Foxo3a和p27kip1在大鼠坐骨神经损伤后背根神经节中的表达

Objective To investigate the expression of Foxo3a and p27kip1 in lumbar dorsal root ganglia (DRG) after injury of sciatic nerve in rats. Methods Adult rats were randomly divided into control group and experimental group. The rats in experimental group were subjected to sciatic nerve clamp.Expression and distribution of Foxo3a and p27kip1 and cellular proliferation and axon regeneration in DRG was detected by western blot and immunohistochemistry. Results Foxo3a protein levels begined to reduce at 1 day (7.0 ± 3.5), reached valley at 2 day (6.0 ± 3.8) after injury, and following Foxo3a downregulation, p27kip1 protein levels begined to decrease at 2 day (29.0 ± 3.5), reached valley at 7 day (21.0± 3.0) after injury. Down-regulation of Foxo3a and p27kip1 was expressed predominantly in neurons and glial cells by double immunolabelling. Foxo3a and p27kip1 were expressed in neurons [(37.8 ± 5.7)%, (43.3 ±4.3)%] and glial [(22.4 ± 3.9)%, (13.8 ± 3.2)%] cells in sections of DRG at 2 day after injury less than neurons [(73.6 ± 2.5)%, (84.1 ± 3.7)%] and glial [(61.3 ± 4.4)%, (68.7 ± 5.6)%] cells in sections of normal DRG. Proliferating cell nuclear antigen (PCNA) and GAP-43 were up-regulation from 2 day, and PCNA reached peak at 7 day after injury.The glial cells were the main type of cellular proliferation.Conclusion Down-regulation of Foxo3a and p27kip1 in lumbar DRG is correlated with the proliferation of glial cells and axonal regeneration after sciatic nerve injury.     

Foxo3a和p27kip1在大鼠坐骨神经损伤后背根神经节中的表达

Objective To investigate the expression of Foxo3a and p27kip1 in lumbar dorsal root ganglia (DRG) after injury of sciatic nerve in rats. Methods Adult rats were randomly divided into control group and experimental group. The rats in experimental group were subjected to sciatic nerve clamp.Expression and distribution of Foxo3a and p27kip1 and cellular proliferation and axon regeneration in DRG was detected by western blot and immunohistochemistry. Results Foxo3a protein levels begined to reduce at 1 day (7.0 ± 3.5), reached valley at 2 day (6.0 ± 3.8) after injury, and following Foxo3a downregulation, p27kip1 protein levels begined to decrease at 2 day (29.0 ± 3.5), reached valley at 7 day (21.0± 3.0) after injury. Down-regulation of Foxo3a and p27kip1 was expressed predominantly in neurons and glial cells by double immunolabelling. Foxo3a and p27kip1 were expressed in neurons [(37.8 ± 5.7)%, (43.3 ±4.3)%] and glial [(22.4 ± 3.9)%, (13.8 ± 3.2)%] cells in sections of DRG at 2 day after injury less than neurons [(73.6 ± 2.5)%, (84.1 ± 3.7)%] and glial [(61.3 ± 4.4)%, (68.7 ± 5.6)%] cells in sections of normal DRG. Proliferating cell nuclear antigen (PCNA) and GAP-43 were up-regulation from 2 day, and PCNA reached peak at 7 day after injury.The glial cells were the main type of cellular proliferation.Conclusion Down-regulation of Foxo3a and p27kip1 in lumbar DRG is correlated with the proliferation of glial cells and axonal regeneration after sciatic nerve injury.     

