Prokaryotic Expression and Identification of Outer Membrane Protein 2 of Chlamydia trachomatis

Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 X His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coli M15, providing an efficient and simple system for assaying the immunological properties of Omp2.

Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T

AIM： To screen the immunogenic membrane proteins of Shigella Aexneri 2a 2457T. METHODS： The routine two-dimensional polyacrylamide gel electrophoresis （2-DE） and Western blotting were combined to screen immunogenic proteins of S. Aexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry （MALDI-TOF-MS） was performed to determine the molecular weight of peptides. Electrospray ionization （ESI-MS/MS） was performed to determine the sequences of the interesting peptides. RESULTS： A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein （YaeT） was detected, which might be a candidate target of vaccine. CONCLUSION： Membrane proteins of S. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.

Recombinant Vaccinia Virus is an Effective and Non-perturbing Vector for Human Dendritic Cells Transfected with Epstein-Barr Virus Latent Membrane Protein 2A

ObjectiveTo study the effects of dendritic cells (DC) transfected with recombinant vaccinia virus encoding Epstein Barr virus (EBV) latent membrane protein 2A(LMP2A) gene,and to provide evidence for further investigation on the therapeutic vaccines against EBV associated malignancies. MethodsMature DC were transfected with EBV LMP2A recombinant vaccinia virus (rVV LMP2A). Before and after the transfection,the expression of surface antigens on mature DC including CD1a,CD83,CD40,CD80,HLA DR was measured by fluorescence activated cell sorter (FACS) and the function of DC to stimulate allogeneic T cells proliferation was measured by mixed leukocyte reactions (MLR). ResultsLMP2A protein was highly expressed (66.1 %) in DC after the transfection of rVV LMP2A. No significant changes in the primary surface antigens expression and in the MLR were detected during the transfection. Transfected DC still had strong potential in stimulating the proliferation of allogeneic T cells. ConclusionRecombinant vaccinia virus was an effective and non perturbing vector to mediate the transfection of LMP2A into DC. The functions of mature DC were not affected significantly by the transfection of Vac LMP2A. This study could provide evidence for the further immunotherapy of EBV associated malignancies,e.g. nasopharyngeal carcinoma (NPC).

Differential expression and regulation of integral membrane protein 2b in rat male reproductive tissues

Aim： To examine the expression and regulation of integral membrane protein 2b （Itm2b） in rat male reproductive tissues during sexual maturation and under different treatments by in situ hybridization. Methods： Testis, epididymis, and vas deferens were collected on days 1-70 to examine Itm2b expression during sexual maturation. To further examine the regulation of Itm2b, adult rats underwent surgical castration and cryptorchidism. Ethylene dimethane sulfonate and busulfan treatments were carried out to test the regulation of Itm2b after destruction of Leydig cells and germ cells. Results： In testis, Itm2b expression was moderately detected in the adluminal area of seminiferous cords on days 1-10, and detected at a low level in the spermatogonia on days 20 and 30. The Itm2b level was markedly increased in Leydig cells from day 20 to day 70. In epididymis and vas deferens, Itm2b was detected from neonate to adults, and the signal gradually increased in accordance with sexual maturation. Itm2b expression was significantly downregulated in epididymis and vas deferens of castrated rats, and strongly stimulated when castrated rats were treated with testosterone. Cryptorchidism led to a significant decline of Itm2b expression in testis and caput epididymis. Itm2b expression in epididymis and vas deferens was significantly decreased after the Leydig ceils were destroyed by ethylene dimethane sulfonate. Busulfan treatment produced no obvious change in Itm2b expression in epididymis or vas deferens. Conelusion： Our data suggested that Itm2b expression is upregulated by testosterone and might play a role in rat male reproduction.

