肾康注射液细菌内毒素检查法的研究

目的建立肾康注射液细菌内毒素检查方法,以期能够替代家兔热原法控制产品的热原,达到快速尧高效尧可靠尧简便的 目的遥方法按叶中国药典2015 年版四部曳通则1143 细菌内毒素检查法进行试验遥采用两个不同厂家的的鲎试剂,对7 个不同 批次的样品开展干扰试验,确定最小不干扰稀释倍数,并对样品进行了细菌内毒素检查遥结果供试品4倍稀释液无干扰作用, 细菌内毒素限值定为1.0 Eu/ml遥结论用细菌内毒素检查法代替热原法是可行的遥     

鲎试验测定注射用头孢雷特中细菌内毒素的方法学研究

目的：检测注射用头孢雷特中的细菌内毒素。方法：供试品干扰试验后,采用凝胶鲎试验对样品中细菌内毒素进行检测。结果：三批注射用头孢雷特浓度为2mg·ml^-1对细菌内毒素的检测无干扰作用,所测样品的头孢雷特细菌内毒素含量均小于0.5EU.mg-1。结论：所建立方法检查注射用头孢雷特中的细菌内毒素可行。     

两种生物材料细菌内毒素检查和热原检查方法比较的研究

目的本试验对样品管腔支架(镍钛)和支架输送系统进行热原检测.方法采用细菌内毒素检查和家兔检查法.结果细菌内毒素试验表明,两样品不合格;热原试验表明,两样品合格.结论导致鲎试剂凝集不一定就能引起生物体的热原反应.出现这种不相一致结果的原因很多.所以在评价生物材料和医疗器械时,建议做热原试验.     

双黄连注射液细菌内毒素检查法的应用

目的:建立双黄连注射液的细菌内毒素检查方法.方法:按照《中国药典》2005年版附录细菌内毒素检查法进行试验.结果:双黄连注射液对鲎试剂与内毒素反应无干扰作用.结论:双黄连注射液采用细菌内毒素检查方法可靠,简单可行,可以考虑代替热原检查.     

鲎试验测定长链脂肪乳注射液中细菌内毒素的方法学研究

目的：确定长链脂肪乳注射液中细菌内毒素鲎试验检查法的可行性。方法：供试品干扰试验后，采用凝胶鲎试验对样品中细菌内毒素进行检测。结果：三批长链脂肪乳注射液稀释至2倍时对细菌内毒素的检测无干扰作用，所测样品的细菌内毒素含量均小于0．5EU·ml^-1。结论：所建立方法检查长链脂肪注射液中的细菌内毒素可行。     

脱细胞粘膜基质的热原试验研究

目的 考察脱细胞粘膜基质的热原试验方法的可行性。方法 依据《中华人民共和国药典》2010年版二部附录XIE细菌内毒素检查法和ISO10993.12 Biological evaluation of medical devices-Part 12:Sample preparation and reference materials,使用离心法消除样品浸提液的悬浮颗粒,使用动态浊度法验证离心条件是否会影响浸提液中细菌内毒素的检测。依据《中华人民共和国药典》2010年版三部附录XIID热原检查法检测脱细胞粘膜基质的热原试验是否合格。结果 本实验中采用的离心条件不会影响样品浸提液中细菌内毒素的检测,热原试验合格。结论该产品可以采用此离心条件进行热原检测。     

家兔法与内毒素法检查生物材料热原的适用性研究

药品与生物材料在生物安全性评价方法上有着很大的区别.临床上广泛运用内毒素法检查药品热原,然而,运用细菌内毒素法进行部分组成成分较为复杂的生物材料的热原试验是否适当有待明确.本研究在2005版药典的基础上,分别运用内毒素法和家兔法对两种组织工程支架材料进行热原试验的比较研究,实验结果表明运用内毒素法得到的试验结果为阴性,...     

