多重巢式RT-PCR技术在急性髓系白血病中的应用

Objective To analyse the fusion genes derived from chromosome structural aberrations in acute myeloid leukemia(AML) and the relationship between fusion genes and the MICM classification, clinical diagnosis, chemotherapy and prognosis. Methods The expression of fusion gene in bone marrow samples was detected with multiplex RT-PCR technique and chromosome karyotypes, immunological phenotypes and clinical data were analyzed in 60 acute myeloid leukemia newly diagnosed. Results 37 cases(61.67 %) of 60 patients carried 5 kinds of fusion genes consisting of MLL-AF9, TLS-ERG, CBFβ-MYH1, AML1-ETO and PML-RARα. The activation of oncogene HOX11 was detected in 13 AML cases, three of them with other chromosome aberration simultaneously.23 cases of 31 patients carrying AML1-ETO or PML-RARα, reached complete remission(CR) after chemotherapy and without relapse. Conclusion Gene typing is the most precise classification method that can direct clinical treatment and evaluate prognosis. Multiplex RT-PCR technique, which can quickly screen 29 kinds of fusion gene derived from chromosome structural aberrations at one time, maybe helpful to improve M1CM classification and guide the choice of treatment.     

环孢素A拮抗急性髓细胞白血病细胞P-糖蛋白功能的实验研究

自从发现盐酸维拉帕米的多药耐药 (MDR)逆转效应以来,许多P 糖蛋白(P gp)功能调节剂被确立,包括几种钙离子拮抗剂、奎宁复合物、FK506、环孢素A(CsA)及其类似物PSC833等。CsA和PSC833联合化疗剂已应用于急性髓细胞白血病(AML)的临床治疗 [1]。本研究即通过体外测定加与不加     

吡格列酮对体外培养大鼠破骨细胞样细胞分化及活性的影响

目的 观察吡格列酮对大鼠破骨细胞样细胞(OLC)分化及活性的影响,探讨PPARγ2活化与破骨细胞之间的关系.方法 用NF-кB受体活化因子配体(RANKL)及巨噬细胞集落刺激因子(M-CSF)协同诱导大鼠骨髓单个核细胞(BMC)向OLC分化,同时加入1、5、10μmol/L盐酸吡格列酮干预.应用RT-PCR分析OLC分化过程中PPARγ2及NF-кB受体活化因子(RANK)mRNA的表达,结合细胞染色及抗酒石酸酸性磷酸酶(TRAP)活性观察吡格列酮对OLC分化的影响,通过骨吸收陷窝分析及整合素β3(CD61)平均荧光强度的检测比较吡格列酮对OLC活性的影响.结果 (1)对OLC分化的影响:培养7 d时10μmol/L吡格列酮组与其它组相比OLC数量及TRAP活性明显减少(P<0.01或P<0.05),表明吡格列酮部分抑制OLC分化且此作用与剂量相关;RT-PCR结果显示在诱导分化早期,不同浓度吡格列酮呈剂量依赖性上调PPARγ2的表达水平,但随OLC的成熟其表达渐减弱,而RANK表达在5及10 p,mol/L吡格列酮干预组培养的各时间点均明显下调,与对照及1μmol/L干预组相比有显著差异(P<0.05或P<0.01);(2)对OLC活性的影响:骨吸收陷窝数量及吸收面积在吡格列酮5、10μmol/L组明显减少,与另两组相比差异显著(P<0.05或P<0.01),培养早期这两组细胞CD61平均荧光强度均明显下调(P<0.05或P<0.01).结论 吡格列酮激活PPAR田γ2转录活性可部分抑制大鼠骨髓OLC的分化及活性.     

忻州地区耳聋患者常见耳聋基因GJB2、GJB3和线粒体DNA 12S rRNA 1555A〉G相关性分析

目的通过对忻州地区83例耳聋患者常见基因GJB2、GJB3和线粒体DNA 12S rRNA 1555A〉G测序分析,从分子水平研究该地区人群聋病的遗传病因和特点,为临床防聋治聋提供策略、依据。方法收集忻州地区83例耳聋患者外周血样本,提取DNA后对目的基因扩增并进行测序分析。结果83例耳聋患者中GJB2基因检测到57例发生突变,9个突变位点,与编码连接蛋白的非综合症耳聋突变数据库（http：／／davinci．crg．OS／deafneSS／index．php？Seccion=mut_db＆db=nonsynd）比对,8个位点已见报道,其中包括3个多态位点c．79G〉A、c．341A〉G、c．368C〉A和5个致病位点c．235delC、c．30-35delC、c．109G〉A、c．176-c．191dell6和c．299-c．300delAT,其中,c．79G〉A和c．341A〉G是主要突变方式,携带率为30．12％（42／174）和23．49％（39／166）。新发现1例未见报道的突变位点c．186C〉T;患者均未检测出GJB3和线粒体DNA12SrRNA1555A〉G基因突变位点。结论通过对忻州地区常见耳聋基因突变位点的研究,了解忻州地区该基因突变谱,为后续国内耳聋基因型分布提供数据支持,同时也为耳聋的早期诊断、治疗提供理论依据。     