胞浆泛素蛋白连接酶2在大鼠脊髓损伤后星形胶质细胞中的表达及意义

目的研究胞浆泛素蛋白连接酶2(Kip1 ubiquitylation-promoting complex 2,KPC2)在大鼠脊髓损伤(spinal cord injury,SCI)过程中的蛋白表达及细胞定位情况,探讨其在SCI修复过程中的生物学功能。方法将成年SD大鼠随机分为2组,对照组7只仅行单纯T9椎板全切除术,实验组49只采用改良Allen法制作T9节段脊髓撞击损伤模型,实验组于伤后6、12 h及1、3、5、7、14 d分别取7只大鼠进行以下检测。采用Western blot检测p27kip1、KPC2、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和细胞周期蛋白A(Cyclin A)在SCI前后的蛋白表达变化,免疫组织化学染色观察KPC2在SCI后的大体定位及表达,免疫荧光双标记染色观察KPC2在SCI过程中与神经元特异性核蛋白(neuronal nuclei,Neu N)、神经胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和PCNA的共定位情况。细胞水平采用体外培养星形胶质细胞增殖模型,Western blot检测KPC2、P27kip1、PCNA表达;免疫共沉淀分析KPC2、KPC1和p27kip1之间在细胞增殖过程中的相互作用。结果 Western blot结果示SCI后3 d,p27kip1显著下调,伴随KPC2、Cyclin A、PCNA表达明显增加。免疫组织化学染色示KPC2阳性信号广泛分布,包括脊髓灰质和白质,实验组KPC2阳性细胞数显著高于对照组(t=10.982,P=0.000)。免疫荧光双标记染色示在脊髓灰质,对照组和实验组KPC2与Neu N双标记阳性细胞数分别为(0.43±0.53)、(0.57±0.53)个/视野,比较差异无统计学意义(t=0.548,P=0.604);在脊髓白质,对照组和实验组KPC2与GFAP双标记阳性细胞数分别为(3.86±0.90)、(0.71±0.49)个/视野,差异有统计学意义(t=7.778,P=0.000);对照组和实验组KPC2与PCNA标记的星形胶质细胞共定位明显,阳性细胞数分别为(0.57±0.53)、(5.57±1.13)个/视野,差异有统计学意义(t=8.101,P=0.000)。体外培养并模拟星形胶质细胞增殖,提取细胞蛋白行Western blot示PCNA和KPC2蛋白表达在血清刺激增殖后即开始增加,24 h达峰值,而p27kip1表达则逐渐减少。免疫共沉淀示KPC2可沉淀p27kip1、KPC1,p27kip1也可沉淀KPC2、KPC1,且在刺激后相互作用明显增加。结论 SCI后KPC2参与介导的p27kip1表达下调,KPC2与SCI后星形胶质细胞的增殖相关。     

大鼠后足切割后脊髓和背根神经节中脑源性神经营养因子表达的变化

目的 观察大鼠后足切割后脊髓和背根神经节( DRG)中脑源性神经营养因子(BDNF)水平的变化及其定位.方法 雄性SD大鼠24只,体重150～250 g,均施行后足切割手术.术后1h、6h、1d、3d各取6只大鼠行行为学检测后处死,分离脊髓和DRG进行免疫组化和双标免疫荧光检测BDNF.结果 大鼠后足切割后,切割侧腰段脊髓背角和DRG中BDNF水平上升(P＜0.05).而对侧腰段和胸段脊髓中BDNF水平均没有明显改变.升高的BDNF主要定位于神经元而非星形胶质细胞或小胶质细胞.结论 大鼠后足切割后切割侧腰段脊髓背角和DRG中BDNF表达上升,BDNF升高主要定位于神经元.     

p27Kip1蛋白在胃腺癌中的表达及基与肿瘤细胞增殖的关系

目的 探讨细胞周期素依赖性激酶抑制蛋白27(p27kip1)在胃腺癌中的表达及其对肿瘤细胞增殖的影响和临床意义.方法 采用免疫组织化学SP法检测100例胃腺癌组织中p27kip1蛋白的表达,并与20例正常胃黏膜组织作对照,同时应用流式细胞仪对胃腺癌S期细胞比(SPF)进行检测.结果 正常胃黏膜组织中p27kip1蛋白高表达率明显高于胃腺癌组(X2=15.45,P＜0.01).在高/中分化组,黏膜层/黏膜下层组和无淋巴结转移的病例中,p27kip1蛋白高表达率分别为65.31%、80.00%、73.53%;SPF值分别为21.6±4.2、20.3±3.9、20.1±4.0.但随着胃癌分化程度的降低,侵及肌层/浆膜层或发生淋巴结转移,p27kip1蛋白高表达率明显降低(41.18%、48.24%、42.42%)(X2值分别为5.84、5.16、8.72,P＜0.05或P＜0.01);而SPF值明显升高(25.9±5.1、24:4±4.8、25.7±5.1)(t值分别为4.592、3.127、5.575,P均＜0.01).p27kip1蛋白高表达与SPF值呈显著负相关(t=3.478,P＜0.01).结论 p27kip1与胃腺癌发生发展密切相关.p27kip1蛋白表达降低和高的SPF值常提示胃腺癌的低分化、高侵袭转移能力,提示p27kip1和SPF值可作为临床判断胃腺癌预后的指标.     

