【摘要】：目的黃體酮(又稱孕酮)是一種類固醇激素,可以在雌性動物的卵巢和胎盤合成,常用于婦產(chǎn)科學(xué)。近幾年研究表明,神經(jīng)系統(tǒng)也可以生成黃體酮,如神經(jīng)膠質(zhì)細(xì)胞,主要是少突膠質(zhì)和星形膠質(zhì)。且有研究表明,在神經(jīng)系統(tǒng)中,黃體酮通過調(diào)節(jié)基因的轉(zhuǎn)錄、細(xì)胞內(nèi)的信號通路和神經(jīng)傳導(dǎo)而發(fā)揮了多效性。近年來有少量文獻(xiàn)報道,黃體酮可以減輕CCI(Chronic constrictive injury)誘導(dǎo)的疼痛行為,并且可以修復(fù)外周的運動及感覺神經(jīng)纖維上電生理的變化,提示黃體酮對于神經(jīng)痛具有治療作用,但其具體機(jī)制并不清楚。本實驗擬用L5脊神經(jīng)結(jié)扎(Spinal Nerve Ligation,SNL)造成的神經(jīng)病理性疼痛模型大鼠,應(yīng)用行為學(xué)和免疫熒光染色法分別檢測孕酮對疼痛行為的影響及對小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞活化的影響,并用ELISA蛋白測定方法對腰骶膨大處脊髓和L5神經(jīng)節(jié)中COX-2(Cyclooxygenase-2)和i NOS(inducible nitric oxide synthase)蛋白表達(dá)的影響,以探討黃體酮的鎮(zhèn)痛機(jī)制。方法實驗一:建造脊神經(jīng)結(jié)扎模型。術(shù)后第3天開始測量大鼠術(shù)側(cè)和對側(cè)的機(jī)械縮足閾值,直到術(shù)后第21天,以檢測大鼠疼痛閾值的經(jīng)時變化過程。取無痛覺過敏和痛覺遲鈍的正常雄性Sprague-Dawley大鼠24只,將其隨機(jī)的分為三組:正常組、假手術(shù)組和手術(shù)組。用von Frey細(xì)絲測定實驗大鼠機(jī)械縮足閾值,以50%的縮足閾值(50%paw withdrawal threshold,50%PWT)表示,繪制模型大鼠的時間-疼痛行為變化曲線。實驗二:黃體酮對大鼠機(jī)械縮足閾值的影響。取健康成年雄性SD大鼠32只,隨機(jī)分為4組:正常組、空白對照組和黃體酮低劑量(8 mg/kg)及高劑量組(16mg/kg)。正常組大鼠不做任何處理;空白對照組大鼠于SNL手術(shù)當(dāng)天開始,皮下注射助溶劑即22.5%環(huán)糊精溶液(注射容積:3ml/kg體重),正常組亦皮下注射22.5%環(huán)糊精溶液,每天一次,連續(xù)21天;實驗組大鼠分別皮下注射低劑量(8 mg/kg/d)和高劑量(16mg/kg/d)的黃體酮。各組大鼠分別于SNL術(shù)前6天開始測量,運用von Frey細(xì)絲檢測大鼠后足的機(jī)械縮足閾值,每天測定一次,直到術(shù)后的第21天。升降法測定大鼠50%機(jī)械性收縮閾值。實驗三:黃體酮對大鼠膠質(zhì)細(xì)胞激活的影響。分別于給藥后3天、7天、14天及21天,行為學(xué)測定后灌流,取脊髓腰骶膨大處,做冰凍切片,對星膠質(zhì)細(xì)胞的標(biāo)志物GFAP(glial fibrillary acidic protein)以及小膠質(zhì)細(xì)胞的標(biāo)志物OX-42進(jìn)行免疫染色,在共聚焦顯微鏡下觀察其表達(dá)變化。實驗四:黃體酮對大鼠腰骶膨大處脊髓和L5神經(jīng)節(jié)內(nèi)COX-2和i NOS蛋白表達(dá)的影響。取健康成年雄性SD大鼠24只,隨機(jī)分為3組:正常組、空白對照組及黃體酮組(16 mg/kg)。正常組大鼠不做任何處理;空白對照組大鼠于SNL術(shù)后開始,皮下注射助溶劑即22.5%環(huán)糊精溶液(注射容積:3ml/kg體重),每天一次,連續(xù)21天;實驗組大鼠皮下注射高劑量(16mg/kg)的黃體酮,每天一次,連續(xù)21天;分別于3天、7天、14天及21天給藥1h后灌流,取脊髓腰骶膨大處和術(shù)側(cè)L5神經(jīng)節(jié),用ELISA試劑盒測定腰骶膨大處脊髓和L5神經(jīng)節(jié)中COX-2和i NOS蛋白表達(dá)量。結(jié)果1.SNL脊神經(jīng)結(jié)扎后機(jī)械性疼痛閾值經(jīng)時變化曲線SNL術(shù)后第3天開始縮足閾值顯著降低,此后縮足閾值基本維持在5g以下。SNL術(shù)后第14天開始,機(jī)械縮足閾值開始逐漸上升,直到第21天,但和假手術(shù)組相比,術(shù)側(cè)機(jī)械縮足閾值依然很低(P0.05),從SNL術(shù)后的第3天開始直到第13天,試驗大鼠的縮足閾值維持在一個較穩(wěn)定的水平(P0.01)。2.SNL脊神經(jīng)結(jié)扎后連續(xù)皮下注射黃體酮對機(jī)械縮足閾值的影響皮下注射黃體酮顯著提高了術(shù)側(cè)后足的機(jī)械縮足閾值。正常組、空白對照組、黃體酮低劑量組(8 mg/kg)和高劑量組(16 mg/kg)的術(shù)側(cè)(左側(cè))機(jī)械縮足閾值術(shù)前分別是14.29±1.63g、14.41±1.01g、14.27±1.02g和14.49±0.95g;給藥3天后分別為14.74±0.73、3.84±1.09g、4.47±0.96g和4.59±1.47g;給藥7天后分別為14.49±0.95、3.47±1.78g、4.76±1.72g和5.41±0.76g;給藥14天后分別為14.32±1.30、4.39±0.61g、5.48±0.54g和7.20±0.81g;給藥21天后分別為14.33±0.93g、5.37±0.98g、8.55±1.10g、11.65±1.83g。結(jié)果表明,術(shù)后第14天和第21天,黃體酮高劑量組(16 mg/kg)、低劑量組(8 mg/kg)和空白對照組相比,機(jī)械縮足閾值顯著增加(分別是P0.05,P0.01),且黃體酮高劑量組(16 mg/kg)和黃體酮低劑量組(8 mg/kg)對比,機(jī)械縮足閾值顯著增加(P0.01)。