冻干人用狂犬病疫苗(Vero细胞)稳定剂配方的筛选

目的筛选出适合冻干人用狂犬病疫苗(Vero细胞)的热稳定性好、无明胶的稳定剂配方。方法以水分、外观、效价、热稳定性效价为指标,尤其是效价和热稳定性效价,对A、B、C、D、E、F、G等6种稳定剂配方进行系统的优化组合筛选,6种稳定剂配方分别含有蔗糖、乳糖、人血白蛋白、甘氨酸、精氨酸、明胶、尿素、甘露醇、右旋糖苷,其中A为现行疫苗生产配方。运用单因素五元设计法,筛选出最优稳定剂组合。结果方差分析结果显示D组配方对冻干人用狂犬病疫苗(Vero细胞)的M-ABT结果 13.95,37℃放置4周M-ABT结果 13.05;NIH效价1.94,37℃放置4周NIH效价1.56。结论 D组配方对冻干人用狂犬病疫苗(Vero细胞)有较好的保护作用。     

冻干干扰素保护剂(稳定剂)的筛选

取同一批重组人干扰素α2a原液,分别加入A,B,C,D,E五种冻干保护剂,按《中国生物制品规程》方法进行冻干,检测其效价,外观,水分含量,内毒素含量,以现行干扰素生产使用的人血白蛋白C保护剂作为对照,结果显示,使用A,D,E保护剂制品在40℃放置4个月,效价降低了45％－60％,使用B保护剂的制品稳定性最好,在40℃放置4个月,效价仅下降了35％,B保护剂配方为最佳保护剂配方。     

两种狂犬病毒IgG抗体检测试剂盒的比较

目的了解员工暴露前接种人用狂犬病疫苗后的免疫效果,并对比使用两种不同试剂盒检测抗体阳转率是否存在差异。方法采集员工接种人用狂犬病疫苗(地鼠肾细胞)以及冻干人用狂犬病疫苗(Vero细胞)共计172例,分别使用两种狂犬病毒抗体检测试剂盒(试剂盒A、B)进行检测,统计血清中狂犬病毒Ig G抗体的水平,计算阳转率并比较差异。结果使用试剂盒A检测接种人用狂犬病疫苗(地鼠肾细胞)者血清样本的阳转率为91.7%,接种冻干人用狂犬病疫苗(Vero细胞)者血清样本的阳转率为51.0%;使用试剂盒B检测接种人用狂犬病疫苗(地鼠肾细胞)者血清样本的阳转率为100.0%,接种冻干人用狂犬病疫苗(Vero细胞)者血清样本的阳转率为74.5%。使用试剂盒B检测接种人用狂犬病疫苗(地鼠肾细胞)者血清样本的阳转率比试剂盒A高8.3%,使用试剂盒B检测接种冻干人用狂犬病疫苗(Vero细胞)者血清样本的阳转率比试剂盒A高23.5%。结论两种不同试剂盒上检测的抗体阳转率都反映出人用狂犬病疫苗(地鼠肾细胞)比冻干人用狂犬病疫苗(Vero细胞)的免疫效果好;使用两种不同试剂盒检测抗体阳转率的差异均具有统计学意义。     

高效价冻干人用狂犬病疫苗暴露后免疫程序的研究

初步确定高效价冻干人用狂犬病疫苗（6．0IU／剂）暴露后免疫程序。制备高效价的冻干人用狂犬病疫苗（6．0IU／剂）,以狂犬病街毒CNX8601和BD06分别攻击小鼠和比格犬的咬肌,接种不同效价的狂犬病疫苗,以RFFIT法检测中和抗体,根据动物死亡情况,计算暴露后疫苗保护率,对不同效价的疫苗进行中和抗体测定和保护率统计分析。在以小鼠为实验动物的疫苗保护率研究中,冻干人用狂犬病疫苗（3．1IU／剂）0／3／7／14／28免疫程序的保护率为40．6％,高效价的冻干人用狂犬病疫苗（6．0IU／剂）0／3／14免疫程序的保护率为56．2％,中和抗体比较,P〈0．05,2组间有显著性差异;在以比格犬为实验动物的保护效果研究中,冻干人用狂犬病疫苗的保护率（3．1IU／剂）为70％;高效价的冻干人用狂犬病疫苗（6．0IU／剂）的保护率为80％,中和抗体的比较,P〉0．05,没有显著性差异。高效价冻干人用狂犬病疫苗暴露后免疫程序可初步确定为0、3、14d免疫。     

