原矛头蝮蛇(Protobothrops mucrosquamatus)主要分布于我国长江以南和台湾地区,原名烙铁头蛇(Trimeresurus mucrosquamatus),属蝰蛇科原矛头蝮属[1].原矛头蝮蛇毒(Protobothrops mucrosquamatus venom,PMV)主要表现为血液毒性,影响心血管及凝血系统等方面,对组织细胞也有毒性作用[2-3].该蛇伤较为常见,临床表现主要为凝血障碍、头昏恶心、视力模糊和胸闷腹痛等,严重者出现皮下出血、血尿、全身内脏出血甚至失血性休克[4-5].迄今,对PMV的研究主要集中在纤溶酶、类凝血酶、丝氨酸蛋白酶及诱导血小板聚集的蛋白等成分[1-2,6].课题组前期研究表明,PMV具有明显的破坏凝血功能的作用,PMV及其组分能诱导血小板聚集引起全血中血小板计数下降,具有酶切底物S-2251活性,能延长凝血酶时间(TT)和部分活化凝血活酶时间(APTT),破坏人微血管内皮细胞[2,7].此前研究更多采用凝血四项等研究方法,为进一步研究和认识PMV对凝血功能的影响,课题组采用血栓弹力图测定方法来开展PMV对大鼠全血凝血功能影响的研究,以期为进一步认识原矛头蝮蛇伤的机制及临床救治方案和新药研究提供参考.

OBJECTIVE To investigate the toxic effect and mechnism of kaempferol on human kidney proximal tubular epithelial cells （HK-2） based on high content analysis technology.METHODS HK-2cells were divided into the control group （final concentration of dimethylsulfoxide＜0.01%）,kaempferol 510,20,50 and 100μmol·L -1 and doxorubicin (Dox,final concentration 10μmol·L -1 ) groups.The count of living cells was detected via Hoechst 33342 staining,the cell viability was determined by MTT assay,the levels of reactive oxygen species （ROS）,mitochondrial membrane potential （MMP） and the intracellular calcium ion were detected by fluorescence probes of DCFH-DA,Mito-Tracker Red CMXRos and Fluo-4 AM.The ATP content was measured with the chemiluminescence method,and apoptosis by flow cytometry.The DNA content was detected by Hoechst 33342 staining.The nuclear translocation of NF-κB p65,the expressions of p38 mitogen-activated protein kinases （p38 MAPK）,p-p38 MAPK,extracellular signal regulated kinase （ERK）,p-ERK and c-Jun N-terminalkinases （JNK）,p-JNK protein in associated mitogen activated protein kinases （MAPK） signal pathway,and those of signal transducer and activator of transcription-3 （STAT3）,p-STAT3 protein in janus kinase-2 （JAK2）/STAT3 signal signa pathway were detected by immunofluorescence assay.RESULTS Compared with the control group,the count of living cells in the kaempferol 5,10,20,50 and 100μmol·L -1 groups and the cell viability in the kaempferol 10,20,50 and 100μmol·L -1 groups were significantly deceased （P＜0.05,P＜0.01）.The levels of ROS were significantly increased in the kaempferol 10,20,50 and 100μmol·L -1 groups （P＜0.05,P＜0.01）,while those of MMP in 5,10,20,50 and 100μmol·L -1 groups were obviously decreased（P＜0.01）.The levels of calcium influx in kaempferol 50,100μmol·L -1 groups were increased significantly（P＜0.05,P＜0.01）,while the ATP content in the kaempferol 50 and 100μmol·L -1 groups was obviously decreased （P＜0.05,P＜0.01）.The apoptotic rates in kaempferol 20,50,100μmol·L -1 groups were up-regulated significantly （P＜0.05,P＜0.01）,but the DNA content hardly changed in all the kaempferol groups.The results of immunofluorescence assay showed that kaempferol 50 and 100μmol·L -1 led to the phosphorylation and nuclear translocation of NF-κB p65,the expressions of p-p38 MAPK,p-ERK,p-JNK and JNK of MAPK signaling pathway were up-regulated significantly,and that the expression of p-STAT3protein of JAK2/STAT3 signaling pathway was also increased （P＜0.05,P＜0.01）.CONCLUSION Kaempferol exerts toxic effect on HK-2 cells,and its mechanism is probably related to oxidative stress injury and apoptosis mediated by the up-regulation of ROS levels.

