探讨热原试验中影响试验数据的因素

目的通过几组试验,分析热原试验中存在常见影响试验数据的几个要素,并提出解决建议。方法对供试品、预选周期、基础体温、家兔的休息周期等多个方面进行统计,总结影响热原数据变化因素。结果家兔升温总和均值随着连续注射批次的增加而有所降低,体温灵敏度变差;合格家兔在4周以内再次预选,合格比例85%以上,6周符合要求的比例为79.1%,9周合格比例降至66.6%;基础体温38.0~38.4℃及39.3~39.6℃区间家兔实验中体温波动幅度≥0.6℃的动物例数较多,38.0~38.4℃区间家兔降温0℃动物数达到50.0%,39.3~39.6℃区间家兔最大升温≥0.4℃的动物数比例较高;家兔冬季基础体温略低于夏季(约0.2℃),休息72~96 h后对家兔基础体温均值、异常体温动物的数量有较好的控制。结论热原检查应尽量采用不同品种供试品交替试验,并通过控制预选兔的使用周期及合理安排家兔实验休息周期,选择基础体温适中的家兔进行试验,以保证热原试验数据的准确性。     

影响家兔热原检查结果的因素分析

目的统计实验数据,分析影响家兔热原检查结果的因素。方法将一定剂量的供试品静脉注入家兔体内,在规定时间内,观察家兔体温升高的情况。结果基础体温≥38.9℃的家兔,其升温≥0.4℃及≥0.6℃的百分比与基础体温<38.9℃家兔相比具有显著性差异(P<0.05),且不同季节家兔升温≥0.4℃的家兔数百分比具有显著性差异(P<0.05),夏秋两季所占百分数高于冬春两季,夏季(6～8月)家兔基础体温较高。结论不同基础体温家兔对热原敏感程度不同,敏感性随家兔基础体温的升高而下降,为保证热原试验结果的准确性,夏季应适当提前热原质检查时间。     

浙江省热原检查用兔敏感性状况调查

目的调查浙江省热原检查用兔对细菌内毒素的敏感性情况,为提高热原检查结果的准确性和可靠性提供参考。方法对全省所有取得生产许可证单位的家兔,用国家颁发的细菌内毒素标准品,＂热原检查法＂进行检查,剂量分别为5EU/Kg和10EU/Kg,记录并比较各单位家兔的平均升温值和升温率。结果对细菌内毒素,各兔场家兔的敏感性有一定的差异。静脉注射5EU/kg,平均升温值为0.40℃～0.87℃,升温率为35%～83%;静脉注射10EU/kg,平均升温值为0.74℃～1.16℃,升温率为70%～94%。结论不同生产单位的家兔对细菌内毒素的敏感性不同,有必要对热原检查用家兔进行细菌内毒素敏感性检查。     

调节性T细胞在健康人群中的表达

目的:观察外周血CD4+CD25+CD127low调节性T细胞(Treg细胞)在不同性别、不同年龄段健康人群中的表达水平,建立该指标应用于临床分析的实验室参考范围。方法:收集180例健康体检成年人EDTA-K3抗凝外周静脉血,用流式细胞术分析CD4+细胞中表达CD25+CD127low细胞的百分率,以SPSS 13.0软件进行统计学分析。结果:Treg细胞在男女性别间的表达百分率无显著性差异(P0.05),在≤49岁的3组之间、≥50岁的2组之间的差异亦无显著性(P0.05);Treg细胞的表达范围在≤49岁为(3.30±1.45)%,≥50岁为(4.69±1.27)%;将≤49岁的3组与≥50岁的2组分别进行两两分析,Treg细胞百分率均存在显著性差异(P0.05)。结论:CD4+CD25+CD127Low调节性T细胞应用于临床时可以忽略性别,须针对≤49岁与≥50岁2个年龄段建立不同的参考范围;本研究建立的Treg细胞参考值范围符合本院健康体检人群特点,该指标可作为本实验室对临床疾病检测及判断预后的依据。     

