大鼠椎间盘累积损伤性炎症反应中大麻素水解酶——FAAH、NAAA表达及意义的初步研究

目的 本研究试图通过检测大麻素（CB）的两种主要水解酶——N-乙酰基乙醇胺水解酸性酰胺酶(NAAA)、脂肪酸酰胺水解酶(FAAH)在鼠累积损伤性椎间盘炎症反应过程中的表达来初步揭示其中大麻素的参与过程及作用机制。方法 清洁级Wistar大鼠随即分成实验组与对照组,根据Jill A. Ulrich制作方法准备实验组,以切皮后即缝合为对照组,在术后第3、7、10天,实验组与对照组每次各取6只,取出相应椎间盘,进行形态学观察,应用Real Time-PCR技术检测 FAAH、NAAA的表达水平。结果 与对照组相比,实验10d组可见刺伤所致纤维环断裂,髓核内出血。在椎间盘累积性损伤7d时, NAAA和FAAH表达含量分别约为对照组的3倍,并形成高峰;而在损伤10d后,其表达量呈减少趋势。结论 作为水解酶的底物CB类物质在第7天时候表达含量呈低谷,而随着时间的延长逐渐增高。CB类物质参与了炎症反应的发展过程,从而抑制骨骼椎间盘的退变。     

大麻素受体及其配体对骨量和骨改建的调节作用

内源性大麻素(CB)系统至少有2种受体,在体内均存在相应的配体和特定的合成及代谢通路,均属G蛋白偶联超家族成员.CB1受体主要分布在中枢神经系统,CB2受体主要分布于外周免疫细胞表面,两者受激动均可产生特定的生理反应.研究表明一些CB类物质可在体内抑制类风湿性关节炎的炎症病变.近来对CB受体基因敲除的小鼠研究显示,CB1受体、CB2受体及其配体,其激动剂可在体内外调节破骨细胞及成骨细胞的功能,从而对骨量和骨改建起调节作用.进一步研究其对骨细胞的作用机制,可为临床应用该类药物提供理论依据.     

内源性大麻素系统与麻醉相关炎症反应的研究进展

炎症是机体的一种防御反应,其主要目的为根除致病因子造成的机体损伤并且恢复受损组织功能.在某些炎症环境中,因致病因子毒性过强或致病因子存在时间过长等导致机体中的炎症因子和炎症介质代谢失衡,从而引起其他系统的疾病;如与麻醉相关的脓毒症和慢性病理性痛.最近研究显示,内源性大麻素系统的代谢与炎症的产生、维持和消退都有关联.本文...     

大麻素受体和配体对胃肠运动及分泌的影响

内源性大麻素系统有几种受体,在体内均存在相应的配体和特定的合成及代谢通路,均属G蛋白耦联超家族成员.近来的一些研究显示,大麻素受体激活后可影响胃肠运动及分泌.本文介绍大麻素的种类,其受体、配体、拮抗剂以及它们对胃肠运动和胃肠分泌的影响,为进一步研究内源性大麻素系统对胃肠运动及分泌的作用机制提供资料,并为临床应用该类药物...     

大麻素1型受体参与疼痛调节机制的研究进展

大麻素1型受体（CB1R）是近年来研究较为广泛的内源性大麻素受体之一,在中枢和外周神经系统均有表达。CB1R位于突触前膜,通过逆行抑制性突触传递调节神经递质的释放,是治疗疼痛的有效靶点。激活CB1R对伤害性、病理性和炎性疼痛均具有镇痛效应,拮抗CB1R可引起疼痛敏化。本文通过对CB1R结构功能、信号转导、镇痛机制方面进行综述,为进一步了解疼痛的病理生理学机制及探索更优疼痛治疗方法提供参考。     