目的 利用高内涵筛选技术研究吴茱萸次碱(rute-acarpine,RUT)的肝毒性及其可能机制.方法 HepG2细胞暴露于不同浓度的RUT后作用不同时间,MTT法检测细胞活力,采用高内涵检测RUT对细胞存活、细胞核面积、线粒体膜电位(MMP)、ROS、钙离子内流、细胞膜完整性(DIR)和MAPK、NF-κB、JAKs-STATs信号通路活化水平的影响,流式细胞检测细胞凋亡.结果 100 μmol·L-1 RUT明显抑制了HepG2细胞的活力(P＜0.01),表现为RUT作用24 h后,细胞核面积减少,核形态不均一,作用48 h后存活细胞数量明显减少(P＜0.01),并伴随着细胞的早期凋亡(P＜0.01);从6 h开始,100 μmol·L-1 RUT组细胞内活性氧和钙离子水平明显升高(P＜0.01),细胞膜完整性明显降低(P＜0.01);100 μmol·L-1 RUT 作用 24 h 后,ERK1/2、JNK、STAT3 和p38的磷酸化水平明显升高(P＜0.01,P＜0.05),总蛋白未见明显变化;在3 h可检测到c-Jun、c-Fos的表达上调,与对照组相比,差异具有显著性(P＜0.01);在3 h时间点,可明显检测到p-NF-κB p65的表达上调(P＜0.01),但NF-κB p65入核不明显.结论 吴茱萸次碱在100 μmol·L-1的条件下对HepG2细胞表现出细胞毒性,其毒性机制主要与氧化应激和炎症反应导致的细胞损伤有关.

<span id="ChDivSummary" name="ChDivSummary" class="abstract-text">目的研究补体旁路途径激活诱导人血管平滑肌细胞（VSMC）增殖活化及其干预作用。方法采用CVF特异激活血浆补体旁路途经。将VSMC与补体旁路激活产物共同孵育,采用倒置相差显微镜和MTT法分别检测细胞形态变化和增殖活性,ELISA法检测培养上清中E-selectin、ICAM-1和VCAM-1含量,Western blot检测p-NF-κB p65、IKK和NF-κB p65蛋白表达水平,双荧光素酶报告基因检测试剂盒检测NF-κB p65转录活性,采用PDTC对上述指标的变化进行干预。结果补体旁路激活导致VSMC增殖活化,上调表达E-selectin、ICAM-1和VCAM-1,其中ICAM-1和VCAM-1含量在6 h达到高峰,E-selectin在12 h出现明显上调;VSMC经补体旁路激活产物刺激后,NF-κB p65磷酸化明显增加,NF-κB p65核内转录活性升高,IKK和NF-κB p65蛋白表达上调,PDTC对上述相关指标的变化有明确的干预作用。结论补体旁路激活可导致VSMC增殖活化,其机制与NF-κB信号通路活化有密切关系,且NF-κB抑制剂PDTC对其增殖活化具有明显的干预作用。</span>

目的 研究黄芩苷对脂多糖(LPS)致人微血管内皮细胞(HMEC)炎症反应的保护作用及作用机制.方法 采用LPS作用HMEC,建立炎症反应模型;以不同浓度的黄芩苷预处理细胞,然后将细胞暴露于LPS,ELISA检测炎症因子ICAM-1、IL-6和MCP-1的含量;荧光观察Ca2+内流并采用流式细胞仪检测细胞内Ca2+水平;免疫荧光法检测NF-κB p65入核情况;双荧光素酶报告基因方法检测NF-κB核内转录活性;Western blot检测NF-κB p65、p-NF-κB p65及TLR4的表达.结果 HMEC暴露于LPS后,出现了明显的Ca2+内流,NF-κB p65发生磷酸化,核内转录活性上调,ICAM-1、IL-6及MCP-1表达上调.不同浓度的黄芩苷均能抑制LPS刺激HMEC后产生的Ca2+内流、NF-κB p65入核及核内转录活性上调,减少ICAM-1、IL-6、MCP-1的表达,下调NF-κB p65、p-NF-κB p65及TLR4的表达,且抑制作用呈现一定的量效关系.结论 黄芩苷能抑制LPS诱导的HMEC炎症反应,其机制与抑制Ca2+内流和NF-κB信号通路的活化,从而降低炎症反应的水平有关.