黃體酮對手術(shù)對側(cè)的痛閾無顯著影響。3.SNL脊神經(jīng)結(jié)扎后連續(xù)皮下注射黃體酮對OX-42表達(dá)的影響免疫染色的結(jié)果表明,脊神經(jīng)結(jié)扎后會使脊髓中的小膠質(zhì)細(xì)胞活化。小膠質(zhì)細(xì)胞激活后,由靜息狀態(tài)的分枝狀分化成活化狀態(tài)的阿米巴狀。SNL術(shù)后小膠質(zhì)細(xì)胞的標(biāo)志物OX-42免疫染色強(qiáng)度經(jīng)過統(tǒng)計分析后表明:正常組中OX-42的活化量很低,處于基線水平。與空白對照組相比,黃體酮組第3天的術(shù)側(cè)光密度值及第7天的術(shù)側(cè)光密度值(IOD)無統(tǒng)計學(xué)意義(P㧐0.05)。與空白對照組相比,黃體酮組第14天術(shù)側(cè)光密度值(IOD)和第21天的術(shù)側(cè)光密度值(IOD)具有統(tǒng)計學(xué)意義(均為P0.01)。黃體酮抑制了SNL后小膠質(zhì)細(xì)胞激活。4.SNL脊神經(jīng)結(jié)扎后連續(xù)皮下注射黃體酮對GFAP表達(dá)的影響免疫染色的結(jié)果表明,脊神經(jīng)結(jié)扎后使脊髓中的星膠質(zhì)細(xì)胞活化。星膠質(zhì)活化后,形態(tài)及數(shù)量會發(fā)生一定的改變,表現(xiàn)為增生、肥大,星膠質(zhì)細(xì)胞的標(biāo)志物GFAP免疫染色的強(qiáng)度在經(jīng)過統(tǒng)計分析后表明,正常組中GFAP的表達(dá)量很低,位于基線水平。與空白對照組相比,黃體酮組第3天的術(shù)側(cè)光密度值和第7天的術(shù)側(cè)光密度值(IOD)無統(tǒng)計學(xué)意義(P㧐0.05)。與空白對照組相比,黃體酮組第14天術(shù)側(cè)光密度值(IOD)和第21天的術(shù)側(cè)光密度值(IOD)具有統(tǒng)計學(xué)意義(均為P0.01)。黃體酮抑制了SNL后星膠質(zhì)細(xì)胞激活。5.SNL脊神經(jīng)結(jié)扎后連續(xù)皮下注射黃體酮對COX-2以及i NOS兩種酶表達(dá)的影響。連續(xù)的皮下注射黃體酮顯著的降低了脊髓背角和神經(jīng)節(jié)中COX-2和i NOS兩種蛋白的表達(dá)量。COX-2和i NOS蛋白在正常組大鼠的腰骶膨大處脊髓背角和L5神經(jīng)節(jié)中的表達(dá)量很低,其平均含量分別為11.24±1.71ng/L、7.71±1.29 ng/L和0.80±0.09μmol/L、0.58±0.11μmol/L。SNL術(shù)后兩種蛋白的濃度急劇升高,COX-2和i NOS蛋白在空白對照組中大鼠的腰骶膨大處脊髓背角和L5神經(jīng)節(jié)中的平均表達(dá)量分別為84.10±8.62 ng/L、88.85±6.91 ng/L和7.40±0.59μmol/L、7.58±0.60μmol/L。連續(xù)皮下注射黃體酮第3天和第7天,與空白對照組相比,COX-2和i NOS兩種蛋白的表達(dá)量沒有統(tǒng)計學(xué)意義(P㧐0.05)。黃體酮治療組中,COX-2第3天在腰骶膨大處脊髓背角和L5神經(jīng)節(jié)中的表達(dá)量分別為84.59±9.23ng/L、87.56±8.26 ng/L,i NOS第3天在腰骶膨大處脊髓背角和L5神經(jīng)節(jié)中的表達(dá)量分別為7.45±0.87μmol/L、7.52±1.25μmol/L;COX-2第7天在腰骶膨大處脊髓背角和L5神經(jīng)節(jié)中的表達(dá)量分別為80.36±5.96 ng/L、85.29±8.41 ng/L,i NOS第7天在腰骶膨大處脊髓背角和L5神經(jīng)節(jié)中的表達(dá)量分別為7.14±0.47μmol/L、7.20±0.91μmol/L;與空白對照組對比,連續(xù)用藥14天和21天后,兩種蛋白表達(dá)量具有統(tǒng)計學(xué)意義(均為P0.01)。黃體酮治療組中,COX-2第14天在腰骶膨大處脊髓背角和L5神經(jīng)節(jié)中的表達(dá)量分別為58.84±5.44 ng/L、61.21±5.43 ng/L,i NOS第14天在腰骶膨大處脊髓背角和L5神經(jīng)節(jié)中的表達(dá)量分別為5.17±0.82μmol/L、5.23±0.58μmol/L;COX-2第21天在腰骶膨大處脊髓背角和L5神經(jīng)節(jié)中的表達(dá)量分別為32.77±5.07 ng/L、31.78±8.37 ng/L,i NOS第21天在腰骶膨大處脊髓背角和L5神經(jīng)節(jié)中的表達(dá)量分別為2.42±0.29μmol/L、2.40±0.36μmol/L。結(jié)論黃體酮可劑量依賴性地抑制大鼠機(jī)械性疼痛行為,機(jī)制可能與抑制膠質(zhì)細(xì)胞的激活以及降低了腰骶膨大處脊髓背角和L5神經(jīng)節(jié)中COX-2和i NOS兩種蛋白的表達(dá)有關(guān)。
