长距离PCR检测重型血友病A病人凝血因子Ⅷ基因倒位

为了筛查山东省重型血友病A(hemophiliaA，HA)凝血因子Ⅶ基因倒位的患并检出携带，采用长距离DNA扩增(LD-PCR)方法，以0．6％琼脂糖凝胶电泳技术，检测临床上确诊的55例重型HA患及其家系成员中是否存在凝血因子Ⅷ(FⅧ)基因倒位。电泳出现11kb带，示凝血因子Ⅷ基因倒位；12kb带，示非倒位；这两条带同时出现为凝血因子Ⅷ基因倒位携带。结果表明：55例无亲缘关系的重型HA患中，发现22例患(或家系成员)有凝血因子Ⅷ基因倒位，占重型HA患的40%；15个家系中查出基因倒位携带5名。结论：运用LD-PCR技术可以准确、简便、快速地检测重型血友病A患是否存在凝血因子Ⅷ基因倒位。     

LD-PCR检测FⅧ基因XbaⅠ位点多态性及其在血友病A携带者诊断中的应用

目的建立高特异性的针对凝血因子Ⅷ(FⅧ)基因内含子22中XbaⅠ多态性位点的连锁分析法,以用于血友病A家系携带者的诊断。方法采用长距离PCR(LD-PCR)方法,特异性地扩增FⅧ基因内含子22中的XbaⅠ位点所在区域,用限制性内切酶XbaⅠ酶切,对5个血友病A家系作家系连锁分析,并对其中可提供多态性信息的家系作携带者诊断。结果家系1中确诊女性携带者1名及1名正常女性成员;家系2中确诊1名女性携带者;而家系3、家系4和家系5该位点无法提供多态性信息。结论基于LD-PCR的FⅧ基因XbaⅠ位点连锁分析方法具有较高的准确性和可靠性,这对临床上诊断血友病A携带者具有积极的意义。     

应用PCR技术对凝血因子Ⅷ基因多态性的研究

凝血因子Ⅷ(FⅧ)是血液凝固过程中起重要作用的一种血浆蛋白,其基因缺陷可导致临床上常见的遗传性出血性疾病——血友病A。本研究应用聚合酶链反应(PCR)技术扩增FⅧ基因的BclⅠ和Xba Ⅰ的特异片段,经酶解后分析酶切位点限制性片段长度多态性(RFLP),BclⅠ的多态信息量(PIC) 为0.36,XbaⅠ为0.49,联合应用则为0.67。在30名受检女性中,BclⅠ杂合频率为40％,XbaⅠ为53％,联合应用则为76％。15个家系中,10个可采用BclⅠ或XbaⅠ进行RFLP分析。对这些家系中19名可疑携带者进行检测,结果诊断出8名基因携带者。     

甜菜碱在提高血清乳酸脱氢酶活性稳定性中的作用

目的 探讨不同浓度甜菜碱在不同保存温度(2～8℃、-20℃)下对血清乳酸脱氢酶(LDH)的稳定作用.方法 将5 mol/L甜菜碱按不同比例加入血清样本中:第1组样本中不添加甜菜碱,第2～5组样本中甜菜碱与样本的比例(V:V)分别为9:1、8:2、7.5:2.5、6:4,甜菜碱浓度为0.5～1.25 mol/L.所有血清...     

甜菜碱对非酒精性脂肪肝大鼠瘦素影响的研究

目的通过甜菜碱对非酒精性脂肪肝（NASH）大鼠瘦素的实验研究,探讨甜菜碱改善NASH的作用。方法采用高脂肪饲料加入诱导脂肪肝药物丙基硫氧嘧啶饲养方式制作NASH大鼠模型,高、低剂量组分别给予甜菜碱400mg/kg、200mg/kg灌胃,持续12周;实验结束后,腹主动脉取血,采用全自动生化仪测定Glu、ALT、AST、TG、TC,采用双抗体夹心法测定INS、LEP,并计算胰岛素抵抗指数（IRI）。结果与正常组比较,模型组大鼠TC、TG、ALT、AST、Glu、INS、IRI、LEP均明显升高（P〈0.01或P〈0.05）;与模型组比较,高剂量组TC、TG、ALT、AST、Glu、INS、IRI、LEP均明显下降（P〈0.01）,低剂量组ALT、AST、IRI、LEP也下降（P〈0.01或P〈0.05）,而TC、TG、Glu、INS有所下降,但差异无统计学意义。结论甜菜碱可降低血脂、血糖水平,可改善胰岛素抵抗,改善肝功能,作用机制可能与瘦素有关;甜菜碱改善NASH的作用机制也可能与瘦素有关。     

各国血友病登记管理工作的现状

血友病属X连锁隐性遗传性出血性疾病,在遗传性出血性疾病中最为常见.近年来,随着诊断治疗水平的提高和患者寿命的延长,其发病率有所上升.建立和完善血友病登记管理制度具有重要的社会意义和临床意义.本文对国内外血友病登记管理工作的进展及现状作一综述.     

