科创中国

目的:构建表达人LIM矿化蛋白-1(LIM mineralization protein-1,LMP-1)基因的腺病毒重组体,体外感染骨肉瘤(Osteosarcoma,OS)细胞系U2OS,并鉴定LMP-1基因在U2OS中的表达.方法:以K562细胞的cDNA为文库,采用PCR方法对LMP-1进行扩增,通过TA克隆与pGEM-T载体连接并DNA测序.双酶切后并将目的基因插入至腺病毒穿梭质粒pAdtrack-CMV,对腺病毒穿梭质粒pAdtrack-CMV-LMP-1行双酶切和DNA测序鉴定,并通过荧光显微镜、RT-qPCR和Western blot检测pAdtrack-CMV-LMP-1在HEK-293T细胞中的表达.线性化pAdtrack-CMV-LMP-1,在BJ5183菌内完成与骨架质粒pAdeasy-1的同源重组,构建重组腺病毒质粒Ad-LMP-1.通过脂质体介导,在HEK-293A细胞内包装出复制缺陷的重组腺病毒Ad-LMP-1,大量扩增、纯化并测定滴度.用Ad-LMP-1感染OS细胞系U2OS,通过荧光显微镜、RT-qPCR和Western blot检测LMP-1在U2OS细胞中的表达.结果:双酶切和DNA测序鉴定穿梭质粒pAdtrack-CMV-LMP-1构建成功,荧光显微镜证实转染了pAdtrack-CMV-LMP-1质粒的HEK-293T细胞内有绿色荧光蛋白表达,RT-qPCR和Western blot验证了LMP-1基因的表达量明显高于对照组.通过扩增、纯化,Ad-LMP-1滴度达到1.5*109pfu/ml.荧光显微镜下观察重组腺病毒感染的U2OS内有绿色荧光蛋白表达,RT-qPCR和Western blot检测发现重组腺病毒感染U2OS后,LMP-1的mRNA和蛋白表达量明显高于对照组.结论:成功构建了人LMP-1基因腺病毒重组体,为进一步的实验研究奠定了基础.

CdSe/CdS semiconductor quantum dots co-sensitized TiO2 nanorod array was fabricated on the transparent conductive fluorine-doped tin oxide (FTO) substrate using the hydrothermal and successive ionic layer adsorption and reaction (SILAR) process. The structural and morphological properties of the samples were characterized by X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM). The results indicate that CdSe/CdS QDs are uniformly coated on the surface of the TiO2 nanorods. The shift of light absorption edge was monitored by taking UV-visible absorption spectra. Compared with the absorption spectra of the TiO2 nanorod array, deposition of CdSe/CdS QDs shifts the absorption edge to the higher wavelength. The enhanced light absorption in the visible-light region of CdSe/CdS/TiO2 nanorod array indicates that CdSe/CdS layers can act as co-sensitizers in quantum dots sensitized solar cells (QDSSCs). By optimizing the CdSe layer deposition cycles, a photocurrent of 5.78 mA/cm2, an open circuit photovoltage of 0.469 V and a conversion efficiency of 1.34 % were obtained under an illumination of 100 mw/cm2.

目的:探讨皖南蝮蛇毒抑瘤组分I(AHVAC-I)对人胃癌细胞株SGC-901生长增殖的影响及作用机制.方法:AHVAC-I处理SGC-7901细胞.CCK-8法检测其生长抑制作用,光镜观察细胞形态变化,TUNEL法检测细胞凋亡情况.结果:AHVAC-I抑制SGC-901细胞的生长增殖,并呈浓度依赖性,半数抑制浓度(IC50)为58.197μg/ml;镜下可见SGC-901细胞悬浮、胞核固缩等典型凋亡形态学变化;TUNEL法显示凋亡指数随浓度增加而增加.结论:AHVAC-I对人胃癌细胞株SGC-7901具有生长抑制作用,诱导凋亡可能是其主要的作用机制之一.

