胞嘧啶脱氨酶的原核表达和多克隆抗体的制备

目的：在原核系统中表达并纯化大肠杆菌胞嘧啶脱氨酶（cytosine deaminase,CD)，制备鼠抗大肠杆菌CD多克隆抗体，方法：亚克隆CD基因到原核表达载体pMAL-c2和pBV222中，并转化入大肠杆菌DH5α内，诱导表达并纯化MBP-CD和6his-CD融合蛋白，用纯化的MBP-CD融合蛋白免疫小鼠制备多克隆抗体。结果：通过重组质粒的酶切筛选出重组阳性克隆，成功地表达和纯化出MBP-CD和6his-CD融合蛋白，用纯化的MBP-CD成功制备了鼠抗CD多克隆抗体，并用6his-CD和GST-CD重组蛋白进行Western印迹分析，证实了抗体的正确性，结论：应用多克隆抗体可以检测体内外CD基因的表达，为临床前和临床上深入开展CD基因的生物治疗研究提供重要的实验材料。     

HLA-DPB1 gene polymorphism and multiple sclerosis: a large case-control study in the southwest of France

The polymorphism at the HLA-DPB1 locus has been characterized in a large number of patients with multiple sclerosis (n = 112) and in healthy controls (n = 115). Both patients and controls lived in the southwest of France (in the Pyrénées Atlantiques) and had similar ethnic background. The typing procedure involved the selective amplification of the second exon of the DPB1 locus by polymerase chain reaction, followed by hybridization of the amplified DNA with 14 sequence-specific oligonucleotide probes. Individual alleles were identified by the pattern of hybridization of the different probes. The distribution of the DPB1 alleles was not significantly different in multiple sclerosis patients and controls (p = 0.11). This does not corroborate the reported association of multiple sclerosis with the primed lymphocyte typing (PLT)-defined DPw4 specificity and is not in favour of a role played by polymorphic residues of the DP molecule in susceptibility to multiple sclerosis.     

Differential Regulation of RGS-2 by Constant and Oscillating PTH Concentrations

PTH has diverse effects on bone metabolism: anabolic when given intermittently, catabolic when given continuously. The cellular mechanisms underlying the varying target cell response are not clear yet. PTH induces RGS-2, a member of the Regulator of G-protein Signaling protein family, via cAMP/PKA, and inactivates PKC-mediated signaling. To investigate intracellular signaling pathways with different PTH concentration-time patterns, we treated UMR 106-01 osteoblast-like cells in a perfusion system. PTH was administered intermittently (4 min/h, 10⁻⁷ M) or continuously at an equivalent cumulative dose (6.6 × 10⁻⁹ M). cAMP was measured using radioimmunoassay, mRNA levels using real-time rtPCR and ribonuclease protection assay, and protein levels using Western immunoblotting. A single PTH pulse transiently increased cAMP levels by 2000% ± 1200%. In contrast to continuous PTH exposure, cAMP induction remained unchanged with intermittent PTH, ruling out desensitization of the PTH receptor. In continuously perfused cells, RGS-2 abundance was three to five times higher than in cells intermittently exposed to PTH for up to 12 h. MKP-1 and -3 were significantly less induced with pulsatile PTH; exposure-mode-dependent differences in MMP-13 and IGFBP-5 were small. Pulsatile but not continuous PTH administration prevents PTHrP receptor desensitization and accumulation of RGS-2 in osteoblasts, which should preserve PKC-dependent signaling.     

Age determines memory for face identity and expression

Background: The recognition of facial expressions is an important component of emotion processing which contributes to interactional behavior. One of the factors highly associated with potential decline of ability in behavioral tasks is age. Methods: We have investigated age‐related changes in facial identity and expression memory of healthy subjects in three age groups: young adults (20–40 years), elderly adults (60–80 years) and, for the first time in the literature, very old adults (over 80 years of age). Using a picture test, photographs of faces with happy or angry expressions were presented to study participants during the encoding task, and the memory for identity and emotional facial expression was investigated in a subsequent recognition task showing emotionally neutral faces. Half of the faces presented in the recognition task were initially shown in the encoding task. Results: Age interacted with the memory process: the ability to recognize both facial identity and emotional expression declined with advanced age. Happy facial expressions were better recognized in all age groups. Although there was a continuous overall decrease in recognition of both happy and angry expressions with advanced age, the effect favoring happy facial expressions was stable also in very old adults. Other factors such as gender or educational level did not affect the memory process for facial expressions. Conclusions: Our findings suggest that age is a significant determinant of memory for facial identity and emotional expression, and that, similar to younger adults, the recognition process of the elderly favors happy emotional facial expressions.     

肝癌多药耐药产生与低糖环境的关系

目的探讨局部微环境低糖与肝细胞癌多药耐药性(MDR)产生的关系及影响机制。方法低糖培养HepG2细胞,应用流式细胞术Annexin V/PI法检测低糖培养的细胞在化疗药物5-氟脲嘧啶(5-Fu)作用后的凋亡情况,分别应用荧光定量聚合酶链反应(PCR)技术和Western blot技术检测低糖培养后HepG2细胞内多药耐药相关基因mdr1、MRP1、LRP的mRNA和蛋白水平的表达。结果在低糖环境下生长时间越长的HepG2细胞对5-Fu的抵抗越强,而且随着低糖培养时间的增加,5-Fu诱导的HepG2细胞的凋亡高峰延迟。低糖培养的HepG2细胞内多药耐药相关基因mdr1、MRP1、LRP在mRNA和蛋白水平的表达随低糖培养时间的延长而升高,以LRP的改变最为显著。结论肝癌生长微环境葡萄糖耐量不足也是肝癌产生MDR的原因之一。低糖可通过上调一组多药耐药相关基因的表达而诱导肝癌的多药耐药性。     

