特发性膜性肾病患者血清PLA2R抗体的检测技术及意义

特发性膜性肾病(idiopathic membranous nephropathy, IMN),即原发性膜性肾病,易发生在40岁以上男性群体中。IMN诊断的主要依据为临床表现及肾活检病理改变,后者是一种有创检测,对患者有一定影响。自肾小球足细胞表面M型磷脂酶A2受体(phospholipase A2 receptor, PLA2R)发现以来,相关领域对IMN的发病机制有了新的认识。随着对PLA2R抗体研究的不断深入,发现其不仅可以作为IMN的诊断指标,而且有助于判断疾病活动情况及疗效监测。该文就抗PLA2R抗体检测技术在IMN中的研究进展作一简要综述。     

鞘内注射P2X7受体拮抗剂BBG对大鼠行为学和脊髓PLA2表达的影响

目的:探讨鞘内注射P2X7受体拮抗剂考马斯亮蓝(BBG)在福尔马林模型大鼠中的镇痛作用及对脊髓磷脂酶A2(PLA2)表达的影响。方法:雄性Wistar大鼠鞘内置管成功后鞘内注射P2X7受体拮抗剂考马斯亮蓝(BBG)后所有的大鼠左侧后肢爪掌背侧皮下注射福尔马林(5%,50μl),观察大鼠在第一相(0~10 min)和第二相(10~60 min)的疼痛行为学比,变化(自发缩足次数)。免疫组织化学方法检测脊髓背角PLA2蛋白的表达与变化。结果:各组间大鼠第一相相比自发缩足次数没有明显的差异;第二相与对照组相比鞘内1μg、5μg及腹腔10μg BBG注射组相比,鞘内10μg BBG注射组大鼠的疼痛行为学明显减少(自发缩足次数,P0.05)。与此同时,鞘内10μg BBG注射组大鼠脊髓背角浅层内PLA_2免疫阳性产物的表达与其它组比较明显减少(P0.05)。结论:鞘内注射10μg BBG可减轻福尔马林诱导的炎性痛觉过敏,可能是通过抑制脊髓背角浅层PLA2的表达而发挥作用。     

冷冻与石蜡肾活检标本中PLA2R1表达的临床诊断意义比较

目的探讨磷脂酶A2受体1(PLA2R1)在肾脏穿刺的冷冻标本及石蜡标本中的表达,分析不同标本中阳性率的临床病理诊断意义。方法收集2013年1月~2019年2月江门市中心医院行肾脏穿刺患者的标本及临床病理资料,其中同时有肾活检冷冻标本及石蜡标本的患者215例,仅有石蜡标本的患者785例。分别利用免疫荧光及免疫组化法检测PLA2R1在肾活检冷冻和石蜡标本中的表达,并分析PLA2R1表达与临床病理特征的相关性。结果在215例冷冻标本中,PLA2R1阳性率为19.1%;在1000例石蜡标本中,PLA2R1阳性率为32.9%,两组中PLA2R1的阳性率差异有统计学意义(P<0.001)。在冷冻和石蜡标本中,PLA2R1表达与患者性别、年龄及临床分期无关,与病理类型相关。在冷冻和石蜡标本中,PLA2R1阳性均集中于特发性膜性肾病(72.0%和79.8%),但非特发性膜性肾病中,则分别为3.0%和19.4%。结论肾活检冷冻及石蜡标本中PLA2R1表达均与特发性膜性肾病相关,但冷冻标本在特发性膜性肾病诊断中更有价值。     

PLA2G6 variant in Parkinson's disease

PLA2G6 was reported recently as the causative gene for PARK14-linked autosomal recessive early-onset dystonia-parkinsonism. In a recent study in Singapore, heterozygous PLA2G6 p.P806R (c.2417C>G) mutation in exon 17 was reported to be a possible Parkinson's disease (PD)-related mutation. To determine the significance of the PLA2G6 mutation, we conducted an association study by performing direct sequencing of PLA2G6 exon 17 in 379 Japanese sporadic PD patients and 310 controls in the Japanese general population. In this group, we found 12 patients (12/379=3.16%) and 10 controls (10/310=3.23%) with a heterozygous p.P806R mutation (P=0.96, χ(2)=0.0019). Therefore, our large case-controlled study suggests that PLA2G6 p.P806R is not a disease-associated polymorphism in PD. Moreover, we performed direct sequencing of all exons and exon-intron boundaries of PLA2G6 in 116 Japanese patients with sporadic PD. Two single heterozygous variants (p.R301C or p.D331N) were found (both frequencies: 1/379 patients vs 0/310 controls) and the roles of their variants were unclear. Finally, combined with the previous report, our findings emphasize that PLA2G6 mutations are unlikely to be the major causes or risk factors of PD at least in Asian populations. However, further large studies in various populations are needed because patients with PLA2G6 mutations can show heterogeneous clinical features.     

