X-连锁肾上腺-脑白质营养不良蛋白3个突变体的制备及其鉴定

目的构建R617C、P508L和G512S突变型表达载体并对其进行鉴定。方法以野生型pGEX-4T-2-NBD原核表达载体为模板,通过体外诱导突变构建突变型表达载体,序列分析鉴定R617C、P508L和G512S突变重组子。结果突变型重组载体测序结果与患者的突变位点完全符合;表达产物的12%SDS-PAGE和蛋白质印迹也显示,约40.5×103的特异性区带能被特异性单克隆抗体所识别,与预期的结果相符;通过GST蛋白纯化试剂盒获得较高纯度的目的蛋白。结论 3个ALDP突变体的成功构建为进一步研究突变对ALDP结构和功能的影响奠定了基础,为了解X-ALD的分子发病机制创造了条件。     

干扰素α-2b联合5－氟尿嘧啶对肝癌细胞株HepG2生物活性的影响

目的观察干扰素α-2b联合5－氟尿嘧啶（5－Fu）对肝癌细胞株HepG2在体外的杀伤抑制作用,同时检测药物作用后肝癌细胞突变型P53的表达情况。方法用不同浓度的干扰素α-2b（100、500、1000、2000U／ml）和5－Fu（10μg／ml）联合对肝癌细胞株作用不同时间（24、48、72h）,用单四唑（MTT）法检测细胞生长抑制率,进而运用PV－9000二步法对突变型P53表达情况进行测定。结果干扰素α-2b和5-Fu联合效果明显优于单用,并与对照组差异有统计学意义（P〈0．05）。经药物处理后,突变型P53表达差异有统计学意义（P〈0．05）。结论干扰素α-2b和5-氟尿嘧啶能有效抑制肝癌细胞的生长,导致突变型P53因子表达的下调而发挥作用。     

野生型和突变型肝豆状核变性蛋白对皮肤成纤维细胞内过多铜的转运作用

目的:将野生型和突变型(R778L)肝豆状核变性基因(ATP7B)转染入Me32aT22/2L细胞株中进行表达,观察野生型和突变型ATP7B的铜转运功能,为以后更深入的基因治疗奠定基础。方法:采用脂质体转染法分别将含野生型和突变型cDNA重组真核表达载体pRc/CMV-WD转染到Me32aT22/2L细胞株中,免疫荧光观察野生型和突变型ATP7B在细胞内的分布,以及铜孵育实验检查野生型和突变型ATP7B的铜转运功能。结果:在转染的Me32aT22/2L细胞株中可检测到ATP7B的表达,ATP7B位于细胞核的周围,在24和48小时后,细胞内铜/蛋白比值分别为335.33±49.86和477.38±30.95,而突变型ATP7B组铜/蛋白比值分别为606.14±45.72和901.84±53.18,两者比较具有统计学差异(P<0.05)。结论:野生型ATP7B具有铜转运功能,而突变型ATP7B则丧失了铜转运的功能。突变R778L基因是肝豆状核变性的致病基因。     

