Real-Time PCR检测Bcl-2和Bax基因在脊髓钝挫伤大鼠中的表达

目的使用Real-Time PCR检测脊髓钝挫伤后不同时间点Bcl-2和Bax基因表达变化。方法 SD成年雌性大鼠72只,随机分为假手术组、脊髓挫伤6h组、脊髓挫伤12h组、脊髓挫伤1d组、脊髓挫伤3d组、脊髓挫伤5d组、脊髓挫伤7d组、脊髓挫伤14d组和脊髓挫伤28d组。大鼠于术后相应各时间点取损伤段脊髓进行RealTime PCR,对基因Bcl-2和Bax表达进行定量分析。结果 Real-Time PCR发现假手术组Bax和Bcl-2基因mRNA水平较高,损伤后均明显下降。Bax基因mRNA在损伤后6h立即下降,12h时明显上升(P<0.01),在损伤后3d又呈现显著性下降(P<0.01),此后一直持续这个水平至28d。Bcl-2基因在损伤后6h也明显下降(P<0.01),12h时立即上升(P<0.01),1d时下降(P<0.01),至损伤后14d又显著性上升(P<0.01)。结论脊髓钝挫伤后大鼠Bax和Bcl-2基因mRNA表达水平于早期显著下降,之后明显上升随后又下降。Bax和Bcl-2基因在脊髓损伤后基因的表达变化为脊髓损伤机制研究提供理论依据。     

白细胞介素3基因疗法和白细胞介素6基因疗法的联合造血调控作用

研究了成纤维细胞介导的ＩＬ－３基因疗法，ＩＬ－６基因疗法以两者联合后对造血系统的影响。结果发现，单用ＩＬ－基因疗法的小鼠白细胞总数，中性粒细胞，骨髓ＣＦＵ－ＧＭ，ＣＦＵ－ＭＫ等显著上升，但血小板上升程度经，单用ＩＬ－６基因疗法的小鼠血小板，中性粒细胞，骨髓ＣＦＵ－ＧＭ，ＣＦＵ－ＭＫ上晚为显著。     

黄精多糖干预骨质疏松性骨折大鼠白细胞介素1和白细胞介素6的表达

背景：骨质疏松性骨折主要是由于雌激素减少引发的高转换型骨质疏松并发症,给予雌激素能够得到有效地纠正。 目的：观察黄精多糖对骨质疏松性骨折大鼠细的胞因子的影响。 方法：6月龄雌性SD大鼠行卵巢切除建立骨质疏松症大鼠模型90 d后,锯断胫骨建立骨质疏松性骨折模型。骨折1周后,分别以400,200,100 mg/kg黄精多糖灌胃和0.066 mg/kg雌二醇肌肉注射给药治疗。 结果与结论：相比于一般性骨折大鼠,骨质疏松性骨折大鼠血清白细胞介素1,6的表达升高;400 mg/kg黄精多糖或雌激素干预治疗后,大鼠血清的白细胞介素1,6的表达明显降低(P < 0.05),但200和100 mg/kg黄精多糖对大鼠血清的白细胞介素1,6的表达没有影响(P > 0.05),提示黄精多糖干预治疗骨质疏松性骨折需要较高浓度     

