Hemolymph coagulation and phenoloxidase activity in Uca tangeri induced by Escherichia coli endotoxin

Uca tangeri is a marine fiddler crab found commonly in the West African coast and is often exposed to Gram-negative pathogens upon injury. The aim of this study was to document the patterns of endotoxin-induced protein coagulation and phenoloxidase (PO) activity in hemolymph fractions of Uca tangeri. Hemolymph from live crabs was obtained by carapace puncture, pooled. and then separated into plasma, hemocyte Lysate (HL), hemocyte lysate supernatant (HLS) and hemocyte lysate debris (HLD). The effect of Escherichia coli (O1111:B4) endotoxin and calcium ion (Ca²⁺) on protein coagulation in the presence/absence of endotoxin and the endotoxin dose-dependence of coagulation and PO activity were each studied in the plasma, HL, HLS and HLD. The results showed Ca²⁺ was required to induce coagulation, and was endotoxin concentration-dependent in the plasma. PO activity was highest in the HLS but PO specific activity was highest in HLD. PO activity remained relatively constant with increased LPS concentration in the range studied 0–10 EU/ml. From the data we conclude that endotoxin-induced protein coagulation occurs in the plasma alone and might be mediated by trans-glutaminases, while PO activity is localized inside hemocytes and cell membranes in Uca tangeri.     

定量测定慢性前列腺炎病人前列腺按摩液和血浆的内毒素水平的临床意义——附45例分析

目的：探讨测定慢性前列腺炎（ＣＰ）病人前列腺按摩液（ＥＰＳ）和血浆内毒素水平的临床价值。方法：对４５例ＣＰ病人行ＥＰＳ显微镜检查和细菌培养,测定其ＥＰＳ和血浆的内毒素含量,并与对照组比较。结果：４５例ＣＰ病人的ＥＰＳ和血浆内毒素水平显著高于对照组（Ｐ〈０．０１）;且革兰阴性菌性ＣＰ病人的ＥＰＳ和血浆的内毒素水平与革兰阳性菌性ＣＰ病人比较均显著升高。结论：测定ＣＰ病人的ＥＰＳ和血浆内毒素的水平对ＣＰ的诊断和治疗均有指导意义,并且方法简便、快速、费用低廉。     

以支气管肺泡灌洗液的内毒素诊断革兰阴性菌肺炎

目的：阐明鲎细胞溶解物试验（ＬＡＬ）检测支气管肺泡灌洗液（ＢＡＬＦ）在诊断革兰阴性杆菌肺炎中的作用。方法：ＬＡＬ测定采用半定量法。ＢＡＬＦ经纤维支气管镜导管法采集。结果：１０例经细菌学确诊患者ＢＡＬＦ内毒素水平均大于１∶１６。细菌学确定的革兰阴性菌肺炎与ＢＡＬＦ内毒素大于或等于１∶１６阳性之间总符合率为９０％（Ｋａｐｐａ系数０．６７）。经细菌学确诊的阴性菌肺炎组ＢＡＬＦ内毒素单位（ＥＵ）为１２．５×１０３ＥＵ／Ｌ,无肺炎组为１．８×１０３ＥＵ／Ｌ,差异有显著性（Ｐ＜０．０１）。结论：以ＬＡＬ检测ＢＡＬＦ为诊断阴性菌肺炎的一种快速、准确、简便的试验方法。     

手术/创伤后外周静脉血内毒素的研究

目的探讨健康成人和创伤病人外周静脉血内毒素水平及其与创伤的关系。方法采用偶氮显色鲎试验检测５０例健康成人及５１例不同手术／创伤病人外周静脉血的内毒素。结果（１）此法检测内毒素可达微量（１０－２Ｅｕ／ｍｌ,ｎ＝５）,重复性较好（ＣＶ＝２．２９％,ｎ＝５）。（２）本法测得健康成人外周静脉血内毒素＜０．０３３Ｅｕ／ｍｌ,与国际水准一致。（３）病人术后３天内毒素升高（Ｐ＜０．０１）。（４）病人外周静脉血内毒素与创伤程度呈正相关,Ｒ２＝０．６６７。结论本法测定血微量内毒素是可行的。外周静脉内毒素水平随创伤程度增加而增加。     

沙赛普肽注射液的细菌内毒素检测

目的：用鲎试剂检测沙赛普肽注射液中的细菌内毒素。方法：采用鲎试法（LT）与家兔法（RT）对沙赛普肽注射液进行细菌内毒素检测。结果：两种方法检查结果一致,用直接稀释法排除细菌内毒素的干扰。结论：以鲎试法用于控制沙赛普注射液的热原方法可行。     

泛影葡胺注射液细菌内毒素检测方法探新

目的：确立用鲎试剂（ＴＡＬ）法检测泛影葡胺注射液中的内毒素。方法：参照《美国药典》２３版及《中国药典》１９９５年版二部收载的细菌内毒素检查方法的要求进行试验。结果：用标示值灵敏度为０．５ＥＵ．ｍｌ＾－１的鲎试剂检查泛影葡胺注射中的细菌内毒素是有效的。结论：０．５ＥＵ．ｍｌ＾－１鲎试剂可以用于泛影葡胺注射液中的内毒素检测。鲎试剂（ＴＡＬ）法可替代泛影葡胺注射液热原检查项目中的家兔（ＲＴ）法。     

Gel clot LAL assay in the initial management of peritoneal dialysis patients with peritonitis: a retrospective study.