[Abstract]:OBJECTIVE Progesterone (progesterone) is a steroid hormone that can be synthesized in the ovaries and placentas of female animals and is often used in obstetrics and gynecology. Recent studies have shown that the nervous system can also produce progesterone, such as glial cells, mainly oligodendrocytes and astrocytes. Gene transcription, intracellular signaling pathways, and nerve conduction play a multipotent role. In recent years, a small number of literatures have reported that progesterone can alleviate pain behavior induced by CCI, and can repair electrophysiological changes in peripheral motor and sensory nerve fibers, suggesting that progesterone has therapeutic effects on neuralgia. The effect of progesterone on pain behavior and the activation of microglia and astrocytes in rats with neuropathic pain induced by L5 spinal nerve ligation (SNL) was studied by behavioral and immunofluorescence staining. Methods To investigate the effects of progesterone on the expression of COX-2 and inducible nitric oxide synthase in the lumbosacral enlarged spinal cord and L5 ganglion, and to explore the analgesic mechanism of progesterone. On the 21st day, 24 normal male Sprague-Dawley rats were randomly divided into three groups: normal group, sham operation group and operation group. Experiment 2: Effect of progesterone on the threshold of mechanical foot contraction in rats. 32 healthy adult male SD rats were randomly divided into 4 groups: normal group, blank control group, low dose progesterone group (8 mg/kg) and high dose progesterone group (16 mg/kg). Rats in the control group were subcutaneously injected with 22.5% cyclodextrin solution (volume: 3ml / kg body weight) for 21 days. Rats in the control group were subcutaneously injected with 22.5% cyclodextrin solution once a day for 21 consecutive days. Rats in the experimental group were subcutaneously injected with low dose (8mg / kg / d) and high dose (16mg / kg / d) progesterone. At the beginning of the day, von Frey filament was used to detect the mechanical shrinkage threshold of the hind foot of rats, once a day until the 21st day after operation. The mechanical shrinkage threshold of 50% was measured by ascending and descending method. Experiment 3: The effect of progesterone on the activation of glial cells in rats. Immunostaining of GFAP (glial fibrillary acidic protein) and OX-42 (microglia marker) was performed in the lumbosacral enlargement area. The expression of COX-2 and I NOS protein in the spinal cord and L5 ganglion of rats with lumbosacral enlargement was observed under confocal microscope. Twenty-four healthy adult male SD rats were randomly divided into three groups: normal group, blank control group and progesterone group (16 mg/kg). Rats in normal group did not receive any treatment; rats in blank control group were subcutaneously injected with 22.5% cyclodextrin solution (injection volume: 3 ml/kg body weight) once a day for 21 days after SNL operation; rats in experimental