ACYW135群脑膜炎球菌多糖疫苗的免疫原性评价

目的评价ACYW135群脑膜炎球菌多糖疫苗在昆明健康人群接种的免疫原性,为流脑防治策略提供依据。方法 2010年对昆明市2岁!3、岁!、4岁!、5岁!、6岁!、10岁!、≥15岁共7个年龄组分层随机抽取筛选出654名健康人,分别采集免前和免后1个月血清。用微量杀菌力试验(TTC法)分别检测血清中抗A、C、Y和W135群脑膜炎球菌杀菌抗体的水平。结果免后1个月抗A、C、Y和W135群脑膜炎球菌的杀菌抗体阳转率分别为96.99%、96.37%、88.43%和87.07%,抗A、C、Y和W135群膜炎球菌血清的杀菌抗体几何平均滴度(GMT)分别为1∶297.991、∶195.80、1∶72.74和1∶45.95。结论 ACYW135群脑膜炎球菌多糖疫苗在≥2岁以上的健康人群中有较好的免疫原性。     

速率比浊法检测ACYW135群脑膜炎球菌结合疫苗中各群多糖含量

目的建立速率比浊法准确检测ACYW135群脑膜炎球菌结合疫苗(group ACYW135 meningococcal conjugate vaccine)的各群多糖含量。方法制备多糖参考品和抗血清,建立检测ACYW135群脑膜炎球菌结合疫苗各群多糖含量的速率比浊法,对建立的方法进行验证及初步应用。结果①建立的方法具备高度的特异性,各群血清仅与本群多糖抗原发生特异性反应,与非本群多糖抗原无交叉反应;②在质量浓度为0.5～5.0μg/mL多糖参考品检测区间线性良好,相关系数r>0.98;③各群血清的最低检测限均低于质量浓度为0.35μg/mL;④批内和批间变异系数(CV)值分别为0.83%～5.34%和2.33%～13.88%,加样回收率为90.5%～118.4%,精密度和准确度均符合常规质量控制要求;⑤初步应用结果显示,建立的方法可准确检测疫苗的各群多糖含量,且与火箭电泳法检测结果差异无统计学意义(P>0.05)。结论建立的方法简便快捷、灵敏准确、自动化程度高,可有效检测ACYW135群脑膜炎球菌结合疫苗的各群多糖含量。     

2～59岁健康人群接种ACYW135群脑膜炎球菌多糖疫苗的免疫原性观察

目的评价ACYW135群脑膜炎球菌多糖疫苗在2～59岁健康人群中的免疫原性。方法 2～59岁健康人群接种者随机抽样(n=60),接种一剂四价脑膜炎球菌多糖疫苗。采集接种前和接种后1个月血清,采用体外杀菌试验(Serum bactericidal assay,SBA)检测血清中抗A、C、Y、W135群脑膜炎球菌的血清杀菌滴度。结果免疫前、后血清抗A群脑膜炎球菌的血清杀菌滴度GMTs(95%CI)分别为1241(736,2091)和7559(5520,10351)(P<0.05);抗C群脑膜炎球菌的血清杀菌滴度GMTs(95%CI)分别为4(9,21)和4787(2947,7775)(P<0.05);抗W135群脑膜炎球菌的血清杀菌滴度GMTs(95%CI)分别为16(9,28)和368(162,883)(P<0.05);抗Y群脑膜炎球菌的血清杀菌滴度GMTs(95%CI)分别为120(58,246)和1373(687,2745)(P<0.05)。免疫前和免疫后血清抗A群脑膜炎球菌的杀菌滴度≥128的比例分别为87(77.4,95.1)%和100(83.2,100)%;抗C群脑膜炎球菌的比例分别为17(8.3,28.5)%和97(88.5,99.6)%;抗W135群脑膜炎球菌的比例分别为13(5.9,24.6)%和68(55.0,79.7)%;抗Y群脑膜炎球菌的比例分别为57(43.2,69.4)%和85(73.4,92.9)%。免疫后较免疫前抗A群、C群、W135群和Y群脑膜炎球菌杀菌抗体滴度≥4倍升高的比例分别为50(27.2,72.8)%、97(88.5,99.6)%、62(43.2,73.9)%和55(41.6,67.9)%。结论虽然免疫前人群由于地方和国家免疫计划的实施已具有较高水平的抗A群脑膜炎球菌的血清杀菌滴度,但接种ACYW135群脑膜炎球菌多糖疫苗后可以使其保护水平进一步提高,并使人群对C群、W135群和Y群脑膜炎球菌的低水平杀菌抗体滴度均显著升高达到保护水平,证明ACYW135群脑膜炎球菌多糖疫苗在2～59岁健康人群中具有比较好的免疫原性。     

