一例凝血因子Ⅷ B区错义突变导致重型血友病A

目的：检测一例重型血友病Ａ患者（ＳＨ９）的基因突变。方法：用ＰＣＲ、变性梯度凝胶电泳（ＤＧＧＥ）和ＤＮＡ测序检测因子Ⅷ基因突变。先用Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ排除内含子２２倒位，然后应用ＰＣＲ对凝血因子Ⅷ基因进行扩增。扩增范围包括所有外显子及其侧翼内含子序列。结果：片段１４２在ＤＧＧＥ中泳动异常。ＤＮＡ测序证实Ｃ２５３５Ａ导致Ｂ区错义突变８２６Ａｓｐ（ＧＡＣ）→Ｇｌｕ（ＧＡＡ）。结论：该突变可能是导致重型血友病Ａ的原因，但有待进一步研究证实。     

急性出血性脑血管病并发多脏器功能失常综合征患者血浆？…

目的 探讨血浆内皮素－１（ＥＴ－１）和降钙素基因相关肽（ＣＧＲＰ）在急性出血性脑血管病（ＡＨＣＶＤ）并发多脏器功能失常综合征（ＭＯＤＳ）发病中的作用。方法 采用放射免疫法分别测定２１例ＡＨＣＶＤ合并ＭＯＤＳ患者（ＭＯＤＳ组）、２０例ＡＨＣＶＤ患者（ＡＨＣＶＤ组）及３０例正常人（正常对照组）血浆中ＥＴ－１和ＣＧＲＰ水平。结果：ＭＯＤＳ组及ＡＨＣＶＤ组血浆ＥＴ－１水平明显高于正常对照组（Ｐ均〈０．０１     

^14C标记合成脂蛋白样颗粒法测定血清中胆固醇酯转运蛋白活性

目的 建立测定血清中胆固醇脂转运蛋白（ＣＥＴＰ）活性的方法。方法 用＾１４Ｃ标记的胆固醇酯合成高密度脂蛋白（ＨＤＬ）样颗粒作为反应基质,检测了４５例健康人,５２例冠心病（ＣＨＤ）患者血清ＣＥＴＰ活性。结果 该方法线性范围为０￣３１．８％,高（２６．４％）、低（６．８３％）活性批内变异系数（ＣＶ）分别为６．１％,７．６％,批间ＣＶ分别为１１．７％、１２．１％,５２例ＣＨＤ患者血清ＣＥＴＰ活性（Ｘ＾－     

SLE患者尿微量蛋白和尿酶检测的临床价值

目的：探讨尿微量蛋白和尿酶检测对狼疮性肾损害的临床价值。方法：用速率散射比浊法检测５１例ＳＬＥ患者随机尿中的白蛋白（Ａｌｂ）、免疫球蛋白Ｇ（ＩｇＧ）、α１微球蛋白（α１ＭＧ）,终点比色法测尿Ｎ－乙酰－β－Ｄ氨基葡萄糖苷酶（ＮＡＧ）,Ｊａｆｆｅ氏速率法测尿肌酐,并按尿蛋白定性分组比较。结果：ＳＬＥ患者尿蛋白阴性组的尿Ａｌｂ／Ｃｒ、ＩｇＧ／Ｃｒ、α１ＭＧ／Ｃｒ、ＮＡＧ／Ｃｒ值明显高于正常对照组（Ｐ〈０     

小儿急性T淋巴细胞白血病tal－1基因缺失分析

根据ｔａｌ－１基因缺失上下游序列设计一对引物，应用多聚酶链反应（ＰＣＲ）技术对１０株人白血病细胞株和７０例小儿急性白血病骨髓标本进行ｔａｌ－１基因缺失分析。结果ＣＥＭ、ＨＢＳ－２和ＲＰＭＩ－８４０２三株Ｔ细胞白血病（Ｔ－ＡＬＬ）细胞株以及３／１６例Ｔ－ＡＬＬ发现ｔａｌ－１基因缺失，５４例其它类型急性白血病均未见基因缺失，而且具ｔａｌ－１基因缺失的Ｔ－ＡＬＬ细胞均为胸腺发育Ｉ期，免疫表型特征为ＣＤ＿２＋、ＣＤ＿７＋、ＣＤ＿１￣－、ＣＤ＿４￣－和ＣＤ＿８￣－。ＰＣＲ方法敏感性达１０￣（－４）。说明ｔａｌ－１缺失发生于部分Ｔ－ＡＬＬ中，可用于微小残留病的检测。     

