荧光定量PCR检测急性白血病患者外周血 WT1基因表达及其临床意义

为研究WT1基因表达与临床疗效和预后的关系,探讨其在白血病微小残留病检测中的作用,用荧光定量RT-PCR方法检测55例初发白血病患者外周血及10例正常人外周血的WT1基因表达,跟踪20例急性白血病患者外周血WT1基因表达。结果显示,白血病初治组(40例ANLL、15例ALL)与正常人外周血WT1基因表达有显著差异(P<0.001)。在急性白血病患者中,WT1≤6.8×10-3组生存期长于WT1>6.8×10-3组(P=0.027);白血病患者初发时WT1呈高度表达,完全缓解后,迅速或缓慢下降至少1个对数级,复发时再次增高。跟踪检测20例急性白血病患者外周血WT1基因表达,结果7例复发,5例在临床复发前2-3月WT1的水平明显增高,至少上升0.8个对数级。结论:荧光定量RT-PCR方法检测白血病外周血WT1表达具有简便易行、准确性高、特异性好的特点。与正常人外周血比较,WT1在各类白血病外周血中呈高度表达,且表达水平与预后负相关;对外周血WT1基因表达进行定量分析,可用于微小残留病的监测。     

正常人骨髓不同细胞群中WT1基因表达及其异构体比例研究

本研究探讨WT1基因在正常人骨髓不同分化阶段造血细胞中的表达程度及异构体间比例关系。采用实时荧光定量RT-PCR方法检测18例正常人骨髓标本中CD34^＋CD38^-、CD34^＋CD38^＋、CD15^＋CD11b^＋、CD33＋CD14^＋、CD20^＋CD5^-和CD20^-CD5^＋细胞群中总WT1、WT1（＋17AA）及WT1（＋KTS）的表达。结果表明：与K562细胞相比,CD34^＋CD38^-、CD34^＋CD38^＋、CD15^＋CD11b^＋和CD33^＋CD14^＋细胞群中均存在WT1基因的低表达,且依次逐渐降低;在CD20^＋CD5^-和CD20^-CD5^＋细胞群中未检测到WT1基因表达。在分化程度较低的CD34^＋CD38^-和CD34^＋CD38^＋细胞群中以＋17AA,＋KTS及WT1（＋/＋）异构体为主,而较成熟的CD15^＋CD11b^＋和CD33＋CD14^＋细胞群中以-17AA、-KTS及WT1（-/-）异构体为主。结论：在正常人骨髓细胞中WT1基因的表达随着细胞分化程度提高而降低,造血细胞可能通过其4种异构体比例的调整而发挥抑制或促进分化的作用。     

Coffee consumption and risk of pancreatic cancer: a systematic review and dose–response meta-analysis

The association between coffee consumption and pancreatic cancer risk has been extensively studied; however, there is no consistent conclusion. Therefore, this meta-analysis study sought to evaluate dose–response relationship between them. A search was conducted using the PubMed and Web of Science databases. Thirteen high-quality cohort studies were identified, involving in 959,992 study participants and 3831 pancreatic cancer cases. Comparing the highest with lowest categories of coffee intake, the pooled relative risk (RR) was 1.08 (95% CI 0.94–1.25). For dose–response analysis, no evidence of a nonlinear dose–response association between coffee consumption and pancreatic cancer (p for nonlinearity =0.171) was found. The risk of pancreatic cancer was increased by 5.87% (RR =1.06, 95% CI 1.05–1.07) with the increment of one cup/day. Coffee consumption was identified to be related with the increasing risk of pancreatic cancer in a dose–response manner. Nevertheless, further mechanistic studies are needed to clarify the concerned issues.     