血清IL-2、sST2表达与特发性膜性肾病免疫抑制剂治疗反应性的相关性

目的探讨特发性膜性肾病患者血清白介素-2(IL-2)、可溶性生长刺激表达基因2蛋白(sST2)表达水平与免疫抑制剂治疗反应性的相关性。方法选取2020年1月至2022年10月于医院接受免疫抑制剂治疗的135例特发性膜性肾病患者,于入院时检测患者血清IL-2、sST2,并于治疗完成后测定24 h尿蛋白定量,依据患者治疗反应性分为缓解组与未缓解组。对比两组患者一般资料及入院时血清IL-2、sST2水平,采用点二列相关性分析血清IL-2、sST2水平与特发性膜性肾病免疫抑制剂治疗反应性的关系,并绘制受试者工作特征(ROC)曲线评估血清IL-2、sST2水平预测特发性膜性肾病免疫抑制剂治疗反应性的价值。结果135例特发性膜性肾病患者中共有132例完成规律治疗,经免疫抑制剂治疗6个月后,101例患者疾病缓解,纳入缓解组,其余31例患者纳入未缓解组。未缓解组年龄、入院时肾功能分级、疾病分期、血清IL-2、sST2水平均高于缓解组,差异有统计学意义(P<0.05);点二列相关性分析显示,血清IL-2、sST2水平与特发性膜性肾病免疫抑制剂治疗反应性不良风险呈正相关(r 1=0.428,P 1<0.001;r 2=0.344,P 2<0.001);绘制ROC曲线,结果显示,血清IL-2、sST2预测特发性膜性肾病免疫抑制剂治疗反应性不良的曲线下面积均>0.7,具有一定预测价值,且联合预测价值更高。结论血清IL-2、sST2表达水平与特发性膜性肾病患者免疫抑制剂治疗反应性密切相关,二者表达水平越高,治疗反应性越差,且联合检测可作为预测特发性膜性肾病患者免疫抑制剂治疗反应性的敏感指标。

穿山龙总皂苷对膜性肾病大鼠肾组织M型PLA2R和IgG4表达的影响及其机制

目的 分析穿山龙总皂苷对膜性肾病大鼠肾组织M型磷脂酶A2受体(Phospholipase A2 receptor, PLA2R)和免疫球蛋白G亚型4(Immunoglobulin G4,IgG4)表达影响及可能机制。方法 将40只SPF级雄性SD大鼠按随机数字表法分为对照组、模型组、贝那普利组(10 mg/kg)、低和高剂量穿山龙总皂苷组(80 mg/kg、160 mg/kg),每组各8只。除对照组,其余4组采用Border法制备膜性肾病模型,造模成功后,贝那普利组灌胃给予贝那普利10 mg/(kg·d),低和高剂量穿山龙总皂苷组分别灌胃给予穿山龙总皂苷80 mg/(kg·d)、160 mg/(kg·d),对照组、模型组灌胃给予10 ml/(kg·d)生理盐水。连续给药4周后,检测24 h尿蛋白、白蛋白、血肌酐、血尿素氮、尿酸水平,HE染色观察肾脏病理改变,蛋白免疫印迹法检测肾脏中M型PLA2R、IgG4、磷酸化磷脂酰肌醇3-激酶(Phosphorylated phosphoinositide 3-kinase, p-PI3K)、磷酸化蛋白激酶B(Phosphorylated protein kinase B,p-AKT)、核因子E2相关因子2(Nuclear factor E2-related factor 2,Nrf2)、血红素加氧酶(Heme oxygenase-1,HO-1)表达水平。结果 与模型组比较,贝那普利组、高剂量穿山龙总皂苷组白蛋白水平明显升高,血肌酐、血尿素氮、尿酸水平明显降低,差异均有统计学意义(P>0.05)。与模型组比较,贝那普利组、低剂量和高剂量穿山龙总皂苷组肾脏病理改变明显改善,24 h尿蛋白水平及肾脏中M型PLA2R、IgG4、p-PI3K、p-AKT表达水平明显降低,肾脏中Nrf2、HO-1表达水平明显增加,差异均有统计学意义(P<0.05)。结论 穿山龙总皂苷对膜性肾病大鼠的肾脏具有保护作用,其机制可能与降低PLA2R、IgG4表达,抑制PI3K/AKT通路,激活Nrf2/HO-1通路相关。

JTE-522-induced apoptosis in human gastric adenocarinoma cell line AGS cells by caspase activation accompanying cytochrome C release,membrane translocation of Bax and loss of mitochondrial membrane potential

AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.

Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting

Objective To identify the sperm membrane proteins that are associated with antisperm antibody Methods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody. Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5～6.2, 4.6,5.1,5.5～5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher. Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two- dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody.

特发性膜性肾病患者血清ROCK2、renalase水平及其诊断价值

目的探讨Rho相关卷曲螺旋形成蛋白激酶2(ROCK2)、肾胺酶(renalase)在特发性膜性肾病(IMN)患者血清中的表达变化及其对IMN的诊断价值。方法选取2019年1月—2020年11月河北以岭医院肾病科收治IMN患者120例为IMN组,另选取同期医院进行健康体检的志愿者120例为健康对照组。酶联免疫吸附法(ELISA)检测受试者血清ROCK2、renalase水平,Pearson法分析ROCK2、renalase水平与部分临床指标的相关性;采用受试者工作特征(ROC)曲线分析血清ROCK2、renalase及二者联合诊断IMN的价值。结果IMN组ROCK2、renalase水平均显著高于健康对照组(t/P=12.837/<0.001、11.066/<0.001)。Ⅰ期、Ⅱ期、Ⅲ期、Ⅳ期IMN患者血清ROCK2逐次升高(F/P=77.154/<0.001),而Ⅰ期、Ⅱ期、Ⅲ期血清renalase水平逐次升高,但Ⅳ期血清renalase水平低于Ⅲ期(F/P=163.042/<0.001)。Alb与血清ROCK2、renalase水平呈负相关(r/P=-0.302/0.009、-0.402/<0.001),PLA2R抗体、BUN、SCr、24 h尿蛋白与血清ROCK2、renalase水平均呈正相关(r/P=0.336/<0.001、0.264/0.011、0.315/0.007、0.320/<0.001,0.359/<0.001、0.284/0.010、0.420/<0.001、0.412/<0.001);ROC曲线显示:血清ROCK2、renalase及二者联合诊断IMN的AUC分别为0.908、0.907、0.965,二者联合优于各自单独预测效能(Z=3.597,3.755,P均<0.001)。结论ROCK2与renalase在IMN患者血清中均高表达,二者联合诊断IMN的价值较高。

Bioabsorbable Barrier Membrane Combined with rhBMP-2 Improved Bone Formation in an Experimental Model of Compromised Healing But Was Not Superior to rhBMP-2 Alone

Objective: Bioabsorbable barrier membranes placed over alveolar ridge bone defects are routinely used in dental surgery to promote bone formation. Combining these osteoconductive membranes with osteoinductive Bone Morphogenetic Proteins could prove useful in long bone fracture treatment. The hypothesis was tested in a clinically relevant model of compromised healing. Methods: Four groups of 8 rabbits underwent unilateral mid-tibial osteotomy, excision of periosteum and endosteum, and plate fixation. One group had rhBMP-2 deposited between the bone ends and Membrane wrapped around the osteotomy, the second group had Membrane wrapped around the osteotomy, the third group had rhBMP-2 placed between the bone ends, and the fourth group received no additional treatment. Results: After 7 weeks, callus size and blood flow were significantly higher in the Membrane+rhBMP-2 group than in the rhBMP-2 treated group, but torsion to failure test showed no significant difference. Membrane treatment and no treatment led to non-union. Conclusion: Absorbable barrier membrane combined with rhBMP-2 enhances bone formation, but has no advantage to rhBMP-2 alone. Membrane alone wrapped around the osteotomy was unable to prevent non-union formation.