急性出血性脑血管病并发多脏器功能失常综合征患者血浆内皮素-1及降钙素基因相关肽水平的观察

目的：探讨血浆内皮素１（ＥＴ１）和降钙素基因相关肽（ＣＧＲＰ）在急性出血性脑血管病（ＡＨＣＶＤ）并发多脏器功能失常综合征（ＭＯＤＳ）发病中的作用。方法：采用放射免疫法分别测定２１例ＡＨＣＶＤ合并ＭＯＤＳ患者（ＭＯＤＳ组）、２０例ＡＨＣＶＤ患者（ＡＨＣＶＤ组）及３０例正常人（正常对照组）血浆中ＥＴ１和ＣＧＲＰ水平。结果：ＭＯＤＳ组及ＡＨＣＶＤ组血浆ＥＴ１水平明显高于正常对照组（Ｐ均＜０．０１），ＭＯＤＳ组ＥＴ１水平又明显高于ＡＨＣＶＤ组（Ｐ＜０．０１）。ＡＨＣＶＤ组血浆ＣＧＲＰ水平高于正常对照组，但无显著性差异（Ｐ＞０．０５）。而ＭＯＤＳ组血浆ＣＧＲＰ水平明显低于正常对照组，ＥＴ１／ＣＧＲＰ（Ｅ／Ｃ）比值明显高于ＡＨＣＶＤ组及正常对照组（Ｐ均＜０．０１）。结论：血浆ＥＴ１水平升高、ＣＧＲＰ水平降低、Ｅ／Ｃ比值严重失衡与ＭＯＤＳ的发生相关；检测血浆ＥＴ１和ＣＧＲＰ水平对评估ＡＨＣＶＤ患者预后有一定意义     

高血压病患者血脂,脂蛋白,载脂蛋白临床研究

目的：探讨高血压病（ＥＨ）患者脂质代谢状况。方法：采用山西医科大学血脂研究室建立的常规方法测定了５０例ＥＨ患者和４５例人血浆ＴＧ、ＴＣ、ＨＤＬ－Ｃ,同时,采用ＥＬＩＳＡ法测定了ａｐｏＡＩ、ａｐｏＢ１００含量。结果：ＥＨ患者ＴＧ、ＴＣ、ＬＤＬ－Ｃ及ａｐｏＢ１００均高于ＣＧ,ＨＤＬ－Ｃ及ａｐｏＡＩ显著低于ＣＧ,以脂蛋白和载脂蛋白变化明显,并且,随高血压病情进展,脂质紊乱加重。结论：ＥＨ本身存在脂质代谢     

新生儿缺氧缺血性脑病红细胞膜 ATP 酶、氧自由基变化及与脑损伤关系的研究

目的：探讨红细胞膜ＡＴＰ酶和氧自由基在新生儿缺氧缺血性脑病（ＨＩＥ）发病中的作用，寻找防治途径。方法：采用孔雀绿分析法检测了３２例新生儿缺氧缺血性脑病时红细胞膜Ｎａ＋Ｋ＋ＡＴＰ酶活性及脂质过氧化代谢产物丙二醛（ＭＤＡ）含量和磷酸肌酸激酶脑型同功酶（ＣＫＢＢ）活性。结果：缺氧缺血性脑病患儿红细胞膜Ｎａ＋Ｋ＋ＡＴＰ酶活性〔（０．１１３±０．０２２）ｎｍｏｌＰｉ·ｈ－１／ｍｇＰｒ〕明显低于正常新生儿〔（０．２９４±０．０３７）ｎｍｏｌＰｉ·ｈ－１／ｍｇＰｒ〕，ＭＤＡ及ＣＫＢＢ活性明显高于正常新生儿，且红细胞膜Ｎａ＋Ｋ＋ＡＴＰ酶活性与ＭＤＡ水平呈明显负相关（ｒ＝－０．３６０，Ｐ＜０．０５），即Ｎａ＋Ｋ＋ＡＴＰ酶活性的降低与脂质过氧化损伤密切相关。结论：氧自由基损伤可能参与了ＨＩＥ的发病和病理过程，进一步损伤细胞膜Ｎａ＋Ｋ＋ＡＴＰ酶功能，使机体生理功能产生不可逆损伤。故对ＨＩＥ治疗中尽早使用抗氧化剂和保护细胞膜Ｎａ＋Ｋ＋ＡＴＰ酶功能的药物十分必要。     

脑脊液CHE、GOT、LDH、α－HBD和TP测定用于脑梗塞的疗效观察

脑脊液ＣＨＥ、ＧＯＴ、ＬＤＨ、α－ＨＢＤ和ＴＰ测定用于脑梗塞的疗效观察山东省交通医院（２５００３１）刘茂林，牛祺麟，刘新风，张怡本文应用神经生长因子（ＮＧＦ）治疗９７例脑梗塞恢复期（ＣＣＩ）患者。治疗前后观察了脑脊液中ＣＨＥ、ＧＯＴ、ＬＤＨ、α－ＨＢ...     