Gfat1基因在2型糖尿病大鼠肺组织中的表达

观察2型糖尿病大鼠不同时期肺组织谷氨酰胺果糖转移酶1(Gfat1)的表达情况.方法 SD大鼠随机分为正常对照组和模型组,对照组18只,模型组28只.模型组高脂饲料喂养2个月后,腹腔注射链脲佐菌素(STZ 15mg/kg)复制糖尿病模型,统计体重变化及空腹血糖值.RT-PCR方法检测造模成功后2周、4周和6周肺组织Gfat1 mRNA表达.结果 模型组大鼠体重增长较快,造模开始第28天,第42天,第56天和第70天高脂模型组与对照组体重差异有显著性(P＜0.05).注射STZ的高脂模型组空腹血糖值较高(FBG≥10.0),和对照组比较,FBG差异有极显著性(P＜0.01).糖尿病大鼠造模成功后2周,模型组肺组织Gfat1的表达低于对照组,与对照组比较差异无显著性(P＞0.05),4周模型组Gfat1的表达高于对照组,模型组与对照组比较差异有显著性(P＜0.05).6周模型组Gfat1的表达高于对照组,但模型组与对照组比较差异无显著性(P＞0.05).结论 大鼠饲喂高脂饲料结合腹腔注射STZ可成功建立2型糖尿病大鼠模型;在不同时期2型糖尿病大鼠肺组织中,Gfat1表达水平发生改变.     

关于建立栀子提取物热原检测方法的考察

目的根据2015版《中国药典》规定的新品种建立热原的指导原则,建立栀子提取物热原的测定方法。方法用国家颁发的细菌内毒素标准品,对设限值剂量的供试品组、10EU/kg内毒素组与10EU/kg内毒素的供试品组同时进行热原检查试验,观察三组家兔的升温情况。结果以上三组同时进行热原检查试验,限值剂量的供试品组结果无异常反应并符合规定,10EU/kg内毒素组与10EU/kg内毒素的供试品组的家兔升温显著且结果为不符合规定。结论以上三组同时进行热原检查试验,限值剂量的供试品组无异常反应并符合规定,10EU/kg内毒素组与10EU/kg内毒素的供试品组的家兔升温显著且结果为不符合规定。说明用该方法对栀子提取物进行热原检查是有效的。     

鲎试验法检测A群流脑多糖菌苗中内毒素的研究

本文应用鲎试验凝胶法定量测定了２１２批Ａ群流脑多糖菌苗中内毒素含量,并与家兔热原试验结果进行了比较,通过试验求得内毒素标准品对家兔体温升温变化关系的线性方程和内毒素对家兔最小致热剂量。结果表明,占检定总批数９４．３％的制品中内毒素含量＜１００ＩＵ／μｇ多糖,家兔热原试验合格率为１００％,５．７％的制品中内毒素含量≥１００ＩＵ／μｇ多糖,家兔热原试验合格率为５８．３％。根据实验结果,可制订用凝胶法替代家兔法进行Ａ群流脑多糖菌苗的热原质试验的判定标准。作者建议：内毒素含量＜１００ＩＵ／μｇ多糖者,热原质试验判为合格,≥１００ＩＵ／μｇ多糖者,用家兔法予以仲裁。     

阿托伐他汀+葡萄籽原花青素预防兔动脉硬化的实验研究

目的:对阿托伐他汀联合葡萄籽原花青素预防兔动脉硬化的效果进行调查。方法:采用电化学发光法对家兔甘油三酯、总胆固醇、低密度脂蛋白固醇、高密度脂蛋白固醇水平进行检测,并采用硫代巴比妥酸法对家兔血清丙二醛含量进行检测。结果:实验前四组家兔的胆固醇、甘油三酯、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇比较无明显差异,P0.05。实验后4组家兔胆固醇、甘油三酯、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇比较存在明显差异,且2组家兔所有指标增长幅度均要明显高于3组、4组。结论:阿托伐他汀联合葡萄籽原花青素能够有效降低兔动脉硬化斑块的发生率。     