Membrane Proteins as Potential Colon Cancer Biomarkers: Verification of 4 Candidates from a Secretome Dataset

Colorectal cancer (CRC) is an important health issue in Taiwan. There were over ten thousand newly diagnosed CRC patients each year. The outcome of late stage CRC still remains to be improved, and tumor markers are expected to improve CRC detection and management. From a colorectal cancer cell secretome database, we chose four proteins as candidates for clinical verification, including tumor-associated calcium signal transducer 2 (TROP2, TACSTD2), transmembrane 9 superfamily member 2 (TM9SF2), and tetraspanin-6 (TSPAN6), and tumor necrosis factor receptor superfamily member 16 (NGFR). Different groups of 30 CRC patients’ tissue samples collected from Chang Gung Memorial Hospital were analyzed by immunohistochemistry (IHC) for the four proteins, and the results were scored by pathologist. For all the four candidate proteins, marked differences of IHC score existed between tumor and adjacent non-tumor counterpart. However, there were only trends between higher protein expression levels and worse outcome. Three proteins (TROP2, TM9SF2 and NGFR) had trends between higher tissue expression and tumor stage or lymph node metastasis. Our study revealed that tissue expression of four proteins (TROP2, TM9SF2, TSPAN6, and NGFR) was markedly different between tumor and adjacent non-tumor counterparts. Overexpression of all these four proteins showed some trends with poorer survival.

磷酸钙复合重组人骨形态发生蛋白2修复和重建胫骨感染性骨缺损

背景:尽管Masquelet技术在临床的应用取得了广泛成功,但目前优化该技术各个环节的研究仍在不断开展,其中加快植骨后骨愈合速度、缩短骨愈合时间是医生关注的焦点。目的:观察磷酸钙复合重组人骨形态发生蛋白2材料修复胫骨感染性骨缺损的效果。方法:选择2017年6月至2022年6月简阳市人民医院收治的感染性胫骨缺损患者31例,均接受Masquelet技术分期治疗,二期手术根据植骨材料的不同分为对照组(n=15)与研究组(n=16),对照组植入异体骨/自体骨颗粒,研究组植入磷酸钙复合重组人骨形态发生蛋白2/自体骨颗粒。二期术后6个月,检测患者外周血白细胞数、C-反应蛋白、血沉等炎性指标,记录影像学骨愈合时间、骨愈合X射线评分、骨缺损愈合分级和邻近关节功能,观察是否存在钉道感染、植入吸收、取骨区疼痛、感染等情况。结果与结论:①两组患者二期术后6个月外周血白细胞计数、血沉、C-反应蛋白水平均低于一期术前(P<0.05),两组间各指标比较差异无显著性意义(P>0.05);②研究组骨愈合时间短于对照组(P<0.05);③研究组患者二期术后6个月的骨愈合X射线评分高于对照组(P<0.05),骨缺损愈合优良率与邻近关节功能优良率均高于对照组(P<0.05);两组间感染复发率与并发症发生率比较差异均无显著性意义(P>0.05);④结果表明,Masquelet技术二期术中应用磷酸钙复合重组人骨形态发生蛋白2治疗胫骨感染性骨缺损的愈合效果良好、安全性高。

LMP-1、Bcl-2表达与鼻咽癌侵袭转移的相关性

目的分析鼻咽癌组织中潜伏膜蛋白(LMP)-1、B淋巴细胞瘤(Bcl)-2基因与其侵袭转移的关系。方法选择120例NPC患者进行研究,使用鼻内镜采集病灶组织,采用免疫组织化学法检测组织中LMP-1、Bcl-2的表达,根据TNM分期评估NPC肿瘤侵袭和转移程度,分析LMP-1、Bcl-2与NPC侵袭和转移的关系。结果120例NPC患者中LMP-1阳性共86例,占71.67%,Bcl-2阳性共90例,占75.00%;LMP-1、Bcl-2阳性患者中EBV-DNA阳性占比居高,与LMP-1、Bcl-2阴性者相比,差异有统计学意义(P<0.05)。TNM不同分期患者LMP-1、Bcl-2阳性表达率相比较,差异有统计学意义(P<0.05)。采用卡方Phi和Cramer's V系数检验发现,NPC组织中LMP-1、Bcl-2表达与肿瘤侵袭和转移均显著相关(P<0.05)。结论LMP-1、Bcl-2在NPC组织中阳性表达率普遍较高,且LMP-1、Bcl-2阳性表达与肿瘤侵袭和转移有关。