血小板膜糖蛋白Ⅱb L721R和Q860X复合杂合突变导致血小板无力症的分子机制

目的 探讨血小板膜糖蛋白Ⅱ b L721R和Q860X复合杂合突变导致血小板无力症的分子机制.方法 应用PCR法对先证者αⅡ b和β3基因所有外显子及其侧翼序列进行扩增;PCR产物纯化后直接测序,检测其突变基因.突变位点经直接测序证实排除基因多态性.采用PCR定点突变法构建αⅡ b L721 R和Q860X突变真核表达载体,测序正确后用脂质体将其分别与表达β3亚基的真核表达载体共转染293T和CHO细胞.采用流式细胞术检测转染细胞膜上αⅡ bβ3的表达,用Westernblot法鉴定αⅡ b L721R和Q860X突变体在转染细胞内的总体表达,采用免疫荧光共聚焦显微镜确定αⅡb L721R和QS60X突变体的细胞内定位.结果 先证者在αⅡ b基因存在T2255G(L721R)和C2671T(Q860x)复合杂合突变.PCDM8 Ⅱ bL721R/PCDM8Ⅲa转染的293T细胞表面αⅡ b为野生型的2.1%,133为野生型的11.0%.PCDM8Ⅱ bQ860X/PCDⅢ8ma转染的293T细胞表面αⅡb为野生型的31.9%,B3为野生型的18.0%.Western blot法证实突变αⅡ b L721R与β3共表达后,可检测到前体αⅡ b(pro-αⅡb),但未检测出成熟αⅡb.αⅡ b Q860X与β3共表达后,检测到截短型αⅡ b蛋白.免疫荧光细胞内共定位研究显示L721R和Q860X突变αⅡ bβ3蛋白大部分分布于内质网,仅有少量在高尔基体中.结论 αⅡb L721R和Q860X突变不影响αⅡ b蛋白的合成和pro-αⅡbβ3复合物的形成,但阻碍了pro-αⅡbβ3复合物由内质网向高尔基体的转运,导致细胞内滞留,从而影响了αⅡbβ3在细胞表面的表达,是导致血小板表面缺乏αⅡbβ3复合物、产生血小板无力症的分子机制.     

突变型与野生型c—myc在非p53依赖性通路中对Bim基因表达的影响

目的探讨突变型与野生型c-myc基因在非p53依赖性通路中对Bim基因表达调控及细胞凋亡的诱导功能。方法分别用携带突变型与野生型c-myc基因慢病毒载体感染p53缺失型乳癌HCCl937细胞，并得到二者过表达的稳定细胞株，以未感染组及感染不携带cmyc基因的慢病毒细胞做空白对照和感染对照，RT-PCR检测突变型与野生型c-myc及Bim基因mRNA表达，MTT、TUNEL检测突变型与野生型c-myc基因过表达乳癌HCCl937细胞增殖与凋亡情况。结果RT-PCR结果显示，突变型组与野生型组cmyc基因mRNA过表达，野生型组Bim基因mRNA表达量与突变型组比较，差异有显著性（F=125．191、157．974，P％0．05）。突变型组细胞增殖活性显著高于野生型组（F=769，64，P％0．05）。突变型组与两对照组细胞凋亡率较低，三者相比差异无显著性，野生型组细胞凋亡率显著高于突变型组与两对照组（F=61．57，P％0．05）。结论非p53依赖性通路中，野生型c-myc基因过表达能明显上调Bim基因表达，通过Bim诱导细胞凋亡，而突蛮后此作用丧失,致瘤件增高。     

野生型与突变型PS1真核表达载体的构建及其在SH-SY5Y细胞中的表达

目的为了对我们发现的中国人家族性阿尔茨海默病早老素-1(presenilin1,PS1)基因新的点突变进行蛋白功能研究,分别构建野生型和突变型PS1(G289T)与绿色荧光蛋白(EGFP)共表达载体,并检测其在SHSY5Y细胞内的表达。方法利用含人全长PS1cDNA的pcDNA3·1(zeo ),采用定点突变技术,构建PS1(G289T)-pcDNA3·1(zeo )载体。采用基因重组技术构建野生型和突变型PS1与绿色荧光蛋白共表达载体。应用脂质体将携带野生型和突变型PS1的质粒转染至SH-SY5Y细胞,检测报告基因表达,RT-PCR检测PS1mRNA表达。结果经限制性内切酶酶切图谱分析及DNA测序证实融合蛋白表达载体构建成功;RT-PCR产物经测序显示突变型PS1mRNA在SH-SY5Y中有表达。结论成功构建了人野生型和突变型PS1与EGFP共表达载体并成功转染至SH-SY5Y细胞,为进一步的研究工作奠定了基础。     