火箭免疫电泳法检测ACYW135群脑膜炎球菌多糖疫苗多糖含量和分子大小的初步验证

目的对火箭电泳法用于检测ACYW135群脑膜炎球菌多糖疫苗的多糖含量和多糖分子大小进行验证。方法用化学法和火箭电泳法分别检测ACYW135群脑膜炎球菌多糖疫苗的多糖含量和多糖分子大小,统计学分析两种方法的检测结果。结果两种方法的检测结果差异无统计学意义。结论火箭电泳法可以用于检测ACYW135群脑膜炎球菌多糖疫苗的多糖含量和多糖分子大小。     

高效阴离子交换色谱-脉冲安培法检测A、C、Y和W135群脑膜炎球菌多糖含量

目的用高效阴离子交换色谱-脉冲安培检测法(HPAEC-PAD)测定A、C、Y和W135群脑膜炎球菌多糖含量,并对该方法进行验证及初步应用。方法采用Carbo Pac~?PA-1 4×250 mm分析柱与Aminotrap 4×50 mm保护柱;使用氢氧化钠/醋酸钠梯度洗脱,以多糖抗原标准溶液质量浓度为横坐标,其相应的峰面积为纵坐标,绘制标准曲线,并对建立的方法进行专属性、精密度和准确性验证;同时与传统方法的检测结果进行比较分析。结果 A、C、Y和W135群脑膜炎球菌多糖质量浓度在0.5～27.0μg/m L范围内与峰面积均具有良好线性关系,r=0.999。该方法专属性高,分析结果重复性良好,RSD均5%;回收率均在95%～105%范围内;与传统的火箭免疫电泳法相比,检测结果差异均无统计学意义(P0.05)。结论 HPAEC-PAD准确、可靠,重复性好,可用于检测脑膜炎球菌多糖原液及疫苗成品中的多糖含量。     

W135群脑膜炎球菌发酵工艺的研究

为研究A、C、Y、W 135群流脑多糖疫苗,针对W 135群脑膜炎球菌的生长特性,采用国产50L发酵罐,针对流脑半综合液体培养基中葡萄糖浓度;培养温度、菌种接种浓度、pH、通氧量及搅拌速度等因素对W 135群脑膜炎球菌生长的影响,选择最适的培养条件。结果表明,选择半综合培养基中补加1%的葡萄糖培养效果良好,菌种接种浓度直接影响多糖复合物产量;最适pH值6.5~7.5的范围可维持W 135群链球菌快速生长;温度控制在36.5±0.2℃可得到较高的培养产物;培养过程中加大通气量及搅拌速度对培养结果有明显改善。确立了W 135群脑膜炎球菌的最适培养条件,在确定培养条件后连续进行3批500L罐放大中试培养,达到规模化生产要求。     

