应用RT/PCR技术检测急性粒细胞白血病M2型AML1/ETO融合基因转录本的研究

目的：搪塞检测ＡＭＬ１－ＥＴＯ融合转录本在ｔ（８;２１）急性粒细胞白血病Ｍ２型「Ｍ２／ｔ（８;２１」的临床诊断和预后判断中的意义。方法：用半筑巢式逆录聚合酶链反应（ＲＴ／ＰＣＲ）技术检测ＡＭＬ１／ＥＴＯ融合基因转录本,应用ＳｏｕｔｈｅｒｎＢｌｏｔ技术及限制性内切酶技术检测ＰＣＲ产物的特生。结果：在１９例ＡＭ加共检出１０例存在ＡＭＬ１／ＥＴＯ融合基因转录本,阳性率为５２．６％,２例ＭＤＳ中１例为阳性     

文摘

文摘００１采用聚合酶链反应在ｔ（８；２１）转录ＡＭＬＩ／ＥＴＯ融合的检测〔英〕／ＫｗｏｎｇＹ…Ｃａｎｃｅｒ．－１９９５，７５（３）．－８２１～８２３急性粒细胞白血病（ＡＭＬ）和。开够增殖症（ＭＤＳ）的八８１ＺＩ）（ｑ２２；ｑ２２），其结果导致２１ｑ２...     

中国版纳猪MHCI类P1分子全长的原核表达与纯化

目的：获得原核表达的中国版纳猪ＳＬＡＩ类Ｐ１蛋白质分子。方法：ＰＣＲ扩增去信号肽的ＳＬＡＩ类Ｐ１ｃＤＮＡ序列，亚克隆至ｐＧＥＭＴ载体，测序。将亚克隆的Ｐ１　ｃＤＮＡ片段插入表达载体ｐＥＴ４２ｂ（＋），构建重组表达质粒ｐＥＴ－４２ｂ（＋）／ｓｌａ－ｐｌ，转化Ｅ·ｃｏｌｉ表达菌 ＢＬ２１－ＣｏｄｏｎＰｌｕｓ（ＤＥ３）－ＲＩＬ，ＩＰＴＧ诱导 Ｐ１－８ ｘ ｈｉｓ融合蛋白表达，经包涵体洗涤，８ ｍｏｌ／Ｌ尿素变性溶解，Ｎｉ２＋亲和层析，梯度透析后，定量保存。ＳＤＳ－ＰＡＧＥ、ｗｅｓｔｅｒｎ－ｂｌｏｔｔｉｎｇ鉴定目的蛋白的表达与纯化。结果：目的蛋白（分子量３９．５ ｋＤ）表达量占细菌总蛋白 １５％，每升表达菌获得纯度９５％的目的蛋白 ４０ ｍｇ～６０ｍｇ。结论：成功建立猪 ＳＬＡ分子全长原核表达、纯化体系，为建立间接识别猪移植抗原ＳＬＡＩ类分子的人Ｔ细胞系及表位分析打下基础。     

HBsAg PreS1肽段的基因工程表达与分离纯化

目的 用基因工程的方法表达和纯化ＨＢｓＡｇ－ＰｒｅＳ１（１～６５）太。方法 以ＨＢＶ全基因为模板,ＰＣＲ扩增ＰｒｅＳ１（１～６５）基因,导入谷胱甘肽巯基转移酶融合表达载体ｐＧＥＸ ４Ｔ－１,构建ｔａｃ启动子控制下的ＧＳＴ－ＰｒｅＳ１（１～６５）融合蛋白表达质粒,转化大肠杆菌ＢＬ２１宿主。结果 工程菌ＢＬ２１ ＰｒｅＳ１（１～６５）产物为可溶性蛋白,表达量约为菌体总蛋白的２０％。用ＮＢＳ－ＢＩＯＦＬ     

BIOLOGIC HEMOSTATIC GLUE IN GENERAL SURGERY

ＢＩＯＬＯＧＩＣＨＥＭＯＳＴＡＴＩＣＧＬＵＥＩＮＧＥＮＥＲＡＬＳＵＲＧＥＲＹＢＩＯＬＯＧＩＣＨＥＭＯＳＴＡＴＩＣＧＬＵＥＩＮＧＥＮＥＲＡＬＳＵＲＧＥＲＹＨｕＹｉ－ｚｅ（ＤｅｐａｒｔｍｅｎｔｏｆＳｕｒｇｅｒｙ，ＳｅｃｏｎｄＡｆｆｉｌｉａｔｅｄＨｏｓｐｉ...     