目的 观察不同剂量亚砷酸钠(NaAsO2)对L-02肝细胞氧化应激性损伤作用及超氧化物歧化酶1(SOD1)、AU碱基富集元件RNA结合因子1(AUF1)表达影响.方法 用0、10、20、40 μmol/L NaAsO2分别处理L-02肝细胞24小时,化学比色法检测谷胱甘肽巯基转移酶(GST)、γ-谷氨酰转肽酶(γ-GT)活力及总胆汁酸(TBA)含量和SOD1、谷胱甘肽过氧化物酶(GPx)活性;荧光探针2,7-二氯二氢荧光素二乙酸酯检测细胞内活性氧族(ROS)相对荧光密度;实时荧光定量PCR和Western Blotting分别检测SOD1和AUF1转录和蛋白表达.结果 (1)与对照组比较,20、40 μmol/L NaAsO2组GST 活性和TBA 含量均升高(P ＜ 0.05);10、20、40 μmol/L NaAsO2组γ-GT 活性也升高(P ＜0.05).(2)与对照组比较,20、40 μmol/L NaAsO2组SOD1活性均下降(P ＜0.05);GPx活性仅在10 μmol/L升高(P ＜0.05);细胞内ROS先降低后升高(P＜0.05).(3)与对照组比较,20和40μmol/L NaAsO2组AUF1和SOD1 mRNA表达均升高(P＜0.05),AUF1 蛋白表达先升高后降低(P＜0.05);10、20 μmol/L NaAsO2组SOD1 蛋白表达升高(P＜0.05),但40μmol/L NaAsO2组SOD1蛋白表达有降低趋势.结论 NaAsO2对L-02肝细胞产生氧化应激性损伤,其机制可能是NaAsO2通过降低AUF1的蛋白表达降低,从转录后调控降低了SOD1 mRNA的稳定性而使其蛋白表达和酶活力降低.

免疫性疾病、急性肺损伤、缺血再灌注损伤等病征的发生与补体异常激活密切相关.抗补体药物研究是新药开发的热点之一.本研究旨在从中华眼镜蛇毒中发现并分离纯化获得新的抗补体活性蛋白质,并对其理化性质和生物学活性加以研究.在抗补体活性追踪的指导下,采用蛋白质层析技术对眼镜蛇毒进行分离纯化;利用MALDI-TOF-MS、SDS-PAGE和葡聚糖凝胶过滤法测定目标蛋白质的纯度及分子量;等电聚焦凝胶电泳法测定其等电点;采用Edman降解法测定目标蛋白质的N-端氨基酸序列;测定目标蛋白质对补体经典途径和旁路途径的抑制活性以及可能的机制;采用MTT法和SRB法检测目标蛋白质对肿瘤细胞的杀伤作用;测定目标蛋白质对多种来源红细胞的溶血活性;采用KB平板扩散法检测抗菌活性.结果 表明,通过SP Sephadex C-25阳离子交换层析和RP-HPLC C18反相层析,从中华眼镜蛇毒中分离纯化获得一个均一的抗补体蛋白质,将其命名为CTX-CI.还原性SDS-PAGE测得CTX-CI的表观分子量为12.7 kD,凝胶过滤法测得分子量为9.7 kD,MALDI-TOF-MS测得精确分子质量为7.0 kD;变性条件下测得CTX-CI的等电点为9.81;N-端氨基酸序列为LKCH.相关活性测定结果表明,CTX-CI能有效抑制人血清补体经典途径,其IC50为0.046 g/L,但对补体旁路途径无明显抑制作用;机制研究表明,CTX-CI能抑制补体经典途径C3转化酶的形成.同时,CTX-CI对肿瘤细胞株A549、K562和MCF-7细胞表现出抑制作用,其IC50分别为0.32 g/L、0.58 g/L、0.63 g/L;对豚鼠红细胞有轻微的溶血作用;能抑制枯草芽孢杆菌和藤黄微球菌的生长.综上所述,本研究从中华眼镜蛇毒中分离纯化出一个新的抗补体蛋白质-CTX-CI,其理化性质和生物学活性表明其属于细胞毒素,CTX-CI能明显抑制补体经典途径,其机制与抑制经典途径C3转化酶的形成有关.