家兔法与内毒素法检查生物材料热原的适用性研究

药品与生物材料在生物安全性评价方法上有着很大的区别。临床上广泛运用内毒素法检查药品热原,然而,运用细菌内毒素法进行部分组成成分较为复杂的生物材料的热原试验是否适当有待明确。本研究在2005版药典的基础上,分别运用内毒素法和家兔法对两种组织工程支架材料进行热原试验的比较研究,实验结果表明运用内毒素法得到的试验结果为阴性,运用家兔法得到的试验结果为阳性。这两种方法分别测定每种材料所得到的热原试验结果不相符合,表明对组成成分复杂的生物材料,含热原的因素较为复杂,用家兔法进行试验检测热原可能更加灵敏。     

鲎试验测定复方氨基酸(15)双肽(2)注射液中的细菌内毒素

目的:检测复方氨基酸(15)双肽(2)注射液中的细菌内毒素。方法:供试品干扰试验后,采用凝胶鲎试验对样品中的细菌内毒素进行检测。结果:三批复方氨基酸(15)双肽(2)注射液稀释至28倍时对细菌内毒素的检测无干扰作用,所测样品的细菌内毒素含量均小于7 EU.ml-1。结论:所建立方法检查复方氨基酸(15)双肽(2)注射液中的细菌内毒素可行。     

肝硬化时内皮素对肾功能的影响

目的：探讨肝硬化时肠源性内毒素血症（ＩＥＴＭ）和血浆内皮素（ＥＴ）在肾功能障碍中所起的作用。方法：采用鲎试剂基质显色法定量测定内毒素，用放射免疫法测定ＥＴ，常规生化法测定血肌酐，同时留２４小时尿液测定尿肌酐与尿钠。采用复合病因复制肝硬化模型。结果：肝硬化患者血浆内毒素水平随肝硬化病变加重而升高，内毒素水平在肝硬化伴功能性肾衰患者中（２０６±１５１ＥＵ／ｍＬ）明显高于肝硬化代偿期（０３２±０２０ＥＵ／ｍＬ）与失代偿期伴腹水（０７３±０２９ＥＵ／ｍＬ），并与肌酐清除率（ＣｒＣｌ），尿钠排泄呈负相关。肝硬化鼠血浆内毒素与肾组织ＥＴ含量（分别０２８±００４ＥＵ／ｍＬ，４６２±０７９ｎｇ／ｇ）明显高于对照组（分别０１４±００２ＥＵ／ｍＬ，２４８±１８８ｎｇ／ｇ），两者呈正相关。结论：ＩＥＴＭ在诱发象征肝功衰竭的ＦＲＦ中起重要作用；ＩＥＴＭ对肾组织ＥＴ的生成和释放有一定作用     

透明质酸钠凝胶预防剖宫产后宫腔粘连的临床应用

背景：透明质酸钠具有预防盆、腹腔粘连的作用,但透明质酸钠宫腔停留时间短,难以达到预防宫腔粘连的作用。国外研究对透明质酸钠进行化学修饰形成自交联透明质酸钠凝胶,它对受损的宫内膜具有很强的黏附性,在宫腔内停留时间可达72 h。 目的：评价透明质酸钠凝胶在剖宫产后预防宫腔粘连的效果及安全性。 方法：由第一作者检索PubMed数据库(http://www.ncbi.nlm.nih.gov/PubMed)及维普数据库(http://www.cqvip.com/)有关透明质酸钠凝胶预防剖宫产后宫腔粘连的文献,检索时限为1990-01/2009-12,中文关键词为“透明质酸钠凝胶,剖宫产,粘连,宫腔粘连”,英文关键词为“sodium hyaluronate,cesarean section,adhesion,intrauterine adhesions”,排除重复研究或综述类文章。 结果与结论：共检索到相关文献150余篇,排除内容重复、综述类文章后筛选纳入16篇文献进行评价,其中中文12篇,英文4篇。结果显示,透明质酸钠凝胶是由N-乙酰葡萄糖醛酸反复交替而形成的一种高分子多糖体生物材料,具有高度的黏弹性、可塑性以及良好的生物相容性,在剖宫产后预防宫腔粘连效果明显,特别是能有效减少分离后再粘连的发生。但有一点十分重要,透明质酸钠凝胶是绝对不能用于血管内的,以免引起肺栓塞并发症。注射过程应缓慢,以免回流入静脉系统。     

过滤和离心对热原实验影响的初步研究

目的了解医疗器械和生物材料的浸提液能否过滤或离心后再进行热原实验。方法采用《中国药典》2010年版(二部)附录检测细菌内毒素的动态浊度法,进行定量测定浓度为0.2 EU/mL的原液中的细菌内毒素含量,并在过滤和离心后分别测定溶液中细菌内毒素的含量。结果溶液中的细菌内毒素含量过滤后大大减少,甚至小于0.040 EU/mL。离心则不会影响溶液中的细菌内毒素含量,与原液中含量相近,为0.260 EU/mL。结论为除去医疗器械和生物材料的浸提液中的悬浮颗粒,可以离心后再进行热原实验。     

