胞嘧啶脱氨酶基因修饰神经干细胞及其表达的离体实验研究

目的 探讨胞嘧啶脱氨酶(cytimidine deaminase，CD)基因修饰神经干细胞及其基因表达。方法 通过构建真核表达质粒pCMVCD，限制性内切酶消化鉴定后，采用Lipofectamine 2000脂质体介导法转染新生大鼠室管膜下区神经干细胞(Neural stem cells，NSCs)，G418筛选阳性克隆，加入不同浓度的5-氟胞嘧啶(5-Flourocytosine，5-FC)，MTT比色法测定NSCs的生存率。结果 本实验成功地培养并鉴定了神经干细胞，并将CD基因成功地转染了神经干细胞，G418阳性NSCs对低浓度5-FC高度敏感。结论 CD基因修饰神经干细胞的离体实验研究为干细胞治疗研究提供依据。     

sFGFR1基因的克隆与在RTS系统中的高效表达

目的：克隆sFGFRl(soluble fibroblast growth factors receptor-1)基因，并在RTS(rapid translation system)系统中高效表达相应蛋白．方法：培养Swiss rat 3T3 fibroblast细胞株，提取总RNA，用RT—PCR方法获取鼠sFGFR1 cDNA片段，酶切后克隆到pIVEX2．3d载体并进行序列分析；采用Roche RTS ProteinMaster500系统，高效表达sFGFR1蛋白并用Western Blot鉴定表达的蛋白．结果：克隆了sFGFR1基因，测序证实序列正确；Western Blot证实sFGFR1蛋白在RTS系统中高效表达．结论：克隆了sFGFR1基因并在RTS系统获得高效表达．     

人Heparanase基因真核和原核表达载体的构建及融合蛋白的表达

目的：构建人Heparanase基因的真核、原核表达载体，大肠杆菌表达其融合蛋白．方法：采用反转录．聚合酶链反应从人肝癌细胞株HepG2cDNA中，分别扩增出Heparanase编码基因，用限制性内切酶BamHI消化后，插入真核表达载体pcDNA3．1中，经酶切鉴定与测序证实后，连接成包括完整的人Heparanase基因ORF区的真核表达载体，以亚克隆法构建于原核表达载体pRSET的相应酶切位点，转化大肠杆菌BL21菌株，异丙基β-D硫代半乳糖苷(IPTG)诱导产生融合蛋白．结果：构建的人Heparanase基因表达载体经序列测定证实，与GenBank登录结果完全一致；双酶切鉴定证实，克隆基因正确插入载体pcDNA3．1及pRSET；SDS-PAGE证实融合蛋白表达成功．结论：成功构建了人Heparanase基因真核、原核表达载体，成功正确表达了6His／Heparanase融合蛋白     

肿瘤坏死因子凋亡相关配体基因的克隆和表达

目的：克隆、表达和鉴定肿瘤坏死因子凋亡相关配体(TRAIL)．方法：从培养的人外周血淋巴细胞中提取总RNA，经RT-PCR获得TRAIL基因．将该基因克隆到pGEM-Teasy载体中，测序鉴定．将TRAIL基因插入pBV220表达载体中，42℃诱导表达4～5h，进行SDS-PAGE分析．免疫印迹法鉴定TRAIL蛋白的表达．结果：DNA测序证明，获得了TRAIL基因，其序列与GenBank中报道序列完全一致．SDSPAGE分析表明，TRAIL蛋白获得高效表达，分子质量为17ku，表达量约占菌体总蛋白的300k，．免疫印迹法鉴定显示，该蛋白可与鼠抗人TRAIL mAb产生阳性反应．结论：成功克隆和表达了TRAIL基因．     

No support for a truncated interferon-α17 allele as risk factor for MS

Cytokines have a central role in multiple sclerosis (MS) pathogenesis and may contribute to the aetiology of MS. A polymorphism in the IFNA17 gene with an allele carrying a pre-mature stop codon has been suggested to convey a 26-fold increased risk for MS. We investigated the possible association between this polymorphism and MS using population-based samples from a genetically well-characterized population. The IFNA17 gene variant was found in 2.8% of 327 MS cases and 3.3% of 698 referents ( P  = 0.64). Thus, our study does not support an association between the IFNA17 allele and risk for MS.     

Screening for SNCA and LRRK2 mutations in Greek sporadic and autosomal dominant Parkinson's disease: identification of two novel LRRK2 variants

Mutations in SNCA and LRRK2 genes, encoding alpha-synuclein and leucine-rich repeat kinase 2, respectively, cause autosomal dominant Parkinson's disease (AdPD). The LRRK2 G2019S (c.6055G > A) and R1441G (c.4321C > G) mutations have also been identified in sporadic PD (sPD). We studied 55 unrelated patients with AdPD, 235 patients with sPD, and 235 healthy age- and gender-matched controls all of Greek origin. Patients with AdPD were screened for SNCA and LRRK2 mutations by direct sequencing. SNCA gene dosage analysis was also performed for AdPD using quantitative duplex polymerase chain reaction of genomic DNA. In addition, we investigated the frequency of the LRRK2 G2019S mutation in sPD. We found no missense mutations or multiplications in the SNCA gene. Here we report two novel variants, A211V (c.632C > T) and K544E (c.1630A > G) in LRRK2 gene in two patients with AdPD that was not present in controls. We identified only one patient with sPD (1/235; 0.4%) carrying the G2019S mutation. LRRK2 mutations are present in AdPD and sPD patients of Greek origin.     