BYHWD对脊髓损伤大鼠脊髓组织Bcl-2和Bax表达的影响

目的:观察补阳还五汤(buyanghuanwu decoction,BYHWD)对脊髓损伤(spinal cord injury,SCI)模型大鼠脊髓神经细胞凋亡相关蛋白Bcl-2、Bax及其mRNA表达的影响,初步探讨BYHWD抑制脊髓神经细胞凋亡的作用机制。方法:Wistar大鼠40只,随机分为四组:正常对照组、SCI模型组、BYHWD组和甲基强的松龙(MP)注射组,每组10只大鼠;后三组动物先建立SCI损伤模型,然后分别给予BYHWD组和MP组BYHWD和MP治疗。四组动物分别于术后1、7、14、21 d和28 d对大鼠后肢神经功能的恢复情况进行BBB(Basso-Beattie-Bresnahan)功能评分。第28 d处死各组大鼠,采用Real-time PCR法检测脊髓组织Bcl-2和Bax mRNA的表达情况,Western Blot法检测脊髓组织Bcl-2和Bax蛋白的表达情况。结果:大鼠麻醉清醒后,SCI组大鼠损伤平面以下完全瘫痪。BBB评分结果显示:术后第7 d~28 d,与正常对照组相比,SCI组BBB评分明显降低(P0.05);与SCI组相比,BYHWD处理组和MP处理组大鼠BBB评分明显增高(P0.05),但BYHWD处理组和MP处理组大鼠相比,BBB评分无明显差异(P0.05)。Real-time PCR和Western Blot结果显示:与正常对照组相比,SCI组Bax蛋白及mRNA的表达明显升高(P0.05),而Bcl-2蛋白及mRNA的表达则明显有所降低(P0.05)。与SCI组相比,BYHWD处理组和MP处理组大鼠脊髓组织Bax蛋白及mRNA表达明显降低(P0.05),而Bcl-2蛋白及mRNA表达则明显有所升高(P0.05)。但BYHWD处理组和MP处理组大鼠脊髓组织Bcl-2、Bax蛋白及mRNA表达之间无明显差异(P0.05)。结论:BYHWD可以改善SCI大鼠后肢的运动功能,通过下调Bax的表达,上调Bcl-2的表达,从而抑制SCI大鼠脊髓神经细胞的凋亡,发挥其保护作用。     

一种快速、简便、敏感、特异的白细胞介素2、3、6生物学检测法：MTS／PMS法的建立

ＭＴＴ的一种新型类似物ＭＴＳ在ＰＭＳ存在情况下可被活细胞还原，形成水溶性甲产物。本实验表明，将ＭＴＳ／ＰＭＳ取代ＭＴＴ用于ＩＬ－２、３、６生物学测定法取得理想结果，ＭＴＳ／ＰＭ５形成的比色产物与细胞数量、ＭＴＳ／ＰＭＳ与细胞孵育时间呈正相关。ＭＴＳ／ＰＭＳ生物学测定法省略了加有机溶剂溶解甲的步骤，ＭＴＳ／ＰＭ５贮存稳定、显色快速，故该法具有快速、简便敏感、特异的优点，适用于细胞因子的生物学测定。     

利塞膦酸钠干预骨质疏松模型大鼠髁突软骨Bcl-2、Bax及Caspase3的表达

BACKGROUND:Bisphosphonates are a kind of traditional antiresorptive drugs, which may influence the growth and development of cartilage tissue. In recent years, there are many data describing how bisphosphonates affect joint cartilage of long bone, but their effects on condylar cartilage remain unclear. OBJECTIVE:To clarify the effects of the third-generation bisphosphonate risedonate on the expression of Bcl-2 (anti-apoptotic), Bax (pro-apoptotic) and caspase-3 in the condylar cartilage of osteoporosis rats. METHODS:Thirty female Sprague-Dawley rats were randomly divided into three groups. In the sham operation group, ovary was exposed but not resected. The model group received a bilateral ovariectomy to establish the models of osteoporosis, and then received saline solution treatment (2.4 μg/kg) subcutaneously every 3 days, once a day, from 3 days pre-surgery. The treatment group received a bilateral ovariectomy to establish the models of osteoporosis, and then received risedronate treatment (2.4 μg/kg) subcutaneously every 3 days from 3 days pre-surgery. Three months after the operation, the condylar cartilages of all animals were harvested. Apoptosis, the expression of Bcl-2, BAX and Caspase3 were observed. RESULTS AND CONCLUSION:(1) The number of apoptotic cells in rat condylar cartilage and subcondylar region: the sham operation group < the treatment group < the model group (all P < 0.05). (2) Expression of Bcl-2: The trend of the model group was lower than that in the sham operation group, although there was no statistically significant difference between the two groups; Bcl-2 expression in the treatment group was statistically higher compared to the model group (P < 0.05). (3) Expression of Bax and Caspase-3: The expression levels of Bax and Caspase-3 were higher in the model group than in the sham operation group (all P < 0.05), while Bax and Caspase-3 expression was lower in the treatment group than that in the model group (all P < 0.05). The results suggested that bisphophonates can regulate apoptosis in condylar cartilage from osteoporosis rats by changing the expression of Bcl-2, Bax and Caspase-3.     

