尿刊酸偶联壳聚糖基因载体的合成和表征

本文将活化的尿刊酸(urocanic acid，UA)与壳聚糖(chitosan，CTS)上的氨基反应合成一种新型非病毒基因载体——尿刊酸偶联壳聚糖(urocanic acid-coupled chitosan，UAC)，通过红外(FT-IR)、核磁共振(¹H NMR)和元素分析对UAC结构进行确证。用正交实验对取代度的影响因素进行考察，结果表明，随着UA投料量的增加和活化时间的延长，UAC的取代度也随之增大；通过琼脂凝胶电泳阻滞实验分析UAC/质粒DNA(plasmid DNA，pDNA)的复合能力及对DNase I酶抗降解能力，结果证明UAC与pDNA有较好的复合能力和抵制DNase I酶降解能力；通过测定UAC/pDNA复合物粒径和对zeta电位的分析，结果证明复合物具有良好的稳定性和易于进入细胞的性质；体外细胞转染是通过荧光显微镜观察UAC介导pDNA在体外培养HepG2细胞中的表达，结果显示，UAC能介导pDNA转染HepG2细胞并在细胞中表达绿色荧光蛋白，其转染效果较CTS明显增强。因而UAC是一种制备工艺简单，具有应用前景的非病毒基因载体。     

注射用核糖核酸Ⅱ通过活性氧介导的PI3K/Akt信号通路诱导人白血病细胞KG1a凋亡

目的研究注射用核糖核酸Ⅱ诱导人急性骨髓白血病细胞系KG1a细胞凋亡与PI3K/Akt信号通路和活性氧(ROS)的关系。方法流式细胞术(FCM)检测细胞周期和细胞内的ROS水平;免疫印迹法(Western blot)检测PI3K/Akt信号通路相关蛋白p-PI3K、p-Akt、p-MDM2、p21和周期相关蛋白cyclin D1、CDK4的表达;加入抗氧化剂N-乙酰半胱氨酸(NAC)后,检测p-PI3K、p-Akt和p53蛋白表达变化。结果 FCM结果显示,注射用核糖核酸Ⅱ可将KG1a细胞阻滞在G0/G1期,且ROS介导了细胞周期的阻滞; FCM检测ROS结果显示,KG1a细胞内ROS水平均随药物浓度增加而升高; Western blot结果显示,注射用核糖核酸Ⅱ下调pPI3K、p-Akt、p-MDM2和cyclin D1、CDK4的表达,上调p21的表达;与仅用注射用核糖核酸Ⅱ相比,经NAC预处理后再使用注射用核糖核酸Ⅱ,明显上调了磷酸化PI3K和Akt的表达,下调了p53的表达。结论注射用核糖核酸Ⅱ通过ROS介导的PI3K/Akt信号通路,诱导人急性骨髓白血病细胞系KG1a细胞凋亡。     

p15基因对人肝癌HepG2顺铂耐药细胞周期和耐药性的影响研究

目的:利用p15-shRNA表达载体干扰细胞周期相关基因p15,探讨p15基因对人肝癌HepG2顺铂耐药细胞周期和耐药性的影响。方法:用梯度浓度的顺铂诱导HepG2细胞耐药并建立动态耐药模型HepG2/顺铂/2.0,以不同浓度(30、60、90nmol·L-1)的干扰质粒p15-shRNAY2和阴性对照质粒(shRNA-F)转染HepG2/顺铂/2.0细胞后48h检测p15荧光阳性细胞,筛选最佳干扰浓度;以筛选结果转染HepG2/顺铂/2.0细胞,与未转染细胞和阴性对照转染细胞比较,检测细胞存活情况、周期分布和p15、Bcl-2、Bax的蛋白表达。结果:最佳干扰浓度为60nmol·L-1;与未转染细胞和阴性对照转染细胞比较,p15-shRNAY2转染细胞的存活率明显增加,G1期细胞明显减少,p15、Bax蛋白表达明显降低,Bcl-2蛋白表达明显增加(P均<0.01)。结论:p15-shRNA可抑制p15基因表达,干扰细胞周期使G1期的比例减少,增加HepG2/顺铂/2.0对顺铂的耐药性。     