不同酸水解酪蛋白对W135群和Y群脑膜炎奈瑟菌荚膜多糖产量的影响

目的 探索不同酸水解酪蛋白对W135群与Y群脑膜炎奈瑟菌(脑膜炎球菌)荚膜多糖产量的影响。方法 分别以NaCl质量分数为37%和14%的酸水解酪蛋白作为有机氮源配制改良半综合高盐培养基和低盐培养基,利用全自动细菌发酵罐分别在两种培养基里培养W135群和Y群脑膜炎球菌,比较这两种菌株在两种培养基中的生长时间、收获液菌密度( A 600 nm 值)、收获液去菌体后与去复合多糖后上清中的荚膜多糖含量,以及纯化后的精糖产量;比较高盐培养基收获液及其2倍稀释液中不同终含量的十六烷基三甲基溴化铵(cetyltrimethylammonium bromide, CTAB)溶液对多糖沉淀效果的影响。结果 W135群与Y群脑膜炎球菌在两种培养基中的培养时间差异无统计学意义( P >0.05),但高盐培养基收获液的菌密度、收获液去菌体后上清液中的多糖含量均高于低盐培养基收获液,差异有统计学意义( P < 0.05),而高盐培养基收获液纯化后的精糖产量低于低盐培养基,差异有统计学意义( P <0.05)。高盐培养液2倍稀释后,在CTAB终体积分数为0.04%时,W135群与Y群脑膜炎球菌多糖全部沉淀,而未稀释高盐培养液即使CTAB终体积分数提高到0.14%,依然也不能完全沉淀 W135群与Y群脑膜炎球菌多糖。结论 不同酸水解酪蛋白对W135群和Y群脑膜炎球菌的生长密度和荚膜多糖产量有影响,用CTAB溶液沉淀荚膜多糖时需控制收获液盐含量。     

检测四价脑膜炎球菌多糖疫苗中A群多糖含量ELISA法的建立

目的建立双抗体夹心ELISA法,对A群流脑多糖抗原进行特异性定量测定。方法制备抗A群多糖的特异性多克隆抗体,所得抗血清经辛酸-硫酸铵沉淀法纯化后,用过碘酸钠法制备辣根过氧化物酶标记多克隆抗体。分别以抗A群多糖多克隆抗体作为包被抗体及酶标二抗,建立双抗体夹心ELISA法,优化反应条件,对A群多糖抗原进行特异性定量测定。结果一系列验证试验表明,该法特异性较好,未检出与C、Y、W135群多糖的交叉反应;1.25～20 ng/mL多糖浓度范围的剂量反应曲线线性最佳,相关系数大于0.98,经实验内10次及不同试验间以16、84、ng/mL测定3次A群多糖中的含量,变异系数在6.3%～11.5%间,回收率在91.8%～105.9%之间,符合常规质控要求,检测限量为4 ng/mL。采用该法测定3批ACYW135群四价脑膜炎球菌多糖疫苗中A群多糖含量、分子大小及回收率的结果均符合规程草案质量标准。结论建立的双抗体夹心ELISA法可尝试用于ACYW135群脑膜炎球菌多糖疫苗中A群多糖的关键质量指标的检测。     

乙醇、丙酮和氯化钙对W135群脑膜炎球菌荚膜多糖唾液酸含量检测的影响

目的研究乙醇、丙酮和氯化钙是否影响间苯二酚法测定W135群脑膜炎球菌荚膜多糖(group W135 meningococcal capsular polysaccharide)唾液酸(sialic acid)含量。方法用苯酚法制备W135群脑膜炎球菌荚膜多糖;分别定量检测唾液酸和W135群脑膜炎球菌荚膜多糖添加不同质量浓度乙醇、丙酮和氯化钙,用间苯二酚法测定其唾液酸含量,检测乙醇、丙酮和氯化钙对间苯二酚测定唾液酸含量的影响。结果添加质量浓度≥39.5μg/mL的乙醇和≥9.9μg/mL的丙酮对唾液酸含量测定均有影响,其中乙醇影响不明显,丙酮的影响极为明显,而不同加入量的氯化钙均对唾液酸含量的测定无影响。结论乙醇和丙酮对间苯二酚法测定唾液酸含量有影响,二者可能会对W135群脑膜炎球菌荚膜多糖质量控制产生影响。     

