脐血的细胞酶类研究

目的：检测脐血的细胞酶类，拟为脐血的临床应用提供有价值的实验室数据。方法：采用组化技术对３０例正常足月产儿脐血细胞的碱性磷酸酶（ＡＬＰ）、过氧化物酶（ＰＯＸ）、非特异性酯酶（ＮＡＥ）、酸性非特异性酯酶（ＡＮＡＥ）进行测定。结果：脐血ＡＬＰ阳性率为４６．６７±１８．５２％，积分６０．７３±２５．７１；ＰＯＸ阳性率为７６．２３±８．３９％，积分１６７．２７±３０．５９；ＮＡＥ阳性率为７．４７±４．７８％；ＡＮＡＥ（单核细胞）阳性率为５．６７±３．０３％，ＡＮＡＥ（淋巴细胞）阳性率为５７．３３±１３．６２％。结论：脐血的细胞酶类与成人骨髓细胞及外周血细胞化学有差异。

细胞酶类的选择性抑制剂或成为科学家研究利器

近日，来自斯克利普斯研究所的研究人员通过研究发现了一系列重要酶类的第一个选择性抑制剂，这种新型的抑制剂以及化学探针可以被用于研究俗称为二酰基甘油脂酶（DAGL）的功能、产物以及调节途径。此前在小鼠巨噬细胞中的研究表明，DAGL的抑制化合物或许存在治疗的效用，因为其可以抑制风湿性关节炎和其相关疾病发病过程中前炎性分子的产生。

血液回收对红细胞ATP含量及葡萄糖6-磷酸脱氢酶活性的影响

目的探讨术中自体血液回收对红细胞ATP含量及葡萄糖6磷酸脱氢酶(G6PD)活性的影响。方法选择心血管外科术中出血量>600ml患者50名,用血液回收机行自体血液回收,取患者术野及回收的红细胞各6ml,另取50份库存2周的异体红细胞悬液6ml,分别检测红细胞ATP含量及G6PD活性。结果回收组和库存组ATP含量、G6PD活性与术野组相比均明显下降(P<0.01),但仍在正常值范围;回收组ATP含量及G6PD活性与库存组相比,差异无显著性。结论血液回收处理可使红细胞ATP含量减少,使G6PD活性降低,但未达影响红细胞功能的程度,回收红细胞与库存红细胞相比差别不明显。

七种临床常用酶细胞化学成套试剂的研制

目的：将国际标准的、稳定可靠的七种酶及糖原细胞化学染色试剂研制为盒式成套试剂。方法：将酶显示试剂分装在安瓿，长期存放，与新鲜配制试剂比较酶显示结果，并在全国２０多家医院使用。结果：与新鲜配制试剂比较、试剂盒经存放６ｍｏ以上，酶显示结果无显著性差异。结论：盒式成套试剂操作简便，省时、省力，提高了实验结果的稳定性，特别是对细胞化学中各种酶的活性检测及质量控制提供了统一的标准。

烟雾暴露对孕大鼠肝脏和胎盘细胞色素P450同工酶表达的影响

目的 研究烟雾暴露对孕大鼠肝脏和胎盘细胞色素P450（CYP）同工酶表达的影响，探讨孕期烟雾暴露的毒理学意义。方法 7只Wistar雌大鼠和14只孕大鼠分为正常未孕组、正常受孕组和吸烟受孕组。从妊娠d9起，吸烟组孕大鼠在特制的半封闭动物饲养箱中熏烟，每日3次（8：00am，12：00am，4：00pm），每次持续15—20min。孕d21剖腹取母体肝脏和胎盘，提取RNA，RT-PCR技术测定CYP同工酶表达的变化。结果 与正常未孕组相比，正常受孕大鼠肝脏CYP1A1，1B1，2B1／2，2E1，3A1mRNA的表达分别为75．1％（P〈0．05），117．2％（P〈0．01）。131．6％。130．5％和85．1％（P〈0．05）；与正常受孕组相比，吸烟孕大鼠肝脏CYP1A1，1B1，2B1／2，2E1，3A1mRNA表达分别为128．9％（P〈0．05），96．4％，127．7％。101．8％及175．5％（P〈0．05）；同时胎盘CYP1A1，1B1，2B1／2及2E1mRNA表达分别为127．1％（P〈0．05），116．5％，90．1％及150．2％（P〈0．01）。结论 孕期烟雾暴露可影响孕大鼠肝脏和胎盘中一些与外源物代谢密切相关的CYP同工酶表达。