力达霉素抑制人纤维肉瘤肺转移的裸鼠活体成像观察

观察力达霉素 (LDM)、LDM与甲氨蝶呤 (MTX) 联合用药对人纤维肉瘤HT-1080^LUC实验性肺转移的抑制作用。构建稳定表达荧光素酶的HT-1080细胞株并扩增培养, 检测HT-1080^LUC细胞的荧光素酶的表达。建立裸鼠人纤维肉瘤HT-1080^LUC实验性肺转移模型, 并采用活体动物成像系统监测肿瘤的生长情况, 观察LDM和MTX的体内抗肺转移瘤活性。结果表明, HT-1080^LUC细胞荧光光子量与细胞数呈线性相关, 最小可检测的细胞数量为100个/孔。活体成像结果显示, 治疗组裸鼠的肺部荧光强度较对照组明显减弱。单独给予LDM 0.025 mg·kg⁻¹、LDM 0.05 mg·kg⁻¹或MTX 0.5 mg·kg⁻¹的肺转移抑制率分别为53.9%、75.9%和70.2%; LDM 0.025 mg·kg⁻¹与MTX 0.5 mg·kg⁻¹联合应用的肺转移抑制率为88.7%, 两药相互作用指数CDI = 0.82。研究表明, LDM对人纤维肉瘤肺转移有明显抑制作用, 与MTX联合用药对肺转移瘤的疗效明显优于单独给药。     

p38丝裂原激活蛋白激酶基因重组慢病毒载体的构建及其在建立人前列腺癌稳定细胞株中的应用

目的构建p38丝裂原激活蛋白激酶基因(MAPK)重组慢病毒载体并建立表达外源性p38基因的人前列腺癌稳定细胞株。方法采用限制性内切酶酶切、T4 DNA连接酶连接等方法,将EGFP/p38融合基因插入慢病毒载体pTYF-EF1α-IRES-EGFP中,构建启动子EF1α调控的EGFP/p38共表达慢病毒载体pTYF-EF1α-EGFP/p38,经酶切鉴定后,利用慢病毒三质粒系统,通过脂质体法转染包装细胞HEK 293T细胞,收集病毒上清后转导人前列腺癌DU145细胞株,经有限稀释法筛选重组EGFP/p38稳定细胞株,用Western blotting检测细胞总p38的表达,用计数法绘制细胞的生长曲线。结果经酶切鉴定,成功构建EGFP/p38重组慢病毒载体,并包装慢病毒,检测病毒悬液的滴度为4.7×106 TU/ml,利用此病毒悬液感染DU145细胞,成功筛选到EGFP/p38稳定表达细胞株,Western blotting显示该细胞株能稳定表达外源性EGFP/p38融合蛋白,这种过表达p38的细胞株的生长速度明显低于对照组。结论成功构建p38 MAPK重组慢病毒载体并建立了表达外源性p38 MAPK基因的稳定细胞株EGFP/p38-DU145,细胞内p38过表达能够抑制DU145细胞的增殖。     

红细胞意外抗体鉴定在输血安全中的重要作用

目的检测分析患者血清中红细胞意外抗体的特异性和分布。方法使用盐水介质法、聚乙二醇间接抗球蛋白法和微柱凝胶法对患者血清进行意外抗体特异性鉴定。结果 149例患者标本中,57例仅存在自身抗体,88例仅存在同种抗体,4例同时存在自身抗体和同种抗体。同种抗体包括5个血型系统,分别为Rh系统（77.1%）、MNS系统（16.3%）、Lewis系统（3.3%）、P系统（2.2%）和Duf-fy系统（1.1%）。Rh系统抗体分布为：抗-E（39.4%）、抗-cE（29.7%）、抗-D（12.7%）、抗-Ce（5.6%）、抗-DC（4.2%）、抗-C（4.2%）、抗-e（2.8%）和抗-c（1.4%）。结论意外抗体的鉴定有助于患者获得相容性血液,保障临床输血安全有效。     

Fabrication and caffeine release from Fe3O4/P(MAA-co-NVP) magnetic microspheres with controllable core-shell architecture