不同高温水平对虾夷扇贝存活率、耗氧率和体腔液免疫酶活力的影响

实验室内营造不同升温模式,研究了2种规格(10和46月龄)虾夷扇贝(Mizuhopecten yessoensis)的存活率、耗氧率及体腔液免疫酶活力对不同高温水平(20、22、24、26、28℃)的响应,分析了2种规格虾夷扇贝在不同升温模式下的耐高温能力。实验设计了缓慢升温和快速升温2种升温模式。缓慢升温模式,将在15℃暂养的2种规格扇贝分别驯化到20、22、24、26和28℃温度水平(升温幅度为1℃·d-1),暂养14 d后进行实验。快速升温模式,将在15℃暂养的2种规格扇贝分别快速升温到20、24及28℃温度水平(升温幅度为4℃·d-1),暂养1 d后进行实验。结果表明:缓慢升温模式时,随温度升高,扇贝存活率逐渐降低,28℃时,46和10月龄贝存活率最低分别为96.7%和77.3%,此外,26~28℃时,各实验处理组,46月龄贝存活率显著高于10月龄贝(P0.05);2种规格扇贝耗氧率随温度升高均呈先上升后下降趋势;15~24℃时,10月龄贝的耗氧率显著高于46月龄贝(P0.05);28℃时,10和46月龄贝的耗氧率降至最低,分别为0.527和0.482 mg·g-1·h-1;另外,扇贝的体腔液免疫酶活力随温度升高也均呈现先上升后下降的变化趋势,且在相同升温模式下,46月龄贝的免疫酶活力显著高于10月龄贝(P0.05)。综上,规格较大的虾夷扇贝在不同高温水平时均表现出较强的耐高温能力。     

重组细胞因子制品热原质试验中的降温现象

中国生物制品规程规定采用家兔试验法进行热原试验。规程要求当家兔降温≥０６℃或２只及２只以上降温在０４～０６℃时应重试。在４２０批各类细胞因子热原质试验中发现２３批出现降温,重试后合格率为１００％。降温原因分析表明降温现象与热原性物质无关,与制品种类不存在显著相关性。因此,热原试验中出现家兔降温时是否需要重试值得商榷     

Studies on the Dutch rabbit for pyrogen test

A study was conducted on Dutch rabbits to examine their applicability for the pyrogen test in comparison with the Japanese white rabbit which, conventionally, has been mainly used for the test in Japan. The following results were obtained. Adult Dutch rabbits, so small as to weight only about 60% of average adult weight of the Japanese white, showed a food consumption as low as 40% of that in the latter. None in the Dutch rabbit group suffered dislocation of the hip-joint during restraint on a neck stock while it was frequently encountered among Japanese white rabbits. The body temperature during restraint was unstable in infantile Dutch rabbits (7-8 weeks old)(, but stable in the young (14 weeks old) and in the adult (20-47 weeks old). Adult Dutch rabbits exhibited greater febrile responses to intravenously injected bacterial pyrogen (LPS) than infantile and young Dutch rabbits and adult Japanese white rabbits. A good linear regression was observed between the dose of LPS (0.01-0.1 microgram/kg, iv) and the febrile response in adult Dutch rabbits as well as in adult Japanese white rabbits. From these data, it is concluded that the Dutch rabbit has an advantage over the Japanese white for the pyrogen test in respect of febrile responsiveness and cost of rearing.     

Evidence for a contribution by brown adipose tissue to the development of fever in the young rabbit

This study was undertaken to determine if brown adipose tissue was involved in heat production during fever produced by S. abortus equi (1 micrograms) in unanesthetized rabbits aged 19-26 days. The fever (0.9-1.6 degrees C) occurred after a delay of 20-30 min and was frequently biphasic. Radiolabelled microspheres for measuring tissue blood flow were injected intraventricularly into three groups of animals: rabbits not given pyrogen, rabbits in which the febrile response to pyrogen was developing, and rabbits in which the febrile response had peaked. Blood flow to brown fat deposits and other organs was calculated from the fractional distribution of the microspheres and the recovery of microspheres in a reference arterial blood sample. At the fever peak, blood flow to brown fat was not significantly different (p greater than 0.05) from the control value (0.9 +/- 0.2), but during the rising phase of the fever the flow increased significantly (p less than 0.01) to 2.6 +/- 0.4 mL min-1 g-1. The blood flow to muscles of the forelimbs and hind limbs was also increased significantly (p less than 0.05) during the rising phase of the fever. No significant change in blood flow to other organs or tissues was found during the rising phase of the fever. These results indicate that both nonshivering as well as shivering thermogenesis contribute to heat production during development of fever in the young rabbit. However, nonshivering thermogenesis was not involved in the maintenance of the elevated body temperature after the fever had peaked.     