神经调节素1基因SNPrs2954041多态性与精神分裂症及其认知功能的关联研究

目的探讨昆明地区汉族人群神经调节素1(NRG-1)基因SNPrs2954041多态性与精神分裂症及认知功能的关系。方法采用聚合酶链反应限制性片段长度多态性(PCR-RFLP)的方法检测141例精神分裂症患者和84名正常对照的NRG-1基因SNPrs2954041多态性;采用韦氏记忆量表(WMS)和威斯康辛卡片分类测验(WCST)评估两组人群的记忆功能和执行功能,并用PANSS量表评定患者的临床症状。结果两组NRG-1基因SNPrs2954041多态性的基因型和等位基因频率差异有统计学意义(2=34.01,P<0.05;2=30.201,P<0.05)。按性别分组后比较,结果仍同前。患者组中各基因型组间认知功能比较结果示:①各基因型组间韦氏记忆量表的理解记忆得分差异有统计学意义(P<0.05),两两比较显示G/G与T/T基因型患者间差异有统计学意义(P<0.05)。②G/G与T/T基因型患者比较,威斯康辛卡片分类测验中的错误数、持续错误数和分类个数差异有统计学意义(P<0.05)。结论NRG1基因SNPrs2954041多态性与精神分裂症存在关联,他也与精神分裂症患者的认知功能相关。     

Two novel gastric cancer-associated genes identified by differential display

AIM To clone novel gastric cancer-associated genes and investigate their roles in gastric cancer occurrence.METHODS A method called differential display was used which allows the identification of differentially expressed genes by using PAGE to display PCR-amplified cDNA fragments between gastric cancer cells and normal gastric mucosa cells. These fragments were cloned into plasmid vector pUC18. Homology analysis was made after sequencing these fragments.RESULTS Two novel genes were identified compared with sequences from GenBank. One was registered with the AD number AF 051783. In situ hybridization showed that these two novel genes expressed specifically in gastric cancer tissues.CONCLUSION The two novel genes obtained by differential display were confirmed to be gastric cancer-associated genes using in situ hybridization.     

Responses of periodontal nerve terminals to experimentally induced occlusal trauma in rat molars: an immunohistochenmical study using PGP 9.5 antibody

The response of periodontal nerves to experimentally induced occlusal trauma in rat molars was assessed by immunohistochemistry for protein gene product 9.5 (PGP 9.5) at light and electron microscopic levels, and by computerized image analysis. The occlusal surface on the left upper first molar of 8-wk-old male Wistar rats was raised approximately 1 min under ether anaesthesia. The rats were perfusion-fixed on d 1, 2, 3, 4, and 7 after bite-raising and then decalcified for 2–3 wk. Frozen sagittal cryostat sections were stained by the avidin-biotin complex method. By the second day after bite-raising many Ruffini endings were swollen and their outline unclear at the light microscopic level. Transmission electron microscopy disclosed PGP 9.5 reaction products within Ruffini endings that had unusually long cytoplasmic projections extending through enlarged slits of the Schwann sheaths and also diffuse extracellular PGP 9.5-immunoreactivity near the Ruffini endings. From d 2 to 4, thin nerve fibres on the pressure side of the periodontal ligament were orientated irregularly and had a prominent beaded appearance. An increase in beaded nerve terminals occurred at d 2–4 post elevation, and decreased later. These results suggest that occlusal trauma induces specific changes in the distribution and shape of nerve terminals in the periodontal ligament.     

Characterization of adenosine deaminase (ADA)-negative B-lymphoblastoid cells cocultured with ADA-positive fibroblasts

Abstract: A cell line, BAD05, derived from B lymphocytes of an adenosine deaminase (ADA; EC 3,5,4,4)-deficient patient could not proliferate in a serum-free medium containing 100 μmol/l deoxyadenosine. When BAD05 was cultured with ADA-positive fibroblasts, the proliferation of BAD05 was improved. BAD05 cell density increased when the initially mixed ratio of fibroblasts/BAD05 was 1/10 or higher, but decreased when the ratio was 1/20 or lower. Deoxyadenosine concentrations in the medium and ATP and deoxyATP (dATP) levels in the BAD05 were measured after 4 hours of coculture at initial BAD05 cell densities of 1 × 10⁵and 1 × 10⁶cells/ml. Deoxyadenosine concentrations in the medium decreased as the density of fibroblasts increased. The dATP level decreased as the mixed ratio rose. The ratio of fibroblasts/BAD05 rather than the cell density of fibroblasts had a larger effect on the dATP levels in BAD05. Under our experimental conditions, ADA-negative cells proliferated well when the ratio of ADA-positive cells/ADA-negative cells was over 1/10.