of the foot. The expression of exon II A and exon VIIA was up-regulated in the contralateral spinal cord. At day 7, the expression of exon II A, exon II B and exon VIIA on the injection side was significantly higher than that on day 1 and significantly higher than that on the contralateral side. The expression of B was still higher than that in normal group, and the expression of exon II A and exon A in contralateral spinal cord was upregulated.
3. The expression of BDNF gene exon I, exon II A, exon II B and exon IV was significantly up-regulated in the DRG injected with CFA1 on the plantar injection side, which was significantly different from that in the control side. The expression was not significantly different from that of the normal control group.
Conclusion:
At different stages of inflammatory pain, the expression of BDNF exons was different in different parts of the body, and the change of BDNF exons was consistent with the change of total BDNF mRNA.
The third part is the expression of acetylated histone 3 in spinal dorsal horn induced by inflammatory pain and the effect of morphine on it.
AIM: To investigate the expression and significance of acetylated histone 3 (ACH3) in the spinal dorsal horn of rats with inflammatory pain induced by Complete Freund's Adjuvant (CFA), and to further clarify the role of histone acetylation in the production and development of inflammatory pain and the effect of morphine intervention on it.
METHODS: Inflammatory pain models were established by subcutaneous injection of CFA and saline into the left plantar of rats. Rats were randomly divided into normal group and experimental group (one day and seven days after injection of CFA). The expression of ACH3 in the dorsal horn of spinal cord was observed by immunohistochemistry and immunofluorescence chemistry. The expression of ACH3 and BDNF in the spinal dorsal horn of rats with early inflammatory pain was detected by Von Frey fiber.
Result:
1. Immunohistochemical results showed that the expression of ACH3 in the dorsal horn of spinal cord was down-regulated one day after injection of CFA, which was significantly different from that in normal rats. The expression of ACH3 gradually recovered 7 days after injection of CFA. The down-regulation mainly occurred in neurons, and the number of ACH3-expressing neurons increased significantly at the 7th day of CFA, and the number of ACH3-expressing astrocytes and microglia also increased.
2. Intraperitoneal injection of morphine (10mg/kg) significantly increased 50% PWT at CFA1d in rats. Morphine inhibited the down-regulation of ACH3 in the dorsal horn of spinal cord in the early stage of inflammatory pain. Morphine increased the number of neurons and glial cells expressing ACH3, especially astrocytes and microglia.
3. The expression of BDNF was weaker in the dorsal horn of spinal cord in normal rats. After injection of CFA1, the expression of BDNF in the dorsal horn of spinal cord was significantly up-regulated, and the expression of BDNF in the injection side was stronger than that in the contralateral side. Morphine intervention had no significant effect on the expression of BDNF in the dorsal horn of spinal cord.
Conclusion:
CFA-induced inflammatory pain can induce a transient down-regulation of ACH3 in the dorsal horn of the spinal cord, which can be reversed by morphine. During the development of inflammatory pain, the expression of ACH3 changes with cell types. Morphine intervention has no significant effect on the expression of BDNF in the dorsal horn of the spinal cord in the early stage of inflammatory pain.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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