实验兔脚皮炎与相对湿度的相关性及对热原筛选实验的影响分析

目的分析实验兔脚皮炎发病率与相对湿度的相关性及脚皮炎实验兔对热原筛选实验合格率的影响。方法将2006、2007年实验室相对湿度记录、实验兔脚皮炎记录及热原筛选实验记录进行汇总统计,并对数据进行生物学分析。结果实验室相对湿度超过70％时,可导致实验兔脚皮炎发病率的升高,二者存在显著相关性（P〈0．01）;有脚皮炎的实验兔可降低筛选实验的合格率,二者存在显著相关性（P〈0．01）;实验兔基础体温的高低,对筛选合格率有显著影响（P〈0．0001）。结论实验兔的脚皮炎发病率与环境相对湿度有显著的正相关性,当实验室相对湿度超过实验兔适宜湿度范围时,可导致其脚皮炎的发病率上升;有脚皮炎的实验兔与无脚皮炎的实验免相比,可显著降低热原筛选实验的合格率。     

Studies on Bacterial Pyrogenicity: III. Specificity of the United States Pharmacopeia Rabbit Pyrogen Test

The specificity of the first or “presumptive” portion of the USP rabbit pyrogen test was investigated by use of a new absolute standard of reference. The reference standard was a 0.9% sodium chloride solution prepared to be pyrogen-free. Details of the preparation were described. The hypothesis was explored that the temperature response of rabbits after intravenous injection of the standard solution was independent of exogenous pyrogen. Reactions observed among the rabbits in our colony allowed a classification of these animals ranging from “consistently reliable” to “consistently unreliable.” Details of the experimental results and implications for pyrogen testing are discussed. The recommendation was made that all rabbit test animals be “screened” in sham and actual tests before being used for pyrogen testing.     

Hyperthermia and disaggregation of brain polysomes induced by bacterial pyrogen.

Hyperthermia induced following injection of bacterial pyrogen to rabbits was associated with a disaggregation of brain polysomes to monosomes. Direct elevation of the body temperature to levels similar to that found after pyrogen administration also resulted in a brain polysome shift. The disaggregation of brain polysomes after either pyrogen injection or elevation of ambient temperature was not due to ribonuclease activation and the phenomenon was associated with a relocalization of polyadenylated mRNA from polysomes to monosomes. Since polysome disaggregation was not found in kidney, it appears that the brain may be more sensitive to elevations in body temperature.     

Differences in endogenous pyrogen fevers induced by iv and icv routes in rabbits

We have compared the characteristics of fevers produced by endogenous pyrogen administered by the intravenous (iv) and by the intracerebroventricular (icv) routes in conscious rabbits. Fevers induced by the intracerebroventricular route have a longer latency to onset, a less steep rise in body temperature, and a longer time to peak elevation in body temperature than do fevers induced by the intravenous route. Furthermore, a dose of indomethacin (2 mg/kg) administered intravenously, which is effective in markedly attenuating fevers produced by the intravenous route, was completely without effect on fevers induced by the intracerebroventricular route. On the other hand, when indomethacin (500 micrograms) was infused intracerebroventricularly, it markedly reduced fevers induced by the subsequent injection of endogenous pyrogen into the contralateral cerebral ventricle, but such pretreatment had little effect on fevers elicited by intravenous injections of endogenous pyrogen. It is concluded that the sites of action of endogenous pyrogen in response to intravenous injections of pyrogen are different from those responding to intracerebroventricular injections of pyrogen and that this is manifest in several distinct differences in the characteristics of the two fevers. These results indicate that the intracerebroventricular model of fever production is not appropriate for the study of the normal pathogenesis of fever.