【摘要】：第一部分炎性痛致大鼠脊髓后角及背根節(jié)ProBDNF及其受體的表達變化 目的：探討完全弗氏佐劑(Complete Freund's Adjuvant, CFA)致炎性疼痛后大鼠脊髓后角和背根節(jié)(Dorsal root ganglion, DRG)內(nèi)ProBDNF及其受體P75NTR和Sortilin的表達變化及意義。 方法：大鼠隨機分為正常對照組和實驗組,實驗組大鼠左側(cè)足底皮下注射CFA和生理鹽水混合溶劑100μl,建立炎性疼痛模型,實驗組分為注射CFA后1d、7d和14d組。在建模前和建模后lh、6h及1d、3d、7d和14d采用Von Frey纖維檢測大鼠50%機械縮足閾值(paw withdrawal threshold, PWT)的變化；采用免疫組織化學及western blot方法觀察不同時間點ProBDNF及P75NTR、Sortilin在脊髓后角及DRG的表達變化。 結(jié)果： 1.足底注射CFA1h后50%PWT即下降,并在6h至1d左右達到最低值,3d后逐漸上調(diào),至14d仍低于基礎(chǔ)值。 2.足底注射CFA1d后,注射側(cè)脊髓后角ProBDNF表達較對側(cè)明顯上調(diào),這種上調(diào)持續(xù)至注射CFA后7d,CFA注射14d后,脊髓后角ProBDNF表達逐漸下降,但仍高于正常組,兩側(cè)表達差異不明顯。脊髓的western blot結(jié)果顯示,ProBDNF表達的明顯增高出現(xiàn)在炎性痛的后期即CFA7d時,且注射側(cè)表達明顯強于對側(cè)。DRG的免疫組化和western blot結(jié)果表明,注射CFA后1d即可導(dǎo)致注射側(cè)DRG內(nèi)ProBDNF的表達上調(diào),注射側(cè)表達強于對側(cè)。與注射CFA1d比,注射CFA后7d時ProBDNF的表達進一步上調(diào),且DRG內(nèi)表達ProBDNF小直徑神經(jīng)元數(shù)量逐漸減少,中、大直徑神經(jīng)元數(shù)量明顯增多。至注射CFA后14d,DRG內(nèi)ProBDNF的表達下調(diào)。 3.脊髓內(nèi)P75NTR的免疫組化和western blot結(jié)果表明,足底注射CFA1d時,P75NTR在脊髓后角的表達達高峰,兩側(cè)表達差異不明顯；至CFA7d,脊髓內(nèi)P75NTR的表達仍較強。注射CFA14d時P75NTR的表達較7d明顯下降。DRG內(nèi)P75NTR的免疫組化和Western blot結(jié)果顯示,在炎性痛早期即CFA1d時,P75NTR的表達即明顯增高,且兩側(cè)表達差異無顯著性；持續(xù)至CFA7d時,P75NTR的表達仍明顯增高,與CFA1d組比差異不明顯,至注射CFA14d時P75NTR的表達下降。 4. Sortilin在正常脊髓后角淺層僅有較弱陽性產(chǎn)物表達,注射CFA后不同時間點脊髓內(nèi)Sortilin的表達與正常對照組比差異不明顯。DRG內(nèi)Sortilin的表達量在炎性痛后不同時間點也無顯著變化,但注射側(cè)DRG內(nèi)表達Sortilin的小直徑神經(jīng)元數(shù)量減少,中直徑神經(jīng)元數(shù)量增多,這與proBDNF的表達模式類似。 結(jié)論： 足底注射CFA能誘導(dǎo)大鼠產(chǎn)生為期2周以上的炎性痛病程；炎性痛時脊髓后角和DRG內(nèi)ProBDNF及P75NTR的表達上調(diào)可能參與了外周痛覺信號的傳導(dǎo)和中樞敏化的形成。DRG內(nèi)表達Sortilin的細胞類型變換可能與proBDNF及BDNF的運輸及釋放有關(guān)。 第二部分炎性痛致大鼠脊髓及背根節(jié)內(nèi)BDNF基因不同外顯子的表達變化 目的：探討完全弗氏佐劑(Complete Freund's Adjuvant, CFA)致炎性疼痛后大鼠脊髓和DRG內(nèi)BDNF總的mRNA及包含不同BDNF外顯子1mRNA的表達變化及意義,從而揭示炎性痛不同時期、不同部位BDNF基因表達調(diào)節(jié)的差異性機制。 方法：大鼠隨機分為正常組和實驗組,實驗組大鼠左側(cè)足底皮下注射CFA和生理鹽水混合溶劑100ul,建立炎性疼痛模型,實驗組包括注射CFA后1d、7d和14d組。采用普通PCR以及Realtime-PCR方法檢測BDNF總的mRNA及BDNF不同外顯子(exon Ⅰ, exon ⅡA, exon ⅡB, exon ⅡC, exon Ⅲ, exon Ⅳ和exon ⅨA)在脊髓和DRG內(nèi)的表達變化。 結(jié)果： 1.