A+C群脑膜炎球菌多糖疫苗稳定性研究

为确定A C群脑膜炎球菌多糖疫苗有效期。对该疫苗在不同储藏温度下的稳定性进行了系统研究,将C群多糖抗原放置在37℃,A C群多糖疫苗分别放置在2-8℃,37℃和56℃,定期取样,用琼脂糖CL-4B凝胶柱层析后,测定Kd值在0.5以前的多糖回收率,结果表明,C群多糖抗原在37℃保存9周,A C群多糖疫苗于2-8℃保存4年,37℃保存88周,56℃保存10个月,多糖抗原或多糖疫苗的Kd值小于0.4,多糖回收率大于75%,符合规程要求;因而A C群脑膜炎球菌多糖疫苗的有效期可定为在2-8℃储藏条件下3年6个月。     

应用生物反应器建立人用冻干狂犬病疫苗(Vero细胞)生产工艺的研究

应用15L生物反应器,采用片状载体对Vero细胞进行高密度培养、制备高毒力滴度的狂犬病毒收获液,经纯化后生产人用冻干狂犬病疫苗。采用15L生物反应器对培养方法(批次培养和连续灌流培养)进行试验,收获高毒力滴度的狂犬病毒收获液并制备人用冻干狂犬病疫苗。结果表明:Vero细胞在接种狂犬病毒后可以连续收获病毒液达到25d以上,冻干狂犬病疫苗的效价可以达到5.54IU/剂。本工艺可以用于进行大规模的人用冻干狂犬疫苗的生产。     

A群C群脑膜炎球菌结合疫苗稳定性

目的评价A群C群脑膜炎球菌结合疫苗原液和成品的稳定性。方法分别将A群、C群脑膜炎球菌结合疫苗原液及A群C群脑膜炎球菌结合疫苗各选取连续3批,分别放置于37℃、20~25℃和2~8℃3种温度下,在一定的时间取样进行主要项目测定,在关键时间点进行全面检测。结果 A群结合疫苗原液于2~8℃保存9个月,20~25℃保存4周,37℃保存4 d;C群结合疫苗原液于2~8℃保存9个月,20~25℃保存6个月,37℃保存4周;A群C群脑膜炎球菌结合疫苗于2~8℃保存2年3个月,20~25℃保存6个月,37℃可以保存9周;各项检测指标均符合质量标准的要求。结论在2~8℃条件下,A群、C群脑膜炎球菌结合疫苗原液存放6个月,A群C群脑膜炎球菌结合疫苗存放2年,其质量稳定。     

保存温度和时间对狂犬病疫苗效力稳定性的影响

对人用精制狂犬病疫苗和浓缩狂犬疫苗分析在－33℃,2－8℃,25℃,37℃保存0－6个月进行效力的稳定性观察,结果表明随存放时间及温度增加,两者的生物活性均有不同程度的降低。在2－8℃条件下疫苗的效力明显比其它温度稳定（P＜0．02）;精制疫苗与浓缩疫苗比较,其效力有更稳定的趋势。     

流感病毒裂解疫苗裂解剂去除工艺改进及稳定性研究

目的改进流感病毒裂解疫苗裂解剂去除工艺,降低残余卵清蛋白和裂解剂含量,提高疫苗质量,降低成本。方法分别将A1、A3和B型流感病毒纯化液用磷酸缓冲液（PB）沉淀法去除裂解剂,经超滤、除菌制备原液,配制6批半成品,其中3批不含硫柳汞,3批含硫柳汞,经全面检定,并观察放置37℃、25℃和2～8℃不同时间的稳定性。结果该疫苗各项指标均符合《中国药典》（2010年版）三部要求,其中卵清蛋白平均为3．83ng／mL,裂解剂平均为57μg／mL,比改进前分别降低97．9％和69％。37℃放置4周、25℃3个月及2-8℃12个月后检定全部合格。结论该工艺步骤简单,去除卵清蛋白和裂解剂效果明显,是进一步提高疫苗质量和降低成本的有效工艺。     