人MBL相关丝氨酸蛋白酶-1 cDNA的克隆与鉴定

目的 获得人甘露聚糖结合凝集素（ＭＢＬ）相关丝氨酸蛋白酶－１（ＭＡＳＰ－１）基因。方法 以人胎肝组织总ＲＮＡ为模板,采用ＲＴ－ＰＣＲ获取目的ｃＤＮＡ片段,克隆入ｐＧＥＭ－Ｔ载体,进行酶切图谱分析和测序鉴定。结果 以ＲＴ－ＰＣＲ方法获得了含信号顺序的全长ＭＡＳＰ－１ｃＤＮＡ,将其与ｐＧＥＭ－Ｔ载体连接,转化大肠杆菌ＴＧ１,建立了ＭＡＳＰ－１的ｃＤＮＡ克隆,酶切图谱与微机分析结果一致。序列分析表明。与     

拮抗内皮素生物学效应对甘油所致大鼠急性肾功能衰竭的影响

目的和方法：在甘油致大鼠急性肾功能衰竭（ＡＲＦ）模型上,观察外源内皮素（ＥＴ）以及心房钠尿肽（ＡＮＰ）、硝苯吡碇（Ｎｉｆ）和ＮＯ前体Ｌ－精氨酸（Ｌ－Ａｒｇ）的作用。结果：甘油致大鼠ＡＲＦ后２４ｈ,肾功能明显受损,血清尿素氮（ＳＵＮ）,肌酐（Ｓｃｒ）,丙二醛（ＭＤＡ）,肾皮质钙含量及血浆内皮素（ＰＥＴ）水平均明显高于正常对照组（Ｐ＜０００１）。外源性ＥＴ可加重ＡＲＦ大鼠肾损伤,使ＳＵＮ、Ｓｃｒ、ＭＤＡ、ＰＥＴ及肾皮质钙含量显著增加,而ＡＮＰ、Ｎｉｆ和Ｌ－Ａｒｇ则使ＡＲＦ大鼠ＳＵＮ、Ｓｃｒ、ＭＤＡ、ＰＥＴ及肾皮质钙含量明显降低,肾组织ｃＧＭＰ含量增加,减轻肾小管损伤。结论：ＥＴ对ＡＲＦ的发生发展具有重要意义,ＡＮＰ、Ｎｉｆ和Ｌ－Ａｒｇ可通过不同途径拮抗ＥＴ的生物学效应,从而对ＡＲＦ的防治起到积极的作用。     

DNA修复蛋白MGMT基因的克隆及转导脐血CD23^＋细胞后耐药特征的研究

目的：增强脐血ＣＤ３４＾＋造血细胞对化疗药物的耐药表型,探讨逆转录病毒介导的基因转移效率和耐药基因特性,以及在脐血造血干细胞保护性基因治疗中的作用和意义。方法：应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）从人肝细胞中获得编码六氧甲基鸟嘌呤－ＤＮＡ－甲基转移酶（Ｏ＾６－ｍｅｔｈｙｌｇｕａｎｉｎｅ－ＤＮＡ－ｍｅｔｈｙｌｔｒａｎｓｆｅｒａｓｅ,ＭＧＭＴ）ｃＤＮＡ;利用基因重组技术,将其克隆于ｐＧＥＭ－Ｔ质粒载体并构建了逆转录病毒载体Ｇ１Ｎａ－ＭＧＭＴ;应用脂质体ＬｉｐｏｆｅｃｔＡＭＩＮＥ基因转移法将后者导入ＧＰ＋Ｅ８６和ＰＡ３１７病毒包装细胞,以卡氮芥１,３－Ｂｉｓ（２－Ｃｈｌｏｒｏｅｔｈｙｌ）－１－Ｎｉｔｒｏｓｏｕｒｅａ（ＢＣＮＵ）加压筛选后的阳性克隆上清经乒乓效应后继而感染脐血ＣＤ３４＾＋细胞。应用ＰＣＲ,Ｓｏｕｔｈ     