应用脐血对血友病A进行产前诊断的研究

目的探讨应用脐带血对血友病A进行产前诊断的可行性。方法24例血友病A携带者妊娠20-24周时在超声引导下抽取脐血,用一期法检测孕妇外周血及胎儿脐血FVIII:C,用ELISA方法检测FVIII:CAg及vWF。结果24例女性携带者血浆FVIII:C,FVII1:CAg及vWF分别为36%～150%(平均值79%±22%),90%～132%(平均值102%±18%),92%～141%(平均值107%±16%);24例胎儿血浆FVIII:C,FVIII:CAg及vWF分别为0.9%～90%(平均41.7%±25%),60%～77%(平均69%±16%),58%～74%(平均66%±13%),其中19例胎儿FVIII:C高于34%,5例胎儿FVIII:C低于5%。结论脐静脉穿刺取血测定FVIII:C、FVIII:CAg及vWF是产前诊断HA的可行性手段。     

甜菜碱对金黄色葡萄球菌生物膜形成抑制与分散的作用

目的研究金黄色葡萄球菌生物膜形成及甜菜碱对其抑制与分散的作用。方法采用结晶紫染色法分别测定终浓度为1.0 g/L的甜菜碱对20株金黄色葡萄球菌生物膜形成的抑制能力和甜菜碱对金黄色葡萄球菌生物膜的分散能力。结果 20株金黄色葡萄球菌均能形成生物膜,其中甲氧西林敏感的金黄色葡萄球菌(MSSA)培养24 h时形成生物膜吸光度值(A_(590 nm))达峰值,为1.99±0.53;耐甲氧西林金黄色葡萄球菌(MRSA)培养48 h时形成生物膜A_(590 nm)达峰值,为1.13±0.47。加入甜菜碱共培养后,MSSA培养24 h后生物膜A_(590 nm)为1.74±0.61,MRSA培养48 h后生物膜A_(590 nm)为0.40±0.12,与对照组相比差异均有统计学意义(t值分别为2.43和5.84,P均0.05)。当生物膜形成后加入甜菜碱,与对照组相比,MSSA和MRSA的生物膜A_(590 nm)差异无统计学意义(P0.05)。结论 1.0 g/L甜菜碱对金黄色葡萄球菌生物膜的形成具有良好的抑制作用,但不能分散已经形成的生物膜。     

血友病A基因突变及其检测方法的进展

血友病 A是由于凝因子 基因缺陷所致的 X-连锁遗传性出血性疾病。 F 基因庞大结构复杂且基因突变异质性高 ,因此对血友病 A的产前诊断和携带者诊断较为困难。近来 ,随着对 F 基因结构研究的深入 ,发现了一些新的突变类型 ,同时突变检测方法也有了较大的发展。     

血友病儿童45例关节健康状况分析及其与步态的相关性研究

目的研究中国血友病儿童关节健康状况,及其对步态的影响。方法分别收集在北京协和医院、广州南方医院、温州市第三人民医院、北京儿童医院就诊的4～18岁的儿童共45例,使用中文版血友病关节健康评估表(HJHS)2.0版评价患者的双膝、双踝、双肘关节的健康状况及步态,对结果进行总结,并将其中的步态分析得分与各关节的总评分及各关节评分做相关性分析。结果 93.3%的患者有至少1个关节受累,91.0%的患者有2个及以上关节受累,84.4%的患者有肘关节受累,93.0%的患者有膝关节受累,93.3%的患者有踝关节受累;年长儿童关节受累更严重;步态异常率为88.9%。步态异常与膝关节、踝关节受累明显相关,与关节疼痛、肌力下降、肌肉萎缩及关节屈曲度和伸展度下降有显著相关,而与关节肿胀、关节肿胀持续时间、关节运动时摩擦音无明显相关性。结论中国血友病儿童关节受累常见,步态异常常见,膝关节与踝关节的受累,以及下肢关节疼痛、肌力下降、肌肉萎缩、关节屈曲度和伸展度下降与步态异常明显相关。     