大麻素受体2激动剂JWH-015对骨癌痛大鼠脊髓背角磷酸化环磷酸腺苷反应元件结合蛋白的影响

目的探讨腹腔注射大麻素受体（cannabinoid receptor,CB）2激动剂对骨癌痛大鼠脊髓背角磷酸化环磷酸腺苷反应元件结合蛋白（phosphorylated cyclic AMP respons eclement binding protein,pCREB）表达的影响。方法运用随机数字表法将63只雌性SD大鼠分为3组：肿瘤给药组（J组,15只）、肿瘤对照组（D组,24只）和假手术对照组（S组,24只）。J组、D组的大鼠左侧胫骨上端骨髓腔被注入5μl Walker256大鼠乳腺癌细胞制备骨癌痛模型;S组则注入等量的生理盐水。在造模后第10天,J组腹腔注射JWH-015（100μg/500μ1）,D组、s组注射等量JWH-015溶剂二甲基亚砜（dimethylsulfoxide,DMSO）。每组大鼠于造模前1d,造模后4、7、10d,腹腔注射后2、6、24、48、72h,检测手术侧机械刺激缩足阈值（paw withdrawal mechanical threshold,pwMT）和行走痛行为学评分。D组和S组大鼠于造模后4、7d,J组、D组和S组大鼠于造模后10d及腹腔注射后6、24、72h,取脊髓腰膨大进行免疫印迹分析。结果与S组比较,J组和D组大鼠造模后7d PWMT开始降低（P〈0.05）,造模后10d行走痛行为学评分增加（P〈0.05）,脊髓背角pCREB表达水平于7、10d上调（P〈0.05）.与D组比较,腹腔注射JWH-015后24h,J组PWMT（8.7±1.6）g显著上升（P〈0.05）,行走痛行为学评分（1.0±0.6）分和pCREB的表达（0.56±0.10）明显下降（P〈0.05）。结论腹腔注射JWH-015可能通过下调脊髓背角pCREB的表达改善骨癌痛大鼠的痛行为。     

大鼠肾脏缺血再灌注损伤中垂体中叶素及降钙素受体样受体的表达

目的 观察垂体中叶素（IMD）及其受体降钙素受体样受体（CRLR）在大鼠肾脏缺血再灌注损伤中的表达变化。 方法 将健康雄性Wistar大鼠随机分为假手术组和手术组,夹闭大鼠双侧肾动脉制作肾脏缺血再灌注损伤（IRI）模型,于缺血再灌注后0、6、12、24、48、72 h 6个时间点各取6只大鼠,留取血清及肾组织标本,对肾脏病理损伤评分并行半定量分析; Western印迹法半定量分析肾组织IMD及其受体CRLR的表达变化;放射免疫法检测血浆中IMD的表达变化。 结果 手术组大鼠发生了急性肾小管坏死（ATN）,以缺血再灌注48 h时病理损伤最重。与假手术组比较,IMD及CRLR在缺血再灌注12、24、48、72 h表达显著增高（均P < 0.01）;血浆中IMD在缺血再灌注12、48、72 h表达显著增高（均P < 0.05）。 结论 IMD及受体CRLR在大鼠肾脏缺血再灌注损伤中表达增加,血浆中IMD表达上调,提示其可能在肾脏缺血再灌注损伤病理生理过程中发挥作用。     

OPN在不同转移潜能肝癌中的表达及意义的初步研究

目的研究骨桥蛋白（OPN）在HCC中的表达与及临床应用价值。方法选取50例HCC手术切除的新鲜标本和50例正常肝组织，10例癌旁组织。做成切片，应用SABC免疫组化法，以OPN单克隆抗体对HCC组织切片进行染色检测。对OPN表达量与HCC的关系进行研究。结果OPN在HCC癌组织中的阳性率70．00％，明显高于癌旁及正常肝组织（P〈0．01）。OPN在高、中、低分化HCC中评分是0．66±1．62、2．24±1．84、2．17±1．65，低分化明显高于高分化（P〈0．05）。OPN在有转移与无转移HCC中的评分是2．65±1．83、1．3±1．71，有转移者高于无转移者（P〈0．01）。结论OPN在HCC癌组织中的表达明显升高，病理分化差者表达明显升高，且伴有转移者高于不伴转移者，提示OPN可能是HCC转移复发的一个潜在指标。     