人狂犬病免疫球蛋白使用效果观察

为了解人狂犬病免疫球蛋白的作用效果,我们将观察对象随机分成了A、B、C三组,分别采用三种措施进行狂犬病的预防治疗,即：A组联合使用狂犬病疫苗与人狂犬病免疫球蛋白;B组联合使用狂犬病疫苗与抗狂犬病血清(马源);C组仅注射狂犬病疫苗,并采用小鼠中和试验对这三组成员在免疫后3、7、14、45天及1年时的中和抗体水平进行检测。结果表明：狂犬病疫苗与人狂犬病免疫球蛋白或抗狂犬病血清联合使用,可使体内更早出现抗狂犬病的中和抗体。注射人狂犬病免疫球蛋白后未发生临床副反应。     

A time course study on dose-response relationship between alcohol exposure and its effects on lipid profile and biomarkers of tissue damage

This present research investigated variations in lipid profiles and important biomarkers of tissue damage in response to graded concentrations of alcohol administration in male Wistar rats. Group A (control) received distilled water while group B, C and D received 30%, 40% and 50% (v/v) alcohol respectively. Five rats each from groups A-D were sacrificed after day(s) 1, 7, 14, 21 and 28 of administration. A significant increase was observed at day 28 for serum cholesterol by 79% (group B), 78% (group C) and 47% (group D) together with serum phospholipid 58% (group B), 50% (group C) and 92% (group D). Serum triacylglycerol increased by 71% (group B), 43% (group C) and 16% (group D) at day 21, while concentration of serum albumin decreased at day 28 by 40.9% (group B), 50.2% (group C), 53.3% (group D) respectively when compared with control (group A). Serum aminotransferases and alkaline phosphatase specific activities, as well as creatinine and uric acid concentration increased in a concentration-dependent manner, following alcohol administration. Though most of these effects induced by alcohol were time- and concentration-dependent, 40% alcohol appear to be more stable, giving results consistent with alcohol-induced damages, with minimal mortality. This study therefore further validated dyslipidemia and imbalance in clinical biomarkers as hallmarks of tissue damage induced by excessive alcohol consumption with an insight on the time- and concentration-response relationship between alcohol consumption and its toxicity.     

Evidence for an essential role of long chain acyl-CoA synthetase in animal cell proliferation. Inhibition of long chain acyl-CoA synthetase by triacsins caused inhibition of Raji cell proliferation

Triacsins A, B, C, and D are new inhibitors of long chain acyl-CoA synthetase (EC 6.2.1.3) and possess different inhibitory potencies against the enzyme (Tomoda, H., Igarashi, K., and Omura, S. (1987) Biochim. Biophys. Acta 921, 595-598). Acyl-CoA synthetase activity in the membrane fraction of Raji cells was also inhibited by triacsins. The same hierarchy of inhibitory potency as that against the enzyme from other sources, triacsin C greater than triacsin A much greater than triacsin D greater than or equal to triacsin B, was observed. When Raji cells were cultivated in the presence of triacsins, cell proliferation was inhibited in a dose-dependent fashion. The drug concentrations required for 50% inhibition of cell growth at day 2 were calculated to be 1.8 microM for triacsin A, much greater than 20 microM for triacsin B, 1.0 microM for triacsin C, and much greater than 15 microM for triacsin D, demonstrating a hierarchy for inhibitory potency of triacsins similar to that against the acyl-CoA synthetase activity. To understand the role of long chain acyl-CoA synthetase in animal cells, the effect of triacsins on the lipid metabolism of Raji cells was studied. When intact Raji cells were incubated with [14C]oleate in the presence of individual triacsins, the incorporation of [14C]oleate into each of the lipid fractions such as phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol was inhibited to an analogous extent. A common hierarchy, triacsin C greater than triacsin A much greater than triacsin D greater than triacsin B, was shown for the inhibition in each synthesis of the three lipids, which was identical with that for acyl-CoA synthetase. These findings indicate that the inhibition of acyl-CoA synthetase is well correlated with the inhibition of lipid synthesis. Taken together, the data strongly suggest that the inhibition of acyl-CoA synthetase by triacsins leads to the inhibition of lipid synthesis and eventually to the inhibition of proliferation of Raji cells.     