雌激素干预去势骨质疏松症大鼠血清白细胞介素8、10的表达

背景：绝经后妇女骨质疏松症患病率高,雌激素能否通过调控细胞因子表达防治骨质疏松？ 目的：观察雌激素对血清细胞因子白细胞介素8、白细胞介素10表达水平影响,以及骨密度与细胞因子表达水平的相关性。 方法：健康雌性SD大鼠30只随机数字表法均分为正常对照组、模型组、雌二醇组。正常对照组行开关术,其余两组行双侧卵巢切除。造模后第3周正常对照组和模型组大鼠每天灌服生理盐水1.5 mL/只;雌二醇组给予戊酸雌二醇1 mg/(kg•d),以1.5 mL生理盐水混悬液形式灌服,共12周。双能X射线骨密度仪测量股骨和胫骨骨密度;ELISA方法定量测定血清白细胞介素8、白细胞介素10及雌二醇表达水平。 结果与结论：模型组和雌二醇组两组血清雌二醇表达水平较正常对照组明显降低,差异有显著性意义  (P < 0.05),表明大鼠去势手术制作成功。模型组血清白细胞介素8表达水平明显高于正常对照组和雌二醇组(P < 0.01),血清白细胞介素10表达水平明显低于两组(P < 0.01);正常对照组血清白细胞介素10表达水平与雌二醇组比较,差异无显著性意义(P > 0.05)。股骨骨密度和胫骨骨密度与血清白细胞介素8表达水平均呈显著负相关(P < 0.001),而与血清白细胞介素10表达水平均呈显著正相关(P < 0.05)。雌激素能够下调白细胞介素8的表达水平,上调白细胞介素10的表达水平。提示雌激素通过调节细胞因子表达水平可能成为防治绝经后妇女骨质疏松症的又一作用机制。     

白细胞介素-6与2型糖尿病发生的研究进展

白细胞介素 - 6 (IL - 6 )是一种多功能的细胞因子。 IL - 6的升高可通过激活急性相反应影响 2型糖尿病的发病 ,并可通过与胰岛素、瘦素的相互作用与 2型糖尿病的发生相关联     

白细胞介素-6受体在大鼠小脑间位核中的表达

我们以往的研究表明小脑皮层颗粒细胞表达白细胞介素-6受体(interleukin-6 receptor,IL-6R),该受体介导了白细胞介素-6(interleukin-6,IL-6)的神经保护作用。然而,大鼠小脑核团是否也表达IL-6R目前尚不清楚。为此,本研究应用RT-PCR、Western blot和免疫组织化学方法分别在基因、蛋白和细胞水平检测IL-6R在大鼠小脑间位核的表达。结果显示:(1)在小脑间位核PCR扩增产物的凝胶电泳图上,在365 bp位置可见一清晰的条带,与IL-6R mRNA预计扩增片断的长度一致;(2)在West-ern blot实验体系中,IL-6R免疫反应阳性条带出现在分子量为80 kD处;(3)免疫组织化学结果显示,小脑间位核中出现大量IL-6R免疫反应阳性细胞。以上结果表明:小脑间位核细胞在mRNA和蛋白水平均表达IL-6R,提示IL-6在小脑间位核内可能具有重要作用。     