COX-2, inflammatory secreted PLA2, and cytoplasmic PLA2 protein expression in small bowel adenocarcinomas compared with colorectal adenocarcinomas.

Cyclooxygenase-2 (COX-2), human synovial inflammatory secreted phospholipase A2 (sPLA2) and cytoplasmic phospholipase A2 (cPLA2) are involved in eicosanoid production and also seem to participate in colorectal tumorigenesis. As there are no data regarding these enzymes in human small bowel tumors, we wanted to determine whether they were involved in human small bowel tumorigenesis, and whether their expression was different in small bowel compared to colorectal adenocarcinomas, as suggested by animal studies. We studied their protein expression by immunohistochemistry in 25 small bowel adenocarcinomas and compared it to 48 colorectal adenocarcinomas. Seventy-six percent of the small bowel and 88% of the colorectal adenocarcinomas had a moderate or strong COX-2 expression. Sixty-eight percent of the small bowel and 67% of the colorectal adenocarcinomas had a moderate or strong sPLA2 expression. Forty-eight percent of the small bowel and 35% of the colorectal adenocarcinomas had a moderate or strong cPLA2 expression. In conclusion, the increased expression of COX-2, sPLA2, and sometimes cPLA2 in both small bowel and colorectal adenocarcinomas is in accordance with the likely eicosanoid involvement in tumor development. The same pattern of protein expression found in both types of adenocarcinoma contradicts experimental results in mice. Moreover, our results strengthen the similarities between these two types of human cancer.     

Phospholipase A2 (PLA2) activity in rabbit chondrocytes

The effects of recombinant interleukin-1 (rIL-1), recombinant tumor necrosis factor (rTNF) and two growth factors, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on PLA₂ activity and prostaglandin E₂ (PGE₂) release were investigated using rabbit chondrocytes. Cellular PLA₂ activity increased 2–10×above controls in the presence of 8×10^–12 M (5 U) rIL-1 or 5×10^–9 M rTNF after 20 hr incubation. PLA₂ activity remained constant with 1–50 ng/ml of either growth factor. PGE₂ release significantly increased (p<0.05) when the chondrocytes were incubated with rIL-1, bFGF and EGF alone, but not with rTNF above. These data suggest PLA₂ activity and PGE₂ release are not coordinately regulated in rabbit chondrocytes.     

Phospholipase A2 (PLA2) activity in undifferentiated U937 cells

PLA₂ activity has been described in U937 cells. The present study characterized PLA₂ activity in these undifferentiated cells. Cells were grown in suspension culture, harvested by centrifugation, and washed and homogenized in a neutral buffer containing standard proteinase inhibitors. A low speed supernatant was fractionated either by acid extraction or by sucrose density gradient centrifugation. PLA₂ activity was measured using either L--1-palmitoyl-2-arachidonoyl [1-¹⁴C]-phosphatidylcholine or heat-inactivated [³H]oleic acid-labeledE. coli as substrates. Substrate-specific PLA₂ activity was found in the acid-extracted and in the 25% sucrose fractions. Standard inhibitors were investigated with these PLA₂ activities. Our results suggest undifferentiated U937 cells contain three distinct PLA₂ activities. This is the first indication that more than one PLA₂ activity is present in undifferentiated U937 cells.     