Insight into the mechanism of dopamine D1-like receptor activation. Evidence for a molecular interplay between the third extracellular loop and the cytoplasmic tail

A chimeric D1A dopaminergic receptor harboring the cytoplasmic tail (CT) of the D1B subtype (D1A-CTB) has been used previously to show that CT imparts high dopamine (DA) affinity and constitutive activity to the D1B receptors. However, the D1A-CTB chimera, unlike the D1B subtype, exhibits a significantly lower DA potency for stimulating adenylyl cyclase and a drastically lower maximal binding capacity (Bmax). Here, using a functional complementation of chimeric D1-like receptors, we have identified the human D1B receptor regions regulating the intramolecular relationships that lead to an increased DA potency and contribute to Bmax. We demonstrate that the addition of variant residues of the third extracellular loop (EL3) of the human D1B receptor into D1A-CTB chimera leads to a constitutively active mutant receptor displaying an increased DA affinity, potency, and Bmax. These results strongly suggest that constitutively active D1-like receptors can adopt multiple active conformations, notably one that confers increased DA affinity with decreased DA potency and Bmax and another that imparts increased DA affinity with a strikingly increased DA potency and Bmax. Overall, we show that a novel molecular interplay between EL3 and CT regulates multiple active conformations of D1-like receptors and may have potential implications for other G protein-coupled receptor classes.     

用皂土法制造型特异性肉毒诊断血清

用皂土为载体与类毒素结合方法及破伤风类毒素抗原抗体絮状反应方法去除A、B、C、D、E、F型肉毒抗血清原料中的异型和异种抗毒素(破伤风抗毒素)。制备的A、B、C、D、E、F型肉毒诊断血清每1m l均能中和相应型的肉毒毒素10000LD50以上,而中和异型肉毒毒素或破伤风毒素均低于5 LD50;A、B、C、D、E、F各型混合后的混合型血清每1m l能中和各型肉毒毒素亦大于10000 LD50,中和破伤风毒素低于5 LD50,即效价和特异性符合规程要求。     

Epidemiology of Streptococcus pneumoniae Serogroup 6 Isolates from IPD in Children and Adults in Germany

This study presents serogroup 6 isolates from invasive pneumococcal disease (IPD) before and after the recommendation for childhood pneumococcal conjugate vaccination in Germany (July 2006). A total of 19,299 (children: 3508, adults: 15,791) isolates were serotyped. Serogroup 6 isolates accounted for 9.5% (children) and 6.7% (adults), respectively. 548 isolates had serotype 6A, 558 had serotype 6B, 285 had serotype 6C, and 4 had serotype 6D. Among children, serotype 6B was most prevalent (7.5% of isolates) before vaccination, followed by 6A and 6C. After the 7-valent pneumococcal conjugate vaccine (PCV7), the prevalence of serotype 6B significantly decreased (p = 0.040), a pattern which continued in the higher-valent PCV period (PCV10, PCV13). Serotype 6A prevalence showed a slight increase directly after the start of PCV7 vaccination, followed by a decrease which continued throughout the PCV10/13 period. Serotype 6C prevalence remained low. Serotype 6D was not found among IPD isolates from children. Among adults, prevalence of both 6A and 6B decreased, with 6B reaching statistical significance (p = 0.045) and 6A showing a small increase in 2011–2012. Serotype 6C prevalence was 1.5% or lower before vaccination, but increased post-vaccination to 3.6% in 2011/12 (p = 0.031). Four serotype 6D isolates were found post-PCV7 childhood vaccination, and two post-PCV10/13. Antibiotic resistance was found mainly in serotype 6B; serotype 6A showed lower resistance rates. Serotype 6C isolates only showed resistance among adults; serotype 6D isolates showed no resistance. Multilocus sequence typing showed that sequence type (ST) 1692 was the most prevalent serotype 6C clone. Thirty-two other STs were found among serotype 6C isolates, of which 12 have not been previously reported. The four serotype 6D isolates had ST 948, ST 2185 and two new STs: 8422 and 8442. Two serogroup 6 isolates could not be assigned to a serotype, but had STs common to serogroup 6.     