电针刺激脊髓损伤大鼠足阳明胃经脊髓受损节段Bax、Bcl-2的mRNA和蛋白的表达

文题释义： 脊髓损伤大鼠模型：用于研究脊髓损伤而制作的大鼠模型,良好的动物模型是实验成功的重要基础,经过不断的研究发展,其造模方法也多种多样,包括钳夹法、打击法、半横切法、完全横切法等,各种方法在稳定性及死亡率方面各有优劣,研究者应当根据自身需求选择合适的造模方式。 细胞凋亡：是细胞的一种基本生物学现象,在多细胞生物去除不需要的或异常的细胞中起着必要的作用。凋亡是多基因严格控制的过程,当脊髓损伤发生时,Bcl-2家族、caspase家族等开始发生调控作用。 背景：中医自《内经》开始就提出了“治痿独取阳明”之理论,但是有关电针干预足阳明胃经治疗脊髓损伤的报道相对较少,该课题研究从细胞凋亡因子Bax、Bcl-2的角度出发,初步探讨电针干预足阳明胃经对脊髓损伤神经修复的相关机制。目的：探讨电针刺激足阳明胃经对脊髓损伤大鼠受损节段Bax、Bcl-2表达的影响。方法：将64只SPF级SD雌性大鼠随机分为4组：对照组、预标记组、损伤后标记组和电针组,每组16只。对照组及预标记组在操作前给予Brdu(5 mg/kg)连续10 d腹腔注射,以标记脊髓内保持分化活力的细胞,对照组于第11天行T₁₀节段椎板切除,预标记组于第11天采用钳夹法造成脊髓损伤缺血模型;损伤后标记组、电针组在制作脊髓损伤模型后连续10 d腹腔注射Brdu(5 mg/kg)标记损伤后活化增殖的细胞,电针组于造模后第3天开始电针干预足阳明胃经足三里、伏兔穴,每周5次。造模后3,10,17,24 d(电针0,7,14,21 d),每个时间点取4只大鼠,采用BBB评分评估运动功能恢复情况,qRT-PCR和Western blot检测脊髓组织Bax、Bcl-2 mRNA及蛋白的表达。结果与结论：①术后3-24 d时,预标记组、损伤后标记组、电针组大鼠神经功能评分低于对照组,差异均有显著性意义(P < 0.05),电针组神经功能评分高于预标记组、损伤后标记组,差异均有显著性意义(P < 0.05);②随着脊髓损伤时间的延长,Bax mRNA及蛋白表达先升高后降低,Bcl-2 mRNA及蛋白表达先降低后升高,Bax/Bcl-2比值随着电针干预时间的推移逐渐减少;③结果表明,电针刺激足阳明胃经可以通过调节Bcl-2、Bax的表达减少神经细胞凋亡,促进脊髓损伤大鼠运动功能的恢复。 ORCID: 0000-0001-6069-4997(魏卫兵) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

急性不完全脊髓损伤模型大鼠相关炎症因子的表达

背景：随着现代交通和工矿事业的发展,脊髓损伤成为临床常见的多发病,严重的影响了人类身心健康和生活质量。 目的：检测在大鼠急性脊髓损伤模型中磷酸化信号转导与转录激活因子3（p-STAT3）介导的IL-1β、IL-6、IL-17等炎症因子的表达变化情况,探讨IL-1β、IL-6、IL-17等炎症因子在急性脊髓损伤后炎症反应过程中的作用。 方法：雄性成年SD大鼠75只,随机分为对照组和脊髓损伤1h、6h、24h和72h四个亚组（n=15只）。采用改良Allen氏法建立脊髓损伤模型,对照组仅行椎板全切除术。在建模之后相应的时间点获取损伤段脊髓、脾脏组织,免疫组织化学染色法检测IL-6和IL-17在损伤段脊髓组织中的分布及表达情况,蛋白免疫印迹法检测p-STAT3在损伤段脊髓组织中的表达变化,采用RT-PCR法检测IL-1βmRNA、IL-6mRNA和IL-17AmRNA在脾脏组织中的表达情况。 结果与结论：脊髓损伤后p-STAT3、IL-1β、IL-6和IL-17的表达明显高于对照组（P＜0.05）。损伤后炎症因子表达量立即升高,IL-1β和IL-6在损伤后6h小时到达高峰,随后开始下降;p-STAT3和IL-17在损伤后24h到达高峰,随后开始下降,至损伤后72h表达量仍高于对照组。损伤早期p-STAT3介导的促炎因子IL-1β和IL-6表达增加,可能导致损伤区域炎症级联放大,诱导继发性脊髓损伤;促炎因子IL-17的异常升高,可能在继发性损伤炎症反应中起着重要作用。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