BACKGROUND: Indiscriminate use of broad-spectrum antibiotic treatment of peritonitis in peritoneal dialysis patients may have either unwanted side-effects or contribute to the development of antibiotic resistance. This may be avoided by improved diagnosis at presentation. The Limulus amoebocyte lysate assay is a convenient test detecting bacterial endotoxins or fungal beta glucans. This study evaluates a qualitative Limulus amoebocyte lysate test as a diagnostic tool used at presentation of a peritoneal dialysis patient with peritonitis. METHODS: One-hundred and eleven episodes of peritonitis in peritoneal dialysis patients have been analysed retrospectively. Limulus amoebocyte lysate results at presentation were compared with culture results. A Limulus amoebocyte lysate assay was performed using a commercial kit by incubating a mixture of dialysate effluent and Limulus amoebocyte lysate reagent at 37 degrees C. The development of a stable solid clot was considered positive. The specificity and sensitivity of the test were calculated. RESULTS: The specificity of the Limulus amoebocyte lysate assay was found to be 98% and the sensitivity 74%. Limulus amoebocyte lysate assay was false-negative in 13 cases of Gram-negative peritonitis (22%). Limulus amoebocyte lysate was positive in three of seven cases of fungal peritonitis. The study included one case each with false-positive Limulus amoebocyte lysate and with culture-negative peritonitis. CONCLUSIONS: The Limulus amoebocyte lysate assay is a convenient and valuable diagnostic tool for excluding Gram-positive peritonitis in peritoneal dialysis patients. This allows more specific antibiotic treatment at presentation and may avoid the development of bacterial resistance. A negative Limulus amoebocyte lysate test is not reliable for the exclusion of Gram-negative peritonitis. In the absence of a positive culture result 48 h after presentation, accompanied by a delayed response to treatment, a positive Limulus amoebocyte lysate assay may indicate the presence of fungus. This justifies early empiric antifungal treatment before definitive culture results are made available. Routine Limulus amoebocyte lysate assay of dialysate effluent from continuous ambulatory peritoneal dialysis patients presenting with peritonitis is recommended.     

凝胶法检测2种中药注射液中内毒素含量

目的:通过特异性及非特异性鲎试剂法对鱼腥草注射液及清开灵注射液的内毒素含量的检测,探讨特异性鲎试剂法检测中药注射剂中内毒素的可行性。方法:采用特异性和非特异性的鲎试剂,对不同批次的鱼腥草注射液、清开灵注射液进行检测。结果:特异性及非特异性的鲎试剂均可检测鱼腥草注射液,其无干扰的最小稀释倍数完全一致;用特异性鲎试剂可以检测清开灵注射液,而用非特异性鲎试剂不能检测。结论:在检测清开灵注射液内毒素含量时,用特异性鲎试剂代替非特异性鲎试剂进行检测是一种较为理想的优化方法。     