比色法与核磁共振法测定A、C、Y、W135群脑膜炎球菌荚膜多糖氧乙酰基含量的比较

目的比较Hestrin比色法(简称比色法)和核磁共振(nuclear magnetic resonance,NMR)法在检测A、C、Y、W135群脑膜炎球菌荚膜多糖氧乙酰基(O-Acetyl,OAc)含量的相关性和精密度。方法用比色法和NMR法测定A、C、Y、W135群脑膜炎球菌荚膜多糖及其多糖衍生物,Y、W135群荚膜多糖水解物的OAc含量,比较分析两种方法的相关性及精密度。结果两种方法检测A、C、Y、W135群脑膜炎球菌荚膜多糖OAc含量的决定系数分别为R~2≥0.954、R~2≥0.960、R~2≥0.969、R~2≥0.972;比色法检测3批C群脑膜炎球菌荚膜多糖(PSC)OAc含量的精密度,SD值分别为0.21、0.21、0.18,对应CV值分别为9.03%、9.01%、8.70%(95%置信区间);NMR法检测3批A群脑膜炎球菌荚膜多糖(PSA)OAc含量的精密度,SD值分别为0.66、0.78、0.83,对应CV值分别为0.72%、0.85%、0.93%(95%置信区间);结论比色法和NMR法在检测A、C、Y、W135群脑膜炎球菌荚膜多糖OAc含量方面相关性良好,精密度良好,核磁法较比色法精密度更高。     

Contents of volume 135 1991

Contents of volume 135 1991     

The Tumor-Suppressive MicroRNA-135b Targets c-Myc in Osteoscarcoma

Osteosarcoma is the most common primary tumor of the bone. It leads to many deaths because of its rapid proliferation and metastasis. Recent studies have shown that microRNAs are important gene regulators that are involved in various cancer-related processes. In this study, we found that miR-135b was down-regulated in both osteoscarcoma patient tumor tissues and osteoscarcoma cell lines in comparison to paired adjacent non-tumor bone tissue. We observed that a lower level of miR-135b was associated with metastasis. The ectopic expression of miR-135b markedly suppressed osteoscarcoma cell proliferation, migration, and invasion. Conversely, the inhibition of miR-135b expression dramatically accelerated cell proliferation, migration, and invasion. The forced expression of miR-135b in osteosarcoma cells resulted in a significant reduction in the protein level of c-Myc and repressed the activity of a luciferase reporter that contained the 3′-untranslated region of the c-Myc mRNA. These effects were abolished by the mutation of the predicted miR-135b-binding site, which indicates that c-Myc may be a miR-135b target gene. Moreover, the ectopic expression of c-Myc partially reversed the inhibition of cell proliferation and invasion that was caused by miR-135b. These data therefore suggest that miR-135b may function as a tumor suppressor to regulate osteosarcoma cell proliferation and invasion through a mechanism that targets the c-Myc oncogene. These findings indicate that miR-135b may play a role in the pathogenesis of osteosarcoma.     

135例无精症患者的细胞遗传学分析：附1例世界首报染色体异常核型

本文对来我室咨询的１３５例无精症患者进行了细胞遗传学分析,发现异常核型３８例,其中４７,ＸＸＹ,ｔ（６;９）（ｐ２１;ｑ２２）为世界首报核型。本文对异常核型与无精症之间的关系进行探讨     