MG132诱导HepG_2细胞凋亡及其对p53表达的影响

目的观察蛋白酶体抑制剂MG132对人肝癌细胞系HepG2的致凋亡作用及其对凋亡相关基因p53蛋白表达的影响。方法采用多个浓度(2,5,10μmol/L)的蛋白酶体抑制剂MG132处理HepG2细胞;流式细胞术和Hoechst荧光染色检测细胞凋亡;免疫细胞化学检测HepG2细胞p53蛋白含量。结果对照组HepG2细胞凋亡率低于5%,在2,5,10μmol/LMG132作用下,细胞凋亡率分别为42.9%,66.1%和72.8%,MG132诱导HepG2细胞凋亡具有剂量—效应关系。经Hoechst荧光染色可见细胞染色质浓缩等凋亡改变。免疫组织化学检测HepG2细胞p53蛋白表达水平增高。结论蛋白酶体抑制剂MG132能够诱导HepG2细胞凋亡,其作用呈剂量—效应关系;p53蛋白表达增加与MG132抑制泛素—蛋白酶体系统降解细胞内p53蛋白有关。     

血管内皮生长因子过表达载体和干扰载体的构建及其对Lewis肺癌细胞的干预

目的:探讨血管内皮生长因子(VEGF)过表达载体和干扰载体对Lewis肺癌细胞的干预作用。方法:构建VEGF过表达载体pIRES2-VEGF-GFP和干扰载体psiRNA-VEGF-GFP,脂质体法介导转染Lewis肺癌细胞;荧光显微镜观察细胞GFP蛋白的表达状态;MTT法和流式细胞术分别检测各组细胞生长差异和细胞周期变化;Western-Blot检测细胞转染前后VEGF蛋白表达量的变化。结果:送检测序的质粒图谱正确,经转染Lewis肺癌细胞后建立了稳定表达模型;过表达VEGF组可见稳定的绿色荧光蛋白表达;过表达VEGF进入S期的细胞达到45.3%,未转染组为29.1%;干扰其表达后细胞的生长受阻,细胞周期阻滞在G0/G1期的比率达74.5%。过表达VEGF组VEGF蛋白的表达量约为未转染Lewis肺癌细胞的3倍;干扰VEGF表达后VEGF蛋白的表达下调。结论:本实验成功构建了VEGF过表达载体和干扰载体,并在Lewis肺癌细胞中建立了稳定表达模型,初步证实VEGF能够促进肺癌细胞恶性进展。     

真核表达载体介导白细胞介素24在HepG2细胞的表达及其体外抗肿瘤效应研究

目的  研究真核表达载体pcDNA3.1(+) 介导白细胞介素（interleukin, IL）24在肝癌细胞系HepG2细胞中表达的可行性,以及IL-24体外对HepG2细胞的抗瘤效应和可能机制。方法 采用脂质体转染法将重组质粒pcDNA3.1(+)-IL-24 及空质粒分组转染HepG2细胞,在倒置显微镜下观察细胞形态变化。用噻唑蓝比色方法测量细胞增殖指数,流式细胞仪研究细胞早期凋亡率及其细胞周期分布。结果  IL-24 mRNA成功表达于HepG2细胞中。重组质粒转染组可见明显的凋亡细胞形态,48 h增殖抑制率（F=27.058,P<0.01）、72 h增殖抑制率( F=63.481,P<0.01)和早期细胞凋亡率( F=326.220,P<0.01)与对照组的差异均有统计学意义。细胞分布呈明显的G2/M周期阻滞。结论 真核表达载体pcDNA3.1(+)可以有效地介导IL-24在HepG2细胞的表达。IL-24对HepG2细胞有明显的增殖阻滞和凋亡诱导效应,G2/M细胞周期阻滞或许是IL-24抗瘤效应的机制。       

葫芦素E通过诱导G_2/M周期阻滞抑制人肝癌细胞增殖

目的研究葫芦素E(cucurbitacin E,CuE)体外对人肝癌BEL7402和HepG2细胞的增殖抑制作用及机制。方法MTT法检测不同浓度CuE体外对BEL7402和HepG2细胞的增殖抑制作用;流式细胞仪检测CuE对细胞周期分布的影响;Western blot法检测CuE对细胞周期调节蛋白cy-clinB1、Cdc25C、Cdk1及p21表达的影响。结果CuE作用72 h可剂量依赖性地抑制BEL7402和HepG2细胞增殖;CuE(100 nmo.lL-1)作用12、24 h后可使BEL7402和HepG2细胞周期阻滞于G2/M期,并可下调Cdk1蛋白的表达,上调p21蛋白的表达。结论CuE体外可抑制人肝癌BEL7402和HepG2细胞的增殖,其增殖抑制作用与诱导G2/M周期阻滞有关。     

芦荟多糖对肿瘤细胞生产周期的影响

目的研究芦荟多糖(A loe Polysaccharides,AP)对接受辐射的正常细胞株和肿瘤细胞株的防护作用是否存在差异性。方法应用流式细胞仪检测细胞周期,W estern b lot法检测细胞p53、p21基因蛋白表达。结果AP预处理可以显著降低293和C.L iver细胞G2/M期阻滞,同时使G0/G1期细胞比例明显上升,对3株肿瘤细胞周期则无显著改变或加重G2/M期阻滞。AP预处理可抑制C.L iver p53蛋白的高表达,对CNE1p53蛋白表达则有提高作用,对293、CNE2和SUNE1p53表达均无影响。p21C IP1/WAF1蛋白在3株肿瘤细胞均无表达,在C.L iver和293细胞均于辐射后高表达。结论AP对正常细胞株有显著辐射防护作用,对肿瘤细胞则无显著影响,这种差异可能与p53、p21介导的细胞周期调节通路有关。     

