HBV-pre-C区段变异患者血清前S1抗原检测的临床价值分析

目的探讨HBV-pre-C区段(nt1896)变异阳性患者血清HBV-pre-S1抗原检测的临床价值.方法选取病毒复制为阳性的乙肝血清76份,其中,HBeAg(-)且HBV-pre-C区段(nt1896)变异阳性的46份;HBV-pre-C区段(nt1896)变异阴性的30份;体检正常对照30例.以ELISA法检测其血清乙肝病毒前S1抗原(HBV-pre-S1),同时检测血清中谷氨酸氨基转移酶(ALT)活性.结果HBV-pre-C区段(nt1896)变异阳性血清中82.6%(38/46)HBV-pre-S1检测为阳性,其中92.1%(35/38)伴血清ALT活性异常增高;HBV-pre-C区段(nt1896)变异阴性的血清中83.3%(25/30)HBV-pre-S1检测为阳性,92.0%(23/25)伴血清ALT活性异常增高;正常对照组中HBV-pre-S1检测为阴性,血清ALT活性均正常.HBV-pre-C区段(nt1896)变异阳性组与阴性组,HBV-pre-S1检出率差异无显著性(P=2.1387,P＞0.05),HBV-pre-S1阳性血清中伴ALT异常增高率在两组间差异也无显著性(P=3.4211,P＞0.05).结论当HBV发生基因变异时,HBeAg不能正常表达,血清中HBV-pre-S1抗原的检测可基本避免基因变异的干扰,大致反映病毒基因的复制及活动.     

慢病毒介导的突变型胸苷激酶对T淋巴细胞杀伤作用的实验研究

目的观察慢病毒介导的突变型单纯疱疹病毒胸苷激酶(HSV-sr39tk)及野生型HSV-tk对T细胞的体外杀伤作用,比较HSV-sr39tk/更昔洛韦(GCV)、HSV-sr39tk/阿昔洛韦(ACV)、HSV-tk/GCV、HSV-tk/ACV对T细胞存活率的影响。方法采用改良的磷酸钙沉淀法将包装质粒、包膜蛋白质粒和含目的基因的转移质粒共转染293T包装细胞,收集的病毒上清感染T细胞后,分别与不同浓度梯度的前体药物GCV和ACV作用4d后,MTT法测定细胞存活率。结果共转染293T细胞后获得较高滴度的慢病毒(2×106IU/ml)。前体药物GCV和ACV浓度在0～10.0μmol/L时转染HSV-sr39tk细胞(39tk+T细胞)存活率下降比较明显,GCV组细胞存活率由(96.04±3.23)%下降为(36.76±4.38)%,ACV组细胞存活率由(97.31±4.61)%下降为(43.75±8.99)%,而GCV和ACV浓度大于10.0μmol/L时,39tk+T细胞存活率下降趋势则减缓。统计显示39tk+T细胞对GCV、ACV均有敏感性(P值均<0.05);转染HSV-tk T细胞(tk+T细胞)对GCV有敏感性(P<0.05),而对ACV不具有敏感性(P>0.05);同一浓度时39tk+T细胞+GCV与tk+T细胞+GCV两组间T细胞存活率差异具有统计学意义(P<0.05)。结论慢病毒载体可高效、稳定地感染T细胞,同时不影响细胞的增殖,与野生型HSV-tk基因比较,表达突变型HSV-sr39tk的T细胞对GCV具有更高的敏感性,而且对ACV也具有敏感性。     

氟喹诺酮药物浓度对金黄色葡萄球菌耐药突变株选择的影响

目的了解氟喹诺酮药物浓度对金葡菌耐药突变株选择的影响。方法将金葡菌ATCC 25923接种于不同浓度环丙沙星及加替沙星琼脂平皿上，根据不同药物浓度平皿上生长的菌落数，计算菌落生长比例。对不同药物浓度筛选出的耐药突变株进行编码拓扑异构酶ⅣC亚单位基因par C（grl A）和促旋酶A亚单位基因gyr A喹诺酮耐药决定区的PCR扩增和测序，并测定外排泵抑制剂利血平对环丙沙星和加替沙星MIC的影响。结果药物浓度对平皿中生长的菌落数有明显影响。环丙沙星在低、中浓度主要选择出主动外排泵介导的非靶位耐药突变株，高浓度选择出Grl A变异的耐药突变株，以Ser-80→Phe为主。加替沙星低浓度选择出非靶位及靶位变异菌株，而中、高浓度均选择出Grl A变异株，也以Ser-80→Phe为主。结论氟喹诺酮药物浓度对金葡菌耐药突变株菌落数以及耐药位点的选择都有明显影响。     