RASSFlA基因对膀胱癌细胞凋亡及化疗药物敏感性的影响

目的探讨RASSFIA基因表达对人膀胱癌细胞凋亡及对化疗药物敏感性的影响。方法采用脂质体介导的基因转染法建立野生型和突变型RASSFlA基因的真核表达载体及空载体转染膀胱癌BIU87细胞株,细胞株分为空白对照组（A组）、空载组（B组）、转染突变型RASSFlA组（c组）与转染野生型RASSFlA组（D组）,B,C,D组采用化疗药物丝裂霉素作用于细胞,四甲基偶氮唑蓝法检测细胞增殖程度,原位凋亡细胞检测技术检测膀胱癌BIU87细胞凋亡。Westernblot测定Caspase-3蛋白的表达水平。结果成功建立稳定表达野生型和突变型RASSFlA基因的膀胱癌细胞株;加入丝裂霉素前、后D组细胞增殖比、凋亡指数及Caspase-3蛋白表达水平与A,B组比较差异均有统计学意义（P〈0．05）,C组与A,B组比较,差异均无统计学意义（P〉0.05）。结论野生型RASSFIA的重表达可促进膀胱癌BIU87细胞凋亡,提高膀胱癌细胞对丝裂霉素的敏感性。     

小细胞肺癌组织中突变型P53与Bcl-2蛋白的表达及意义

目的探讨小细胞肺癌组织中突变型P53与Bcl-2蛋白的表达及意义。方法用SP免疫组化法检测40例小细胞肺癌组织中突变型P53与Bcl-2蛋白的表达情况,并将结果进行相关分析。结果小细胞肺癌组织中突变型P53与Bcl-2蛋白的阳性表达率明显高于正常肺组织（P〈0.05）。突变型P53表达与淋巴结转移和TNM分期有关（P〈0.05）,而Bcl-2表达与之无关（P〉0.05）。突变型P53与Bcl-2蛋白的表达有相关性（P〈0.05）。结论小细胞肺癌组织中突变型P53蛋白与预后有关,Bcl-2蛋白与预后无关。     

小细胞肺癌组织中突变型P53与Bcl-2蛋白的表达及意义

目的探讨小细胞肺癌组织中突变型P53与Bcl-2蛋白的表达及意义。方法用SP免疫组化法检测40例小细胞肺癌组织中突变型P53与Bcl-2蛋白的表达情况,并将结果进行相关分析。结果小细胞肺癌组织中突变型P53与Bcl-2蛋白的阳性表达率明显高于正常肺组织（P〈0.05）。突变型P53表达与淋巴结转移和TNM分期有关（P〈0.05）,而Bcl-2表达与之无关（P〉0.05）。突变型P53与Bcl-2蛋白的表达有相关性（P〈0.05）。结论小细胞肺癌组织中突变型P53蛋白与预后有关,Bcl-2蛋白与预后无关。     

胃腺癌突变型P53蛋白和PCNA的表达及对比研究

目的：研究突变型 P53蛋白和增殖细胞核抗原（PCNA）在胃腺癌组织中表达情况,探讨突变型 P53蛋白和PCNA 在胃癌发生发展中作用。方法应用免疫组化 EnVision 法检测42例胃腺癌及正常胃黏膜组织中突变型 P53蛋白和 PCNA 的表达水平,分析不同分化程度胃腺癌中突变型 P53蛋白和 PCNA 的表达的相关性。结果免疫组化方法检测到胃腺癌组织中突变型 P53蛋白和 PCNA 的表达水平显著高于正常胃黏膜组织（P ＜0．05）。胃腺癌组织中突变型 P53蛋白与 PCNA 表达水平与胃腺癌细胞分化程度、有无淋巴结转移有关（P ＜0．05）,与患者的性别、年龄、胃腺癌浸润深度无关（P ＞0．05）。胃腺癌组织中变型 P53蛋白与 PCNA 表达呈正相关（P ＜0．01）。结论突变型 P53蛋白和 PCNA 的表达与胃癌分化程度关系密切,可作为预测胃腺癌恶性程度和评估患者预后的指标。     