云南省中性粒细胞明胶酶相关脂质运载蛋白参考区间的建立

目的建立云南省红河哈尼族彝族自治州蒙自地区采用胶乳免疫比浊法测定血浆中性粒细胞明胶酶相关脂质运载蛋白(NGAL)时的参考区间。方法选取2018年1-12月该院健康体检者1085例,按年龄分为小于18岁组(48例)、18~<30岁组(148例)、30~<40岁组(212例)、40~<50岁岁组(298例)、50~<60组(237例)和大于或等于60岁组(142例)。分析各组不同年龄、不同性别间NGAL水平变化,建立各年龄段NGAL水平参考区间。结果<18岁组NGAL水平分别与其他组比较,差异均有统计学意义(P<0.05)。≥60岁组NGAL水平分别与18~<30岁、30~<40岁、40~<50岁、50~<60岁组比较,差异均有统计学意义(P<0.05)。各组不同性别间NGAL水平比较,差异均无统计学意义(P>0.05)。未成年人、成年人和老年人血浆NGAL的参考区间依次为17.89~88.93、25.85~91.17、23.13~110.49ng/mL。结论健康人群血浆NGAL水平与年龄相关,不同地区实验室应按照年龄确定本地区的参考区间,以满足临床要求。

高原鼠兔肺动脉血管功能及形态变化

目的 :研究肺循环对慢性缺氧的适应机理。方法 :在 430 0m的高度捕捉到高原适应动物鼠兔 ,带到 2 2 6 0m的高度并和 10只Wistar大鼠在模拟 430 0m和 5 0 0 0m高度的低压仓内进行了肺动脉压的测定 ,观察肺组织学和组织免疫化学的改变。结果 :在 2 2 6 0m ,鼠兔的Ppa明显低于Wistar大鼠 ,二者分别为 (1.5± 0·0 7)kPa和 (2 .9±1.1)kPa(P <0 .0 1)。随着海拔高度的增加 ,鼠兔的Ppa上升不明显 ,而Wistar大鼠增加显著。左右心室比重鼠兔为 0 .2 2 ,而Wistar大鼠为 0 .45。鼠兔的Hb ,Hct和 2 .3 DPG均低于大鼠。大鼠肺小血管周围可见肥大细胞 (7.1±0 .33)mm2 ,免疫组化染色mastcelltyptase颗粒呈阳性 ,鼠兔未发现肥大细胞及此种免疫反应。肺小动脉中层较鼠兔厚 ,分别为 2 7.2 1%和 9.2 2 % ,壁的厚度和Ppa有很好的 (r =0 .76 3)。 结论 :鼠兔无低氧性肺血管收缩 ,是一种遗传性适应。大鼠肥大细胞通过激活某些生长因子 。

复方丹参对全脑缺血后再灌注损伤的影响

为观察复方丹参对全脑缺血后再灌注损伤的影响,给大鼠腹腔注射复方丹参注射液(0.2ml/100g),15min 后阻断基底动脉和两侧颈总动脉60min,随后恢复血液供应连续观察60min。结果表明,与再灌组比较,丹参组脑组织 Na^+含量显著降低(P<0.01),K^+含量显著增高(P<0.01),乳酸脱氢酶和磷酸肌酸激酶活性显著增高(P<0.01,P<0.01),乳酸积聚和丙二醛含量显著降低(P 均<0.01)。病理检查发现,脑细胞肿胀及间质水肿与神经元变性显著减轻。表明复方丹参对全脑再灌注损伤有明显保护作用。