足底注射CFA1d后,注射側(cè)脊髓內(nèi)BDNF總的mRNA的表達明顯升高,注射側(cè)強于對側(cè)；上調(diào)持續(xù)至注射CFA后7d左右達高峰,對側(cè)表達也有增高,但注射側(cè)表達仍強于對側(cè)；至14d時,BDNF mRNA表達下調(diào),但仍強于正常對照組。注射側(cè)DRG內(nèi)BDNF mRNA的表達在注射CFA1d后明顯升高,注射側(cè)強于對側(cè)；上調(diào)持續(xù)至注射CFA后7d；至14d時,BDNF mRNA表達下調(diào),與正常對照組相比差異沒有顯著性。 2.足底注射CFA1d時,exon Ⅰ在注射側(cè)脊髓的表達明顯上調(diào),與對側(cè)相比有明顯差異；exon ⅡA和exon ⅨA在對側(cè)脊髓表達有上調(diào)。至注射CFA7d時,注射側(cè)exon ⅡA、exon ⅡB和exon ⅨA的表達較1d時明顯增高,并明顯高于對側(cè)。注射CFA14d時,脊髓兩側(cè)exon ⅡB的表達仍高于正常組,對側(cè)脊髓內(nèi)exon ⅡA和exon ⅨA的表達有上調(diào)。 3.足底注射CFA1d時,注射側(cè)DRG內(nèi)BDNF基因exon Ⅰ、exon ⅡA、exon ⅡB和exon Ⅳ的表達明顯上調(diào),與對側(cè)相比有明顯差異。至注射CFA7d時,exon Ⅰ的表達較CFA1d時進一步增高,且明顯高于對側(cè),其他外顯子的表達無明顯變化。至注射CFA后14d時,各外顯子的表達與正常對照組比差異均不明顯。 結(jié)論： 在炎性痛的不同時期、不同部位BDNF外顯子出現(xiàn)差異性的表達變化,BDNF外顯子的變化與總的BDNF mRNA的變化趨勢基本相符。 第三部分炎性痛致大鼠脊髓后角乙�；M蛋白3的表達變化及嗎啡的干預(yù)影響 目的：探討完全弗氏佐劑(Complete Freund's Adjuvant, CFA)致大鼠炎性疼痛后脊髓后角乙�；M蛋白3(Acetylated Histone3,ACH3)水平的表達變化及意義,進一步說明組蛋白乙酰化在炎性疼痛產(chǎn)生和發(fā)展過程中發(fā)揮的作用以及嗎啡干預(yù)對其的影響。 方法：大鼠左側(cè)足底皮下注射CFA和生理鹽水混合溶劑100ul,建立炎性疼痛模型。大鼠隨機分為正常組和實驗組(注射CFA后1d、7d組),后部分實驗增加CFA1d+嗎啡干預(yù)組。采用免疫組織化學和免疫熒光化學方法觀察不同組脊髓后角ACH3的表達變化以及ACH3與神經(jīng)元、星形膠質(zhì)細胞和小膠質(zhì)細胞的雙標情況；采用Von Frey纖維檢測嗎啡對炎性痛早期大鼠50%PWT的變化,并觀察嗎啡干預(yù)對炎性痛早期脊髓后角ACH3及BDNF表達的影響。 結(jié)果： 1.免疫組織化學結(jié)果顯示,注射CFA后1d,脊髓后角ACH3的表達明顯下調(diào),與正常大鼠相比差異顯著；至注射CFA后7d,ACH3表達水平逐漸恢復(fù)。免疫熒光雙標結(jié)果表明,正常大鼠脊髓后角ACH3的表達主要在神經(jīng)元,部分在膠質(zhì)細胞。注射CFA1d后,ACH3的下調(diào)主要發(fā)生在神經(jīng)元；而至CFA7d時表達ACH3的神經(jīng)元數(shù)量明顯增加,且表達ACH3的星形膠質(zhì)細胞和小膠質(zhì)細胞數(shù)量也增加。 2.腹腔注射嗎啡(10mg/kg)可明顯增高大鼠CFA1d時的50%PWT。嗎啡可抑制炎性痛早期脊髓后角ACH3的下調(diào),其作用在于增加了表達ACH3的神經(jīng)元及膠質(zhì)細胞的數(shù)量,尤其是星形膠質(zhì)細胞和小膠質(zhì)細胞。 3.正常大鼠脊髓后角可見較弱BDNF表達,注射CFA1d后注射側(cè)脊髓后角BDNF的表達明顯上調(diào),且注射側(cè)表達強于對側(cè)；嗎啡干預(yù)對脊髓后角BDNF的表達無明顯影響。 結(jié)論： CFA誘導(dǎo)的炎性痛可導(dǎo)致脊髓后角短暫的ACH3下調(diào),這種下調(diào)可被嗎啡逆轉(zhuǎn)。在炎性痛的發(fā)生發(fā)展過程中,ACH3的表達變化有細胞類型的轉(zhuǎn)換。嗎啡干預(yù)對炎性痛早期脊髓后角BDNF的表達無明顯影響。
[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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R363

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7 呂慎金;楊燕;;梅花鹿BDNF基因與行為性狀的相關(guān)分析[A];第十二次全國畜禽遺傳標記研討會論文集[C];2010年

8 王迎新;張向榮;張志s，

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