多重巢式RT-PCR技术在急性髓系白血病中的应用

Objective To analyse the fusion genes derived from chromosome structural aberrations in acute myeloid leukemia(AML) and the relationship between fusion genes and the MICM classification, clinical diagnosis, chemotherapy and prognosis. Methods The expression of fusion gene in bone marrow samples was detected with multiplex RT-PCR technique and chromosome karyotypes, immunological phenotypes and clinical data were analyzed in 60 acute myeloid leukemia newly diagnosed. Results 37 cases(61.67 %) of 60 patients carried 5 kinds of fusion genes consisting of MLL-AF9, TLS-ERG, CBFβ-MYH1, AML1-ETO and PML-RARα. The activation of oncogene HOX11 was detected in 13 AML cases, three of them with other chromosome aberration simultaneously.23 cases of 31 patients carrying AML1-ETO or PML-RARα, reached complete remission(CR) after chemotherapy and without relapse. Conclusion Gene typing is the most precise classification method that can direct clinical treatment and evaluate prognosis. Multiplex RT-PCR technique, which can quickly screen 29 kinds of fusion gene derived from chromosome structural aberrations at one time, maybe helpful to improve M1CM classification and guide the choice of treatment.     

多重巢式RT-PCR技术在急性髓系白血病中的应用

Objective To analyse the fusion genes derived from chromosome structural aberrations in acute myeloid leukemia(AML) and the relationship between fusion genes and the MICM classification, clinical diagnosis, chemotherapy and prognosis. Methods The expression of fusion gene in bone marrow samples was detected with multiplex RT-PCR technique and chromosome karyotypes, immunological phenotypes and clinical data were analyzed in 60 acute myeloid leukemia newly diagnosed. Results 37 cases(61.67 %) of 60 patients carried 5 kinds of fusion genes consisting of MLL-AF9, TLS-ERG, CBFβ-MYH1, AML1-ETO and PML-RARα. The activation of oncogene HOX11 was detected in 13 AML cases, three of them with other chromosome aberration simultaneously.23 cases of 31 patients carrying AML1-ETO or PML-RARα, reached complete remission(CR) after chemotherapy and without relapse. Conclusion Gene typing is the most precise classification method that can direct clinical treatment and evaluate prognosis. Multiplex RT-PCR technique, which can quickly screen 29 kinds of fusion gene derived from chromosome structural aberrations at one time, maybe helpful to improve M1CM classification and guide the choice of treatment.     

80例儿童急性淋巴细胞白血病的染色体分析

目的与方法本文对80例儿童急性淋巴细胞白血病（急淋）进行了细胞遗传学研究。结果发现正常核型为50%,染色体数量异常占40%,结构异常的染色体包括t（9;22）（q34;q11）,（8;14）（q24,q32）,6q占10%等。结论探讨认为细胞形态学,免疫表型和细胞遗传学的联合分析（MIC）有助于儿童急淋的诊断和分型,尤其细胞遗传学对于儿童急性淋巴细胞白血病的诊断和预后判断具有重要意义。     

白血病患者线粒体基因组D-loop区基因不稳定性研究

目的:探讨白血病患者线粒体基因组D-loop区基因的不稳定性。方法:采用PCR扩增24例患者D-loop区HV-1和HV-2两个高变区并测序,其结果与剑桥标准序列(rCRS)和mtDB数据库进行比对,分析其突变情况。采用SPSS 22.0统计学软件对数据进行分析。结果:23例患者D-loop区发生突变,突变率95.83%(23/24);共检测到82个基因突变,其中HV-1区47个(57.32%),HV-2区35个(42.68%)。急性髓系白血病患者中未治疗组(UT)与治疗组(T)对比:突变率分别为(2.37±0.82)×10~(-3)和(4.76±2.45)×10~(-3),其差异有统计学意义(P0.01)。急性髓系白血病治疗组中部分缓解组(PR)和完全缓解组(CR)突变率比较显示,突变率分别为(5.10±2.56)×10~(-3)和(4.51±2.51)×10~(-3),其差异无统计学意义(P0.05)。M3组间比较显示,未治疗组(UT)、部分缓解组(PR)和完全缓解组(CR)突变率分别为(2.55±0.63)×10~(-3),(5.37±3.41)×10~(-3)和(3.71±1.65)×10~(-3),其差异无统计学意义(P0.05)。结论:D-loop区中存在较高的突变率和多种突变类型,具有较强的不稳定性;化疗可能加剧D-loop区基因的不稳定性。     

bcr-abl基因真核表达载体的构建及其在COS-7细胞中的表达

目的 构建慢性粒细胞白血病bcr-abl融合基因的真核表达载体,并在哺乳动物细胞COS-7中高效表达.方法 提取慢性粒细胞白血病K562细胞的RNA,经RT-PCR技术扩增得到bcr-abl基因后,将其克隆入真核表达载体pEGFP-N3中,并进行PCR和酶切鉴定.鉴定为阳性的克隆,再进一步进行测序分析.将构建成功的真核表达载体转入COS-7细胞中进行瞬时表达.结果 反转录PCR扩增后的产物,经琼脂糖凝胶电泳可以在约874 bp处看见一特异性的条带,序列分析证明扩增产物与GeneBank中的bcr-abl基因序列相同.将pEGFP-bcr-abl真核表达载体转入COS-7细胞后,经RT-PCR、Western blotting检测证明表达产物正确.结论 成功构建了含有bcr-abl融合基因的真核表达质粒,从而为进一步研究其结构和功能,寻找CML诊疗的新方法奠定了基础.