Tagging-SNP haplotype analysis of the secretory PLA2IIa gene PLA2G2A shows strong association with serum levels of sPLA2IIa: results from the UDACS study

Recent prospective analysis identified secretory phospholipase A(2)-IIa (sPLA(2)IIa) as a coronary artery disease (CAD) risk predictor. This study aimed to examine the relationship between serum levels of sPLA(2)IIa and variation in the sPLA(2)IIa gene (PLA2G2A) in a cohort of patients with Type II diabetes (T2D) mellitus. Six tagging single nucleotide polymorphisms (tSNPs) accounting for > 92% of the genetic variability in PLA2G2A were identified and distinguished six common haplotypes (frequencies > 5%). In the 523 Caucasian T2D patients, levels of sPLA(2)IIa, independent of CRP, were negatively correlated with total antioxidant status (P = 0.003) and high-density lipoprotein cholesterol (P = 0.006) in men and correlated with CAD status in women (P = 0.002) (Odds ratio of top two tertiles versus bottom = 2.50) [95% CI (1.13-5.53) P = 0.024]. Overall, tSNP haplotypes showed a highly significant association with sPLA(2)IIa levels (P < 0.0001), explaining 6.3% of the variance. The most common haplotype (frequency 14.2%) was associated with 53% higher sPLA(2)IIa levels [3.25 ng/ml (+/- 0.14)] compared with the combined other haplotypes [2.13 ng/ml (+/- 0.09), P < 0.00001]. Five of the six tSNPs were associated with significant effects on sPLA(2)IIa levels but the raising haplotype could not be distinguished by a single tSNP and none are likely to be functional. These data confirm the relationship between elevated sPLA(2)IIa levels and CAD risk reported in both cases: control and prospective analyses. The strong impact of PLA2G2A haplotypic variation on sPLA(2)IIa levels will help clarify the causality of this association.     

Phospholipase A2 (PLA2) activity in bovine pulmonary artery endothelial cells

We have investigated the role of recombinant human interleukin-1 (rIL-1) and recombinant human tumor necrosis factor (rTNF-) on PLA₂ activity, protein synthesis and eicosanoid production in bovine pulmonary artery endothelial cells. Cellular PLA₂ activity increased 4-fold and production of PGE₂ increased 3-fold at 1–2 hrs in the presence of 10 units/ml rIL-1. PLA₂ activity increased 3-fold at 30 min and PGE₂ production increased 2-fold with 5×10^–9 M rTNF-. The data show that endothelial cells respond more rapidly to rIL-1 (2–6 hr) and rTNF- (30 min) than do chondrocytes and synovial cells (6–16 hrs), suggesting endothelial cells may play a primary role in initiating the inflammatory response.     

Novel PLA/Chitosan Composite Materials Prepared by SCF-CO2 Technique

Because of the incomparable merits （nontoxicity, non-remainder, fast transfer mass） of supercritical carbon dioxide fluid technique（SC-CO2）, it was used to developed a series of novel biodegradable tissue engineering scaffold materials in this research. The novel PLA/chitosan composite materials could be molded to different shapes, and the porosity of the materials were over 200 lam and connected. Chondrocyte cultivation, subcutaneous and intramuscular implantation were mainly discussed this paper. The results showed that the cells could well adhere, grow and multiplicate on the surface of the materials, which indicated good biocompatibility of the composite materials. The plantation test revealed that the PLA materials had already dismissed 2 month late in the body, while the composite materials could still keep certain strength and shape, and the most important things is the response of the tissue toward the implanted PLA/chitosan composite materials was mild and had far less inflammation than PLA materials. 8 to 16 weeks later, fiber membrane was stable; degradation of the materials was seen clear and tissue had already spread into it.     

CoA-independent transacylase has characteristics distinct from those of PLA2 enzymes

CoA-independent transacylase (CoA-IT) catalyzes the transfer of arachidonic acid from acyl- to alkyl-linked phospholipids. The removal of arachidonic acid from the sn-2 position of the donor phospholipid is a PLA₂-like reaction. However, examination of CoA-IT in U937 cells demonstrated that CoA-IT has many characteristics that are distinct from those of PLA₂ enzymes, including activity in the absence of Ca²⁺, activity that was heat and acid unstable and stable in 10 mM 2-mercaptoethanol and that was inhibited by detergents. Compounds that inhibit PLA₂ activity did not inhibit CoA-IT activity, including quinacrine, aristolochic acid and arachidonic acid. All of these characteristics of CoA-IT are in contrast to those of most PLA₂ enzymes. These data suggest that CoA-IT is biochemically different from, and has a mechanism of action unique from, PLA₂ enzymes.     