Artificial Nucleic Acid Tractor-Directed Simultaneous Depletion of Oncogenic Membrane Proteins Without Hijacking Proteolysis-Specific Actuator

Targeted protein degradation(TPD)is an emerging tool for degrading proteins of interest,which affords an attractive modality for cancer therapy.However,the present TPD technologies must engage a proteolysis-specific actuator to initiate degradation of targeted proteins in the proteasome or lysosome.Herein,we report an artificial tractor that can induce endocytosis-mediated protein depletion without hijacking a proteolysis-specific actuator.In this design,bispecific aptamer chimeras(BSACs)are established,which can bridge human epidermal growth factor receptor 2(ErbB-2),an important biomarker in a common important biomarker in cancer,with membrane proteins of interest.Taking advantage of the property of aptamer-induced endocytosis and digestion of ErbB-2,another membrane protein is translocated into the lysosome in a hitchhike-like manner,resulting in lysosomal proteolysis along with ErbB-2.This strategy frees the TPD from the fundamental limitation of proteolysis-specific actuator and allows simultaneous regulation of the quantity and function of two oncogenic receptors in a cell-type-specific manner,expanding the application scope of TPD-based therapeutics.

The endoplasmic reticulum membrane protein complex subunit Emc6 is essential for rhodopsin localization and photoreceptor cell survival

The endoplasmic reticulum(ER)membrane protein complex(EMC)is responsible for monitoring the biogenesis and synthetic quality of membrane proteins with tail-anchored or multiple transmembrane domains.The EMC subunit EMC6 is one of the core members of EMC and forms an enclosed hydrophilic vestibule in cooperation with EMC3.Despite studies demonstrating that deletion of EMC3 led to rhodopsin mislocalization in rod photoreceptors of mice,the precise mechanism leading to the failure of rhodopsin trafficking remains unclear.Here,we generated the first rod photoreceptor-specific knockout of Emc6(RKO)and cone photoreceptor-specific knockout of Emc6(CKO)mouse models.Deficiency of Emc6 in rod photoreceptors led to progressive shortening of outer segments(OS),impaired visual function,mislocalization and reduced expression of rhodopsin,and increased gliosis in rod photoreceptors.In addition,CKO mice displayed the progressive death of cone photoreceptors and abnormal localization of cone opsin protein.Subsequently,proteomics analysis of the RKO mouse retina illustrated that several cilium-related proteins,particularly anoctamin-2(ANO2)and transmembrane protein 67(TMEM67),were significantly down-regulated prior to OS degeneration.Detrimental rod photoreceptor cilia and mislocalized membrane disc proteins were evident in RKO mice.Our data revealed that in addition to monitoring the synthesis of rhodopsin-dominated membrane disc proteins,EMC6 also impacted rod photoreceptors'ciliogenesis by regulating the synthesis of membrane proteins associated with cilia,contributing to the mislocalization of membrane disc proteins.

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

Associations of oxidative stress and inflammation and their role in the regulation of membrane fluidity of red blood cells in hypertensive and normotensive men: An electron spin resonance investigation

There is evidence showing that increased levels of oxidative stress and C-reactive protein (CRP) might be associated with obesity, hypertension, atherosclerosis and other cardiovascular diseases. This study was undertaken to investigate possible relationships among plasma 8-iso-prostaglandin F2α (8-iso-PG F2α: an index of oxidative stress), high-sensitivity (hs)-CRP and membrane fluidity (a reciprocal value of microviscosity) in hypertensive and normotensive men using an electron spin resonance (ESR)-method. The order parameter (S) for the spin-label agents (5-nitroxide stearate) of red blood cell (RBC) membranes in the ESR spectra was significantly higher in hypertensive men than in normotensive men, indicating that membrane fluidity was decreased in hypertensive men. Both plasma 8-iso-PG F2α and hs-CRP levels were significantly increased in hypertensive men compared with normotensive men. In addition, plasma plasma 8-iso-PG F2α levels were correlated with plasma hs-CRP levels. In contrast, plasma nitric oxide (NO)-metabolites were lower in hypertensive men than in normotensive men, and inversely correlated with plasma 8-iso-PG F2α and hs-CRP. The order parameter(S) of RBCs was correlated with plasma 8-iso-PG F2α and plasma hs-CRP, and inversely correlated with plasma NO-metabolites, suggesting that reduced membrane fluidity of RBCs might be associated with increased oxidative stress, inflammation and endothelial dysfunction. Multivariate regression analysis also showed that, after adjusting for general risk factors, both plasma 8-iso-PG F2α and hs-CRP were significant determinants of membrane fluidity of RBCs. The ESR suggests that associations of oxidative stress and inflammation might have a close correlation with impaired rheologic behavior of RBCs and microcirculatory dysfunction in hypertensive men.