Biological and metabolic response in STS-135 space-flown mouse skin

Abstract

There is evidence that space flight condition-induced biological damage is associated with increased oxidative stress and extracellular matrix (ECM) remodeling. To explore possible mechanisms, changes in gene expression profiles implicated in oxidative stress and in ECM remodeling in mouse skin were examined after space flight. The metabolic effects of space flight in skin tissues were also characterized. Space Shuttle Atlantis (STS-135) was launched at the Kennedy Space Center on a 13-day mission. Female C57BL/6 mice were flown in the STS-135 using animal enclosure modules (AEMs). Within 3–5 h after landing, the mice were euthanized and skin samples were harvested for gene array analysis and metabolic biochemical assays. Many genes responsible for regulating production and metabolism of reactive oxygen species (ROS) were significantly (p < 0.05) altered in the flight group, with fold changes >1.5 compared to AEM control. For ECM profile, several genes encoding matrix and metalloproteinases involved in ECM remodeling were significantly up-/down-regulated following space flight. To characterize the metabolic effects of space flight, global biochemical profiles were evaluated. Of 332 named biochemicals, 19 differed significantly (p < 0.05) between space flight skin samples and AEM ground controls, with 12 up-regulated and 7 down-regulated including altered amino acid, carbohydrate metabolism, cell signaling, and transmethylation pathways. Collectively, the data demonstrated that space flight condition leads to a shift in biological and metabolic homeostasis as the consequence of increased regulation in cellular antioxidants, ROS production, and tissue remodeling. This indicates that astronauts may be at increased risk for pathophysiologic damage or carcinogenesis in cutaneous tissue.     

Glucose and streptozotocin stimulate p135 O-glycosylation in pancreatic islets

Streptozotocin has been widely used to create animal models of diabetes. Structurally, streptozotocin resembles N-acetylglucosamine, with a nitrosourea group corresponding to the acetate present in N-acetylglucosamine. Streptozotocin has recently been shown to inhibit O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase, which removes O-linked N-acetylglucosamine from proteins. Compared to other cells, beta-cells express much more of the enzyme O-GlcNAc transferase, which catalyzes addition of O-linked N-acetylglucosamine to proteins. This suggests why beta-cells might be particularly sensitive to streptozotocin. In this report, we demonstrate that both streptozotocin and glucose stimulate O-glycosylation of a 135 kD beta-cell protein. Only the effect of glucose, however, was blocked by inhibition of fructose-6-phosphate amidotransferase, suggesting that glucose acts through the glucosamine pathway to provide UDP-N-acetylglucosamine for p135 O-glycosylation. The fact that both glucose and streptozotocin stimulate p135 O-glycosylation provides a possible mechanism by which hyperglycemia may cause streptozotocin-like effects in beta-cells and thus contribute to the development of type 2 diabetes.     

The poliovirus 135S particle is infectious.

The molecular mechanism of cell entry by unenveloped viruses is poorly understood. The picornaviruses poliovirus, human rhinovirus, and coxsackievirus convert to an altered form (the 135S or A particle) upon interaction with receptors on susceptible cells at 37 degrees C. The 135S particle is thought to be a necessary intermediate because it accumulates inside susceptible cells soon after infection and drugs which inhibit conversion of the virus to this form also prevent infection. However, since a variable fraction of the altered 135S particles is reported to elute unproductively from the surface of susceptible cells, their precise role remains unclear. We have found that poliovirus 135S particles can infect Chinese hamster ovary (CHO) and murine L cells, neither of which are susceptible to infection by native poliovirus. The infectivity of the particles in tissue culture appears to be between 10(3) to 10(5) times less than that of poliovirus on HeLa cells. The 135S particle infectivity was not sensitive to RNase but was greatly reduced by proteolytic treatment. Proteolysis specifically removed the newly exposed N terminus of VP1, a feature which has previously been shown to mediate interactions of the particle with lipid membranes. These results demonstrate that although the infectivity of the 135S particle appears to be receptor independent, it nonetheless requires some property associated with the protein coat. In particular, the N terminus of VP1 plays an important role in the infection process. Our findings are consistent with the hypothesis that the 135S particle is an intermediate in the normal cell entry pathway of poliovirus infection.