胆固醇酯转运蛋白基因15显子D442G突变的检测

应用聚合酶链反应－限制性片段长度多态性分析（ＰＣＲ－ＲＥＬＰ）的方法检测１１０例脑动脉粥样硬化患者胆固醇酯转运蛋白（ＣＥＴＰ）基因１５外显子Ｄ４２Ｇ错义突变。首先用聚合酶链反应行异性扩增包含人ＣＥＴＰ基因１５外显子第１５０６位碱基的基因序列，扩增产和用限制性内切酶ＭｓｐⅠ酶切，聚丙烯酰胺凝胶电泳后分析结果。１１０例脑动脉粥样硬化患者中发现４例杂俣突变。突变率３．６％，与对照组４％比较无明显差异。该     

Particle size analysis of high density lipoproteins in patients with genetic cholesteryl ester transfer protein deficiency

We investigated the detailed distribution of high-density lipoproteins (HDL) particle size in patients with cholesteryl ester transfer protein (CETP) deficiency. Serum samples pre-stained with Sudan black B were electrophoresed using 4-30% polyacrylamide gradient gels, and the Stokes diameter of HDL particles was determined in 23 patients with genetic CETP deficiency, nine patients with hyperalphalipoproteinemia and seven subjects with normal HDL cholesterol concentrations. The mean Stokes diameter of HDL particles in CETP deficient patients (11.2+/-0.6 nm) was significantly greater than hyperalphalipoproteinemia (10.7+/-0.3 nm, P<0.05) and normal subjects (9.5+/-0.4 nm, P<0.01). A significant relationship was found between mean HDL size and serum CETP mass concentrations (P<0.05). When the particle size of all detected HDL bands was investigated, extra-large HDL particles larger than 12 nm were found in 14 of the 23 patients with CETP deficiency, which were not found in any of the hyperalphalipoproteinemia patients or normal subjects. Serum low-density lipoproteins (LDL) cholesterol and total cholesterol concentrations were lower in CETP deficiencies with extra-large HDL particles than those in non-carriers (P<0.01). These results indicate that extra-large HDL may be an index to clarify the relationship between genetic CETP deficiency and atherosclerosis.     

云南德宏地区傣族人群珠蛋白生成障碍性贫血社区群体筛查和基因诊断结果分析

目的 了解该地区傣族人群珠蛋白生成障碍性贫血(Thal)患病率和基因缺失突变类型.方法 对710例傣族人群进行红细胞指数、微量血红蛋白(Hb)电泳、HbA2定量检测初筛,以聚合酶链发应(PCR)和反向点杂交技术鉴定β-Thal基因突变类型,以跨跃断裂位点PCR(GAP-PCR)技术和凝胶电泳鉴定α-Thal基因缺失类型.结果 α-和β-Thal检出率分别为25.35%(180/710)、14.51%(103/710),β-Thal复合α-Thal检出率为29.13%(30/103).结论 该地区傣族人群Thal检出率较高,应积极制订有效的干预和控制措施,降低Thal患儿的出生率.     

原发性肝细胞癌患者肝组织中乙型肝炎病毒X基因的变异

目的:对原发性肝细胞癌(hepatocellular carcinoma,HCC)患者肝组织中乙型肝炎病毒(hepatitis Bvirus,HBV)X基因及其常见变异位点进行检测分析,以探讨HBVX基因及其变异与HCC发生发展的关系。方法:采用聚合酶链反应(polymerase chain reaction,PCR)方法检测22例乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)阳性的HCC患者肝组织中HBVX基因,并对PCR产物进行基因测序分析,同时检测5例正常肝脏组织中HBVX基因。结果:22例HCC患者癌组织及癌周组织中HBVX基因检出率分别为68%和77%,5例正常肝脏组织中均未检测到HBVX基因,癌组织及癌周组织中HBVX基因检出率均很高(均P<0.0125),而癌组织与癌周组织的阳性率没有明显差异(P>0.0125)。癌组织及癌周组织中HBVX基因发生1762T/1764A双突变率分别为93%和47%,肝癌组织中双突变率要明显高于癌周组织(P<0.05),未发现1762T及1764A单独发生突变。结论:HCC患者肝组织中HBVX基因检出率很高,肝癌组织中发生1...     