塞来昔布上调Cyclin D1基因甲基化水平对食管癌细胞增殖和凋亡的抑制作用

目的探究塞来昔布通过上调Cyclin D1基因甲基化水平对食管癌细胞增殖和凋亡的作用及机制。方法将细胞分为对照、转染和塞来昔布组,分别转染阴性对照载体、Cyclin D1过表达载体和Cyclin D1转染后给予60μmol/L塞来昔布。采用MTT法、流式细胞术分别检测细胞增殖、细胞周期和细胞凋亡率的变化,Western blotting法检测细胞周期和细胞凋亡相关蛋白表达水平;甲基化特异性PCR(MS-PCR)、qRT-PCR法用于检测Cyclin D1甲基化特异性扩增片段和Cyclin D1 mRNA表达水平。结果塞来昔布能够抑制Cyclin D1诱导的细胞体外增殖,阻滞细胞周期向S期转化,并促进食管癌细胞凋亡;MS-PCR结果显示塞来昔布能够上调Cyclin D1基因甲基化水平,在转录水平抑制细胞内Cyclin D1 mRNA的表达。结论塞来昔布能够通过上调Cyclin D1基因甲基化水平发挥抑制食管癌细胞增殖、促进其体外凋亡的作用。     

过表达SATB1对人原发性肝癌细胞HepG2增殖的促进作用

目的研究核基质结合蛋白SATB1对人原发性肝癌HepG2细胞增殖的影响。方法脂质体介导pEGFP1 SATB1真核表达全长基因质粒转染人原发性肝癌HepG2细胞,分为实验组（pEGFP1 SATB1）、对照组（pEGFP1空载体）及空白对照组。RT PCR、Western blot检测转染后SATB1mRNA和蛋白表达变化情况,噻唑蓝（MTT）法、流式细胞仪检测SATB1全长基因转染对人原发性肝癌HepG2细胞生长、增殖的影响。结果与空白对照组及对照组比较,转染pEGFP1 SATB1后,HepG2中SATB1mRNA和蛋白表达明显增加,细胞生长曲线检测结果表明转导入pEGFP1 SATB1细胞增殖明显加快,细胞数明显增多;流式细胞仪检测结果表明,细胞周期中G0/G1期细胞明显减少,S、G2/M期细胞明显增多。结论SATB1能明显促进HepG2细胞增殖,其机制可能与参与调控细胞周期各期DNA复制、转录有关。     

wt—P53基因对人膀胱移行细胞癌细胞系转染及其生物学特性影响的研究

目的：探讨膀胱肿瘤的治疗途径。方法：利用Ｌｉｐｏｆｅｃｔａｍｉｎｅ将外源性野生型ｐ５３（ｗｔ－ｐ５３）基因导入人膀销移行细胞癌细胞系ＴＢＣ－１细胞中,并得到表达,结果;细胞在体外标准条件下,生长增殖率降低,流式细胞计检测显示细胞周期Ｇ０＋Ｇ１细胞比例明显增高,凋亡指数升高,细胞生长速度减低,代表细胞增殖能力的ＡｇＮＯＲｓ计数下降,结论：增加ｗｔ－ｐ５３的基因表达具有抑制恶性肿瘤生长的作用。     

Nanoparticle-mediated wild-type p53 gene delivery results in sustained antiproliferative activity in breast cancer cells