Recombinant glycoprotein based vaccine for Chandipura virus infection

Chandipura virus (CHPV) has emerged as an important pediatric encephalitis-causing pathogen with very high mortality in India. No specific vaccine or treatment is available till date. We attempted to prepare a candidate vaccine employing recombinant CHPV Glycoprotein (rGp). The Glycoprotein gene (G-gene) of CHPV was expressed using Baculovirus expression system. The rGp was purified by HPLC and used for mice immunization, 3 doses, and 4 weeks apart. One microgram rGp was found to be optimum. Sero-conversion was observed as early as 2nd week by detecting anti-CHPV IgG antibodies. Antibody titres were immunogen-concentration dependent. Intracerebral challenge of the immunized mice with 100 LD₅₀ of the homologous strain demonstrated 90% protection. In in vitro neutralization, antibodies from the immunized mice were able to neutralize heterologous viruses. There was 60% T cell proliferation observed against rGp in immunized mice. The study shows that rGp induces both arms of immune response and represents an ideal vaccine candidate for further evaluations.     

瘦蛋白的分子生物学特性研究进展

瘦蛋白 (leptin)是由肥胖基因编码、脂肪细胞分泌的一种分子量为 16kDa的蛋白质 ,是一种重要的代谢调节信号 ,对人和动物的采食量、能量代谢、繁殖性能等具有重要调节功能 ,对人肥胖病的治疗和改善动物的生产性能、胴体组成等有良好的潜在应用前景 .主要概述了瘦蛋白基因的克隆、转录、表达调控以及瘦蛋白受体与信号转导等分子生物学特性     

Synergistic effect of low dose Cyclosporine A and human interleukin 10 overexpression on acute rejection in rat lung allotransplantation

Objective: Electroporation mediated transfer of plasmid DNA into peripheral muscle results in high transfection efficiency. The aim of this study was to investigate the effect of gene transfer of human IL-10 (hIL-10) into the tibialis anterior muscle (MTA) in combination with low dose Cyclosporine A (CsA) on acute rejection of lung allografts in the rat. Methods: Lung allotransplantation was performed from male BN donor to male Fisher F344 rats. Gene transfer was achieved by intramuscular injection into the MTA of the recipient followed by electroporation (4×20 ms impulses at 200 V/cm) 24 h prior to the transplantation. Group A (n=5) received CsA (2.5 mg/kg bw ip) for 5 days post-transplant and group B (n=5) 2.5 μg of PCIK hIL-10 (plasmid expression vector containing human CMV immediate early gene promoter and enhancer) and a low dose CsA (2.5 mg/kg bw i.p.). Graft function was assessed by blood gas at day 5 after exclusion of the native lung. Animals were sacrificed and blood was drawn to measure serum hIL-10 levels (ELISA) and tissue was sampled for histological grading of rejection. Results: Local expression of hIL-10 was confirmed at the mRNA level by in situ hybridization. All group A control animals showed severe signs of rejection. At day 5 all grafts in group B showed good gas exchange mean PaO2 233±123 mmHg, vs 44±8 mmHg in group A. Histological examination revealed moderate to severe rejection in all animals in group A (IIIB, ISHLT) in contrast to low moderate rejection in group B (II–IIIA). hIL-10 serum levels on day 5 were 14±7 pg/ml in group B vs. 0 in group A. Conclusions: Electroporation mediated hIL-10 overexpression in a peripheral muscle of the recipient in combination with low dose CsA reduces acute rejection in this model of rat lung allotransplantation.     

性别差异对脓毒症大鼠肝脏Toll样受体4和髓样分化蛋白-2基因表达的影响

目的探讨性别差异对脓毒症大鼠肝脏组织Toll样受体4(TLR4)和髓样分化蛋白- 2(MD-2)基因表达的影响。方法以脂多糖(LPS)按5 mg/kg体重由大鼠腹腔注射制作脓毒症动物模型,注射后2 h留取肝脏组织检测TLR4、MD-2和肿瘤坏死因子-α(TNF-α)基因表达,同时测定各组大鼠血浆中丙氨酸氨基转移酶(ALT)及雌二醇含量。结果正常雌雄性大鼠肝脏组织均可表达少量TLR4、MD-2、TNF-α基因,其中雌性组分别为0．175±0．034、0．211±0．044、0．201±0．068; 雄性组分别为0．205±0．061、0．243±0．049、0．243±0．063,两组数据差异无统计学意义(P> 0．05),但LPS刺激后雌性大鼠肝脏组织上述指标分别为0．615±0．089、0．708±0．181、0．730± 0．118,血浆中ALT含量为(81．07±10．72)U／L;雄性组分别为0．723±0．091、1．123±0．272、 0．881±0．156,ALT含量为(106．39±14．21)U／L,雌性组各项指标均明显低于雄性大鼠(P< 0．05)。相关分析表明雌性及雄性脓毒症大鼠肝脏组织TLR4及TNF-α基因表达与相应性别大鼠血浆中雌二醇含量呈显著负相关(P<0．05)。结论 LPS刺激后大鼠肝脏组织TLR4、MD-2及 TNF-α基因表达存在性别差异,内源性雌激素的作用可能导致雌性脓毒症大鼠肝脏组织损伤较雄性轻。