大鼠坐骨神经损伤后许旺细胞源神经营养因子表达的实验研究

目的 观察坐骨神经在正常和损伤后许旺细胞源神经营养因子的表达。方法 将75只SD大鼠坐骨神经地夹伤、切断、切断加缝合等,术后第5、7、14、30、60d取材,做常规免疫组织化学研究。结果 （1）正常坐骨神经和脊髓中许细胞源神经营养因子有较弱表达;（2）切断组坐骨神经近段术后第14d许旺细胞源神经营养因子表达最高,远段第7d最高,脊髓术后第5d最高;切断缝合组和夹伤组神经和脊髓部以术后第60d最高;（3）损伤侧灰质后角表达强于对照侧。结论 (1)许旺细胞源神经营养因子在正常神经和脊髓中有较弱表达;(2) 旺细胞源神经营养因子在坐骨神经不同损伤情况下表达不同,但都高于正常组;（3）许旺细胞对神经损伤后修复作用可能通过许旺细胞源神经营养因子实现。     

不同类型坐骨神经损伤对相应脊髓节段基因表达的影响

目的：比较坐骨神经夹伤与坐骨神经结扎后神经元胞体基因表达的差异,探讨外周神经损伤后再生与否与基因表达差异的关系。方法：根据坐骨神经损伤基因表达谱分析的结果,选择部分表达变化的基因;通过RT-PCR的相对定量的方法检测其在坐骨神经夹伤与坐骨神经结扎后相应脊髓节段的表达水平。结果：比较两种模型差异表达的基因类别可以发现,可再生神经元胞体上调表达的基因与组织细胞的增殖(PCNA)、生长(FRAGl,NCAM)及抗凋亡(Bcl-2)有关;而不可再生的神经元胞体这些基因的表达下调。结论：外周神经元胞体对不同类型神经损伤的反应存在差异,这种差异可能与神经再生能力的不同有关。     

氯胺酮对CCI大鼠背根神经节GAP-43 mRNA表达的影响

目的 观察氯胺酮对坐骨神经慢性压迫损伤(CCI)模型大鼠背根神经节(DRG)生长相关蛋白(GAP-43) mRNA表达的影响.方法 36只成年雄性SD大鼠.体重180～220g,随机均分为三组.氯胺酮组(K组)和CCI组(C组)均采用CCI模型,假手术组(S组)仅暴露坐骨神经,不结扎.K组大鼠于CCI后3～14d每天腹腔注射氯胺酮10mg/kg,C组和S组腹腔注射等容量生理盐水.在术前(To)、术后3d(T1)、7d(T2)、14d(T3)时测定机械痛阈和热痛阈值;T1～T3时测定痛阈后,每组随机取4只大鼠取腰段脊髓背根神经节(DRG)检测GAP-43 mRNA的水平.结果 与S组比较,K组和C组术后机械痛阈和热痛阈降低,GAP-43 mRNA表达升高(P<0.05);与NP组比较,T2、T3时K组机械痛阈和热痛阈升高,GAP-43 mRNA表达降低(P<0.05).结论 腹腔注射氯胺酮10mg/kg可减轻CCI大鼠疼痛,其机制可能与抑制DRG中GAP-43 mRNA表达升高有关.     