四引物扩增受阻突变体系快速检测Wilson病基因Arg778 Leu突变

目的 建立一种不需要限制性片段长度多态性或直接测序的新方法检测肝豆状核变性（WD）病人突变热点ATP7B基因的Arg778Leu突变基因型。方法 用四引物扩增受阻突变体系聚合酶链反应（tetra-primer amplification refractory mutation system-PCR,tetra-primer ARMS—PCR）检测47例WD患者和30例正常对照的ATP7B基因Arg778Leu突变,并用测序进行验证。结果 47例WD患者中检出Arg778Leu纯合突变4例,杂合突变14例,总检出率38．3％（18／47）;30例正常对照未发现突变;DNA测序结果与四引物ARMS—PCR结果完全一致。结论 ATP7B基因Arg778Leu突变是中国人WD突变热点;四引物ARMS—PCR法检测Arg778Leu突变有快速、简便、准确的优点,可以区分等位基因是否纯合,适于大样本的人群筛查。此法也可用于检测其他点突变。     

High density lipoprotein cholesterol in secondary hyperlipoproteinemias

Cholesterol of high density lipoproteins (Ch HDLP) was assayed in primary hyperlipoproteinemia (HLP) in patients with coronary heart disease (CHD) and in secondary HLP that developed in the presence of chronic glomerulonephritis with the nephrotic syndrome, hypothyrosis, diabetes mellitus, and lipoprotein metabolism disturbances innduced by longterm oral contraceptive adminnistration. In patients with primary HLP and the nephrotic syndrome, the Ch HDLP level was found to be reduced, this reduction being more manifest in hypertriglyceridemia; the reverse correlation between Ch HDLP and triglyceride content in the blood was observed. In patients with diabetes mellitus and hypothyrosis, Ch HDLP concentration was found to grow parallel with hypertriglyceridemia degree. Simultaneously, hypertriglyceridemia and hyper-Ch-HDLP were found in the blood under the effect of OCs. High levels of Ch HDLP during OC intake were found to depend on accumulation of cholesterol ethers in them, whereas in diabetes mellitus and hyperalphalipoproteinemia, detected in male subjects during prophylactic examinations, high levels of Ch HDLP were found to be related to the accumulation of free cholesterol. Study of the ratio of free and bound cholesterol in HDLP allows a differentiation between physiologic and pathologic hyper-Ch-HDLP; in physiologic hyperalphaproteinemia, cholesterol ethers were found to accumulate in HDLP, in pathologic ones, free cholesterol. At the same time, study of the cholesterol/phospholipid ratio gives an idea of the ability of HDLP to take hold of cholesterol from the peripheral cell membranes. Except for estrogen-induced lipoprotein metabolism disturbances, hyperalphalipoproteinemia in HLP is a sign or progressing lipoprotein metabolism disturbances. (author's modified)     

Use of polymerase chain reaction for diagnosis of inherited disorders

The polymerase chain reaction (PCR) is a rapid method for generating a 10(6)- to 10(7)-fold increase in the number of copies of a discrete DNA or RNA sequence. The technique is being used for rapid prenatal diagnosis and carrier testing of several inherited disorders. After PCR, mutations producing single-gene disorders can be detected by several different methods, including endonuclease digestion and gel electrophoresis (applicable when a mutation affects an endonuclease recognition site), gel electrophoresis (used for detection of deletions), and hybridization to an oligonucleotide probe specific for a mutation. Less often, gene sequencing of a PCR product is used to rapidly identify a mutation. In addition, the PCR technique can be applied to polymorphism analysis to provide diagnosis by linkage analysis. In other areas, PCR is being used to detect and characterize microbial pathogens and to characterize mutations associated with carcinogenesis. The PCR method is useful in situations in which the amount of DNA sample is limited, such as in forensics and prenatal testing, or in which the quality of the DNA sample is poor.     