High sensitivity pyrogen testing in water and dialysis solutions

BACKGROUND: The dialysis patient is confronted with hundreds of litres of dialysis solution per week, which pass the natural protective barriers of the body and are brought into contact with the tissue directly in the case of peritoneal dialysis or indirectly in the case of renal dialysis (hemodialysis). The components can be tested for living specimens or dead pyrogenic (fever-inducing) contaminations. The former is usually detected by cultivation and the latter by the endotoxin-specific Limulus Amoebocyte Lysate Assay (LAL). However, the LAL assay does not reflect the response of the human immune system to the wide variety of possible pyrogenic contaminations in dialysis fluids. Furthermore, the test is limited in its sensitivity to detect extremely low concentrations of pyrogens, which in their sum result in chronic pathologies in dialysis patients. The In vitro Pyrogen Test (IPT) employs human whole blood to detect the spectrum of pyrogens to which humans respond by measuring the release of the endogenous fever mediator interleukin-1beta. Spike recovery checks exclude interference. The test has been validated in an international study for pyrogen detection in injectable solutions. METHODS: In this study we adapted the IPT to the testing of dialysis solutions. RESULTS: Preincubation of 50 ml spiked samples with albumin-coated microspheres enhanced the sensitivity of the assay to detect contaminations down to 0.1 pg/ml LPS or 0.001 EU/ml in water or saline and allowed pyrogen detection in dialysis concentrates or final working solutions. CONCLUSIONS: This method offers high sensitivity detection of human-relevant pyrogens in dialysis solutions and components.     

Prostaglandin E2 and thromboxane B2 in cerebrospinal fluid of afebrile and febrile cat

Levels of prostaglandin (PG) E2 and thromboxane (TX) B2, the stable metabolite of TXA2, were measured by radioimmunoassay in cerebrospinal fluid (CSF) collected from the third ventricle and the cisterna magna of conscious cats. In the absence of fever, PGE2 was usually below the threshold of the assay (0.05-0.37 ng/ml), while TXB2 was measurable in the majority of cases and its concentration was greater in the third ventricle (about 0.7 ng/ml) than in the cisterna magna (about 0.2 ng/ml). At either site, TXB2 content rose if any manipulation was required for the collection of samples. PGE2 levels increased to measurable values (max 1.1-1.4 ng/ml) during fever produced by intrathecal or intravenous administration of leucocytic pyrogen. In contrast, TXB2 concentration rose to an average of 2.2-4 ng/ml only when pyrogen (bacterial or leukocytic) was given intrathecally. Moreover, TXB2 elevation, unlike PGE2 elevation, was limited to the uprise phase of the fever. Imidazole, given either intraperitoneally (50 mg/kg) or intrathecally (3 mg), attenuated the pyrogen fever and suppressed any rise in TXB2 levels. At the same time, the drug tended to increase the PGE2 content of the CSF. Evidence was also obtained suggesting that a fraction of PGE2 is bound to CSF protein, and this event may be important to the inactivation of the compound. These findings are consistent with the concept that PGE2 is involved in the sequence of events underlying pyrogen fever. A role for thromboxane A2 in this process remains to be established.     

Accumlation of Hyaluronate in Human Lung Carcinoma As Measured by A new Hyaluronate Elisa

We have developed a new ELISA to quantify hyaluronate. This ELISA takes advantage of an anti-keratan sulfate antibody to differentiate between the coated aggregating rat chondrosarcoma proteoglycan which captures the hyaluronate and the keratan sulfate-bearing aggregating proteoglycan added subsequently. Absorbance in this assay was shown to be linear to the logarithmic concentration of hyaluronate in the range of 15 to 1000 ng/ml. Pre-treatment of hyaluronate with papain or protease did not interfere with its quantification; in contrast, pre-treatment with 0.1N NaOH significantly interferes with the subsequent measurement of the hyaluronate molecules. The size of the hyaluronate molecule was found to be an important factor in quantification: only large size hyaluronate molecules could be quantified accurately. The ELISA was used to show that human lung carcinomas contain 2 to 500 times as much hyaluronate as normal lung tissue from the same patient.     