PLGA-5-氟尿嘧啶缓释微球治疗结直肠癌裸鼠的探讨

目的研究PLGA-5-氟尿嘧啶缓释微球瘤周给药对结直肠癌移植瘤的治疗效果。方法将60只结直肠癌荷瘤鼠随机分为6组,每组10只。A、B组瘤周注射PLGA-5-氟尿嘧啶缓释微球,5-氟尿嘧啶剂量分别为200mg/kg和100 mg/kg;C、D组瘤周注射5-氟尿嘧啶注射液,5-氟尿嘧啶剂量分别为200 mg/kg和100 mg/kg;E组瘤周注射PLGA微球,800 mg/kg;F组不给予任何治疗。于0、3、6、9、12、15d观察裸鼠生存状况、称体重、测量肿瘤大小。15d时处死动物,称瘤重,计算抑瘤率,绘制肿瘤生长曲线;取血行白细胞计数、肝肾功能检查。结果A、B组肿瘤生长曲线平缓,15 d时A、B组肿瘤体积与C、D、E、F组比较,结果差异有显著性,C、D组肿瘤体积与E、F组比较差异无显著性;A、B组抑瘤率分别为75%和62%,与C、D组比较,结果差异有显著性;A、B、C、D、E组体重在0 d及15 d与F组比较,差异无显著性,15 d时各组白细胞计数及肝肾功能检查值均在正常范围。结论PLGA-5-氟尿嘧啶缓释微球瘤周给药能有效抑制结直肠癌移植瘤的生长,且无明显的毒副作用。     

双歧杆菌对新生大鼠坏死性小肠结肠炎肠损伤及肠道菌群影响

目的建立新生SD大鼠坏死性小肠结肠炎（NEC）模型,探讨添加双歧杆菌对新生大鼠NEC模型肠损伤的保护作用及肠道菌群的影响,为应用双歧杆菌防治NEC提供依据：方法SD新生大鼠出生48h开始给予鼠配方奶人工喂养,100％氮气缺氧90s,4℃冷刺激10min,每天2次,连续3d,建立新生SD大鼠NEC模型。按析因设计,32只新生SD大鼠随机分成4组,每组动物各8只。A组为NEC模型组并在出生48h起每日给予长双歧杆菌灌胃（1×10^8CFU／d）;B组为NEC模型组,C组为对照组,都未添加双歧杆菌;D组为对照组并给予长双歧杆菌灌胃（1×10^8CFU／d）。在最后一次缺氧、冷刺激后24h空腹断头处死大鼠,留取回盲部近端肠管组织进行肠组织损伤评分,组织学评分≥2确定为NEC;实验前后留取各组新生鼠粪便,按照张秀荣方法进行肠道菌群检测。应用Kruskal-Wallis H检验等方法进行统计学分析,α=0．05为显著性检验标准。结果开始造模后,A、B组新生SD大鼠相继出现腹泻、腹胀、萎靡、活动减少,A组程度较轻。A、B、C和D组肠组织损伤评分（^-x±s）分别为1．88±0.84、3．13±0．84、0．63±0．52、0．50±0.54,各组间肠组织损伤评分差异有显著性,与B组相比,A组肠组织损伤评分明显降低,但仍高于C、D两组,差异均有显著性。各组实验前肠道细菌总数,杆菌、球菌数,G^＋杆菌、G^＋球菌数差异均无显著性,肠道菌群中杆球菌比值在正常范围中。实验结束时,各组间新生鼠肠道细菌总数,杆菌、球菌数,杆球菌比值,G^＋杆菌、G^＋球菌数及其占肠道总菌群数的比率差异均有显著性;除B组杆菌数外,与实验前相比,各组新生大鼠实验结束后肠道细菌总数、球菌数、杆菌数、G^＋杆菌数、G^＋球菌数均有显著增加,差异均有显著性;A、B组肠道杆球菌比值和G^＋杆菌数占肠道总菌群数比     

Effect of energy source during culture on in vitro embryo development, resistance to cryopreservation and sex ratio