多重PCR技术在Y染色体微缺失快速筛查中的应用

目的探讨多重聚合酶链反应(PCR)技术在Y染色体微缺失快速筛查中的应用价值。方法以38例健康男性和46例无(少)精症男性外周血淋巴细胞基因组DNA为模板,采用针对Y染色体无精子因子(AZF)4个亚区(AZFa～d)15个序列标签位点(STS)的多重引物进行PCR检测,并以琼脂糖凝胶电泳分析PCR产物。结果 84例受试者多重PCR检测成功率为100%,且特异性好,扩增效率高。健康男性标本可检出15个STS。患者中检出AZFc+d微缺失1例。结论多重PCR是快速筛查Y染色体微缺失的有效工具。     

A seminested PCR test for simultaneous detection of two common mutations (35delG and 167delT) in the connexin-26 gene.

BACKGROUND: Several mutations described in the connexin-26 gene cause nonsyndromic autosomal recessive deafness (NARD). The prevalence of two frame-shift mutations, known as 35delG and 167delT, was relatively high in patients with NARD from different populations. METHODS AND RESULTS: A seminested PCR test has been developed for simultaneous detection of two common mutations in the connexin-26 gene. The test is based on PCR amplification of a 285-bp DNA fragment that covers both the 35delG and 167delT mutations. The latter mutation destroys a Pst I site and is easily detected by Pst I digestion of the 285-bp DNA fragment. However, the 35delG mutation does not destroy or create a restriction site. To create a site, we designed a mismatched primer that generated an EcoN I site in an 87-bp DNA fragment, but only if the 35delG mutation was present. The test was validated using five DNA samples previously characterized for the connexin-26 mutations. After validation, we screened 45 unrelated patients with NARD and 280 healthy Omani subjects for the presence or absence of the 35delG and 167delT mutations. Neither mutation was found to be present in patients or control subjects. CONCLUSION: We developed a seminested PCR test for the simultaneous detection of both common mutations in the connexin-26 gene. In our analysis of 45 patients and 280 control subjects, the 35delG and 167delT mutations were absent in both groups.     

HIV-1辅助受体CXCR4配体暨趋化因子SDF-1α转录子基因多态性研究

目的 研究趋化因子SDF-1α转录子基因突变,分析突变发生频率,以及其对HIV感染的影响.方法 研究对象包括居住在中国香港地区的278名HIV阴性健康中国人,49例HIV阳性白种人和13名高危未感染中国人.分别提取外周血单个核细胞基因组DNA和总RNA.建立PCR及逆转录PCR法,对SDF-1α启动子(promoter)、开放读码区(ORF)和3'端非编码区(3'UTR)基因进行序列分析.用人工引入点突变的错配引物双重PCR法对一新发现突变进行筛查,分析其在研究对象中的分布及意义.结果 对包括13名高危中国人、49例白种人和38名随机选取的健康中国人在内的100份标本测序分析显示,在SDF-1α启动子及ORF区内未见任何突变;在SDF-1α3'UTR发现一"GA"插入式基因突变,命名为SDF-1-3'GA(+),在GenBank中的序列号为AY874118.该突变在3组研究对象中均有发生,在健康中国人中的频率为15.1%(84/556),但在13名有HIV接触而未感染的高危人群中发生频率为30.7%(8/26).结论 与本身重要生理功能相对应,SDF-1α基因十分保守;在其3'UTR新发现突变SDF-1-3'GA(+)是否影响HIV感染,值得进一步研究.     

Use of polymerase chain reaction to detect heterozygous familial hypercholesterolemia

We used a modification of the polymerase chain reaction (PCR), involving two pairs of oligonucleotide primers, to detect a mutation in the low-density lipoprotein (LDL) receptor gene, commonly occurring among patients with familial hypercholesterolemia (FH) in Finland. This mutation, called FH-Helsinki, involves a large (about 9500 base pairs, bp) deletion in the LDL receptor gene extending from intron 15 to exon 18. For the PCR, one pair of primers was designed to cover both sides of the deletion in its immediate vicinity. In the presence of the deletion, the primers were brought close enough to each other to allow the amplification and electrophoretic detection of a 300-bp amplification product. In the absence of the deletion, no amplification occurred and this band accordingly was not visible in the gel. To render the interpretation of the results unequivocal, we designed a second pair of oligonucleotide primers. This pair of primers allowed another amplification product (158 bp) to appear in samples containing a normal exon 17, i.e., in DNA specimens from healthy subjects and FH heterozygotes with or without the FH-Helsinki deletion. The technique is easy to perform, avoids the use of radioactive reagents, and is applicable to the detection of any extensive DNA deletion.