丝氨酸蛋白酶抑制剂与肥胖、胰岛素关系的研究进展

长期以来,脂肪组织一直被认为是仅供能量贮备的终末分化器官。但是自从1994年发现瘦素以来,脂肪组织的内分泌功能越来越受到人们关注。随着抵抗素（resistin）、脂联素（adiponectin）、内脏脂肪素（visfatin）、海帕西啶（hepcidin）、apelin等脂肪细胞因子的发现,脂肪组织旺盛的内分泌功能亦逐渐为人们所认识,     

冠心病患者血清脂肪特异性丝氨酸蛋白酶抑制剂与炎症因子的相关性分析

目的观察冠心病(CHD)患者血清脂肪特异性丝氨酸蛋白酶抑制剂(vaspin)与炎症因子的相关性。方法选取2010年4月至2015年4月该院CHD患者114例作为CHD组,按疾病类型又分为稳定性心绞痛组、不稳定性心绞痛组、急性心肌梗死组,按冠状动脉病变支数又分为1支病变组、2支病变组、3支及以上病变组。同时选取健康人群45例作为健康对照组。采用酶联免疫吸附法检测vaspin、肿瘤坏死因子(TNF-α)、白细胞介素(IL)-6、IL-10和C反应蛋白(CRP)水平变化。结果 CHD组vaspin水平明显低于健康对照组,而TNF-α、IL-6、IL-10和CRP水平明显高于健康对照组,差异有统计学意义(P0.05)。稳定性心绞痛组、不稳定性心绞痛组和急性心肌梗死组中vaspin水平依次降低,CRP、IL-6、IL-10和TNF-α水平依次升高;1支病变组、2支病变组和3支及以上病变组中vaspin水平依次降低,CRP、IL-6、IL-10和TNF-α水平依次升高;各组间比较差异有统计学意义(P0.05)。CHD病变严重程度与CRP、IL-6、IL-10和TNF-α水平呈正相关(P分别为0.007、0.003、0.004、0.005),与vaspin水平呈负相关(P=0.002)。结论 vaspin水平与CHD患者的炎症因子紧密相关,与CHD血管病变严重程度有关。     

血清丝氨酸蛋白酶抑制剂与肥胖及2型糖尿病的相关性

目的 探讨2型糖尿病患者血清中脂肪特异性丝氨酸蛋白酶抑制剂(vaspin)、瘦素、脂联素的变化及其与2型糖尿病和肥胖的关系.方法 对80例2型糖尿病患者,69例空腹糖耐量受损患者,73例健康对照者采集病史.进行体格检查并留取血清测定其血糖、vaspin、瘦素、脂联素水平.结果 2型糖尿病组vaspin、瘦素水平显著高于对照组和空腹糖耐量受损组(P<0.05),脂联素水平显著低于对照组和空腹糖耐量受损组(P<0.05).空腹糖耐量受损组vaspin、瘦素水平显著高于对照组(P<0.05),脂联素水平显著低于对照组(P<0.05).两组中肥胖亚组的vaspin、瘦素水平均显著高于非肥胖亚组(P<0.05),脂联素水平均显著低于非肥胖亚组(P<0.05).2型糖尿病组vaspin与瘦素水平呈显著正相关(r分别0.362,P<0.05),与脂联素呈负相关(r=-0.397,P<0.05).结论 Vaspin水平与肥胖、2型糖尿病密切相关,在2型糖尿病的发生、发展中具有重要的作用.     

丝氨酸蛋白酶抑制剂在卵巢癌发展和血管生成中的作用机制研究

目的研究丝氨酸蛋白酶抑制剂(mammary serpin,MASPIN)在卵巢癌的发展和血管生成中的作用。方法采用免疫组化LsAB法对31例正常卵巢组织、35例上皮性卵巢癌(epithelial ovarian cancer,EOC)标本进行MASPIN、血管内皮生长因子(vascular endothelial growth factor,VEGF)表达检测,分析它们与卵巢癌临床病理特点的关系;MASPIN真核表达质粒体外转染卵巢癌SKOV3细胞,用细胞免疫化学及半定量逆转录聚合酶链反应(RT-PCR)方法检测转染后卵巢癌细胞中VEGF的蛋白质和mRNA表达变化。结果 MASPIN、VEGF在卵巢癌中的表达与正常卵巢组织相比明显上调(P<0.05);MASPIN蛋白表达与非浆液性卵巢癌、低临床分期、无腹水形成差异有统计学意义((P<0.05);VEGF蛋白表达与卵巢癌腹水形成差异有统计学意义(P<0.05);卵巢癌中MASPIN蛋白表达与VEGF呈负相关(r=-0.738,P<0.05);细胞免疫组化及半定量逆转录聚合酶链反应(RT-PCR)方法均证实转染MASPIN cDNA的SKOV3细胞中VEGF的表达明显弱于转染空载体组和未转染组(P<0.05),而后两者之间差异无统计学意义(P>0.05)。结论 MASPIN在卵巢癌的发展过程中可能仍发挥着肿瘤抑制基因的作用。MASPIN能从蛋白质和mRNA水平下调卵巢癌中VEGF的表达,并可能通过这一机制抑制卵巢癌血管生成。     

