鲨烯、苔条、紫菜、花菜对大鼠血清胆固醇的作用

通过喂养试验,观察了11种食物和1种食物提取物(鲨烯),对大鼠血清胆固醇浓度的作用。结果发现,鲨烯、苔条、紫菜、花菜均有降低血清T-C的作用,其中鲨烯和苔条还能促进HDL-C的增高。与对照组相比,鲨烯组大鼠血清T-C,LDL-C和VLDL-C分别下降50.5％、60.8％和53.7％,而HDL-C则增高56.5％。苔条组大鼠血清T-C,LDL-C和VLDL-C分別下降34.9％,42.7％、和31.4％,而HDL-C增高58.3％。紫菜组和花菜组大鼠血清T-C则分别下降29.4％和36.7％。     

刺五加法尼基焦磷酸合酶、鲨烯合酶、鲨烯环氧酶基因启动子CpG岛的预测与功能验证

目的获得刺五加法尼基焦磷酸合酶(FPS)、鲨烯合酶(SS)、鲨烯环氧酶(SE)基因启动子的CpG岛分布情况和功能活性。方法针对已克隆的FPS、SS、SE基因启动子序列,使用EMBOSS和Li Lab进行CpG岛预测,同时利用GUS基因作为报告基因,p CAMBIA1301质粒作为表达载体,利用根癌农杆菌转化拟南芥进行功能验证。结果发现刺五加FPS、SS启动子含有2个CpG岛,长度分别是520、218 bp和108、103 bp,SE启动子含有3个CpG岛,长度为290、119、149 bp。FPS、SS、SE基因启动子均具有启动活性,但强度不一,SS启动子活性最强。结论研究首次获得刺五加FPS、SS、SE基因启动子区域的CpG岛分布情况,以及其功能活性,为后续进一步FPS、SS、SE甲基化分析及其在刺五加中的表达调控研究奠定了基础。     

太子参鲨烯环氧酶基因的克隆及其表达分析

目的对太子参三萜类皂苷生物合成关键调控酶鲨烯环氧酶1(SQE1)基因进行全长c DNA克隆和功能分析。方法基于其他植物SQE基因的同源序列设计简并引物,以太子参块根总RNA为模板,RT-PCR结合RACE技术克隆太子参SQE1基因的全长c DNA,并进行生物信息学分析。利用农杆菌叶盘转化法将SQE1基因转化烟草,研究其对烟草总三萜量的影响。结果获得太子参SQE1基因全长c DNA序列2 038 bp,该序列包含1 554 bp的开放阅读框,编码517个氨基酸,相对分子质量为5.67×104,等电点为8.8,与其他药用植物的SQE蛋白具有较高的同源性,含有FAD结合结构域和4个跨膜区域,转太子参SQE1基因的烟草的总三萜量明显高于非转基因植株。结论首次克隆获得太子参SQE1基因的全长c DNA,该基因的异源表达可一定程度提高转基因植物的总三萜量,为阐明与应用太子参三萜类成分生物合成途径提供科学依据。     

滇重楼鲨烯环氧酶基因的克隆及原核表达研究

目的克隆滇重楼Paris polyphylla var.yunnanensis三萜皂苷生物合成途径中的关键酶鲨烯环氧酶(squalene epoxidase,SE)基因,并进行原核表达。并用Real-time PCR法检测cDNA样本中SE1基因和SE2基因的相对量。方法以滇重楼根为材料提取总RNA并反转录为cDNA。以cDNA为模板,根据转录组数据中的2组SE基因全长序列设计特异性引物,对SE基因进行克隆后转入到pEASY-T1 Simple Cloning Vector中,经测序鉴定正确后,构建pEASY-E1-SE表达载体,利用Art Media protein Expression/Amp+培养基自动诱导表达。Real-time PCR法检测cDNA样本中SE1和SE2基因的相对量。结果获得了2条滇重楼SE基因,命名为ppSE1和ppSE2。ppSE1的全长为1 932 bp,开放阅读框(ORF)为1 578 bp,编码525个AA;ppSE2的全长为1 828 bp,ORF长为1 548 bp,编码515个氨基酸。荧光定量PCR结果显示ppSE1基因和ppSE2基因在茎和叶中的表达具有显著差异,ppSE1的表达在叶中最为显著。酶切和测序结果表明,原核表达载体pEASY-E1-SE构建成功;SDS-PAGE分析显示,在BL21(DE3)表达感受态细胞中成功诱导表达了SE基因的2种融合蛋白。结论克隆了滇重楼的SE基因,获得了在体外具有生物学活性的SE蛋白。ppSE1基因和ppSE2基因在滇重楼中具有不同的表达模式,在滇重楼次生代谢产物的合成中起着不同的作用。     

Adjuvant oil induces waves of arthritogenic lymph node cells prior to arthritis onset

