A novel DDC gene deletion mutation in two Chinese mainland siblings with aromatic l-amino acid decarboxylase deficiency

Background

Aromatic l-amino acid decarboxylase (AADC) deficiency (OMIM #608643) is a rare and severe disorder of biogenic amine synthesis caused by mutations in the DDC gene. The phenomenology of the movement disorder includes intermittent oculogyric crises and limb dystonia, generalized athetosis, and impaired voluntary movement.

缺氧对乳腺癌细胞E-cadherin、cytokeratin、vimentin表达的影响

目的:研究体外缺氧对乳腺癌细胞系MCF7浸润能力及其细胞表面黏附分子E-cadherin和细胞骨架蛋白犤细胞角蛋白(cytokeratin)、波形蛋白(vimentin)犦表达的影响。方法:模拟体外缺氧环境,观察缺氧对乳腺癌细胞MCF7浸润穿透Matrigel的能力;以及采用半定量RT-PCR检测细胞表面黏附分子E-cadherin和cytokeratin、vimentin表达情况。结果:缺氧状态下MCF7细胞的浸润能力明显增强;且在缺氧条件下E-cadherin基因表达下降、cytokeratin、vimentin基因表达升高。结论:缺氧状态下MCF7细胞转移能力和其表面的E-cadherin基因、cytokeratin、vimentin基因表达存在一定关系。     

四环素调控系统修饰永生化大鼠星形胶质细胞株的构建及鉴定

目的构建四环素及其衍生物强力霉素诱导表达目的基因的永生化大鼠星形胶质细胞株。方法用脂质体法将逆转录病毒载体四环素调控系统中的质粒pRevTet-On转染病毒包装细胞 PT67,经筛选培养后获得病毒载体RevTet-On,采用RT-PCR进行鉴定,并应用NIH313细胞测定病毒滴度。将RevTet-On感染永生化大鼠星形胶质细胞,用有限稀释法挑选阳性细胞单克隆后扩大培养,每个克隆瞬时转染含萤光素酶报告基因的质粒pRevTRE-Lue,加入强力霉素48h后分别检测其萤光素酶活性值,挑选出强力霉素诱导表达高、背景表达低的细胞株并检测其诱导表达的时效、量效关系。结果 RT-PCR结果显示逆转录病毒包装成功,病毒最高滴度为7．4×105CFU／ml。RevTet-On转染永生化大鼠星形胶质细胞后挑选出48个单克隆,瞬时转染pRevTER-Luc后筛选出1株高表达低背景细胞株, 其诱导表达值为876．1 RLU,背景表达值为42．5 RLU,诱导倍数为20．6。该细胞株在加入诱导因子强力霉素1 h后目的基因即开始表达,在24 h时达到高峰,在强力霉素浓度100-2 000 ng/ml的范围内, 其诱导表达活性与药物浓度呈剂量依赖性。结论含四环素调控系统的永生化大鼠星形胶质细胞株诱导活性可靠,可用于调控表达外源基因的研究。     

腺病毒介导MDA-7/IL-24选择性杀伤肝癌 细胞HepG2的研究

目的 ：观察MDA-7/IL-24基因对肝癌的选择性杀伤作用，为肝癌的基因治疗提供理论基础。 方法 ：将携带人MDA-7/IL-24基因的腺病毒Ad.mda-7感染人正常肝细胞L02和肝癌细胞HepG2;用RT-PCR法观察MDA-7/IL-24基因的表达;ELISA方法检测细胞培养上清液中MDA-7/IL-24蛋白的浓度;4甲基偶氮唑蓝染色法（MTT）及Hoechst染色观察MDA-7/IL-24对肝癌细胞的生长抑制和杀伤作用;Annexin-V和PI双染后流式细胞仪检测2种细胞的凋亡;用流式细胞仪检测细胞周期。 结果 ：复制缺陷型腺病毒能介导外源基因MDA-7/IL-24在肝癌细胞株HepG2和正常细胞L02中的高效表达;细胞培养上清液中有MDA-7/IL-24蛋白的表达; MDA-7/IL-24能明显抑制肝癌细胞生长并可促进肝癌细胞的凋亡;MDA-7/IL-24阻滞肝癌细胞于G2/M期，能选择性杀伤肝癌细胞而对正常的肝细胞无阻滞作用和毒性作用。结论 ：复制缺陷型重组腺病毒载体Ad.mda-7能介导MDA-7/IL-24基因在人肝癌细胞中高效表达，促使细胞增殖阻滞及诱导肿瘤细胞凋亡，选择性地杀伤肝癌细胞HepG2，而对正常肝细胞L02无任何毒性作用。     

IL-1Ra和IL-10真核表达质粒载体的构建及其表达检测

目的:构建人IL-1Ra和人IL-10真核表达质粒载体并检测其表达。方法:用双酶切方法切取PCDI-IL-1Ra和PCDI-IL-10质粒中包含人IL-1Ra和人IL-10 CDS全长序列的cD-NA片段,并分别连接到真核表达质粒pcDNA3.1上,然后用壳聚糖转染上述质粒到原代软骨细胞,RT-PCR检测其mRNA水平的表达。结果:成功将人IL-1Ra和IL-10 CDS全长序列的cDNA片段克隆到真核表达载体,mRNA水平检测到目的基因的表达明显提高。结论:真核表达质粒可以用于外源基因的原代软骨细胞导入和表达,为进一步的基因治疗研究提供依据。     