Functions and mechanisms of cytosolic phospholipase A_(2)in central nervous system trauma

Central nervous system(CNS)trauma,including traumatic brain injury and spinal cord injury,has a high rate of disability and mortality,and effective treatment is currently lacking.Previous studies have revealed that neural inflammation plays a vital role in CNS trauma.As the initial enzyme in neuroinflammation,cytosolic phospholipase A_(2)(cPLA2)can hydrolyze membranous phosphatides at the sn-2 position in a preferential way to release lysophospholipids andω3-polyunsaturated fatty acid dominated by arachidonic acid,thereby inducing secondary injuries.Although there is substantial fresh knowledge pertaining to cPLA2,in-depth comprehension of how cPLA2 participates in CNS trauma and the potential methods to amelio rate the clinical res ults after CNS trauma are still insufficient.The present review summarizes the latest understanding of how cPLA2 participates in CNS trauma,highlighting novel findings pertaining to how cPLA2 activation initiates the potential mechanisms specifically,neuroinflammation,lysosome membrane functions,and autophagy activity,that damage the CNS after trauma.Moreover,we focused on testing a variety of drugs capable of inhibiting cPLA2 or the upstream pathway,and we explored how those agents might be utilized as treatments to improve the results following CNS trauma.This review aimed to effectively understand the mechanism of cPLA2 activation and its role in the pathophysiological processes of CNS trauma and provide clarification and a new referential framework for future research.

支气管哮喘急性期患儿血清VAMP2水平表达及其临床意义

目的 探讨支气管哮喘急性期患儿血清中囊泡相关膜蛋白2(vesicle-associated membrane protein 2,VAMP2)表达水平及其临床意义。方法 选取2017年6月~2020年6月在西安医学院第二附属医院儿科就诊的94例支气管哮喘急性期患儿(研究组),以及同期入院复查的30例支气管哮喘缓解期患儿(对照组)进行研究。比较两组的一般临床资料,并检查两组患儿的呼气峰流量(peak expiratory flow,PEF)、第1秒用力呼气量(forced expiratory volume in 1second,FEV1)和用力肺活量(forced vital capacity,FVC)。采用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测血清VAMP2水平,并根据支气管哮喘急性期患儿的严重程度分为轻度组(n=36)、中度组(n=31)和重度组(n=27),比较三组血清VAMP2水平;支气管哮喘急性期患儿血清VAMP2水平与PEF,FEV1,FVC的关系采用Pearson法分析;受试者工作特征(receiver operating characteristic,ROC)曲线分析血清VAMP2水平对支气管哮喘急性期患儿发作的诊断价值。结果 研究组的血清VAMP2水平(8.01±2.12 ng/L)高于对照组(5.23±1.21 ng/L),差异具有统计学意义(t=6.824,P<0.05)。研究组中的PEF(73.45%±7.12%),FEV1(65.34%±8.34%)和FVC(69.34%±9.14%)均低于对照组(87.34%±7.23%,82.45%±10.31%,89.89%±10.03%),差异具有统计学意义(t=9.269,9.222,10.471,均P<0.05)。重度组、中度组和轻度组血清VAMP2水平依次为9.50±3.04ng/L,8.01±1.21 ng/L和6.85±1.01 ng/L,差异具有统计学意义(F=15.412,P<0.05);重度组VAMP2水平高于中度组、轻度组(t=4.269,7.851,均P<0.05),中度组血清VAMP2水平高于轻度组,差异具有统计学意义(t=3.571,P<0.05)。支气管哮喘急性期患儿血清VAMP2水平与PEF,FEV1和FVC呈负相关(r=-0.506,-0.487,-0.399,均P<0.05)。血清VAMP2水平诊断支气管哮喘急性期患儿发作的曲线下面积为0.909,截断值为6.802 ng/L,敏感度和特异度分别为77.7%,93.3%。结论支气管哮喘急性期患儿血清VAMP2水平升高,可以作为辅助诊断支气管哮喘急性发作的有效指标。