Accumulation of hyaluronate in human lung carcinoma as measured by a new hyaluronate ELISA

We have developed a new ELISA to quantify hyaluronate. This ELISA takes advantage of an anti-keratan sulfate antibody to differentiate between the coated aggregating rat chondrosarcoma proteoglycan which captures the hyaluronate and the keratan sulfate-bearing aggregating proteoglycan added subsequently. Absorbance in this assay was shown to be linear to the logarithmic concentration of hyaluronate in the range of 15 to 1000 ng/ml. Pre-treatment of hyaluronate with papain or protease did not interfere with its quantification; in contrast, pre-treatment with 0.1N NaOH significantly interferes with the subsequent measurement of the hyaluronate molecules. The size of the hyaluronate molecule was found to be an important factor in quantification: only large size hyaluronate molecules could be quantified accurately. The ELISA was used to show that human lung carcinomas contain 2 to 500 times as much hyaluronate as normal lung tissue from the same patient.     

International validation of novel pyrogen tests based on human monocytoid cells

It is a requirement that parenteral medicines be tested for pyrogens (fever causing agents) using one of two animal-based tests: the rabbit pyrogen test and the bacterial endotoxin test. Understanding the human fever reaction has led to novel non-animal alternative tests based on in vitro activation of human monocytoid cells in response to pyrogens. Using 13 prototypic drugs, clean or contaminated with pyrogens, we have validated blindly six novel pyrogen tests in ten laboratories. Compared with the rabbit test, the new tests have a lower limit of detection and are more accurate as well as cost and time efficient. In contrast to the bacterial endotoxin test, all tests are able to detect Gram-positive pyrogens. The validation process showed that at least four of the tests meet quality criteria for pyrogen detection. These validated in vitro pyrogen tests overcome several shortcomings of animal-based pyrogen tests. Our data suggest that animal testing could be completely replaced by these evidence-based pyrogen tests and highlight their potential to further improve drug safety.     

Observations on the site & mode of action of pyrogens in the rabbit brain

1. Leucocyte pyrogen has been injected bilaterally into various parts of the rabbit brain. It caused fever when injected into the pre-optic area and the anterior hypothalamus, but not when injected into the posterior hypothalamus, the mid-brain, the pons, the cerebellum or the cerebral cortex.

2. The mean time which elapsed between a leucocyte pyrogen injection into the anterior hypothalamus and the onset of fever was 7·8 min. For similar injections of bacterial pyrogen the time lag was 24·8 min. The mean time lag between bilateral injections of noradrenaline into the anterior hypothalamus and the onset of fever was 7·4 min.

3. The amount of leucocyte pyrogen required to cause fever when injected into the anterior hypothalamus was less than 1/100 of that required to cause a similar fever on intravenous injection. The quantity of bacterial pyrogen injected into the hypothalamus was of the same order as that which would cause a similar fever on intravenous injection.

4. Control injections of saline, plasma, cerebrospinal fluid, heated leucocyte pyrogen and red cells into the anterior hypothalamus did not cause fever.

5. After attempts to deplete the hypothalamus of its monoamine stores by intraventricular injections of reserpine, the rabbit had fever as a result of an intravenous injection of bacterial pyrogen.

6. We conclude that the anterior hypothalamus and the pre-optic area are sites at which leucocyte pyrogen acts to cause fever in the rabbit. The mechanism of this febrile response is not clear, but it appears that part, at least, of the response could be mediated by a mechanism other than release of noradrenaline or failure to release 5-HT.

     

几丁糖和透明质酸钠在产科防粘连应用中的生物相容性比较

背景：几丁糖和透明质酸钠是目前临床常用的预防粘连材料,但目前关于二者在产科患者中预防粘连的相关报道相对较少。 目的：观察几丁糖和透明质酸钠在产科患者中的防粘连效果。 方法：纳入180例剖宫产妇,年龄23-39岁,按照治疗方法分为对照组、几丁糖组、透明质酸钠组,每组60例,对照组剖宫产后常规关闭腹腔,几丁糖组、透明质酸钠组剖宫产后关闭腹腔前,在子宫手术切口表面及手术部位附近肠管和腹膜分别涂抹几丁糖与透明质酸钠。术后1 d,检测3组血清白细胞介素6、白细胞介素10、肿瘤坏死因子α及C-反应蛋白水平;随访1个月,观察3组术后粘连及并发症发生情况。 结果与结论：几丁糖组、透明质酸钠组粘连发生情况及粘连发生率均低于对照组(P < 0.05),血清白细胞介素6、白细胞介素10、肿瘤坏死因子α及C-反应蛋白水平均低于对照组(P < 0.05),术后感染、出血、疼痛等的并发症发生率均低于对照组(P < 0.05);几丁糖组与透明质酸钠组粘连发生情况、血清指标水平及并发症发生情况比较差异均无显著性意义。表明几丁糖和透明质酸钠均可有效抑制剖宫产术后的粘连及炎症反应,减少并发症的发生。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程