A novel route was proposed to design and construct a magnetic composite microsphere consisting of Fe(3)O(4) nanoparticles chemically-covalently encapsulated with pH-smart poly(methacrylic acid-co-N-vinyl pyrrolidone) (P(MAA-co-NVP)) cross-linked co-polymers by a surface-initiated radical dispersion polymerization route. The multistep surface treatment was employed to improve the dispersity and surface-chemical reactivity of Fe(3)O(4) nanoparticles, involving introduction of active -NH(2) groups, coupling of 1,1-methylene bis-(4-isocyanato-cyclohexane) and immobilization of 2,2'-azobis[2-methyl-N-(2-hydroxyethyl) propionamide]. The structure and morphological characterization was carried out by FT-IR, TEM, SEM and XRD. The chemically covalent interactions were investigated by FT-IR, TEM, TGA and DSC. The neat Fe(3)O(4) nanoparticles took on an aggregated spherical shape with an average diameter of about 12 nm, while Fe(3)O(4)/P(MAA-co-NVP) magnetic microspheres assumed controllable and monodispersed spheres with a mean dimension of ca. 0.8 μm. The microspheres exhibited superparamagnetic properties. The in vitro caffeine release behavior under varying pH environment was investigated to evaluate the potential of Fe(3)O(4)/P(MAA-co-NVP) magnetic microspheres as a magnetic drug targeting carrier. The results indicated that the microspheres have a faster drug-release rate at pH 7.4 than at pH 1.4, corresponding to their pH swelling. The kinetic modeling demonstrated that the drug release is controlled by a balance between co-polymer chain relaxation and Fickian diffusion process, and the proposed carrier is suitable for a magnetic targeting drug-delivery system.     

人腹膜间皮细胞对卵巢癌SKOV3细胞黏附、迁移和侵袭力的影响

目的：探讨人腹膜间皮细胞（HPMC）对卵巢癌SK-OV3细胞黏附、迁移、侵袭力的影响及可能机制.方法：建立HPMC原代培养的方法;采用免疫细胞组织化学法鉴定原代HPMC;验证HPMC中部分细胞因子的表达;采用黏附、迁移及侵袭实验观察HPMC与卵巢癌SKOV3细胞相互作用时对SKOV3细胞转移力的影响,及其作用机制是否与CXCL12-CXCR4轴有关.结果：成功培养出了HPMC.鉴定分离出的细胞符合间皮细胞的特征.HPMC中表达CXCL12、间皮素,不表达CXCR4.SKOV3与HPMC共同培养组的吸光度、迁移和侵袭细胞数显著高于单独SKOV3培养组,差异有统计学意义（P〈0.05）.稳定高表达CXCR4的SKOV3细胞（SKOV3-CX-CR4-cDNA）与HPMC共同培养组的吸光度、迁移和侵袭的细胞数目显著高于其余3组,差异均有统计学意义（P〈0.05）.结论：HPMC与SKOV3细胞相互作用时,增强了SKOV3细胞黏附、迁移和侵袭力,其作用机制与CXCL12-CXCR4轴有关.     

Mechanism of apoptotic effects induced selectively by ursodeoxycholic acid on human hepatoma cell lines

AIM: To investigate the effects of ursodeoxycholic acid (UDCA) on apoptosis and proliferation of hepatoma cell lines. METHODS: Human hepatoma cell lines HepG2 and BEL 7402 were cultured in medium supplemented with different concentrations of UDCA, normal human hepatic line L-02 was used as control. Cell proliferation, apoptosis and gene expression were detected using methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, Western blot, DNA ladder assay, electron microscopy, and immunocytochemistry. RESULTS: Ursodeoxycholic acid inhibited the proli- feration of HepG2 and BEL7402 cell lines in a dose- dependent manner. Ursodeoxycholic acid can change cell cycle distribution of HepG2 and BEL7402, the proportion of cells in G0-G1 phase increased whereas the proportion of S phase cells and G2-M phase cells decreased. Ursodeoxycholic acid arrested the cell cycle in G0-G1 phase by down-regulating the cell cycle related proteins cyclin D1, D3 and retinoblastoma protein (pRb). The apoptotic rates of HepG2 and BEL7402 treated with UDCA (1.0 mmol/L) were significantly higher than those of control. In the HepG2 and BEL7402 treated with UDCA, expression of bcl-2 decreased whereas expression of Bax increased, the nuclear fragmentation and chromosomal condensed, cells shrank and lost attachment, apoptotic bodies and DNA ladders appeared. UDCA had no effect in inducing apoptosis on L-02 cell lines. CONCLUSION: UDCA can selectively inhibit proliferation and induce apoptosis of HepG2 and BEL7402 cell lines by blocking cell cycle and regulating the expression of Bax/bcl-2 genes.