中国人遗传性高铁血红蛋白血症NADH—细胞色素b5还原酶基因L …

目的：鉴定导致中国人遗传性高铁血红蛋白血症（ＲＣＭ）的ＮＡＤＨ－细胞色素ｂ５还原酶（ｂ５Ｒ）基因突变类型，探讨ＲＣＭ发病的分子基础。方法：逆转录－聚合酶链反应产物直接测序和ｃＤＮＡ克隆测定分析先证者的ｂ５Ｒ编码基因，限制性酶切分析其基因组ＤＮＡ。结果；发现一例ＲＣＭ患者ｂ５Ｒ基因第７２密码子存在新的错义突变（ＣＴＣ→ＣＣＣ）。结论：该突变导致ｂ５Ｒ蛋白第７２位亮氨酸被脯氨酸替换（Ｌ７２Ｐ）是该先证     

Concentration of NADH-cytochrome b5 reductase in erythrocytes of normal and methemoglobinemic individuals measured with a quantitative radioimmunoblotting assay.

The activity of NADH-cytochrome b5 reductase (NADH-methemoglobin reductase) is generally reduced in red cells of patients with recessive hereditary methemoglobinemia. To determine whether this lower activity is due to reduced concentration of an enzyme with normal catalytic properties or to reduced activity of an enzyme present at normal concentration, we measured erythrocyte reductase concentrations with a quantitative radioimmunoblotting method, using affinity-purified polyclonal antibodies against rat liver microsomal reductase as probe. In five patients with the "mild" form of recessive hereditary methemoglobinemia, in which the activity of erythrocyte reductase was 4-13% of controls, concentrations of the enzyme, measured as antigen, were also reduced to 7-20% of the control values. The concentration of membrane-bound reductase antigen, measured in the ghost fraction, was similarly reduced. Thus, in these patients, the reductase deficit is caused mainly by a reduction in NADH-cytochrome b5 reductase concentration, although altered catalytic properties of the enzyme may also contribute to the reduced enzyme activity.     

中国人遗传性高铁血红蛋白血症二个家系所见Gytb5R基因Arg^57…

目的：探明遗传性高铁血红蛋白血症ＮＡＤＨ－细胞色素ｂ５还原酶（Ｃｙｔｂ５Ｒ）缺陷的分子机制。方法：用逆转录－多聚酶链反应（ＲＴ－ＰＣＲ）方法，克隆３个遗传性高铁血红蛋白血症家系Ｃｙｔｂ５Ｒ９２１ｂｐ的ｃＤＮＡ编码序列，并用限制性酶切ＰＣＲ产物分析３个家系的基因组ＤＮＡ。结果：２个家系存在Ａｒｇ＾５７→Ｇｌｎ突变，３个家系均未发现Ｇｌｕ＾２２２→ｇｌｙ突变。结论：Ａｒｇ＾２２２→Ｇｌｎ突变是中国人民     

In vitro characterization of missense mutations associated with quantitative protein S deficiency

OBJECTIVE: To elucidate the molecular consequences of hereditary protein S (PS) deficiency, we investigated the in vitro synthesis of the PS missense mutants in COS-1 cells and their activated protein C (APC) cofactor activities. PATIENTS: Four patients with quantitative PS deficiency suffering from venous thrombosis were examined. RESULTS: We identified three distinct novel missense mutations, R275C, P375Q and D455Y, and two previously reported missense mutations, C80Y and R314H. The P375Q and D455Y mutations were found in one patient and observed to be in linkage on the same allele. The R314H mutant showed the lowest level of expression (32.7%), and the C80Y, P375Q + D455Y, and R275C mutants exhibited a moderate impairment of expression, that is, 43.8%, 49.5%, and 72.3% of the wild type, respectively. Furthermore, pulse-chase experiments demonstrated that all mutants showed impaired secretion and longer half-lives in the cells than the wild type PS. In the APC cofactor assays, the C80Y mutant showed no cofactor activity, and the R275C mutant showed reduced activity, 62.3% of the wild type PS, whereas the R314H and P375Q + D455Y mutants exhibited normal cofactor activity. CONCLUSION: These data indicate that the C80Y and R275C mutations affect the secretion and function of the PS molecule, and that the R314H and P375Q + D455Y mutations are responsible for only secretion defects, causing the phenotype of quantitative PS deficiency observed in the patients.     