重组日本血吸虫亲环素B的克隆、表达及免疫分析

目的克隆、表达日本血吸虫亲环素B(SjCyPB)基因,鉴定并分析重组蛋白的免疫性。方法根据Genbank中日本血吸虫序列设计一对特异性引物,以日本血吸虫cDNA为模板扩增SjCyPB基因,酶切后连接到表达载体pET28,构建pET28a(+)-SjCyPB重组质粒,转化入感受态大肠杆菌BL21/DE3后对质粒进行双酶切和测序鉴定。IPTG诱导表达后,经亲和层析纯化重组蛋白。采用Western Blotting分析鉴定重组蛋白的抗原性。将纯化的重组蛋白免疫大鼠,获得免疫血清,ELISA检测抗SjCyPB特异性抗体滴度。结果构建的pET28a(+)-SjCyPB重组质粒经双酶切和测序鉴定证实SjCyPB基因成功连接到pET28a(+)质粒中。经原核表达后获得纯化的重组蛋白,Western Blotting结果显示,该重组蛋白可与感染日本血吸虫的兔血清结合形成明显的条带。采用ELISA检测重组蛋白免疫后大鼠免疫血清的特异性IgG抗体滴度为1∶51 200。结论成功克隆了日本血吸虫CyPB基因,并获得大量纯化的重组蛋白,重组SjCyPB具有免疫原性和抗原性。     

2型糖尿病患者血清脂肪特异性丝氨酸蛋白酶抑制剂与糖脂代谢的相关性研究

目的探讨2型糖尿病患者血清中脂肪特异性丝氨酸蛋白酶抑制剂(vaspin)与糖脂代谢的关系。方法用ELISA法测定80例2型糖尿病,69例空腹糖耐量受损患者,73例健康对照者血清中vaspin的水平,同时测定空腹血糖(FBG)、总胆固醇(TCHO)、甘油三酯(TG)、空腹胰岛素(FINS)、糖化血红蛋白(HBA1C)、超敏CRP(hsCRP),并做统计学处理。结果 2型糖尿病组vaspin水平显著高于健康对照组和空腹糖耐量受损组(P<0.05),空腹糖耐量受损组vaspin水平高于健康对照组(P<0.05)。同组患者中肥胖亚组vaspin水平高于非肥胖亚组(P<0.05),2型糖尿病组中血清vaspin与体质指数(body mas index,BMI)、FBG、FINS、HOMA-IR、TG、hsCRP呈正相关(r值分别为0.472、0.464、0.302、0.347、0.268、0.311,P<0.05)。结论 Vaspin是联系2型糖尿病糖脂代谢的重要脂肪因子,与BMI、FBG、FINS、HOMA-IR、TG、hsCRP呈正相关,可为2型糖尿病治疗提供新思路。     

有氧运动对肥胖儿童血清脂肪特异性丝氨酸蛋白酶抑制剂及网膜素-1水平的影响

目的：探讨有氧运动对肥胖儿童血清脂肪特异性丝氨酸蛋白酶抑制剂（ Vaspin ）及网膜素-1（Omentin-1）水平的影响。方法选取45例肥胖儿童作为观察组,对其采取有氧运动减肥治疗,2个月为一疗程。以30例健康体检儿童作为正常对照。酶联免疫法检测患者治疗前后血清Vaspin、Omentin-1水平,并观察治疗前后体重指数（BMI）、腰围（WC）、臀围（HC）和腰臀比（WHR）变化。结果治疗前观察组肥胖儿童体重、BMI、WC、HC、WHR及血清Vaspin、TG、TC、LDL-C水平均显著高于正常组（ P＜0.05）, Omentin-1及HDL-C水平低于正常组（ P＜0.05）;治疗后观察组儿童体重、BMI、WC、HC、WHR及血清Vaspin、TG、TC、LDL-C水平显著降低（ P＜0.05）,Omentin-1及HDL-C水平显著升高（ P＜0.05）。相关性分析显示肥胖儿童血清Vaspin水平与BMI指数、WC、WHR、TG呈正相关（ P＜0.05）;而Omentin-1与BMI指数、WC、TG呈负相关,与HDL-C呈正相关（ P＜0.05）。结论肥胖儿童存在血清Vaspin、Omentin-1水平异常;有氧运动可以显著调节二者水平,减轻体重。     