Secretory PLA2-IIA: a new inflammatory factor for Alzheimer's disease

Secretory phospholipase A₂-IIA (sPLA₂-IIA) is an inflammatory protein known to play a role in the pathogenesis of many inflammatory diseases. Although this enzyme has also been implicated in the pathogenesis of neurodegenerative diseases, there has not been a direct demonstration of its expression in diseased human brain. In this study, we show that sPLA₂-IIA mRNA is up-regulated in Alzheimer's disease (AD) brains as compared to non-demented elderly brains (ND). We also report a higher percentage of sPLA₂-IIA-immunoreactive astrocytes present in AD hippocampus and inferior temporal gyrus (ITG). In ITG, the majority of sPLA₂-IIA-positive astrocytes were associated with amyloid β (Aβ)-containing plaques. Studies with human astrocytes in culture demonstrated the ability of oligomeric Aβ_1–42 and interleukin-1β (IL-1β) to induce sPLA₂-IIA mRNA expression, indicating that this gene is among those induced by inflammatory cytokines. Since exogenous sPLA₂-IIA has been shown to cause neuronal injury, understanding the mechanism(s) and physiological consequences of sPLA₂-IIA upregulation in AD brain may facilitate the development of novel therapeutic strategies to inhibit the inflammatory responses and to retard the progression of the disease.     

Cytosolic PLA2 genes possibly contribute to the etiology of schizophrenia.

The present study detected three single nucleotide polymorphisms (SNPs), BanISNP at the PLA2G4A locus, rs1648833 at the PLA2G4B locus, and rs1549637 at the PLA2G4C locus, to investigate a genetic association between the cytosolic PLA2 (cPLA2) genes and schizophrenia. A total of 240 Chinese parent-offspring trios of Han descent were recruited for the genetic analysis. The transmission disequilibrium test (TDT) showed allelic association for rs1549637 (chi(2) = 5.68, uncorrected P = 0.017), but not for BanISNP and rs1648833. The conditioning on genotype (COG) test revealed a disease association for the BanISNP-rs1648833 combination (chi(2) = 12.54, df = 3, P = 0.0057) and for the BanISNP-rs1549637 combination (chi(2) = 9.72, df = 2, P = 0.021), but the conditioning on allele (COA) test did not show such an association for the above two combinations. Neither the COA test nor the COG showed a disease association for the rs1648833-rs1549637 combination. In the combination of all three SNPs, the COG test, but not the COA test, showed a strong association (chi(2) = 22.93, df = 6, P = 0.0008). These findings suggest that these three cPLA2 genes may all be involved in contributing to the etiology of schizophrenia although their effect size appears to be relatively small.     

Activation of PLA2 isoforms by cell swelling and ischaemia/hypoxia

Phospholipase A2 (PLA2) activity is increased in mammalian cells in response to numerous stimuli such as osmotic challenge, oxidative stress and exposure to allergens. The increased PLA2 activity is seen as an increased release of free, polyunsaturated fatty acids, e.g. arachidonic acid and membrane-bound lysophospholipids. Even though arachidonic acid acts as a second messenger in its own most mammalian cells seem to rely on oxidation of the fatty acid into highly potent second messengers via, e.g. cytochrome P450, the cyclo-oxygenase, or the lipoxygenase systems for downstream signalling. Here, we review data that illustrates that stress-induced PLA2 activity involves various PLA2 subtypes and that the PLA2 in question is determined by the cell type and the physiological stress condition.     

利用噬菌体肽库确定IL-2Rα表位序列①

目的确定IL-2Rα表位,为研制高效特异性强的小分子肽类免疫抑制剂奠定基础。方法用抗IL-2Rα单克隆抗体5G1对噬菌体六肽库进行筛选,选择出与5G1结合较强的克隆,经DNA序列分析,获得保守序列。对含保守序列的克隆进行生物学活性鉴定。结果选择出31个与5G1结合较强的克隆。获得Ser-Ser-Phe和Ser-Ser-Arg两种保守序列。生物学活性鉴定表明含Ser-Ser-Arg序列克隆较含Ser-Ser-Phe被抑制效果好。结论Ser-Ser-Arg是IL-2Rα表位的关键序列。以此表位序列合成的小分子肽可作为IL-2Rα的拮抗剂而成为免疫抑制剂。