坐骨神经预损伤后miR-124-3p调节GAP-43表达并促进轴突生长的实验研究

 目的 通过研究坐骨神经预损伤后背根神经节中microRNA表达谱变化,探讨坐骨神经预损伤促进脊髓后索损伤修复机制。方法 利用微阵列芯片技术和生物信息学方法,研究与坐骨神经预损伤促进脊髓后索损伤修复有关的microRNA,并用RT-qPCR技术、Western blot技术、免疫荧光染色、反义miRNA寡核苷酸抑制剂等技术验证。结果 单纯脊髓后索损伤组miR-124-3p在大鼠脊髓后索损伤后7 d、14 d明显上调;坐骨神经预损伤组,脊髓后索损伤后7 d、14 d背根神经节中miR-124-3p明显下调。与正常背根神经节神经元相比,抑制miR-124-3p后GAP-43表达增加,神经元轴突延长。与对照组相比,各组各时间窗STAT3 mRNA表达差异无统计学意义,坐骨神经预损伤组STAT3蛋白在脊髓后索损伤后7 d和14 d表达上调而单纯脊髓后索损伤组脊髓后索损伤后7 d和14 d STAT3蛋白表达下调。与正常背根神经节神经元相比抑制miR-124-3p的神经元STAT3免疫荧光增强、轴突延长,p-STAT3、GAP-43蛋白表达明显上调。与正常背根神经节神经元相比AG490+AMO-124共同处理的神经元轴突长度无明显差异,STAT3表达明显上调,而p- STAT3和GAP-43表达无明显差异。结论 背根神经节神经元中miR-124-3p下调通过STAT3蛋白的上调促使GAP-43蛋白表达增加是坐骨神经预损伤促进脊髓后索损伤修复的机制之一。     

Skp2、p27kip1在大肠癌中的表达及其临床意义

目的研究Skp2、p27kip1在大肠癌组织中的表达,探讨它们之间的关系及其临床意义。方法采用免疫组化方法检测Skp2、p27kip1蛋白在83例大肠癌、18例大肠腺瘤和20例大肠正常黏膜中的表达。结果大肠癌组织中Skp2蛋白阳性率(28.65±12.60)%,显著高于大肠腺瘤组织(17.28±10.66)%(P<0.01)和大肠正常黏膜组织(6.60±5.54)%(P<0.01)。Skp2蛋白表达与细胞分化程度、肿瘤分期及淋巴结转移呈正相关(P<0.01)。大肠癌组织中p27kip1蛋白阳性率(24.61±11.27)%,显著低于大肠腺瘤组织(49.94±13.22)%(P<0.01)和大肠正常黏膜组织(65.40±15.74)%(P<0.01)。p27kip1蛋白表达与细胞分化程度、肿瘤分期及淋巴结转移呈负相关(P<0.01)。大肠癌组织中Skp2与p27kip1表达呈负相关(r=-0.430,P<0.01)。结论大肠癌中Skp2蛋白的过度表达与p27kip1蛋白降解有关,提示Skp2蛋白可能在大肠癌的发生发展中起重要作用。     

神经病理性疼痛大鼠背根神经节神经元中小白蛋白的分布特征及表达变化

目的 观察神经病理性疼痛大鼠背根神经节(DRG)神经元中小白蛋白(PV)的分布特征及表达变化.方法 成年雄性SD大鼠42只,随机分为空白对照组(Control组)、假手术组(Sham组)、CCI术后1、3、5、7、14 d组,6只/组.所有动物隔日进行热、机械痛觉过敏痛觉阈值测定,行为学测试完成后取出术侧L5 DRG作冰冻切片,用免疫荧光双标技术经PV单克隆抗体和MAP-2多克隆抗体检测PV神经元的分布特征和表达变化规律.结果 (1)与Control组和Sham组比较,CCI组大鼠分别于术后1、3 d出现明显的机械、热痛觉过敏(P＜0.05),术后7 d 50%机械缩爪阈值(50%PWT)和热缩爪潜伏期(PWL)均降至最低(P＜0.01),术后14 d有所恢复,但仍低于术前水平.(2)在正常大鼠L5DRG中仅有少量PV阳性神经元,多以大型、中型神经元为主,少数为小型神经元.PV阳性神经纤维交织成网环绕在PV阴性神经元的胞体周围.(3)手术侧PV阳性神经元数目在CCI 1、3 d未见明显改变.在术后5、7 d明显减少(P＜0.05),术后14 d恢复至正常水平(P＞0.05).结论 CCI模型大鼠DRG中PV的表达水平呈时间依赖性改变,这种改变与疼痛行为学变化在时相上基本保持一致,因此推测外周感觉神经元中PV表达下降可能参与神经病理性疼痛的发展过程.     