锤头状核酶对肝癌突变基因p53的抑制作用

目的 探讨p53核酶对肝癌细胞突变型抑癌基因p53的抑制作用。方法 应用计算机设计并合成针对突变型p53(249位密码子AGG→AGT)的锤头状核酶RZ，构建其体外转录和真核表达载体，检测核酶对突变型p53(mtp53)的体外切割作用，并在Lipofect AMINE^TM2000的介导下转染肝癌细胞MHCC97，应用逆转录聚合酶联反应(RT—PCR)检测核酶对肝癌细胞突变型p53的抑制作用。结果 测序证实核酶基因被正确克隆人体外转录载体pBSKU6和真核载体pEGFPC1中。体外切割效率为42％，而野生型p53(wtp53)没有被切割。在Lipofect AMINETM2000的介导下成功转染肝癌细胞MHCC97，RT—PCR检测证实突变型p53的mRNA水平明显下降，细胞内的切割效率为69％。结论 p53核酶可成功抑制肝癌细胞中突变型p53的表达，为肝癌的基因治疗提供了一个新的选择。     

Decreased ethanol sensitivity and tolerance development in gamma-protein kinase C null mutant mice is dependent on genetic background

Initial sensitivity and tolerance development to the sedative-hypnotic and hypothermic effects of ethanol were investigated in gamma-protein kinase C (PKC) null mutant mice. Null mutants from a C57BL/6J x 129/SvJ mixed genetic background demonstrated decreased ethanol sensitivity and failed to develop chronic tolerance after 10 days of ethanol liquid diet. However, when the null mutation was introgressed onto a C57BL/6J background for six generations, the "no tolerance" phenotype for sedative-hypnotic and hypothermic effects of ethanol was no longer apparent Outcrossing the gamma-PKC null mutation to a C57BL/6J x 129/SvEvTac mixed background restored the "no tolerance" phenotype to ethanol-induced sedation after chronic ethanol diet; however, as measured by hypothermia, tolerance was still evident in the null mutant mice. These observations and the results of tests of chronic tolerance in the C57BL/6J, 129/SvJ, and 129/SvEvTac background inbred strains indicate that gamma-PKC plays an important role in initial sensitivity and tolerance to ethanol. However, the impact of gamma-PKC is modulated by the background genotype. These results stress the importance of including the effect of genetic background when evaluating the effects of single gene mutations on quantitative behavioral traits.     

The emergence of ‘hypervirulence’ in Clostridium difficile

The impact of Clostridium difficile-associated disease (CDAD) in healthcare settings throughout the developed world is considerable in terms of mortality, morbidity, and disease management. The incidence of CDAD has risen dramatically since the turn of this century, concomitant with the emergence of so-called hypervirulent strains which are thought to cause a more severe disease, higher relapse rates, and increased mortality. Pre-eminent amongst hypervirulent strains are those belonging to ribotype 027, which were first reported in Canada in 2003 and shortly thereafter in the UK. Since its arrival in Europe, it has spread rapidly and has now been reported in 16 member states and Switzerland. The physiological factors responsible for the rapid emergence of hypervirulent C. difficile strains remain unclear. It is known that they produce a binary toxin (CDT) in addition to toxins A and B, that they are resistant to fluoroquinolones due to mutations in gyrA, and that they are resistant to erythromycin. Representative strains have been suggested to produce more toxin A and B in the ‘laboratory flask’ (most likely due to a frameshift mutation in the repressor gene tcdC), to be more prolific in terms of spore formation, and also exhibit increased adherence to human intestinal epithelial cells due to altered surface proteins. However, the contribution of these and other as yet unidentified factors to the rapid spread of certain C. difficile variants (e.g., ribotypes 027 and 078) remains unclear at present. The advent of ClosTron technology means that it is now possible to construct genetically stable isogenic mutants of C. difficile and carry out reverse genetic studies to elucidate the role of specific gene loci in causing disease. The identification of virulence factors using this approach should help lead to the rational development of therapeutic countermeasures against CDAD.     

Construction and characterization of an auxotrophic ctxA mutant of O139 Vibrio cholerae

Cholera caused by the O139 serogroup still remains a public health concern in certain regions of the world and the existing O1 vaccines do not cross-protect cholera caused by this serogroup. An aminolevulinic acid (ALA) auxotroph vaccine candidate against the O139 serogroup, designated as VCUSM2, was recently developed. It was found to be immunogenic in animal model studies but showed mild reactogenic effects due to the presence of two intact copies of Vibrio cholerae toxin (CTX) genetic element. In the present study we have modified the ctx operon by systematic allelic replacement methodology to produce a mutant strain, designated as VCUSM14. This strain has two copies of chromosomally integrated and mutated ctxA gene, encoding immunogenic but not toxic cholera toxin A subunit (CT-A). The amino acids arginine and glutamic acid at position 7th and 112th, respectively, in CT-A of VCUSM14 were substituted with lysine (R7K) and glutamine (E112Q), respectively. Two copies of the ace and zot genes present in the ctx operon were also deleted. Cholera toxin-ELISA using GM1 ganglioside showed that the both wild type CT and mutated CT were recognized by anti-CT polyclonal antibodies. VCUSM14 produced comparatively less amount of antigenic cholera toxin when compared to the VCUSM2 and Bengal wild type strain. VCUSM14 did not elicit fluid accumulation when inoculated into rabbit ileal loops at doses of 10⁶ and 10⁸ CFU. The colonization efficiency of VCUSM14 was one log lower than the parent strain, VCUSM2, which can be attributed to the ALA auxotrophy and less invasive properties of VCUSM14. VCUSM14, thus a non-reactogenic auxotrophic vaccine candidate against infection by O139 V. cholerae.