超活化血小板裂解液对类风湿关节炎患者成纤维细胞样滑膜细胞影响的实验研究

目的 探讨超活化血小板裂解液(sPL)对类风湿关节炎患者成纤维细胞样滑膜细胞(RA-FLS)的影响。方法 选取哈尔滨医科大学附属第一医院2018年1月—2019年3月6例类风湿关节炎患者作为研究对象,其中男3例、女3例,年龄26～49岁、中位年龄33岁,受累关节功能分级均为Ⅱ级。收集患者静脉血标本,先进行两次离心分离获得富血小板血浆(PRP);再加入0.08 mmol/L CaCl₂, 37 ℃孵育激活血小板;然后经过冷冻、融化、离心、过滤除菌、去除纤维蛋白原,获得sPL。培养RA-FLS,分为对照组和2.5%、5%、10% sPL组,分别与含有0、2.5%、5%、10% sPL的培养液培养48 h。采用酶联免疫吸附试验(ELISA)检测各组细胞中炎症因子白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α、IL-1β的浓度;细胞计数试剂盒(CCK)-8法测定各组细胞增殖活性;流式细胞术测定各组细胞的凋亡率;体外小管生成实验检测各组细胞血管生成能力;Transwell实验检测各组细胞迁移能力和侵袭能力;Western blot法测定各组细胞增殖细胞核抗原(PCNA)、细胞周期蛋白D1(CyclinD1)、B细胞淋巴瘤/白血病-2蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)、基质金属蛋白酶(MMP)2、MMP-9、血管内皮生长因子(VEGF)和VEGF受体-2 (VEGFR2)的表达情况。结果 (1)对照组、2.5%sPL组、5%sPL组、10%sPL组细胞培养基中IL-6浓度分别为(80.18±11.67)、(59.94±9.50)、(46.60±8.04)、(60.67±9.24)pg/mL,TNF-α浓度分别为(70.75±9.14)、(54.56±7.81)、(43.27±6.30)、(53.99±8.60)pg/mL,IL-1β浓度分别为(64.18±9.90)、(46.97±8.79)、(36.28±7.44)、(47.66±8.15)pg/mL,组间比较差异均有统计学意义(F=12.186、11.934、10.709,P值均＜0.01);2.5%sPL组、5%sPL组、10% sPL组细胞中IL-6、TNF-α、IL-1β的表达水平均明显低于对照组,差异均有统计学意义(P值均＜0.05);5% sPL组炎症因子表达水平低于2.5% sPL组和10% sPL组,差异均有统计学意义(P值均＜0.05)。(2)对照组、2.5%sPL组、5%sPL组、10%sPL组RA-FLS增殖率分别为87.33%±10.98%、76.17%±8.18%、60.83%±7.99%、75.83%±8.45%,细胞凋亡率分别为6.51%±1.16%、16.23%±2.75%、29.69%±3.80%、16.37%±2.29%,小管形成数分别为(41.00±7.55)、(26.67±4.16)、(11.67±3.51)、(26.00±6.56)个,迁移细胞数目分别为(443.00±54.06)、(282.33±31.66)、(154.64±23.18)、(292.00±35.03)个,侵袭细胞数目分别为(243.30±27.39)、(67.00±12.53)、(22.33±8.74)、(79.33±14.98)个,组间比较差异均有统计学意义(F=8.795、38.095、34.278、29.352、94.772, P值均<0.05);2.5%sPL组、5%sPL组、10% sPL组RA-FLS细胞增殖率、血管管腔形成数量、迁移细胞数目和侵袭细胞数目均低于对照组,细胞凋亡率均高于对照组,差异均有统计学意义(P值均＜0.05);5% sPL组中细胞增殖率、小管形成数、迁移细胞数目和侵袭细胞数目均明显低于2.5% sPL组和10% sPL组,细胞凋亡率则高于2.5% sPL组和10% sPL组,差异均有统计学意义(P值均＜0.05)。(3)对照组、2.5%sPL组、5%sPL组、10%sPL组RA-FLS PCNA、CyclinD1、Bax、Bcl-2、MMP-2、MMP-9、VEGF和VEGFR2蛋白相对表达量比较,差异均有统计学意义(P值均＜0.01);2.5%sPL组、5%sPL组、10% sPL组细胞中PCNA、CyclinD1、Bcl-2、MMP-2、MMP-9、VEGF和VEGFR2蛋白相对表达量均低于对照组,Bax蛋白相对表达量均高于对照组,差异均有统计学意义(P值均＜0.05);5% sPL组PCNA、CyclinD1、Bcl-2、MMP-2、MMP-9、VEGF和VEGFR2蛋白相对表达量均低于2.5% sPL组和10% sPL组,而Bax蛋白相对表达量则高于2.5% sPL组和10% sPL组,差异均有统计学意义(P值均＜0.05)。结论 sPL对RA-FLS的增殖、迁移、侵袭、血管生成以及炎症因子的产生具有抑制作用,对RA-FLS凋亡具有促进作用,且SPL对RA-FLS的生长抑制效果与SPL浓度有关。     

Platelet lysate as a serum replacement for skin cell culture on biomimetic PCL nanofibers

Platelets are a popular source of native growth factors for tissue engineering applications. The aim of the study was to verify the use of platelet lysate as a fetal bovine serum (FBS) replacement for skin cell culture. The cytokine content of the platelet lysate was characterized using the Bio-Plex system. The cells (fibroblasts, melanocytes, and keratinocytes) were cultured on PCL nanofibrous scaffolds to mimic their natural microenvironment. The cytokine content of the platelet lysate was determined, and to the cells, a medium containing platelet lysate or platelet lysate in combination with FBS was added. The results showed that 7% (v/v) platelet lysate was sufficient to supplement 10% (v/v) FBS in the culture of fibroblasts and keratinocytes. The combination of platelet lysate and FBS had a rather inhibitory effect on fibroblasts, in contrary to keratinocytes, where the effect was synergic. Platelet lysate did not sufficiently promote proliferation in melanocytes; however, the combination of FBS and platelet lysate yielded a better outcome and resulted in bipolar morphology of the cultured melanocytes. The data indicated that platelet lysate improved cell proliferation and metabolic activity and may be used as an additive to the cell culture media.