重组腺病毒介导LacZ基因在大鼠肺脏的表达

目的探讨重组腺病毒介导的β-半乳糖苷酶(LacZ)基因在大鼠肺脏的转基因表达。方法Wistar大鼠70只,随机分为Ad-Null组和Ad-LacZ组(n=35),分别应用1．67×10~9pfu／ml复制缺陷型重组腺病毒AdCMV和AdCMVLacZ各600μl,经气管导管滴入；各组于滴人病毒后2、5、7、14、21、28和35 d行肺组织X-gal染色。另取大鼠20只,随机分为C组、L组、M组和H组(n=5),分别应用病毒保存液和低滴度(1．67×10~8pfu／ml)、中滴度(1．67×10~9pfu／ml)、高滴度(5×10~9pfu／ml)的AdCMVLacZ各600μl,滴入病毒后7 d行肺组织X-gal染色和HE染色。结果滴入病毒后2 d,Ad-LacZ组肺组织内即有转基因表达,滴人后7 d达高峰,并维持至35 d；转基因表达位于气管、支气管上皮细胞和肺泡细胞。H组、M组转基因阳性细胞率明显高于L组和C组(P＜0．01)。HE染色显示,H组中3只大鼠肺组织有轻度炎性浸润。所有动物均未见远隔器官转基因表达。结论气管内滴人重组腺病毒可呈剂量依赖性介导LacZ基因在大鼠肺内表达,但过高剂量可诱发机体炎性反应。     

hTERT反义cDNA对人脑胶质瘤细胞的抑制作用

目的探讨人端粒酶催化亚基(hTERT)反义cDNA(pAdeasy-hTERT)在体外对胶质瘤细胞生长的抑制作用。方法将腺病毒介导的pAdeasy-hTERT作用于人胶质瘤细胞系U251,用MTT法检测细胞存活率、端粒酶重复序列扩增法测定端粒酶活性、Westem杂交鉴定hTERT蛋白的表达、RT-PCR法检测hTERT cDNA水平、PCNA观察肿瘤细胞的凋亡情况以及用流式细胞仪对转染后肿瘤细胞的周期进行分析。结果pAdeasy-hTERT在体外明显抑制肿瘤细胞生长,降低端粒酶活性,抑制hTERT表达。结论pAdeasy-hTERT显著抑制人胶质瘤细胞生长,可成为恶性胶质瘤基因治疗的靶基因。     

表达谱基因芯片筛选体外循环前后心肌细胞因子的变化

Objestive Systemic inflarmmation may be triggered by injury, hypothermia, ischemia-reperfusion and the contact of the blood with foreign body during cardiopulmonary bypass (CPB). To determine the application values of gene chip technique in the clinical practice and the study of cardiovascular stagery, as well as to provide clues to the study of inflammatory responess during CPB, microarry for gene expression profiles was used to identify the differences in the gene expression of myocardium between pre-and post- CPB. Methods Six adult patients who underwent CPB from March to May in 2003 were involved. Samples of right atrium were col- lected before and at immediate end of CPB. BD AtlasTM cDNA Expression Arrays was used to identify the differences in the gene ex- pression of cytokines. The results were compared with that of semi-quantative RT-PCR. Resellts The mean age of 6 patients (5 males and 1 female) was (32.67± 11.72) years. The baseline heart function was gradeⅡin 3 cases and grade Ⅲ in 3 other cases. The baseline left ventricular ejection fraction(LVEF)was (58.17±7.91)%. The mere duration was (91.67±43.88) minutes for CPB and was (58.67±43.46) minutes for aorta blocking. The minimum nasopharynx/rectal temperture was (29.37±1.90)℃/ (32.15±1.52)℃. Gene expression profiles of cytokines in the myocardium pre- and post-CPB were analysed successfully. The ex- pression of IL-6, IFN-γ,Wnt5a, TNFRSF1B, a member of tumor necrosis factor receptor superfamily, PIGF and MFNG in the myo- cardium were unpregulated after CPB. Conclusion Microarray technique is applicable in the study of cytokines changes dying CPB. cDNA microarray identified pleliminarily the differences in the gene expression between pre- and post-CPB. These genes may be in- valved in inflammation and other psthophysiological responses incuced by CPB. The myocardiym is probably one of the major sources of cytokines during CPB. Further study may be helpful in understanding the llngthe development of inflammation during CPB, and eventually, reducing the post-operative complications.     

热缺血再灌注损伤影响肝脏细胞周期检查点调控的基因表达分析

目的动态观察肝脏热缺血再灌注损伤（WIRI）对细胞周期检查点基因的表达影响，研究其分子机制帮助了解那些与热缺血再灌注相关的疾病。方法采用成组对照的实验设计，利用微阵列芯片和逆转录-聚合酶链反应（PCR）联合分析技术，分析SD大鼠热缺血再灌注损伤处理后的基因表达变化（其中芯片分析8只、RT-PCR定量分析16只）；运用系统生物学分析方法对功能基因进行聚类。结果分别得到了热缺血再灌注损伤处理后肝脏的细胞周期各检查点基因及早期即刻基因表达数据，将其中与细胞周期密切相关的的功能基因（上调的223条、下调的62条变化幅度均大于3倍）做进一步分析。结论肝脏热缺血再灌注损伤通过细胞周期／检查点控制基因影响细胞周期G1／S、G2／M的运行，抑制DNA的合成和染色体的分裂。推测可能影响受损的DNA双链自我修复和后期的组织细胞再生、凋亡。