应用YAC探针进行荧光原位杂交检测慢性粒细胞白血病BCR基因重排

为检测慢性粒细胞白血病（ＣＭＬ）中ＢＣＲ基因重排，采用酵母人工染色体（ＹＡＣ）ＤＮＡ的Ｉｎ－ｔｅｒ－Ａｌｕ－ＰＣＲ产物为探针进行荧光原位杂交（ＦＩＳＨ）研究了１０例ＣＭＬ，其中包括初诊患者２例，ＣＭＬ急变并接受化疗２例，α干扰素治疗２例，自体骨髓移植术（ＡＢＭＴ）后３例和Ｐｈ染色体阴性ＣＭＬ１例。同时进行细胞遗传学和ＲＴ－ＰＣＲ检测。结果：９例ＣＭＬ的４６％～１００％的可分析核型显示ｔ（９；２２）易位，其中携带ｔ（９；２２）细胞最少者为１例自体骨髓移植术后８个月的患者，其６４％的分裂相存在ｔ（９；２２），３６％为正常核型；１例Ｐｈ（－）ＣＭＬ未见ＢＣＲ基因易位，而ＲＴ－ＰＣＲ（＋），提示ＡＢＬ基因片段插入２２ｑ１１，造成隐匿性Ｐｈ染色体。结果表明：应用ＹＡＣ探针进行原位杂交的定位明确。ＦＩＳＨ检测微小残留病（ＭＲＤ）比常规细胞遗传学方法更敏感，而且可以完成ＰＣＲ方法不易进行的定量分析。     

STUDY ON SOLUBILITY OF APATITE CERAMIES IN VITRO

ＳＴＵＤＹＯＮＳＯＬＵＢＩＬＩＴＹＯＦＡＰＡＴＩＴＥＣＥＲＡＭＩＥＳＩＮＶＩＴＲＯＳＴＵＤＹＯＮＳＯＬＵＢＩＬＩＴＹＯＦＡＰＡＴＩＴＥＣＥＲＡＭＩＥＳＩＮＶＩＴＲＯＬ．ＮｉｎｇＭ．Ｘｕｅ（ＳｈａｎｇｈａｉＳｅｃｏｎｄＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ...     

Cryptic chromosomal aberrations leading to an AML1/ETO rearrangement are frequently caused by small insertions

The translocation t(8;21)(q22;q22), which results in the fusion of the AML1 (RUNX1) and ETO (CBFA2T1) genes, is a recurrent aberration in acute myeloid leukemia (AML), preferentially correlated with FAB M2, and has the highest incidence in childhood AML. Because of the favorable prognosis, the evidence of the t(8;21) or the AML1/ETO fusion gene is mandatory in most of the therapy trials, allowing the stratification of the patients to the correct risk group in terms of treatment. Here we present six out of 59 children with AML who were positive for AML1/ETO by RT-PCR, but showed no evidence of the classical t(8;21)(q22;q22) by conventional cytogenetics. Because of the discrepancies between molecular and cytogenetic analyses, these six patients were further investigated by fluorescence in situ hybridization analysis. Small hidden interstitial insertions resulting in an AML1/ETO rearrangement were detected in five (8.5%) of the 59 patients, whereas the sixth patient showed a cryptic three-way translocation. The insertions could be characterized as ins(21;8) in three patients and ins(8;21) in the remaining two. Additionally, three of the patients showed secondary chromosome aberrations leading to a higher complexity of the karyotype. In conclusion, the combination of more than one standard technique in the analysis of AML1/ETO is useful to reveal the overall frequency of cryptic chromosome rearrangements and permits a better understanding of the mechanisms involved in the generation of this fusion gene.     

