本文選題：甲醛炎性痛 + 膠質(zhì)細(xì)胞�。� 參考：《河北醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的:一氧化氮(nitric oxide,NO)是調(diào)節(jié)疼痛的重要介質(zhì),NO合酶(NO synthase,NOS)是生成NO的限速酶。已有證據(jù)表明,外周傷害性刺激可誘導(dǎo)脊髓NOS表達(dá)增多,產(chǎn)生大量NO參與疼痛的產(chǎn)生與維持。我室既往應(yīng)用免疫組化和NADPH-d等方法研究表明,甲醛炎性痛時(shí),脊髓后角nNOS和iNOS表達(dá)增多,進(jìn)一步證實(shí)了NO在外周炎性痛和痛過敏的產(chǎn)生和維持中的作用。但是,這些NOS陽性細(xì)胞的細(xì)胞構(gòu)成尚不清楚。 越來越多的證據(jù)表明,脊髓膠質(zhì)細(xì)胞的激活與病理性痛和痛覺過敏的產(chǎn)生和維持有密切關(guān)系。例如,可逆性膠質(zhì)細(xì)胞代謝抑制劑可以抑制許多疼痛模型中的自發(fā)痛和痛覺過敏的產(chǎn)生和維持。在許多類型的神經(jīng)病理性痛,如脊神經(jīng)損傷(冷凍,結(jié)扎)、坐骨神經(jīng)結(jié)扎等神經(jīng)病理性痛,以及甲醛、酵母多糖等引起的炎性痛過程中,均可見脊髓星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞增殖活化。這些增殖活化的膠質(zhì)細(xì)胞可釋放多種化學(xué)物質(zhì)和細(xì)胞因子促進(jìn)痛和痛覺過敏的產(chǎn)生和維持。由此可見,除神經(jīng)元以外,脊髓膠質(zhì)細(xì)胞可能也參與甲醛炎性痛時(shí)脊髓NOS表達(dá)的上調(diào)。 因此,本實(shí)驗(yàn)采用免疫組織化學(xué)雙重標(biāo)記GFAP(星形膠質(zhì)細(xì)胞特異性標(biāo)記蛋白)和iNOS、以及連續(xù)切片免疫組化對(duì)比觀察OX42(小膠質(zhì)細(xì)胞特異性標(biāo)記蛋白)和iNOS的表達(dá)等方法,觀察大鼠足底注射甲醛引起的急性炎性痛過程中,脊髓后角星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞在脊髓iNOS表達(dá)增多中的作用,以期進(jìn)一步闡明脊髓膠質(zhì)細(xì)胞和NO在外周炎癥性痛和痛過敏中的作用。 方法:將30只雄性Sprague-Dawley大鼠隨機(jī)分為生理鹽水組和甲醛組,分別足底注射生理鹽水和甲醛,每組按足底注射后取材時(shí)間的不同又分為1 h、24 h和48 h組(n=5)。各組于相應(yīng)時(shí)間點(diǎn)斷頭取腰5脊髓節(jié)段(L5),石蠟切片上行iNOS/GFAP免疫組化雙重染色。 另取30只大鼠隨機(jī)分為生理鹽水組和甲醛組,每組按足底注射后取材時(shí)間的不同分為1 h、1 d、3 d、7 d和14 d組。各組于相應(yīng)時(shí)間點(diǎn)灌注取材,冰凍連續(xù)切片,免疫組化對(duì)比觀察L5脊髓后角OX42和iNOS的表達(dá)。 對(duì)以上各組動(dòng)物均于取材前行相應(yīng)的行為學(xué)觀測(cè):1 h組動(dòng)物于取材前行痛反應(yīng)評(píng)分;其他組動(dòng)物于取材前測(cè)定熱輻射縮足反射潛伏期和機(jī)械縮足反射閾值。 結(jié)果:大鼠右后足底注射5%甲醛后,注射足出現(xiàn)典型的雙向性自發(fā)縮足反應(yīng),持續(xù)1 h以上。注射部位熱輻射縮足潛伏期延長和機(jī)械刺激縮足反射閾值升高,而非注射足相應(yīng)部位的熱輻射縮足潛伏期縮短和機(jī)械刺激縮足反射閾值降低。注射后甲醛后1 h,24 h和48 h,在注射足同側(cè)的L5脊髓后角和中央管周圍均可觀察到少量iNOS/GFAP雙標(biāo)細(xì)胞散在分布,各時(shí)間點(diǎn)之間無顯著差異。 連續(xù)切片免疫組化對(duì)比觀察發(fā)現(xiàn),足底注射甲醛后,L5脊髓后角iNOS與OX42陽性細(xì)胞從形態(tài)、分布、表達(dá)時(shí)相上均有顯著差異,并未呈現(xiàn)明顯一致的趨勢(shì),iNOS與OX42的表達(dá)沒有顯著的相關(guān)性。 結(jié)論:大鼠足底注射甲醛后,注射部位對(duì)熱和機(jī)械刺激感覺減退,而非注射足相應(yīng)部位出現(xiàn)痛覺過敏現(xiàn)象。足底注射甲醛誘導(dǎo)脊髓后角iNOS表達(dá)上調(diào)的過程中,脊髓膠質(zhì)細(xì)胞發(fā)揮一定的作用,但不起主要作用;脊髓后角表達(dá)上調(diào)的iNOS可能主要由神經(jīng)元所介導(dǎo)。
[Abstract]:Objective: nitric oxide (NO) is an important mediator for regulating pain, and NO synthase (NO synthase (NOS) is a speed limiting enzyme for NO. The evidence shows that peripheral nociceptive stimulation can induce the increase of NOS expression in the spinal cord and produce a large number of NO involved in the production and maintenance of pain. The increase in the expression of nNOS and iNOS in the posterior horn of the spinal cord further confirms the role of NO in the production and maintenance of peripheral inflammatory and painful hypersensitivity, however, the cell composition of these NOS positive cells is not yet clear.