磷酸弗林酸性簇分选蛋白2对血管紧张素Ⅱ诱导的心肌肥大的影响及其机制

目的探究磷酸弗林酸性簇分选蛋白2(PACS-2)对血管紧张素Ⅱ(AngⅡ)诱导的心肌肥大的影响及其机制。方法剪取并消化出生1~2 d的SD大鼠乳鼠的心脏组织,采用差速贴壁法获取乳鼠心肌细胞(NRCMs),原代培养24 h。以浓度为1.5μmol/L的AngⅡ处理NRCMs 0、3、6、12、24 h,采用Western blot方法检测细胞中FUN14域蛋白1(FUNDC1)、PACS-2、三磷酸肌醇受体蛋白(IP3R)的表达水平,采用实时荧光定量PCR(RT-qPCR)方法检测细胞中心钠肽(ANP)、脑钠肽(BNP)、β-肌球蛋白重链(β-MHC)mRNA的表达水平。将NRCMs分为A~C组,A组使用无血清培养基培养,B、C组分别转染si-NC和si-PACS-2,采用Western blot方法检测各组细胞中PACS-2蛋白的表达水平。将NRCMs分为D~G组,D组使用无血清培养基培养,E组以浓度0.15μmol/L的AngⅡ培养,F、G组分别转染si-NC、si-PACS-2后再以浓度0.15μmol/L的AngⅡ培养,采用TRITC-鬼笔环肽染色技术检测各组心肌细胞表面积,RT-qPCR检测各组细胞ANP、BNP、β-MHC mRNA的表达水平,以Fluo-4,AM探针检测各组细胞胞质Ca^(2+)的水平。将NRCMs分为H~K组,H组使用无血清培养基培养,I、J组分别转染si-NC、si-PACS-2,K组转染si-PACS-2并且以浓度1μmol/L的钙调蛋白(CaM)拮抗剂处理后,采用RT-qPCR方法检测各组细胞ANP、BNP、β-MHC mRNA的表达水平。结果以浓度1.5μmol/L的AngⅡ处理NRCMs 0、3、6、12、24 h,NRCMs中ANP、BNP、β-MHC mRNA的表达水平呈时间依赖性上调(F=25.73~58.30,P<0.05),并且处理第24小时时相较于第0小时时均显著上调(t=5.35~37.50,P<0.05)。以浓度1.5μmol/L的AngⅡ处理NRCMs 0、3、6、12、24 h,NRCMs中IP3R、PACS-2以及FUNDC1蛋白的表达水平均呈时间依赖性下调(F=5.37~9.07,P<0.05),并且处理第24小时时相较于第0小时时显著下调(t=6.55~7.42,P<0.05)。与B组相比,C组NRCMs中PACS-2蛋白的表达水平显著下调(t=5.92,P<0.05)。与F组相比,G组NRCMs中心肌细胞表面积增大,ANP、BNP、β-MHC mRNA的表达水平及胞质Ca^(2+)水平均上调(t=3.50~26.60,P<0.05)。与J组相比,K组NRCMs中ANP、BNP、β-MHC mRNA表达水平均显著下调(t=3.27~5.13,P<0.05)。结论敲低PACS-2可增加NRCMs中胞质Ca^(^(2+))水平,并且可能以Ca^(^(2+))-CaM依赖的方式加重AngⅡ诱导的心肌肥大的发生。