日本血吸虫童虫cDNA文库免疫学筛选及H1基因的克隆

目的　获取可能编码新的血吸虫诊断和候选疫苗分子的 c DNA克隆。　方法　利用血吸虫病患者的血清筛选日本血吸虫童虫 c DNA文库 ,获得一批阳性克隆 ,并对 H1克隆插入片段的核苷酸进行序列测定。根据结果 ,一方面进行同源性分析 ;另一方面设计合成引物 ,应用 PCR法进行扩增 ,并将其克隆入 p GEM-T载体。　结果　用抗血清初筛约 7.2× 1 0 3个重组的噬菌体 ,得到 1 2个持续阳性反应克隆 ,其中 H1克隆的插入片段长度为 1 1 86bp,具有一个 5 91 bp的完整开放阅读框 ,与现有的基因无任何同源性。利用 PCR法成功地扩增了该基因 ,并克隆入载体 p GEM-T。　结论　从 c DNA文库中筛选的对血吸虫感染可能具有保护作用的分子克隆是可行的 ,为进一步研究提供了基础。     

表达序列标签法寻找日本血吸虫新基因

目的 运用表达序列标签(expressed sequence tag，EST)技术，从日本血吸虫成虫eDNA文库中筛选和鉴定日本血吸虫新基因，为寻找日本血吸虫新的候选疫苗奠定基础。方法 转化日本血吸虫成虫eDNA文库，随机挑取重组阳性克隆进行测序，获得Esr；用NCBI站点的GenBank对所获得的新基因序列进行同源性检索；利用BLASTP程序对同源性高的基因进行核苷酸和氨基酸水平的同源性比较。结果 发现xzw000008克隆的eDNA序列为日本血吸虫新基因并获得GenBank登录号。该eDNA序列与曼氏血吸虫polyubiquitin基因高度同源，核苷酸水平的同一性为87％；氨基酸水平的同一性为98％。结论 用EST寻找日本血吸虫新基因是一种可行的方法，所筛选到的日本血吸虫新基因长575bp，编码191个氨基酸，与曼氏血吸虫polyubiquifin基因高度同源，为进一步研究该基因的全序列及功能作准备。     

日本血吸虫候选疫苗原肌球蛋白的表达载体构建、原核表达及免疫原性研究

目的 将日本血吸虫原肌球蛋白基因(tropomyosin)克隆到原核表达载体pET-28a(+)裁体上,在BL-21(DE3)型大肠埃希菌中表达其产物,并对其进行免疫原性分析.方法 从日本血吸虫的Expressed Sequence Tag(EST)文库中筛选出包含tropomyosin全长基因的EST,然后用高保真酶PCR扩增,构建到pET-280(+)裁体上,最终在BL-21(DE3)型大肠埃希菌中用异丙基-β-D-硫代半乳糖苷(IFTG)诱导表达.表达的his-tropomyosin融合蛋白用镍螯舍树脂蛋白纯化柱(Ni-NTA resin)亲和层析纯化,得到纯度较高的tropomyosin后,通过Western Blotting技术,用感染日本血吸虫的兔血清作为天然抗体来研究tropomyosi的免疫原性.结果 pET-28a(+)-tropomyosin重组质粒在BL-21(DE3)型大肠埃希菌中成功表达,使用SDS-PAGE分析得到实际Mr约为40 000的his-tropomyosin融合蛋白,Ni-NTA resin的纯化效果很明显,获得的相对纯的目的 蛋白经过Western BIotting方法 检潮显示:tropomyosin融合蛋白能够与感染日本血吸虫6 w的兔血清有较强的免疫反应.结论 Tropomyosin能够在BL-21(DE3)型大肠埃希菌中高效表达,且抗原免疫原性强,日本血吸虫感染的兔血清能够识别体外表达的tropomyosin,具有与天然的tropomyosin相同的免疫原性.     

Secretory leukocyte protease inhibitor suppresses the inflammation and joint damage of bacterial cell wall-induced arthritis.

Disruption of the balance between proteases and protease inhibitors is often associated with pathologic tissue destruction. To explore the therapeutic potential of secretory leukocyte protease inhibitor (SLPI) in erosive joint diseases, we cloned, sequenced, and expressed active rat SLPI, which shares the protease-reactive site found in human SLPI. In a rat streptococcal cell wall (SCW)-induced model of inflammatory erosive polyarthritis, endogenous SLPI was unexpectedly upregulated at both mRNA and protein levels in inflamed joint tissues. Systemic delivery of purified recombinant rat SLPI inhibited joint inflammation and cartilage and bone destruction. Inflammatory pathways as reflected by circulating tumor necrosis factor alpha and nuclear factor kappaB activation and cartilage resorption detected by circulating levels of type II collagen collagenase-generated cleavage products were all diminished by SLPI treatment in acute and chronic arthritis, indicating that the action of SLPI may extend beyond inhibition of serine proteases.     

Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis

Serine protease gene fragments approximately 480 nucleotides in length were amplified from Ctenocephalides felis larval and adult cDNA libraries using degenerate oligonucleotide PCR primers. Partial clones of thirty-eight distinct serine protease encoding sequences were isolated, and nineteen different full-length cDNAs encoding mature serine proteases were subsequently cloned and sequenced. All of the mature proteases contained the histidine, aspartic acid and serine amino acids of the catalytic triad characteristic of serine proteases. The mature C. felis serine proteases had amino acid sequences that were at most 29–53% identical to those known insect and arachnid serine proteases. Two of the C. felis gene sequences had similarity with the Drosophila melanogaster developmental genes snake and stubble. mRNA expression of selected serine protease genes was examined in different life stages, tissues, genders, and in response to bloodfeeding.     

血清内皮细胞特异分子-1、脂肪特异性丝氨酸蛋白酶抑制剂水平对2型糖尿病大血管病变诊断的临床价值

目的 探讨血清内皮细胞特异分子-1(Endocan-1)、脂肪特异性丝氨酸蛋白酶抑制剂(Vaspin)水平对2型糖尿病(T2DM)大血管病变(MVC)的临床诊断意义.方法 选取72例患者,根据是否合并大血管疾病,分为T2DM组36例和MVC组36例;此外,选同期36例健康者作为对照组.利用酶联免疫吸附检测技术(ELIS...     

Amblyomma americanum tick saliva serine protease inhibitor 6 is a cross‐class inhibitor of serine proteases and papain‐like cysteine proteases that delays plasma clotting and inhibits platelet aggregation

We previously demonstrated that Amblyomma americanum tick serine protease inhibitor 6 (AamS6) was secreted into the host during tick feeding and that both its mRNA and protein were ubiquitously and highly expressed during the first 3 days of tick feeding. This study demonstrates that AamS6 is a cross‐class inhibitor of both serine‐ and papain‐like cysteine proteases that has apparent antihaemostatic functions. Consistent with the typical inhibitory serpin characteristics, enzyme kinetics analyses revealed that Pichia pastoris‐expressed recombinant (r) AamS6 reduced initial velocities of substrate hydrolysis (V₀) and/or maximum enzyme velocity (V_max) of trypsin, chymotrypsin, elastase, chymase, and papain in a dose–response manner. We speculate that rAamS6 inhibited plasmin in a temporary fashion in that while rAamS6 reduced V₀ of plasmin by up to ～53%, it had no effect on V_max. Our data also suggest that rAmS6 has minimal or no apparent effect on V₀ or V_max of thrombin, factor Xa, and kallikrein. We speculate that AamS6 is apparently involved in facilitating blood meal feeding in that various amounts of rAamS6 reduced platelet aggregation by up to ～47% and delayed plasma clotting time in the recalcification time assay by up to ～210 s. AamS6 is most likely not involved with the tick's evasion of the host's complement defense mechanism, in that rAamS6 did not interfere with the complement activation pathway. Findings in this study are discussed in the context of expanding our understanding of tick proteins that control bloodmeal feeding and hence tick‐borne disease transmission by ticks.     

A serine protease homologue Bombyx mori scarface induces a short and fat body shape in silkworm

Body shape is one of the most prominent and basic characteristics of any organism. In insects, abundant variations in body shape can be observed both within and amongst species. However, the molecular mechanism underlying body shape fine‐tuning is very complex and has been largely unknown until now. In the silkworm Bombyx mori, the tubby (tub) mutant has an abnormal short fat body shape and the abdomen of tub larvae expands to form a fusiform body shape. Morphological investigation revealed that the body length was shorter and the body width was wider than that of the Dazao strain. Thus, this mutant is a good model for studying the molecular mechanisms of body shape fine‐tuning. Using positional cloning, we identified a gene encoding the serine protease homologue, B. mori scarface (Bmscarface), which is associated with the tub phenotype. Sequence analysis revealed a specific 312‐bp deletion from an exon of Bmscarface in the tub strain. In addition, recombination was not observed between the tub and Bmscarface loci. Moreover, RNA interference of Bmscarface resulted in the tub‐like phenotype. These results indicate that Bmscarface is responsible for the tub mutant phenotype. This is the first study to report that mutation of a serine protease homologue can induce an abnormal body shape in insects.