The IκB kinase- a bridge between inflammation and cancer

A potential link between inflammation and cancer has been suspected for over a century, but the exact molecular mechanisms connecting the two remained nebulous. We proposed that NF-κB transcription factors regulated via the IκB kinase （IKK） complex play a critical role in coupling inflammation and cancer and have set out to test this hypothesis in mouse models of cancer. Using mice bearing mutations in the genes coding for the IKKβ and IKKα catalytic subunits we obtained evidence supporting a critical role for IKKβ in tumor promotion and more recently identified the involvement of IKKα in metastatogenesis. Whereas the major pro-tumorigenic function of IKKβ is mediated via NF- κB, the proetastatic function of IKKα is NF-κB-independent. In addition to illustrating the critical roles of the two IKK molecules in linking inflammation and cancer and providing an explanation for increased cancer risk in response to persistent infections and inflammation, these results also identify new targets for development of novel anti-cancer therapies and preventive strategies. Instead of targeting the cancer cell itself, as done by conventional anti-cancer drugs, the new therapeutics will target processes that occur within inflammatory cells that are essential for cancer development and progression. Unlike cancer cells, inflammatory cells retain a normal and stable genome and therefore are unlikely to become genetically resistant to therapeutic intervention.

Legalon-SIL downregulates HCV core and NS5A in human hepatocytes expressing full-length HCV

AIM: To determine the effect of Legalon-SIL (LS) on hepatitis C virus (HCV) core and NS5A expression and on heme oxygenase-1 (HMOX-1) and its transcriptional regulators in human hepatoma cells expressing full length HCV genotype 1b. METHODS: CON1 cells were treated with 50 μmol/L or 200 μmol/L LS. Cells were harvested after 2, 6 and 24 h. HCV RNA and protein levels were determined by quantitative real-time polymerase chain reaction and Western blotting, respectively. RESULTS: HCV RNA (core and NS5A regions) wasdecreased after 6 h with LS 200 μmol/L (P < 0.05). Both 50 and 200 μmol/L LS decreased HCV RNA levels [core region (by 55% and 88%, respectively) and NS5A region (by 62% and 87%, respectively) after 24 h compared with vehicle (dimethyl sulphoxide) control (P < 0.01). Similarly HCV core and NS5A protein were decreased (by 85%, P < 0.01 and by 65%, P < 0.05, respectively) by LS 200 μmol/L. Bach1 and HMOX-1 RNA were also downregulated by LS treatment (P < 0.01), while Nrf2 protein was increased (P < 0.05).CONCLUSION: Our results demonstrate that treatment with LS downregulates HCV core and NS5A expression in CON1 cells which express full length HCV genotype 1b, and suggests that LS may prove to be a valuable alternative or adjunctive therapy for the treatment of HCV infection.

Bcl-2 cleavages at two adjacent sites by different caspases promote cisplatin-induced apoptosis

The protein encoded by bcl-2 proto-oncogene plays an important role in the mitochondria-mediated apoptotic pathway. Although the general role of Bcl-2 is anti-apoptotic, previous work showed that Bcl-2 fragments cleaved by caspases could promote apoptotic process. We report herein that Bcl-2 protein was cleaved to produce two fragments of around 23 kDa in human hepatocarcinoma BEL-7404 cells or in Bcl-2 overexpressing CHO cells induced by cisplatin. Treating cells with the general caspase inhibitor z-VAD-fmk blocked the induced cleavage of Bcl-2. Mutagenesis analyses showed that Bcl-2 was cleaved by caspases at two adjacent recognition sites in the loop domain （YEWD31↓AGD34↓V）, which could be inhibited by caspase-8 and -3 inhibitors, respectively. Overexpression of the carboxyl terminal 23 kDa fragments increased the sensitivity of CHO cells to cisplatin-induced apoptosis. These results indicate that Bcl-2 can be cleaved into two close fragments by different caspases during cisplatin-induced apoptosis, both of which contribute to the acceleration ofapoptotic process.

The Influence of Magnetite Nanoparticles on Human Leukocyte Activity

In this contribution the influence of chemically synthesized magnetite particles coated by sodium oleate and PEG （MPEG）, and magnetosomes （MS） was gradually tested on the process of phagocytosis and the metabolic activity （lysozyme and peroxidase activity） in leukocyte. Lysozyme activity is oxygen-independent liquidation mechanisms of engulfed microorganism, peroxidase activity is oxygen-dependent one. The both tested samples MS and MPEG lysed leukocyte cells during incubation. MPEG with concentration 10 and 20 μg/mL lysed almost all leukocytes and their cell viability was in the 14 ± 0.05% range. On the other hand, MS begin to influence leukocytes activity at the concentration of 1 μg/mL and this influence grows with increasing concentration up to 20 μg/mL. MS are more suitable for biological applications than MPEG which are more aggressive material than MS and their using is unavailable for these types of the test mainly for the concentration 10 - 20 μg/mL.