Osteoclasts play a part in pain due to the inflammation adjacent to bone

Bone disorders with increased osteoclastic bone resorption are frequently associated with bone pain and inhibitors of osteoclasts reduce bone pain. Osteoclasts degrade bone minerals by secreting protons through the vacuolar H+-ATPase, creating acidic microenvironments. Because acidosis is a well-known cause of pain, we reasoned that osteoclasts cause pain through proton secretion. We explored this using an animal model in which a single subcutaneous injection of the complete Freund's adjuvant (CFA) in the hind-paw caused inflammatory hyperalgesia (hyper-responsiveness to noxious stimuli). Osteoclastic bone resorption was increased in the metatarsal bones in the CFA-injected hind-paws. CFA-induced hyperalgesia was significantly suppressed by the bisphosphonates, zoledronic acid (ZOL) and alendronate and osteoprotegerin. c-src-deficient mice in which osteoclasts are inherently dysfunctional exhibited reduced CFA-induced hyperalgesia. Repeated subcutaneous injections of parathyroid hormone-related protein into the hind-paw also induced hyperalgesia with increased osteoclastic bone resorption. The hyperalgesia was associated with increased mRNA expression of acid-sensing ion channel (ASIC) 1a, 1b and 3 in the ipsi-lateral dorsal root ganglions (DRGs) by RT-PCR and c-Fos in the ipsi-lateral spinal dorsal horn by immunohistochemistry. Of note, ZOL decreased the ASIC1a mRNA expression and c-Fos. Treatment of the DRG cell line F-11 with acid (pH5.5) increased ASIC1a, 1b and 3 mRNA expression and nuclear c-Fos expression. The ASIC blocker amiloride inhibited acid-induced c-Fos expression in F-11 cells. Moreover, F-11 cells transfected with the transient receptor potential channel vanilloid subfamily member 1 (TRPV1) showed increased acid-induced nuclear c-Fos expression compared with parental F-11 cells. Finally, bafilomycin A1, an inhibitor of the vacuolar H+-ATPase, reversed the hyperalgesia and down-regulated ASIC1a mRNA expression in the DRGs. These results led us to propose that osteoclasts play a part in CFA-induced inflammatory pain through an activation of the acid-sensing receptors including ASICs and TRPV1 by creating acidosis.     

Reduction in bone formation and elevated bone resorption in ovariectomized rats with special reference to acute inflammation

Changes in bone modeling and remodeling in the tibia of growing rats within 30 days of ovariectomy (ovx) were evaluated by histomorphometric, mechanical; and biochemical means. Three days after ovx, suppressed bone formation was seen. This was shown by reduced osteoid volume, osteoblast surface, and bone formation rate in the secondary spongiosa, and a reduced longitudinal growth rate in the growth plate. In addition, the alkaline phosphatase and tartrate-resistant acid phosphatase activity in bone marrow supernatants was suppressed in conjunction with elevated serum sialic acid levels, indicating inflammation. Although estrogen deprivation itself may provoke the inflammatory process, the serum sialic acid level in the ovx group returned to the baseline level within 5 days after surgery, while that of estradiol in the ovx group remained consistently lower. This suggests that surgical stress, not estrogen deprivation, is the primary cause of the inflammatory response shortly after ovx. A significant difference (p < 0.01) between the ovx and sham rats was seen in the osteoclast surface, which peaked on day 7 in the ovx rats. On day 14 postovariectomy, the bone formation rate peaked and remained constant until day 30. In the ovx rats, there was a sustained reduction in the serum albumin level until day 30. Estrogen deprivation may be the primary cause of these changes, because both surgical ovx and medical oophorectomy with gonadotropin-releasing hormone agonist (G(nRHa) reduce the serum albumin level. In numerous studies dealing with changes after ovx in rats, we have observed: 1) a transient reduction in bone formation in relation to inflammatory changes evoked by ovx surgery, and 2) a sustained reduction in the serum albumin level for at least 30 days after ovx that is possibly due to estrogen deprivation.     

脓毒症大鼠多器官Toll样受体4基因表达的改变及意义

目的　探讨脓毒症大鼠肝、肺、肾及小肠组织中Toll样受体4(TLR4)基因表达的变化 规律及其意义。方法　雄性Wistar大鼠100只,动物随机分为正常对照组、假手术组、盲肠结扎穿孔 (CLP)致脓毒症组和杀菌/通透性增加蛋白(BPI)治疗组。分别检测肝、肺、肾、小肠组织TLR4、TNF αmRNA表达以及组织、血浆中内毒素水平的改变。结果　CLP后6～12h肝、肺、肾及小肠组织 TLR4mRNA表达显著升高达峰值(P<0.05或P<0.01);BPI早期治疗可显著降低各组织内毒素 水平,CLP后12h肝、肺、肾、小肠组织和24h肾组织TLR4mRNA表达亦明显抑制(P<0.05或 P<0.01)。相关分析显示,肝、肺、肾组织TLR4mRNA表达分别与相应组织及血浆内毒素水平、组 织TNF αmRNA水平均呈显著正相关。结论　CLP后细菌内毒素可迅速侵入血循环和多种组织, 它对于诱导体内TLR4基因表达上调具有显著影响,而TLR4基因广泛表达可能参与了脓毒症的病 理生理过程。     