高效液相色谱法测定人血清甜菜碱方法的建立

目的 采用柱前衍生高效液相色谱法-紫外检测法建立一种测定人血清甜菜碱浓度的方法.方法 以对溴苯乙酰溴和18-冠醚-6溶于乙腈制成衍生液,将其与甜菜碱反应形成的衍生物用Supelcosil LC-SCX色谱柱进行分离.流动相为乙腈∶水体积比90∶10,含16 mmol/L的氯化胆碱,等度洗脱,流速为0.8 ml/min,检测波长为259 nm,利用甜菜碱标准品绘制标准曲线,外标法定量检测20名健康大学生志愿者血清甜菜碱.结果 甜菜碱的测定线性范围为6.25～200.00 μmol/L,回归方程Y=1 568.1X-2 747.5,R2=0.999 8.最低检测限为3.0 μmol/L.批内不精密度为1.88%～3.79%(平均3.24%),批间不精密度为3.14%～6.76%(平均4.39%).方法 回收率为95.89%～102.86%(平均99.16%).结论 成功建立一种检测人血清甜菜碱浓度的方法,适用于实验室及临床常规检测.     

单细胞四重巢式荧光聚合酶链反应进行血友病A基因诊断和性别鉴定的研究

目的 建立快速有效检测血友病A基因诊断和性别鉴定的单细胞聚合酶链反应（PCR）方法。方法 联合6个与凝血因子Ⅷ（FⅧ）紧密连锁的STR位点（DXS15、DXS9901、G6PD、DXS1073、DXS1108和F8Civs13）通过多重荧光PCR进行正常人群多态性分析和血友病A家系基因诊断，从中选取杂合度和诊断率较高的STR位点联合性别位点牙釉蛋白基因（Amelogenin gene）建立单淋巴细胞多重巢式荧光PCR的检测方法。结果 选择了3个杂合度和诊断率较高的STR位点（F8Civs13、DXS1073和DXS15）联合性别位点建立了单细胞四重巢式荧光PCR的检测方法。Amelogenin、DXS15、F8Civs13和DXS1073在男性的单细胞扩增成功率分别为94．3％、91．4％、100％和100％，在女性为100％、97．1％、97．1％和97．1％。该女性的DXS1073和DXSl5位点呈杂合子，均未发现等位基因脱失现象。扩增皆未发现假阳性和假阴性的情况。结论 建立的单细胞四重巢式荧光PCR方法快速有效，有望应用于临床血友病A非创伤性产前基因诊断。     

Short and long-term variation of plasma glycine betaine concentrations in humans

BACKGROUND: Glycine betaine is important in cell volume regulation and in remethylating homocysteine, a vascular risk factor. OBJECTIVE: We investigated changes in circulating glycine betaine concentrations in human volunteers both under acute osmotic stress and over longer time scales. DESIGN: Plasma glycine betaine concentrations were measured in normal human volunteers in three studies: (1) during acute diuresis and antidiuresis; (2) during prolonged diuresis for 5 days, and antidiuresis for 5 days followed by further diuresis for the final 5 days; (3) repeated samples taken 3 years apart. RESULTS: Circulating glycine betaine concentrations remained almost unchanged for several hours after acute diuretic or antidiuretic stresses. There was more (3-10-fold) interindividual variation than intraindividual variation. A similar pattern was found on day 15 of the study. In a 3-year follow-up, plasma glycine betaine concentrations on the two occasions were highly correlated with no systematic change, showing that individual set points remain stable for years. In contrast, there was no relationship among plasma proline betaine concentrations at these times. Urinary glycine betaine excretions measured 3 years apart were also found to correlate once the perturbing effect of dietary proline betaine excretion was allowed for. CONCLUSIONS: Human circulating glycine betaine is homeostatically controlled with a distinct control value for each individual. In contrast, peripheral blood concentrations of proline betaine, which is present in the diet (and has no known metabolic or physiological role in mammals), is not controlled.     