NADH cytochrome b5 reductase activity in lymphoid cell lines. Expression of the defect in epstein Barr virus transformed lymphoblastoid cell lines from patients with recessive congenital methemoglobinemia.

Recessive congenital methemoglobinemia (RCM) is due to the homozygous deficiency of NADH-cytochrome b5 reductase (EC 1.6.2.2.). In type I disease, in which the patients are only methemoglobinemic, the enzyme defect is fully expressed in the erythrocytes, whereas the leukocytes are much less affected. In type II disease, in which the patients are, in addition, mentally retarded, the defect is generalized to all the tissues including cultured fibroblasts. In the present study we have investigated Epstein-Barr virus (EBV) transformed lymphoid cell lines (LCL) derived from patients with both types of cytochrome b5 reductase deficiency and from nondeficient individuals. The total cytochrome b5 reductase activity of the control LCL was found to be similar whatever the LCL origin, except for one lymphoma line (Daudi). The enzyme from the control LCL (c 252/B 95) was found to be immunologically related to the human soluble erythrocyte cytochrome b5 reductase, indicating that it is the product of the same gene: the DIA1 (diaphorase) locus. The LCL derived from one patient with the type I disease and two patients with the type II disease were investigated.l In the former the defect was expressed to a lesser degree than in the cases with mental retardation in which the defect was much pronounced, and involved both the mitochondrial and the microsomal fraction. This indicated that all the subcellular forms of the cytochrome b5 reductase are under the same genetic control. Altogether, these data show that the LCL are a favorable material for studying both types of cytochrome b5 reductase deficiency and for investigating in depth the molecular aspects of this metabolic disease.     

A missense mutation in the cholesteryl ester transfer protein gene with possible dominant effects on plasma high density lipoproteins.

Plasma HDL are a negative risk factor for atherosclerosis. Cholesteryl ester transfer protein (CETP; 476 amino acids) transfers cholesteryl ester from HDL to other lipoproteins. Subjects with homozygous CETP deficiency caused by a gene splicing defect have markedly elevated HDL; however, heterozygotes have only mild increases in HDL. We describe two probands with a CETP missense mutation (442 D:G). Although heterozygous, they have threefold increases in HDL concentration and markedly decreased plasma CETP mass and activity, suggesting that the mutation has dominant effects on CETP and HDL in vivo. Cellular expression of mutant cDNA results in secretion of only 30% of wild type CETP activity. Moreover, coexpression of wild type and mutant cDNAs leads to inhibition of wild type secretion and activity. The dominant effects of the CETP missense mutation during cellular expression probably explains why the probands have markedly increased HDL in the heterozygous state, and suggests that the active molecular species of CETP may be multimeric.     

A compound heterozygote for a novel missense mutation (G105R) in exon 3 and a missense mutation (D204E) in exon 5 of the lipoprotein lipase gene in a Japanese infant with hyperchylomicronaemia