The aim of this work was to evaluate whether minimizing the glucose concentration during culture or replacing the hexose with other energy substrates and/or embryotrophic compounds would affect the in vitro development, the resistance to cryopreservation and the sex ratio of bovine embryos. In vitro matured and fertilized oocytes were randomly assigned to 4 groups for in vitro culture, that differed in the energy substrates included: group A) 1.5 mM glucose, as in standard SOF; group B) 0.15 mM glucose; group C) 0.125 mM G3P, in the presence of 0.15 mM glucose and group D) 0.34 mM citrate, in combination with 2.77 mM myo-inositol. Blastocysts were evaluated on day 7, then vitrified by cryotop in 16.5% DMSO, 16.5% EG and 0.5 M sucrose and warmed in decreasing concentration of sucrose (0.25 to 0.15 M sucrose). The survival rates were assessed after 24 h in vitro culture. Finally, the blastocysts produced were sexed by PCR. An increased blastocyst rate was recorded in groups B, C and D, i.e., when glucose concentration was reduced, compared to group A (28.2, 41.0, 35.7 and 35.8, respectively in groups A, B, C and D; P < 0.01). However, the embryos cultured in group D showed the slowest developmental speed, indicated by the lowest percentage of advanced stage-embryos (expanded and hatched blastocysts) out of the total blastocysts (56.1, 45.8, 56.9 and 31.8 %, respectively in groups A, B, C and D; P < 0.01). Furthermore, survival rates after 24 h culture of vitrified-warmed blastocysts also decreased in group D (73.3, 73.1, 71.4 and 58.4%, respectively in groups A, B, C and D; P < 0.01). Interestingly, in group D a higher percentage of female embryos was obtained compared to group A, with intermediate values in groups B and C (45.6, 53.4, 50.0 and 61.5%, respectively in groups A, B, C and D; P < 0.05). In conclusion, it was demonstrated that the energy substrate during in vitro culture affects both the production and the viability of blastocysts. Furthermore, manipulating the metabolic profile of embryos during in vitro culture may have an impact on sex ratio.     

哈尔滨地区假丝酵母菌DNA异质性及药物敏感性分析

研究假丝酵母菌的DNA异质性及药物敏感性,为预防和监控院内假丝酵母菌感染奠定基础。将临床分离的假丝酵母菌菌株,用科玛嘉显色培养基鉴定菌种,经纸片扩散法进行药敏试验,应用随机扩增多态性DNA（RAPD）技术对这些菌株进行基因分型。结果显示：93株假丝酵母菌中白假丝酵母菌68株,非白假丝酵母菌25株,所有菌株对制霉菌素,两性霉素B两种药物的敏感率最高（100％）,酮康唑其次（70．9％）,氟康唑的敏感率最低（50．5％）,引物1和引物2将来源不同的68株白假丝酵母菌分别分成4型（A1、B1、C1、D1）和6型（A2、B2、C2、D2、E2、F2）。哈尔滨地区的假丝酵母菌感染以白假丝酵母菌为主,且主要为A1、B1型（引物1）或A2、B2型（引物2）;基因型与药敏谱无明显相关性。     

Embryos produced in vitro from bulls carrying 16;20 and 1;29 Robertsonian translocations: efficiency and kinetics of oocyte fertilization and embryo development

The present experiments were designed to study the effects of Robertsonian translocations on the efficiency and kinetics of in vitro fertilization and early and advanced embryo development. Spermatozoa from bulls with rob(16;20), rob(1;29) and normal karyotype (A, B and C, respectively) were used. Oocytes were matured, fertilized and cultured by the standard protocol described previously. Twenty-four hours after fertilization, adequate numbers of oocytes were fixed, stained and examined. The development of embryos was evaluated on days 2 (D2), 7 (D7) and 8 (D8) after fertilization. The rate of normally fertilized oocytes was significantly lower (p < or = 0.01) for bull A than for bulls B and C. However, no significant differences in the kinetics of fertilization were found between bulls A, B and C. The D2 cleavage rate of embryos was significantly lower (p < or = 0.01) for bull A than for bulls B and C. Both D7 and D8 blastocyst rates for bull A or bull B were significantly lower (p < or = 0.01 or p < or = 0.05) than those for bull C. The percentages of both D7 advanced blastocysts and D8 expanded blastocysts were significantly lower (p < or = 0.01) for bulls A and B than for bull C. In conclusion, for rob(16;20), the efficiency of fertilization was strongly reduced; it resulted in low early and advanced embryo development. On the other hand, for the rob(1;29), neither fertilization nor early embryo development were affected and only advanced embryo development was decreased. But for both translocations, blastocyst formation was significantly delayed.