GDNF和HSV-GDNF对培养的大鼠脊髓运动神经元缺氧后的Bcl-2表达的影响

目的:观察细胞胶质源性神经营养因子(Glial cell line-derived neurotrophic factor,GDNF)和单纯疱疹病毒介导的GDNF(GDNF transformed by herpes simplex virus vector,HSV-GDNF)对体外培养的大鼠脊髓运动神经元缺氧-复氧时的作用和Bcl-2表达的影响。方法:观察缺氧2h、4h和缺氧4h后恢复供氧24、72h时,脊髓运动神经元存活数,并用抗Bcl-2抗血清行免疫组织化学染色,对Bcl-2免疫反应阳性神经元作平均光密度分析。结果:经GDNf、HSV-GDNF孵育的脊髓运动神经元缺氧-复氧后Bcl-2表达较对照组明显增强,神经元损伤程度减轻,神经元存活数明显高于对照组,且HSV-GDNF组的效果更好。结论:GDNF、HSV-GDNF对缺氧-复氧的大鼠脊髓运动神经元有保护作用,HSV-GDNF比GDNF更能增强缺氧-复氧后脊髓运动神经元Bcl-2的表达,提高神经元存活数,抑制缺氧后神经元的死亡。     

Effects of interleukin-1 and dexamethasone on interleukin-6 production and growth in human meningiomas

Interleukin-6 (IL-6) has been shown to be released by cultured human meningioma cells and may be a positive or negative regulator of tumour growth. IL-6 protein and mRNA levels have been examined in a series of meningiomas. In 14 cases, the results are compared with the effects of IL-6 and dexamethasone on growth and IL-6 secretion in vitro. Tumours with the highest in vivo IL-6 mRNA expression also showed maximum induction of IL-6 and increased cellular proliferation on IL-1 stimulation in vitro. Dexamethasone decreased the IL-1-stimulated IL-6 release in all cases. Meningiomas which had little or no IL-6 message were refractory to IL-1 control of IL-6. Remarkably, these formed the group of meningiomas that increased their growth rate in response to dexamethasone. © 1997 John Wiley & Sons, Ltd.     

雌激素对大鼠心内神经节免疫因子白细胞介素-6表达的影响

目的 观察白介素-6(IL-6)在成年雌性大鼠、卵巢切除大鼠、卵巢切除后雌二醇替代大鼠心内神经节的表达及变化.方法 免疫组织化学方法.结果 各组大鼠心内神经节均存在IL-6阳性神经元,但卵巢切除组大鼠心内神经节IL-6阳性神经元明显增多,表达明显增强.结论 心房后壁心内神经节存在IL-6,并且受雌激素影响,提示雌二醇可能影响心内神经节IL-6的表达.     

大鼠脊髓半横断损伤后脊髓神经细胞凋亡与caspase-3的表达

为研究损伤脊髓神经细胞凋亡和相联系的caspase-3的表达,用原位末端标记法(TUNEL)检测了成年大鼠胸脊髓(T9)半横断损伤(hSCI)后脊髓神经细胞的凋亡变化,并用免疫组织化学染色方法观察了caspase-3的表达。结果显示:hSCI后6h损伤侧脊髓腹角即出现较多的TUNEL阳性神经细胞,并持续至5周。损伤脊髓的吻侧段于手术后4d达高峰,尾侧段脊髓于伤后1d达高峰。与假手术组相比,在所有观察的时间点TUNEL阳性神经细胞的数量在损伤侧均有明显增加。caspase-3免疫组化染色显示,与假手术组相比,伤后6h损伤侧脊髓腹角caspase-3表达也明显增加,并持续至5周。以上结果提示hSCI后,脊髓可出现神经细胞凋亡,且此凋亡的发生可能与caspase-3的表达有关。     