甜菜碱对血清酶ALT活性的稳定作用及其基质效应的研究

目的 研究甜菜碱(betaine)在不同温度下对人血清中ALT酶活性的稳定作用,并评估添加甜菜碱的人血清在各检测系统中的基质效应.方法 血清中添加终浓度为1 mol/L的甜菜碱,检测37 ℃、25 ℃、4 ℃、-20 ℃下放置不同时间的血清中的ALT酶活力.根据美国临床和实验室标准协会(CLSI)EP14-A文件进行基质效应实验,以4个被评价检测系统为被比较系统,以罗氏公司试剂-日立仪器检测系统为比较系统,用比较系统和被评价系统检测40个患者血清样品以及添加了1 mol/L甜菜碱的3个血清(高、中、低浓度)样本的ALT酶活力;用直线回归方法统计数据,以回归直线的(-Y)估计值(^/Y)的95%可信区间估计这3个添加了甜菜碱的样品在4种检测系统中的基质效应.结果 37 ℃添加甜菜碱的血清48 h后ALT酶活力稳定为99%,对照血清降至42%;25 ℃添加甜菜碱的血清4 d后ALT酶活力稳定为98%,对照血清降至86%;4 ℃添加甜菜碱的血清4周后ALT酶活力稳定为97%,对照血清1周后已降至71%;-20 ℃添加甜菜碱的血清4个月后ALT酶活力稳定为99%,对照血清1个月后已降至79%.添加了甜菜碱的3个样本的值均在回归直线的(-Y)估计值(^/Y)的95%可信区间内,即其在4个被评价系统和比较系统中均无明显基质效应.结论 甜菜碱可作为一个较好的血清ALT酶活力的稳定剂用于血清酶ALT的校准品的研制中.

Abstract：

Objective To investigate the stabilization of betaine on the enzyme activity of ALT in human serum at different temperatures, and to evaluate the matrix effects of human serum with betaine among different analytical systems. Methods The enzyme activity of ALT in human serums with l mol/L betaine stored at 37 ℃, 25 ℃, 4 ℃ and - 20 ℃ respectively were measured. The matrix effect experiment was carried out according to the procedures specified in Clinical and Laboratory Standards Institute (CLSI)document EP14-A. Four analytical systems were the candidates for evaluation, and Roche Diagnostics (Roche)-Hitachi analytical system served as the reference system. The enzyme activity of ALT of 40 patient specimens and 3-level specimens ( high, medium and low) with 1 mol/L betaine were measured using the evaluated methods and the reference method respectively. The linear regression analysis was performed to compare the mean y value of 3-level specimens with the statistically defined limits ( the two-tailed 95% prediction interval) derived from 40 patient specimen data. Results The enzyme activity of ALT could be stabilized with betaine in serum, which remained to be 99% for 48 h at 37 ℃, 98% for4 d at 25 ℃, 97%for 4 weeks at 4 ℃, and 99% for 4 months at - 20 ℃. The enzyme activity of control serum decreased to 42%, 86%, 71%, 79% respectively. The mean y values of three specimens with the betaine were within the 95% prediction interval of the estimated mean y values, which suggested no obvious matrix effects in serum. Conclusion Betaine can be used in the development of enzyme ALT calibrator as its stabilizer.     

An abnormal urinary excretion of glycine betaine may persist for years

OBJECTIVES: Does abnormal betaine excretion persist? DESIGN AND METHODS: Patients (10) with abnormal excretion in 1998 were recalled in 2005. Subsequent urine samples were collected on 4 days from persistently abnormal subjects. RESULTS: Half the 1998 abnormal patients were abnormal in 2005. Only 1/20 controls was abnormal (p=0.015). All patients with abnormal excretion in 1998 and 2005 had abnormal excretion on successive days while no controls did. CONCLUSIONS: High betaine excretion may be chronic and a health risk.     