We systematically investigated the molecular defects resulting in primary lipoprotein lipase (LPL) deficiency in a Japanese male infant (hereafter called 'the patient') with severe fasting hypertriglyceridaemia (type I hyperlipoproteinaemia). The primary LPL deficiency was diagnosed on the basis of the findings that no LPL activity was detected in post-heparin plasma (PHP) and that the immunoreactive LPL mass in PHP was less than 2% of the control level. The patient was a compound heterozygote for a novel missense mutation (G(568)GA-->AGA/Gly(105)-->Arg; G105R) in exon 3 and a missense mutation (GAC(867)-->GAG/Asp(204)-->Glu; D204E) in exon 5 of the LPL gene. The biological significance of both missense mutations was examined by an in vitro study of the expression of the mutant G105R LPL cDNA and D204E LPL cDNA in COS-1 cells. Both mutant LPLs were catalytically inactive and were barely released by heparin from the expressing COS-1 cells. These findings explain the failure to detect LPL activity and immunoreactive LPL mass in the patient's PHP. The G105R allele could be detected by digestion with the BsmAI restriction enzyme, and the D204E allele by digestion with HincII. The patient inherited the G105R allele from his mother and the D204E allele from his father. His parents were heterozygotes for the corresponding mutant allele, but normolipidaemic. The novel G105R missense mutation could not be detected by conventional analysis of single-strand conformation polymorphism, but it was identified by extensive sequencing of the entire exons and their flanking regions in the LPL gene.     

乳铁蛋白多肽嵌合体对铜绿假单胞菌PAO1及PAO-JP2型菌株QS毒力因子的作用

目的观察乳铁蛋白多肽嵌合体（LFchimera）对铜绿假单胞菌PAO1及PAO-JP2型菌株QS毒力因子的影响。方法以PAO1和PAO-JP2为试验菌株,分为A、B、C、D四组。A组：未作任何干预的铜绿假单胞菌PAO1;B组：LFchimera（1μmol/L）干预的铜绿假单胞菌PAO1;C组：未作任何干预的铜绿假单胞菌PAO-JP2;D组：LFchimera（1μmol/L）干预的铜绿假单胞菌PAO-JP2。分别测定毒力因子绿脓菌素（氯仿萃取法）、弹性蛋白酶（弹性蛋白-刚果红法）、胞外蛋白水解酶（偶氮酪蛋白法）。结果和A组各毒力因子（绿脓菌素、弹性蛋白酶、蛋白水解酶）的活性表达（7.03±0.14、0.149±0.009、0.808±0.038）比较,B组铜绿假单胞菌PAO1菌株的表达（3.07±0.13、0.046±0.004、0.325±0.022）明显下降,差异有统计学意义（P〈0.05）。C组铜绿假单胞菌PAO-JP2菌株各毒力因子（绿脓菌素、弹性蛋白酶、蛋白水解酶）的活性表达（0.76±0.04、0.015±0.003、0.054±0.006）和D组（0.74±0.05、0.014±0.002、0.053±0.005）比较,差异无统计学意义（P〉0.05）。结论 LFchimera可能通过抑制铜绿假单胞菌QS系统减弱其相关毒力因子的活性,从而发挥抗感染作用。     

Activating and deactivating mutations in the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2

Here we describe 2 mutations in growth and differentiation factor 5 (GDF5) that alter receptor-binding affinities. They cause brachydactyly type A2 (L441P) and symphalangism (R438L), conditions previously associated with mutations in the GDF5 receptor bone morphogenetic protein receptor type 1b (BMPR1B) and the BMP antagonist NOGGIN, respectively. We expressed the mutant proteins in limb bud micromass culture and treated ATDC5 and C2C12 cells with recombinant GDF5. Our results indicated that the L441P mutant is almost inactive. The R438L mutant, in contrast, showed increased biological activity when compared with WT GDF5. Biosensor interaction analyses revealed loss of binding to BMPR1A and BMPR1B ectodomains for the L441P mutant, whereas the R438L mutant showed normal binding to BMPR1B but increased binding to BMPR1A, the receptor normally activated by BMP2. The binding to NOGGIN was normal for both mutants. Thus, the brachydactyly type A2 phenotype (L441P) is caused by inhibition of the ligand-receptor interaction, whereas the symphalangism phenotype (R438L) is caused by a loss of receptor-binding specificity, resulting in a gain of function by the acquisition of BMP2-like properties. The presented experiments have identified some of the main determinants of GDF5 receptor-binding specificity in vivo and open new prospects for generating antagonists and superagonists of GDF5.