Decreased mitochondrial deoxyribonucleic acid and increased oxidative damage in chronic hepatitis C

AIM： To determine whether alteration of the mito- chondria DNA （mtDNA） copy number and its oxidative damage index （mtDNA△CT） can be detected by analysis of peripheral blood cells in hepatitis C virus （HCV）- infected patients. METHODS： This study enrolled two groups of pa- tients aged 40-60 years： a control group and an HCV- infected group in Department of Gastroenterology and Hepatology in Changhua Christian Hospital. Patients with co-infection with hepatitis B virus or human im- munodeficiency virus, autoimmune disease, malignant neoplasia, pregnancy, thyroid disease, or alcohol con- sumption 〉 40 g/d were excluded. HCV-infected pa- tients who met the following criteria were included： （1） positive HCV antibodies for 〉 6 mo; （2） alanine aminotransferase （ALT） levels more than twice the upper lim- it of normal on at least two occasions during the past 6 mo; and （3） histological fibrosis stage higher than F1. The mtDNA copy number and oxidative damage index of HCV mtDNA （mtDNA△CT） were measured in periph- eral blood leukocytes. The association between mtDNA copy number and mtDNA△CT was further analyzed using clinical data. RESULTS： Forty-seven normal controls （male/female： 26/21, mean age 50.51 ± 6.15 years） and 132 HCV- infected patients （male/female： 76/61, mean age 51.65 ± 5.50 years） were included in the study. The geno- types of HCV-infected patients include type 1a （n = 3）, type 1b （n = 83）, type 2a （n = 32）, and type 2b （n = 14）. Liver fibrosis stages were distributed as follows： F1/F2/F3/F4 = 1/61/45/25 and activity scores were A0/ A1/A2/A3 = 7/45/55/25. There were no age or gender differences between the two groups. HCV-infected pa- tients had higher hepatitis activity （aspartate transami- nase levels 108.77 ± 60.73 vs 23.19 ± 5.47, P 〈 0.01; ALT levels 168.69 ± 93.12 vs 23.15 ± 9.45, P 〈 0.01） and lower platelet count （170.40±58.00 vs 251.24 ± 63.42, P 〈 0.01） than controls. The mtDNA copy num- ber was lower in HCV-infected patients than in controls （173.49 vs 247.93, P 〈 0.05）. The mtDNA△CT was higher in HCV-infected patients than in controls （2.92 vs 0.64, P 〈 0.05）. To clarify the clinical significance of these results in HCV-infected patients, their association with different clinical parameters among HCV-infected pa- tients was analyzed. A negative association was found between mtDNA copy number and elevated aspartate transaminase levels （r = -0.17, P 〈 0.05）. Changes in mtDNA copy number were not associated with HCV RNA levels, HCV genotypes, liver fibrosis severity, or inflammatory activity in the liver biopsy specimen. How- ever, a correlation was observed between mtDNA△CT and platelet count （r = -0.22, P 〈 0.01）, HCV RNA level （r = 0.36, P 〈 0.01）, and hepatitis activity （r = 0.20, P = 0.02）. However, no difference in the change in mtDNA△CT was observed between different fibrosis stages or HCV CONCLUSION： Oxidative stress and mtDNA dam- age are detectable in patient＇s peripheral leukocytes. Increased leukocyte mtDNA△CT correlates with higher HCV viremia, increased hepatitis activity, and lower platelet count.

Genetic identification of two species of Pleuronichthys by DNA barcoding

DNA barcoding is a new method for biological taxonomy, offering the ability to identify species from fragments in any life-history stage. Pleuronichthys cornutus and P. japonicus are two morphologically similar species. Pleuronichthys japonicus has never been found previously in China. However, in this study, we identified both species using DNA barcoding （cytochrome c oxidase subunit I （COI））, the mtDNA control region and cytochrome b. The results reveal that： l） intraspecific variation in the DNA barcode is much less than interspecific variation; 2） the two morphologically similar species were placed into separate clades distinguishable by high bootstrap values; 3） COI barcodes are more powerful for identifying the two species than the other two mtDNA fragments.