Inflammation-mediated osteopenia (IMO) during acute inflammation in rats is due to a transient inhibition of bone formation

Summary Local inflammation was induced in rats through the subcutaneous injection of magnesium silicate. Trabecular bone volume of the tibia decreased progressively during a 3 week observation period following the inflammatory stimulus. The trabecular bone surface covered with osteoblasts was strikingly reduced during the first week but had normalized by the end of the third week. Calcification rate in the cortical bone of the tibia was reduced with a parallel reduction in endosteal osteoid seam width. Both calcification rate and tetracycline double-labeled surface of vertebral trabecular bone were reduced during the first 2 weeks. Neither total bone resorption surface nor active bone resorption surface were increased. There was a decrease in osteoclast numbers/mm² bone tissue associated with decreasing bone volume. Our data demonstrate a transient inhibition of bone formation during acute inflammation in the rat and indicate that changes in osteoblast function are part of the acute phase response following local inflammation.     

双膦酸盐及其联合甲氨蝶呤对CIA大鼠炎症与骨破坏影响的研究

目的建立模拟类风湿关节炎(rheumatoid arthritis,RA)发病的胶原诱导性关节炎(collagen-induced arthritis,CIA)大鼠模型,探讨双膦酸盐(bisphosphonate,BP)及双膦酸盐联合甲氨蝶呤(methotrexate,MTX)、自拟补肾强骨方对CIA大鼠关节炎症及局部骨破坏的作用及机制,为临床提供实验依据。方法建立CIA大鼠模型,将大鼠随机分为5组:(1)灭菌用水治疗(8只);(2)BP+自拟补肾强骨方治疗组(8只);(3)BP+MTX治疗组(8只);(4)BP治疗组(8只);(5)恩利+MTX治疗组(8只)。每只大鼠均在炎症分值达到2分及以上时开始治疗,从治疗开始隔天进行关节炎症评分,绘出炎症变化曲线,评价各治疗方法对炎症的抑制作用;治疗4w后,处死大鼠,左侧股骨和第5腰椎行骨生物力学检查,胫骨上段行micro-CT扫描和制作硬组织切片,观察股骨及腰椎的骨生物力学的变化,胫骨上段骨小梁变化及骨量变化。结果 BP+自拟补肾强骨方组和BP+MTX组对CIA大鼠关节炎症均具有明显抑制作用,二组比较差异无统计学意义。BP+自拟补肾强骨方、BP+MTX组和恩利+MTX组均具有明显的抑制CIA大鼠炎症关节周围骨量丢失的能力,且BP+自拟补肾强骨方、BP+MTX组抑制骨量减少的作用主要是通过抑制骨小梁数量减少及增加骨小梁宽度来实现的。而在骨生物力学检查中,BP+自拟补肾强骨方与MTX+恩利组对于皮质骨和松质骨强度的改善明显。而BP组则对于皮质骨强度和松质骨强度均无明显改善作用。因此BP联合自拟补肾强骨方与BP联合MTX对于促进骨小梁数量的增殖有较好的作用,改善皮质骨和松质骨的强度,效果较单独使用BP好。结论 BP联合MTX或自拟补肾强骨方均能明显抑制CIA大鼠关节炎症反应、关节侵蚀及关节周围骨丢失的作用。     

GrB/Bid蛋白在胃癌中表达及临床意义的初步研究

目的 探讨胃癌组织中颗粒酶B(GrB)和Bid蛋白的表达情况及与胃癌演进的关系,了解二者在肿瘤免疫耐受机制中的作用.方法 采用免疫组织化学S-P法检测GrB和Bid在胃癌和切缘组织中的表达状况.结果 GrB在胃癌组织中阳性表达率为47.62％,切缘组织中的阳性率为40.48％,二者相比差异无统计学意义(P ＞0.05);Bid在胃癌组织中的阳性表达率(26.19％)明显低于胃切缘上皮组织(76.19％),二者相比有统计学意义(P ＜0.05);GrB的表达水平与胃癌临床分期、淋巴转移有关(P＜0.05),Bid的表达水平与胃癌分化程度、浸润深度、临床分期及淋巴结转移有关(P ＜0.05);GrB和Bid在42例胃癌中表达呈负相关(rs=-0.311,P＜0.05).结论 胃癌组织中GrB和Bid表达可能共同参与胃癌演进,胃癌组织中Bid的表达下调或缺失可能引起胃癌细胞逃逸内源或外源性GrB介导的细胞凋亡,获得免疫耐受潜能,逃逸机体免疫应答.