t(8;21)急性髓细胞白血病发病机制的研究进展

t(8;21)(q22,q22)是急性髓细胞白血病最常见的染色体易位，易位形成的AML1/ETO融合基因在白血病中起重要作用。AML1/ETO可抑制AML1靶基因的活化，也可通过多种途径影响多种转录因子，干扰细胞正常的增殖与分化而致细胞转化。     

Identification of ins(8;21) with AML1/ETO fusion in acute myelogenous leukemia M2 by molecular cytogenetics

A high percentage of cases of acute myelogenous leukemia (AML) of the M2 subtype show a rearrangement between the AML1 and ETO genes. The detection of the AML1/ETO fusion has clinical relevance because patients with this subtype have a good prognosis. We present the results of conventional and molecular cytogenetic studies in a patient with acute myelogenous leukemia French-American-British M2 classification, who had a complex karyotype involving chromosomes 8 and 21. Dual-color fluorescence in situ hybridization (FISH) using the AML1/ETO probe demonstrated a recombination of both genes on an add(8) chromosome. The use of other FISH probes (CEP8, c-myc and TEL21) and spectral karyotyping indicated that AML1/ETO fusion occurred as a consequence of a previously undescribed ins(8;21)(q22;q22.1q22.3).     

Molecular analysis of 12 patients with the t(8;21) translocation and M2 acute myelogenous leukemia.

The t(8;21)(q22;q22) is a nonrandom cytogenetic abnormality associated with acute myelogenous leukemia of the M2 subtype (FAB classification). The 8q- and 21q+ derivative chromosomes have previously been isolated in somatic cell hybrids and used to map the anonymous sequences D21S65 and D21S17, which were proximal and distal, respectively, to the breakpoint on chromosome 21. DNA from a series of 12 t(8;21) patients and 7 controls was analyzed by pulsed field gel electrophoresis. Physical linkage of probes D21S65 and D21S17 on a 2100 kb NruI fragment was established by partial digestion experiments. In all the patients, the translocation generated a rearranged D21S65 NruI fragment of 650 to 750 kb, suggesting heterogeneity in the breakpoints. This heterogeneity was confirmed by using BssHII, SacII, and EagI enzymes. Our results are consistent with the presence of a 100 Kb breakpoint cluster region on chromosome 21 encompassing the AML1 gene. Interestingly, in half of the patients, demethylation of an NruI site located 7 kb proximal to the last exon of the AML1 gene occurred on the nontranslocated chromosome 21.     

t(8;21)儿童急性髓细胞性白血病临床和生物学特征

目的了解儿童t(8;21)急性髓细胞白血病(acute myeloid leukemia, AML)的临床和生物学特征.方法对41例儿童t(8;21)AML作了回顾性分析,取同期诊治的19例t(8;21)阴性AML作为对照组.分析临床、形态学、染色体、免疫表型和分子生物学等资料.结果本组t(8;21)AML占同期连续的60例儿童急性AML的68.3%,其中典型易位29例、变异易位2例、单纯8q-各2例、t(8;21)为特征的近四倍体2例和隐匿易位6例.37例(80.4%)为M2型AML,大多有下述形态学改变白血病细胞有核凹陷、近核浅染区、胞浆嗜碱性、伴有成熟分化和核浆发育不平衡等;有CD13高表达抗原;绘逆转录-聚合酶链反应检测的23例均检出AML1/ETO融合基因转录本,包括正常核型的6例;t(8;21) AML与对照组相比,完全缓解率差异无显著性(82.4% vs 75%,P>0.05),但复发率的差异有显著性(10.7% vs 41.7%,P<0.05).结论 t(8;21) AML是儿童AML中最常见的类型,主要和M2型有关,具有独特的形态学、免疫学和临床特征.