Role of cyclooxygenase-2 in lipid metabolism in hepatic stellate cells

Hepatic stellate cells(HSCs) are a kind of adipocytes. In HSCs lipids mainly exist in the form of lipid droplets. They are abundantly found in the cytoplasm and their main constituents are triglycerides. Lipid metabolism in HSCs is closely related to its biological activity, however the mechanism of lipid droplets disappearance after HSC activation is not clearly established yet. Recent research shows that, cyclooxygenase-2 plays an important regulatory role in the lipid metabolism of HSCs. This paper seeks to review the subject based on studies that have been conducted so far to understand the role of cyclooxygenase-2 in the metabolism of lipids in HSCs.

Increased Activity of the Human Telomerase Catalytic Subunit Promoter by the VEGF Enhancer in Human Cancer Cells

We attempted to improve the activity of hTERT promoter by fusing the vascular endothelial growth factor （VEGF） enhancer. To determine the potential as cancer specific promoters, we measured the reporter gene transfection assay driven by the hTERT promoter and the VEGF enhancer in human cancer cells. We found that the hTERT promoter containing VEGF enhancer conferred strong expression of the reporter gene only in different cancer cell lines but not in normal human cells. Retrovirus vector expressing HSV-TK controlled by the hTERT promoter and the VEGF enhancer was constructed. A549 cells infected with LN-enh-hT-TK was significantly suppressed and induced to apoptosis more than those infected with LN-hT-TK. The apoptosis ratio ofA549 cell infected with two kinds of retrovirus cell with GCV in lower concentration is 20.94% and 50.7%. It suggested that there is significant differentiation between the assay groups. Our results demonstrated the possible application of hTERT promoter and the VEGF enhancer in targeted cancer gene therapy.

Functional study of p38 mitogen-activated protein kinase based on cell-penetrating peptide delivery system

Objective p38 Mitogen-activated protein kinase （MAPK） is a crossing center of various pathways. In this study, protein transduction system based on human immunodeficiency virus （HIV）-1 transactivator of transcription （TAT）, which is an efficient delivery peptide of the foreign proteins into cells, was employed to study p38 MAPK functions in eukaryotic cells. Methods p38 And its dominant negative form, p38AF, were constructed into pET-His-TAT vector correctly to verify that the recombinant plasmids were well-founded through restriction enzyme digestion and DNA sequencing. The two proteins, His-TAT-p38 and His-TAT-p38AF, were expressed and purified in Escherichia coli by SDS-PAGE. Then they were incubated with ECV304 cells respectively and readily transduced into cells in a time-dependent and dose-dependent manner. The cells were stimulated by sorbitol. Activating transcription factor （ATF） 2 phosphorylation level was checked using Western blot to assess the activity of endogenous p38. Results Compared with controls, it was found that His-TAT-p38 increased the level ofATF2 phosphorylation in sorbitol-stimulated ECV304 cells, while His-TAT-p38AF inhibited it, indicating p38 MAPK protein delivery system based on TAT was constructed successfully. TAT-p38 and its dominant negative form possessed high biological activity after transduction into ECV304 cells by TAT protein delivery system. The results showed that p38AF fused with TAT could inhibit the transduction of endogenous p38 signal pathway in part, and other pathway might regulate p38 phosphorylation. Conclusions Our study provides a novel pathway to inhibit p38 signal pathway and establish a new method to study p38 function.

首诊为Usher综合征的轻型Zellweger谱系障碍1例

患儿女性,5岁,因“夜盲、听力差1年”就诊,因视网膜色素变性合并感音神经性耳聋,首诊为Usher综合征。目标区域捕获测序分析发现患儿携带Zellweger谱系障碍致病基因PEX1复合杂合变异c.5G>A,p.W2*/c.3022C>T,p.P1008S。后期随访发现患儿恒牙釉质发育不良,指甲白斑,过氧化物酶体功能生化异常,诊断为轻型Zellweger谱系障碍。