group were subcutaneously injected with progesterone for 21 days. High dose progesterone (16mg/kg) was injected once a day for 21 consecutive days, and then perfused at 3, 7, 14 and 21 days for 1 hour. The expression of COX-2 and I NOS in the lumbosacral enlargement and the L5 ganglion was measured by ELISA kit. After SNL operation, the threshold of mechanical foot contraction began to rise gradually from the 14th day to the 21st day, but compared with sham operation group, the threshold of mechanical foot contraction was still very low (P 0.05). From the 3rd day to the 13th day after SNL operation, the threshold of mechanical foot contraction began to rise gradually. Effects of continuous subcutaneous injection of progesterone after spinal nerve ligation on the threshold of mechanical foot contraction in rats (left side) mechanical constriction threshold of the left side (left side) was 14.29 [(1.63 g, 14.41 [(1.63 g, 14.41 [1.01 g, 14.41 [1.01 g, 14.27 [1.02g, 14.27 [1.02g and 14.49 [0.02g] and 14.49 [0.49 [0.73, 3.84 [1.09g, 4.84 [1.09g, 4.47 [0.96 g and 4.47 [0.47 [0.96g and 4.59 [4.59 [1.47] 1.47 [1.47g, 3.47 [1.78 g, 4.76 [1.72g and 5.76 [5.48 [0]. The results showed that on the 14th and 21st days after operation, the mechanical foot shrinkage thresholds of high dose progesterone group (16 mg/kg), low dose progesterone group (8 mg/kg) and blank control group were significantly increased (P 0.05, P 0.01, respectively). Compared with the low dose progesterone group (8 mg/kg), the mechanical foot shrinkage threshold increased significantly (P 0.01). Progesterone had no significant effect on the contralateral pain threshold. 3. The effect of continuous subcutaneous injection of progesterone after spinal nerve ligation on the expression of OX-42. The immunostaining results showed that spinal cord microglia were activated after spinal nerve ligation. OX-42 immunostaining intensity, a marker of microglia after SNL, was statistically analyzed. The activation of OX-42 in the normal group was very low at baseline level. Compared with the control group, the operative side optical density on day 3 and the operation on day 7 in the progesterone group were significantly lower. There was no significant difference in the lateral optical density (IOD) between the progesterone group and the control group (P? 0.05). Compared with the control group, the IOD of the progesterone group on the 14th day and the IOD of the 21th day were statistically significant (all P 0.01). Progesterone inhibited the activation of microglia after SNL. 4. Progesterone injected subcutaneously after spinal nerve ligation inhibited the expression