A single intradermal injection of the adjuvant‐oil squalene induces T cell mediated arthritis in DA rats. The chain of events leading from nonspecific provocation of the immune system to arthritis is largely unknown. Previous studies have demonstrated that lymph node (LN) cells are of pathogenic importance, i.e. cells from LNs draining the injection site can transfer arthritis to naïve DA rats. Recently we have demonstrated cellular uptake of adjuvant oil in draining lymph nodes but also that nondraining LNs become hyperplastic and harbour arthritogenic cells. Here, we aimed to determine from which time‐point prior to arthritis onset arthritogenic cells appear in draining inguinal and nondraining axillary/brachial LNs, respectively. We demonstrated that the ability to transfer arthritis was strongly dependent on the time‐point after adjuvant‐injection with clear‐cut differences between draining and nondraining LN cells. Cells harvested at day 5 postinjection (p.i) were not able to transfer arthritis, while at day 8 p.i, a first wave of arthritogenic cells appeared in draining LNs. The ability to transfer arthritis was associated with a pro‐inflammatory cytokine profile as indicated by the IL‐1β and IFNγ expression in cells from draining LNs. Subsequently, at day 11 p.i., just before arthritis onset, arthritogenic cells appeared also in nondraining LNs. These results shed new light on the induction of arthritic diseases, implicating a two step mechanism for the development of pathogenic cells. Firstly, a pro‐inflammatory burst in responding lymphoid organs leading to a local pool of arthritogenic cells and, secondly, a transmission of arthritogenecity to other LNs and precipitation of disease in peripheral joints.     

噪声性听力损伤的治疗观察

对具有不同程度听力损伤患者（共２０例耳）,在不脱产情况下,口服赛鲨力补氧胶囊２５天（１１．２ｇ／ｄ）观察治疗前后其听力和静脉血中的铜蓝蛋白酶、超氧化物珐化酶（ＳＯＤ）活性及过氧化脂质（ＬＰＯ）含量的变化,结果表明,口服赛鲨力补氧胶囊后,患者静脉血中的铜蓝蛋白酶、超氧化物歧化酶活性显著升高：语频段听力恢复的有效率为７１．４％,高频有效率为７８．９％。因此,鲨烯不仅可以改善体内的代谢物清除水平,而且能     

鲨烯复合剂延缓衰老作用的研究

目的:研究鲨烯复合剂的延缓衰老作用。方法:采用自制的鲨烯复合剂,进行高、中、低3个剂量组的果蝇生存试验和SD大鼠生化指标测定。结果:鲨烯复合剂能明显增加SD大鼠超氧化物歧化酶(SOD)的活性,高剂量组SOD活力为(181.6±19.9)mg/L,与老年对照组的(117.6±18.8)mg/L相比,差异显著;能显著降低血中丙二醛(MDA)含量,高剂量组MDA为(1.39±0.28)mmol/L,与老年对照组(2.63±0.48)mmol/L相比,差异显著;能延长果蝇的半数死亡时间、平均寿命和平均最高寿命。半数死亡时间可延长至53-65d,平均寿命延长至(53±12)-(63±12)d,平均最高寿命延长至(72±3)-(81±2)d。结论:鲨烯复合剂具有明显的延缓衰老作用。     

New applications of squalene synthase inhibitors: Membrane cholesterol as a therapeutic target

Squalene synthase (SQS) inhibitors, mostly known as antihyperlipidemic agents for controlling blood cholesterol levels, have been increasingly used to study alterations of the cholesterol content in cell membranes. As such, SQS inhibitors have been demonstrated to control cellular activities related to cancer cell proliferation and migration, neuron degeneration, and parasite growth. While the mechanisms behind the effects of cellular cholesterol are still being revealed in detail, the evidence for SQS as a therapeutic target for several seemingly unrelated diseases is increasing. SQS inhibitors may be the next promising candidates targeting the three remaining primary therapeutic areas, beyond cardiovascular disease, which still need to be addressed; their application as anticancer, antimicrobial, and antineurodegenerative agents appears promising for new drug discovery projects underway.     

甘草鲨烯合酶基因多态性对其编码酶催化效率影响的研究

目的:分析甘草鲨烯合酶(squalene synthase,SQS)基因多态性及其对编码酶催化效率的影响,为揭示优质甘草形成的分子机制奠定基础.方法:提取甘草总RNA; PCR扩增其SQS基因编码序列;测序后分析SQS序列的多态性;将具有明显差异的4个甘草SQS序列(SQS1C,SQS1F,SQS2A,SQS2B)连入表达载体pET-32a(+),转化大肠杆菌BL21;IPTG诱导蛋白表达,纯化后用于体外酶促反应;GC-MS鉴定产物并比较相对含量.结果:甘草SQS1基因存在单核苷酸多态性(single nuclear polymorphisms,SNPs)、插入与缺失多态性(InDel)、选择性剪接(AS)多态性;SQS2基因只存在SNPs.氨基酸序列分析显示,SQS1非保守替换占53.94％,结构域中有2个位点突变;SQS2非保守替换占60％,结构域中有1个位点突变.在4种编码酶的体外酶促反应中,利用GC-MS均能检测到产物生成;SQS1F编码酶积累鲨烯的能力高于其他序列.结论:甘草SQS基因具有丰富的多态性,其编码酶催化效率差异显著,这可能是优质甘草形成的分子基础.