Primary sensory neurons with dichotomizing axons projecting to the facet joint and the sciatic nerve in rats.

STUDY DESIGN: Dorsal root ganglion (DRG) neurons that have dichotomizing axons to the lumbar facet joint and to the sciatic nerve were investigated in rats using a double fluorescent labeling technique. OBJECTIVES: To clarify the existence of DRG neurons with dichotomizing axons projecting to the lumbar facet joint and to the sciatic nerve in rats. SUMMARY OF BACKGROUND DATA: DRG neurons having dichotomizing axons have been reported in several species and are considered to be related to referred pain. However, such DRG neurons have not been investigated in the lumbar spine. Clinically, pain from the lumbar facet joint is sometimes referred to the lower extremities innervated by the sciatic nerve. METHODS: Two kinds of neurotracers (DiI and FG) were used in the present double-labeling study. DiI crystals were placed in the left L5-L6 facet joint, and FG was applied to the ipsilateral sciatic nerve or along the midline of the L5 dermatome. Bilateral DRGs T13-S1 were observed by fluorescence microscope. RESULTS: DRG neurons double labeled with DiI and FG were recognized only in the ipsilateral DRGs from L3 to L6 levels. Approximately 3% of DRG neurons innervating the L5-L6 facet joint had other axons to the sciatic nerve. By contrast, no double-labeled neurons were observed after FG was applied to the L5 dermatome. CONCLUSIONS: In rats approximately 3% of DRG neurons innervating the lumbar facet joints have dichotomized axons projecting to the sciatic nerve.     

The time-dependent difference of GAP-43 expression between sensory neurons and motoneurons after peripheral nerve transection.

The L5 dorsal root ganglion (DRG) cells and L5 anterior horn (AH) cells of rats were studied and examined immunocytochemically after transection of the sciatic nerve to find out whether there would be time-dependent differences in the increase of growth-associated protein (GAP-43) expression between sensory neurons and motoneurons. On the seventh day after transection at mid-thigh level, the levels of GAP-43 in the DRG cells significantly increased, while those in the AH cells began to increase gradually from the 14th day onward. Transection at the piriform muscle level induced a significant increase in immunoreactivity of GAP-43 on the third day in the DRG cells, and on the seventh day in the AH cells. These results showed that sensory neurons expressed GAP-43 immunoreactivity earlier than motoneurons after peripheral nerve transection regardless of the site, suggesting that sensory neurons might start to produce cytoskeletons for axonal elongation earlier than motoneurons after nerve transection.     

THE TIME-DEPENDENT DIFFERENCE OF GAP-43 EXPRESSION BETWEEN SENSORY NEURONS AND MOTONEURONS AFTER PERIPHERAL NERVE TRANSECTION

The L5 dorsal root ganglion (DRG) cells and L5 anterior horn (AH) cells of rats were studied and examined immunocytochemically after transection of the sciatic nerve to find out whether there would be time-dependent differences in the increase of growth-associated protein (GAP-43) expression between sensory neurons and motoneurons. On the seventh day after transection at mid-thigh level, the levels of GAP-43 in the DRG cells significantly increased, while those in the AH cells began to increase gradually from the 14th day onward. Transection at the piriform muscle level induced a significant increase in immunoreactivity of GAP-43 on the third day in the DRG cells, and on the seventh day in the AH cells. These results showed that sensory neurons expressed GAP-43 immunoreactivity earlier than motoneurons after peripheral nerve transection regardless of the site, suggesting that sensory neurons might start to produce cytoskeletons for axonal elongation earlier than motoneurons after nerve transection.     