of GFAP. The results of immunostaining showed that the astrocytes in spinal cord were activated after ligation of spinal nerve. After activation of astrocytes, the morphology and number of astrocytes changed to a certain extent, showing hyperplasia and hypertrophy. The intensity of GFAP immunostaining, a marker of astrocytes, showed that the expression of GFAP in the normal group was very low and located in the base. There was no significant difference between the progesterone group and the control group on the 3rd day and the 7th day (P? 0.05). Compared with the control group, the progesterone group had significant difference on the 14th day and the 21st day (both P 0.01). Progesterone inhibited SNL. Posterior astrocyte activation. 5. Effect of progesterone on the expression of COX-2 and I NOS after spinal nerve ligation. Progesterone significantly decreased the expression of COX-2 and I NOS proteins in spinal dorsal horn and ganglion after continuous subcutaneous injection. COX-2 and I NOS proteins in lumbosacral enlargement of normal rats. The expression levels of COX-2 and I NOS in spinal dorsal horn and L5 ganglion of rats in blank control group were 84.10% and 84.10% respectively. The expression of COX-2 and I NOS in spinal dorsal horn and L5 ganglion of lumbosacral enlargement in progesterone treatment group was not significantly different from that in control group (P The expression levels of I NOS in the spinal dorsal horn and L5 ganglion of lumbosacral enlargement on the 3rd day were 7.45 (+ 0.87) and 7.52 (+ 1.25) respectively. The expression levels of COX-2 in the spinal dorsal horn and L5 ganglion of lumbosacral enlargement on the 7th day were 80.36 (+ 5.96 ng/L), 85.29 (+ 8.41 ng/L) and 80.29 (+ 8.41 ng/L) respectively. The expression levels of COX-2 in spinal dorsal horn and L5 ganglion were 7.14 (+ 0.47) and 7.20 (+ 0.91) respectively. Compared with the control group, the expression levels of COX-2 in spinal dorsal horn and L5 ganglion on day 14 and 21 were statistically significant (both P 0.01). The expression levels of COX-2 in spinal dorsal horn and L5 ganglion of lumbosacral enlargement on the 14th day were 5.17 (+ 0.82) and 5.23 (+ 0.58) respectively. The expression levels of COX-2 in spinal dorsal horn and L5 ganglion of lumbosacral enlargement on the 21st day were 32.77 (+ 5.07) ng/L, 31.78 (+ 8.37) ng/L, and I NOS in lumbosacral enlargement on the 21st day. The expression levels of COX-2 and I NOS in spinal dorsal horn and L5 ganglion were 2.42.29 and 2.40.36 0.366550 Close.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R965

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