Bcl-2和Bax蛋白在人胚胎早期脊髓发育中的表达及意义

为探讨人胚胎发育早期脊髓内神经细胞增殖与凋亡的变化规律及其相关蛋白Bcl-2和Bax在脊髓中的分布规律及其表达意义,本研究应用免疫组织化学ABC法检测了第2、3、4月胎龄段的人胚胎脊髓组织中央管、前角和后角处Bcl-2和Bax蛋白的表达状况。结果显示:在第2、3、4月胎龄段,脊髓中央管、前角和后角处均有Bcl-2和Bax蛋白阳性表达。本文结果提示,Bcl-2和Bax蛋白在人胚胎脊髓的生长发育过程中起重要的调节作用。     

脊髓压迫性损伤大鼠海马和脊髓内BDNF的表达变化

目的探讨脑源性神经营养因子(BDNF)在脊髓压迫性损伤后的表达变化及作用。方法用自行设计的方法制作脊髓压迫模型,免疫荧光检测BDNF在星形胶质细胞、神经元和上下行轴突的表达;WB检测BDNF在大鼠海马及脊髓的表达。结果随着时间延长,受损部位的BDNF+-GFAP荧光强度逐渐增强;BDNF+-Tuj1荧光强度逐渐减弱;受损部位相邻上下段的BDNF+-NF200比受损部位的明显增强。海马和脊髓受损中心相邻的上、下段的BDNF蛋白表达在损伤后1d达到峰值,然后逐渐下降(P0.05);而脊髓受损中心的BDNF蛋白表达逐渐下降(P0.05)。结论脊髓损伤后,BDNF的表达下调,随伤后时间呈一定规律变化,是引起受损部位神经元表达下降的原因之一。     

Combination of dexamethasone and etanercept reduces secondary damage in experimental spinal cord trauma

The aim of our study was to evaluate the therapeutic efficacy of combination therapy with etanercept and dexamethasone (DEX) in vivo in experimental murine model of spinal cord trauma, which was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and cytokine production followed by recruitment of other inflammatory cells, production of inflammation mediators, tissue damage, apoptosis and disease. Treatment of the mice with etanercept (1.25 mg/kg) and DEX (0.025 mg/kg) when administered as a combination therapy but not as a single treatment significantly reduced the degree of (1) spinal cord inflammation and tissue injury (histological score), (2) infiltration of neutrophils (MPO evaluation), (3) inducible nitric oxide synthase, nitrotyrosine, and cytokines expression (tumor necrosis factor-alpha and interleukin-1 beta), (4) and apoptosis (Terminal deoxynucleotidyltransferase-mediated UTP end labeling staining, Fas-ligand expression and Bax and Bcl-2 expression). In a separate set of experiments we have also clearly demonstrated that the combination therapy significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate for the first time that strategies targeting multiple proinflammatory pathways may be more effective than a single effector molecule for the treatment of spinal cord trauma.     

Effects of dantrolene on apoptosis and immunohistochemical expression of NeuN in the spinal cord after traumatic injury in rats

Dantrolene has been shown to be neuroprotective by reducing neuronal apoptosis after brain injury in several animal models of neurological disorders. In this study, we investigated the effects of dantrolene on experimental spinal cord injury (SCI). Forty-six male Wistar rats were laminectomized at T13 and divided in six groups: GI (n = 7) underwent SCI with placebo and was euthanized after 32 h; GII (n = 7) underwent laminectomy alone with placebo and was euthanized after 32 h; GIII (n = 8) underwent SCI with dantrolene and was euthanized after 32 h; GIV (n = 8) underwent SCI with placebo and was euthanized after 8 days; GV (n = 8) underwent laminectomy alone with placebo and was euthanized after 8 days; and GVI (n = 8) underwent SCI with dantrolene and was euthanized after 8 days. A compressive trauma was performed to induce SCI. After euthanasia, the spinal cord was evaluated using light microscopy, TUNEL staining and immunochemistry with anti-Caspase-3 and anti-NeuN. Animals treated with dantrolene showed a smaller number of TUNEL-positive and caspase-3-positive cells and a larger number of NeuN-positive neurons, both at 32 h and 8 days (P ≤ 0.05). These results showed that dantrolene protects spinal cord tissue after traumatic SCI by decreasing apoptotic cell death.