狼疮抗凝物伴凝血因子Ⅷ、Ⅸ和Ⅺ缺乏的检验诊断:附13例报告

目的 探讨狼疮抗凝物伴凝血因子(Ⅷ、Ⅸ和Ⅺ)缺乏的检验诊断方法.方法 检测13例狼疮抗凝物伴凝血因子(Ⅷ、、Ⅸ和Ⅺ)缺乏患者(包括长期服用氯丙嗪的精神分裂症5例、抗磷脂综合征2例、天疱疹1例、原因未明5例)、1O例血友病A患者和48名健康志愿者的凝血因子活性、凝血因子抗体、抗心磷脂抗体(ACA)、狼疮抗凝物(LA)以及凝血酶的生成.结果 13例狼疮抗凝物伴凝血因子(Ⅷ、Ⅸ和Ⅺ)缺乏患者的活化部分凝血活酶时间(APTT)延长,凝血因子Ⅷ、Ⅸ、Ⅺ活性(FⅧ∶C、FⅨ∶C、FⅪ∶C)降低,针对这些凝血因子的抗体及LA均为阳性,6例ACA为阳性.其中LA阳性、伴凝血因子(Ⅷ、Ⅸ和Ⅺ)缺乏组中,11例无出血患者的凝血酶生成试验(TGT)的延迟时间值为(5.60±1.18)min,高于正常对照组的(1.88±0.25)min,差异具有统计学意义(t=10.05,P＜0.01);达峰时间值为(8.11±1.91)min,高于正常对照组的(4.10±0.36)min,差异具有统计学意义(t=8.83,P＜0.01);凝血酶生成潜力(ETP)值为(1640.87±279.80)nmol·L-1·min-1,高于遗传性血友病A组的(734.59±118.30)nmol·L-1·min-1,差异具有统计学意义(t=9.77,P＜0.01);ETP比值为(86.00±14.66)%,高于遗传性血友病A组的(38.50±6.20)%,差异具有统计学意义(t=9.77,P＜0.01).遗传性血友病A组的达峰时问值为(8.01±1.81)min,高于正常对照组的(4.10±0.36)min,差异具有统计学意义(t=8.20,P＜0.01);而延迟时间值为(2.01±0.84)min,与正常对照组的(1.88±0.25)min比较,差异无统计学意义(t=1.26,P＞0.05).结论 狼疮抗凝物伴凝血因子(Ⅷ、Ⅸ和Ⅺ)缺乏和遗传性血友病是以APTT、PT、PLT和APTT纠正试验作为筛选试验;以LA、FⅧ∶C、FⅨ∶C、FⅪ∶C和它们的抗体检测作为确诊试验;检测LA和ACA可为寻找病因提供依据;ETP值和ETP比值是判断LA阳性伴凝血因子(Ⅷ、Ⅸ和Ⅺ)缺乏患者临床是否出血的较敏感指标.     

Simultaneous determination of betaine and N,N-dimethylglycine in urine

A method is described for the determination of betaine and its metabolite N,N-dimethylglycine in human urine. The method involves a deamination step and a solid-phase extraction to isolate both substances from urine followed by high performance liquid chromatography (HPLC) separation on a 5-μm SCX column with UV absorbance detection. Limit of detection is about 0.9 μg for both substances. Recoveries were > 85%. The method appears to be suitable for monitoring betaine and its metabolite in urine of patients undergoing therapy with betaine.     

同时存在血友病A及血友病B患者家系的基因诊断

目的:对1个同时存在血友病A(HA)及血友病B(HB)患者的家系进行分子发病机制研究,并对家系女性成员进行携带者检测。方法:采用活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)、纤维蛋白原(Fg)、凝血因子Ⅷ(FⅧ)活性(FⅧ:C)、FⅨ活性(FⅨ:C)及血管性血友病因子(vWF)等测定进行HA及HB表型诊断;用长链聚合酶链反应(LD-PCR)进行F8基因内含子22倒位检测,2组序列特异的PCR对F8基因内含子1倒位进行检测;采用直接核苷酸测序法对F9基因各外显子及其侧翼序列进行检测,联合F9基因相关的6个微卫星位点(DXS8094、DXS1211、DXS1192、DXS102、DXS1227及DXS8013)进行家系遗传连锁分析。结果:先证者1是由F8基因内含子22倒位引起的HA。先证者2是由F9基因突变nt32772A→T突变(p.Ser365Cys,GI:22385320)引起的HB,其突变基因遗传自其母亲,来源于先证者2外公的X染色体,其外公为该突变的体细胞和生殖细胞嵌合体,其阿姨口腔细胞DNA未检出该突变。结论:该家系2名先证者的基因诊断结果与表型诊断相符。单碱基延伸法可用于突变嵌合率的检测,并为血友病散发家系的突变来源提供可靠的信息。     

Diagnosis of hemophilia a in a female subject by using restriction fragment length polymorphisms linked to the factor VIII gene

Summary A 6-year-old girl, daughter of a male patient with moderate hemophilia A (factor VIII 3%), was referred to our Center because she also had very low levels of factor VIII (4%). The proband’s brother has mild hemophilia A (7%); the mother (29%) is a possible carrier, no other case of hemophilia A being reported in her family. Hence, the factor VIII deficiency found in the girl is consistent either with a carrier state with extreme lyonization in favour of the hemophilic gene or with homozygosity for the hemophilia gene. To distinguish these possibilities, we studied the segregation of three restriction fragment length polymorphisms (RFLPs) linked to the factor VIII gene in the 4 members of the family. Employing one intragenic (FVIII-BclI) and two extragenic (St14-TaqI and Dx13-Bg1II) RFLPs, we showed that the proband has inherited from both parents the defective gene, being therefore homozygous for the hemophilia gene.