Objective To detect the cell viability and the expressions of stem cell surface markers after chemotherapeutic drug treatment.Methods We observed the cytotoxic effects of three chemotherapeutic agents[epirubicin(Epi),fluorouracil(5-FU)and cyclophosphamide(Cyc)]in three cell lines,and the cell viabilities after removed these chemotherapeutic agents.Expressions of stem cell surface markers CD44,CD24,CD90,CD14 and aldehyde dehydrogenase1(ALDH1)in breast cancer cells were analyzed by real-time PCR.The post hoc analysis(Tukey's tests)in conjunction with one-way ANOVA was used for statistical analysis.Results The initial cytotoxic efficacy was most notable.After the treatment of the same therapeutic agents,cell viability was decreased by 64.8%35.14%,32.25%in BT-483 cells,66.4%,22.94%and 45.88%in MDA-MB-231 cells,97.1%,99.5%and 76.4%in MCF cells.The difference was significant compared with that before treatment(P=0.000).However,the inhibitory effects were diminished after chemotherapeutic agent withdrawal.Cell viabilities were increased to 167.9%,212.04%and 188.66%in MDA-MB-231 cells at48 h after withdrawal.At 72 h after withdrawal,cell viability was increased with a significant difference in three cell lines(all P values=0.000).Expressions of CD44 and ALDH1 were most prevalent for MDA-MB-231,BT-483 and MCF-7 cells.ALDH1 mRNA level was significant higher in BT-483(HER-2 overexpression cell line)than MDA-MB-231(triple negative cell line)(P=0.012).CD14 mRNA level in MCF-7 cells were significantly lower than that in MDA-MB-231 and BT-483(P=0.003,0.001).BT-483 showed significantly higher level of CD44 than MDA-MB-231 and MCF-7 cell line(P=0.013,0.020),and no significant difference was detected between MDA-MB-231 and MCF-7 breast cancer cells(P=0.955).CD90 mRNA expressions were detected in MDA-MB-231 cells and MCF-7 cells,but not in BT-483 cells.Conclusion Some malignant cells could survive in vitro and begin to proliferate again between cycles of chemotherapy.

目的:研究从白屈菜乙醇提取物中分离出的白屈菜碱在诱导HeLa细胞凋亡中的作用及参与其作用的主要信号转导通路.方法:细胞先以不同浓度白屈菜碱处理48h,用噻唑蓝法分析确定半数致死量(medianlethaldose,LD50).用4',6-二脒基-2-苯基吲哚染色,追踪分析核浓染以及DNA损伤和碎片的形态学变化,并用流式细胞术分析检测活性氧(reactive oxygen species,ROS)的产生以及细胞周期阻滞和线粒体膜电位的变化.用圆二色光谱分析寻找白屈菜碱和小牛胸腺DNA可能的相互作用.用逆转录聚合酶链反应和蛋白免疫印迹法测定p38、p53、蛋白激酶B(proteinkinase B,AKT)、磷脂酰肌醇3激酶(phosphatidylinositol 3-kinases,PI3K)、Janus激酶3(Janus kinase 3,JAK3)、信号转导及转录激活因子3(signal transducer and activator of transcription 3,STAT3)等的mRNA和蛋白表达,以及E6、E7癌基因和促凋亡基因、抗凋亡基因的mRNA和蛋白表达.结果:根据白屈菜碱的LD50(30μg/mL),选定3种实验剂量,即22.5、30和37.5μg/mL.结果显示,白屈菜碱抑制了HeLa细胞增殖,诱发其细胞凋亡,表现为ROS的产生,细胞亚G1和G0/G1周期阻滞,线粒体膜电位变化和DNA碎片产生.圆二色光谱分析结果显示白屈菜碱和小牛胸腺DNA间存在有效的相互作用.信号通路的研究显示白屈菜碱通过上调p38、p53和其他促凋亡基因的表达,以及下调AKT、P13K、JAK3、STAT3、E6、E7和其他抗凋亡基因的表达,有效诱发细胞凋亡.结论:从白屈菜中分离出的自屈菜碱能通过改变p38-p53及AKT/P13激酶信号转导通路有效地诱发HeLa细胞凋亡.

目的:通过检测梅毒血清固定患者外周血CD4+CD25+调节性T细胞(regulatoryTcells,Treg)比例和功能的改变,探讨Treg在梅毒血清固定现象形成中的作用.方法:收集梅毒血清固定患者26例,正常对照23例,利用流式细胞术分别检测外周血Treg比例及Treg内Foxp3、细胞毒性T淋巴细胞抗原(cTLA)-4及白介素(IL)-10的定量表达情况.结果:梅毒血清固定组患者外周血Treg比例明显高于正常对照组(P〈0.01);且Treg内转录因子Foxp3及功能性分子cTLA-4和IL-10的表达量也明显高于正常对照组(P〈0.05或P〈0.01).结论:梅毒血清固定患者外周血Treg比例和功能的异常,可能是导致该现象形成的重要原因之一.

目的:构建含变形链球菌表面蛋白A区编码基因(pacA)、葡糖基转移酶催化区编码基因(gtfB-cat)及霍乱毒素B亚单位编码基因(ctxB)的嵌合表达质粒并表达目的蛋白.方法:将目的基因pacA、gtfB-cat、ctxB克隆至原核克隆载体pET32-a(+)构建嵌合质粒pET-pacA-cat-ctxB,将其转入表达宿主菌E.coliBL21(DE3),并测量其表达情况.结果:构建的重组质粒经酶切、PCR鉴定及测序,证实目的基因均正确插入到载体pET'32a(+)中,插入的相位正确,未改变目的基因的阅读框架,经诱导后表达目的蛋白,分子质量大小与预期-致.结论:成功构建了嵌合质粒pET-pa-cA-cat-ctxB,且它们均在原核细胞内获得正确表达.