There is growing evidence that the activation of spinal glial cells is closely related to the formation and maintenance of pathological pain and hyperalgesia. For example, reversible glial metabolic inhibitors can inhibit the production and maintenance of pain and hyperalgesia in many of the pain models. In many types of neuropathic pain, such as spinal nerve injury (spinal nerve injury). The proliferation and activation of astrocytes and microglia in the spinal cord can be seen during the process of inflammatory pain caused by the ligature of the sciatic nerve and the ligature of the sciatic nerve. These proliferating and activated glial cells can release a variety of chemicals and cell factors to promote the production and maintenance of pain and hyperalgesia. It can be seen that in addition to neurons, spinal glial cells may also participate in upregulation of NOS expression in spinal cord during formalin induced pain.
Therefore, the experimental immunohistochemical double labeling GFAP (astrocyte specific labeling protein) and iNOS, and the continuous slice immunohistochemical staining were used to observe the expression of OX42 (microglia specific marker protein) and iNOS, and to observe the acute inflammatory pain during the acute inflammatory pain caused by the injection of formaldehyde in the foot of the rat, and the posterior horns of the spinal cord. The role of glial cells and microglia in the increase of iNOS expression in the spinal cord is expected to further elucidate the role of spinal glial cells and NO in peripheral inflammatory pain and pain allergy.
Methods: 30 male Sprague-Dawley rats were randomly divided into normal saline group and formaldehyde group. The normal saline and formaldehyde were injected into the plantar. Each group was divided into 1 h, 24 h and 48 h group (n=5) after the plantar injection. Each group was divided into 5 spinal segments at the corresponding time point (L5), and the paraffin section was performed by iNOS/GFAP immunohistochemistry. Dyed.
Another 30 rats were randomly divided into normal saline group and formaldehyde group. Each group was divided into 1 h, 1 D, 3 D, 7 d and 14 d groups according to the plantar injection time. Each group was perfused at corresponding time points, frozen continuous slices and immunohistochemical staining was used to observe the expression of OX42 and iNOS in the posterior horn of spinal cord by immunohistochemistry.
The animals in all of the above groups were observed before they were taken. 1 h groups were used to score the pain response before taking wood. The other animals measured the latent period of radiation contraction reflex and the threshold of mechanical contraction reflex before taking wood.
Results: after the injection of 5% formaldehyde in the right posterior foot of the rat, the injection foot had a typical two-way spontaneous contraction reaction, lasting more than 1 h. The latent period of heat radiation contraction in the injection site and the increase of the reflex threshold of mechanical stimulation were increased, but the latent period of heat radiation contraction in the corresponding part of the foot of the injection foot was shortened and the threshold of the mechanical stimulation contraction reflex was reduced. After 1 h, 24 h and 48 h after formalin, a small amount of iNOS/GFAP double standard cells scattered around the posterior horn of the L5 spinal cord and around the central canal were observed, and there was no significant difference between each time point.
The contrast observation of continuous slice immunohistochemical staining showed that after the injection of formaldehyde in the plantar, there were significant differences in the morphology, distribution and expression of iNOS and OX42 positive cells in the posterior horn of the L5 spinal cord, and did not show a significant consistent trend. There was no significant correlation between the expression of iNOS and OX42.
Conclusion: after the injection of formaldehyde in the foot of the rat, the sensation of heat and mechanical stimulation is reduced, and the hyperalgesia occurs in the corresponding part of the foot. The spinal glial cells play certain roles in the process of inducing the up-regulated expression of iNOS in the posterior horn of the spinal cord by the injection of formaldehyde. The expression of iNOS in the posterior horn of the spinal cord may be up-regulated. It is mainly mediated by neurons.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 葛偉,陳軍,李源,陳會(huì)生;老齡大鼠對(duì)外周不同化學(xué)組織損傷的痛行為反應(yīng)特點(diǎn)[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2002年05期

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