<span id="ChDivSummary" name="ChDivSummary" class="abstract-text">&lt;正&gt;疼痛是诸多病征共有的症状,是动物的神经系统对伤害刺激的感觉。疼痛研究的焦点在于疼痛发生的机制以及干预治疗的策略和药物研究^[1-2]。补体旁路激活会导致炎症和组织损伤^[3]。有研究表明,补体与某些神经病理性疼痛关系密切^[4-5],但其与非神经性疼痛的关系尚不清楚。本研究用眼镜蛇毒因子（cobra venom factor,CVF）特异抑制补体旁路途径,观察小鼠对疼痛的反应。1材料与方法</span>

<span id="ChDivSummary" name="ChDivSummary" class="abstract-text">目的研究川芎嗪（TMP）对补体旁路激活致人微血管内皮细胞（HMEC）炎症反应的干预作用及其可能的分子机制。方法以眼镜蛇毒因子（cobra venom factor,CVF）来激活正常人血清的补体旁路,用激活产物作用于HMEC,作用前加入不同浓度TMP预处理细胞,然后将细胞暴露在该激活产物中,采用ELISA方法检测细胞培养上清中可溶性黏附分子（ICAM-1、VCAM-1、E-selectin）和炎症介质（IL-6、TNF-α）的含量变化;双萤光素酶报告基因检测试剂盒测定NF-κB核内转录活性。结果 HMEC受补体旁路激活产物刺激后,引起黏附分子和炎症介质的表达上调,以及NF-κB核内转录活性的上调。不同浓度的TMP对上述炎症反应相关指标的上调均有干预作用,且表现出剂量依赖性。结论 TMP对补体旁路特异激活HMEC炎症反应有明显的干预作用,其机制可能与抑制NF-κB的核内转录活性有关。</span>

Aim To explore the protective effect and mechanism of icariin ( ICA ) on acute lung injury ( ALI) in mice induced by activation of the comple-ment alternative pathway. Methods 32 healthy KM mice were randomly divided into four groups: the nor-mal group, the model group, the PDTC group and the Icariin group, which received 7-day intragastric admin-istration respectively. Then cobra venom factor ( CVF) was used to activate specifically complement alternative pathway to induce acute lung injury in mice by intrave-nous injection. Myeloperoxidase ( MPO ) activity of lung homogenate, the cell count and the protein con- tent of bronchoalveolar lavage fluid ( BALF ) were measured. The concentration of IL-6, TNF-α, P-selec-tin and ICAM-1 in BALF and serum were determined by ELISA. The pathological change of lung tissue was observed by HE staining. The phosphorylation of NF-κB p65 in lung tissues was checked by immunohisto-chemistry. The effect of the transcriptional activity of NF-κB signal pathway in microvascular endothelial cells was measured by employing dual-luciferase re-porter assay system. Results ICA reduced MPO ac-tivity of lung homogenate, the cell count and the con-tent of IL-6, TNF-α, P-selectin in BALF obviously. The level of TNF-α, P-selectin and ICAM-1 in serum was decreased, the pulmonary inflammatory cell infil-tration was reduced, the phosphorylation of NF-κB p65 in lung was inhibited significantly and the transcrip-tional activity of NF-κB was also down-regulated. Con-clusion ICA can alleviate acute inflammatory re-sponse of ALI mice induced by activation of the com-plement alternative pathway. The mechanism may be highly related to the inhibition of inflammatory cell in-filtration in lung tissue, the down-regulation of phos-phorylation of NF-κB p65 and nuclear transcriptional activity.