环化酶激活剂对大鼠感觉神经元保护作用的实验研究

目的探讨环化酶激活剂（前列腺素E1、腺苷及Zn^2＋离子）对周围神经损伤后感觉神经元的保护作用.方法在大鼠坐骨神经夹毁模型术后21 d,取背根节用图像分析法观测前列腺素E1（PGE1）、腺苷（Ade）和高Zn^2＋饲料（Zn^2＋）对背根节细胞数、核偏心率及核等圆径的影响,并与人胎盘神经生长因子（hNGF）的作用相比较.结果NGF及环化酶激活剂组与夹毁对照组相比都能增进背根节细胞的存活和减轻核偏移（P＜0.05）;NGF组和高Zn^2＋饲料组在背根节细胞存活数、核偏心率及核等圆径方面与假手术组无差别（P＞0.05）.结论PEG1、Ade及Zn^2＋离子等环化酶激活剂对DRG神经元均有保护作用,其中Zn^2＋离子的保护效应与NGF无差别.     

神经病理性痛大鼠背根神经节JNK的表达

目的 探讨神经病理性痛大鼠背根神经节C-Jun氨基末端蛋白激酶(JNK)的表达.方法 雄性SD大鼠88只,体重200～250 g.随机分为2组(n=44):假手术组(SH组)和慢性压迫性损伤组(CCI组).结扎大鼠右侧坐骨神经建立CCI模型.各组取8只大鼠,于CCI前、CCI后1、3、5、7、14 d时,测定热刺激缩足反射潜伏期(TWL).各组于CCI前、CCI后1、3、5、7、14 d时,各取6只大鼠测定背根神经节磷酸化JNK(p-JNK)的表达.结果 与CCI前比较,CCI组CCI后各时点TWL降低,p-JNK表达增加(P＜0.01);与SH组比较,CCI组CCI后3、5、7、14 d时TWL降低,p-JNK表达增加(P＜0.01).结论 背根神经节JNK表达升高可能参与了大鼠神经病理性痛的形成.     

咪唑立宾对大鼠肾小球系膜细胞增殖的抑制作用

目的探讨咪唑立宾（MZ）对肾小球系膜细胞增殖的抑制作用及其对细胞周期蛋白依赖性蛋白激酶抑制物p27^kip1表达的影响。方法大鼠系膜细胞同步后分为无血清组、20％FCS刺激组、20％FCS＋MZ组。四甲基偶氮唑盐（MTT）法检测细胞数。BrdU标记法检测S期细胞。流式细胞仪检测细胞周期。Western印迹检测p27^kip1蛋白的表达。结果MZ可抑制20％FCS刺激所致的系膜细胞增殖，呈剂量依赖性。与20％FCS刺激组比较，20％FCS＋MZ组S期细胞的百分比显著降低[（10．28±1．26）％比（21．04±5．38）％，P〈0．05]。MZ可改善由20％FCS刺激诱导的GO／G1期细胞减少[（83．53±2．50）％比（71．15±1．25）％]和S期细胞增加[（12．22±1．22）％比（23．19±0．38）％，P〈0．051。20％FCS刺激后p27^kip1蛋白表达显著下降，MZ上调p27^kip1蛋白表达。结论MZ能够有效抑制系膜细胞增殖，作用时相在G1／S期转化，其抑制系膜细胞增殖的作用部分是通过上调p27^kip1蛋白表达实现的。     

复方中药对神经损伤后感觉神经元超微结构变化的研究

目的 研究复方中药对大鼠坐骨神经损伤后感觉神经元超微结构的变化。方法 雄性ＳＤ大鼠１２只,根据术后用药的不同随机分成中药组与对照组,每组６只大鼠。选择左侧坐骨神经切断加近端神经双重结扎的模型。于术后７、１４、２８ｄ取Ｌ５背根神经节,按常规方法制成切片后,在电镜下观察感觉神经元的变化。结果 对照组的背根神经节感觉神经元,其超微结构随着时间的推移,线粒体肿胀,及线粒体嵴、基质丢失。细胞核逐渐变小,核质