Gene expression with nonviral vectors is usually transient and lasts for only a few days. Therefore, repeated injection of the expression vector is required to maintain a therapeutic protein concentration in the target tissue. Biodegradable nanoparticles (approximately 200 nm diameter) formulated using a biocompatible polymer, poly(D,L-lactide-co-glycolide) (PLGA), have the potential for sustained gene delivery. Our hypothesis is that nanoparticle-mediated gene delivery would result in sustained gene expression, and hence better efficacy with a therapeutic gene. In this study, we have determined the antiproliferative activity of wild-type (wt) p53 gene-loaded nanoparticles in a breast cancer cell line. Nanoparticles containing plasmid DNA were formulated using a multiple-emulsion-solvent evaporation technique. To understand the mechanism of sustained gene expression with nanoparticles, we monitored the intracellular trafficking of both the nanoparticles and the nanoparticle-entrapped DNA, and also determined p53 mRNA levels over a period of time. Cells transfected with wt-p53 DNA-loaded nanoparticles demonstrated a sustained and significantly greater antiproliferative effect than those with naked wt-p53 DNA or wt-p53 DNA complexed with a commercially available transfecting agent (Lipofectamine). Cells transfected with wt-p53 DNA-loaded nanoparticles demonstrated sustained p53 mRNA levels compared to cells which were transfected with naked wt-p53 DNA or the wt-p53 DNA-Lipofectamine complex, thus explaining the sustained antiproliferative activity of nanoparticles. Studies with fluorescently labeled DNA using confocal microscopy and quantitative analyses using a microplate reader demonstrated sustained intracellular localization of DNA with nanoparticles, suggesting the slow release of DNA from nanoparticles localized inside the cells. Cells which were transfected with naked DNA demonstrated transient intracellular DNA retention. In conclusion, nanoparticle-mediated wt-p53 gene delivery results in sustained antiproliferative activity, which could be therapeutically beneficial in cancer treatment.     

siRNA沉默肝癌细胞报告基因瞬时表达效应的定量分析

目的研究小分子干扰RNA(siRNA)特异性抑制肝癌细胞基因瞬时表达的可行性和基因沉默效率。方法凝胶电泳成像观察siRNA体外孵育后的降解性,利用Cy3示踪观察其转染HepG2细胞后动力学变化。将报告基因(GFP)和siRNA共同瞬时转染至HepG2细胞,流式细胞仪、Western blot RT-PCR等分析特异性siRNA抑制报告基因瞬时表达的效应。结果siRNA在空白培养液中和转染细胞内可较长时间保持相对稳定。与空白和非特异siRNA相比,GFPsiR-NA明显抑制GFP蛋白产物和mRNA的表达(P<0·05)。结论特异性siRNA能高效沉默人肝癌细胞转染基因的瞬时表达,为基因治疗提供了一种新的治疗策略。     

bFGF对兔视网膜缺血再灌注损伤中wt-p53蛋白表达的影响

目的 研究碱性成纤维细胞生长因子(bFGF)对兔视网膜缺血再灌注损伤(RIRI)后野生型p53(wt-p53)蛋白表达的影响.方法 56只健康大耳白兔随机分为正常对照组(8只)与损伤组(48只);损伤组同一大耳白兔在RIRI后左眼玻璃体腔注入生理盐水(损伤对照组),右眼玻璃体腔注入bFGF(损伤治疗组);损伤组按照RIRI后处死时间不同,分为6组(每组8只),并对各组大耳白兔取视网膜做切片并进行免疫组化分析.结果 正常对照组视网膜组织基本无wt-p53蛋白表达,损伤对照组与损伤治疗组均于RIRI后6 h发现wt-p53蛋白表达,24 h达到高峰,之后随着RIRI时间的延长逐渐减弱,两组RIRI后6、12、24、48 h wt-p53蛋白阳性表达比较差异有统计学意义(P＜0.05,P＜0.01).结论 wt-p53蛋白的规律表达说明视网膜细胞凋亡与RIRI密切相关,而bFGF在抑制视网膜细胞凋亡及维持视网膜神经节细胞存活再生有重要作用.     

Cell cycle changes mediated by the p53/miR-34c axis are involved in the malignant transformation of human bronchial epithelial cells by benzo[a]pyrene

Characterization of aberrant microRNA (miRNA) expression during carcinogen-induced cell transformation will lead to a better understanding of the role of miRNAs in cancer development. In this investigation, we evaluated changes in p53 function and its downstream target miRNAs in benzo[a]pyrene (BaP)-induced transformation of human bronchial epithelial (HBE) cells. Chronic exposure to BaP induced malignant transformation of cells, in which there were increased levels of mutant p53 (mt-p53) and reduced expression of wild-type p53 (wt-p53) and phosphorylated p53 (p-p53). With acute (12 h) exposure to BaP, p-p53 was increased, and with increasing time of exposure (24 h), the increase in p-p53 at a concentration of 1 μM BaP was followed by a decline with increasing concentrations; wt-p53 and mt-p53 did not change. With prolonged exposure (48 h), p-p53 and wt-p53 decreased, but mt-p53 increased. At different exposure times, the levels of miR-34c were consistent with p-p53. Over-expression of miR-34c resulted in inhibition of the BaP-induced G1-to-S transition and diminished up-regulation of cyclin D. Further, up-regulation of miR-34c or silencing of cylin D prevented BaP-induced malignant transformation. Thus, changes in the cell cycle mediated by the p53/miR-34c axis are involved in the transformation cells induced by BaP.     

Curcumin up-regulates LDL receptor expression via the sterol regulatory element pathway in HepG2 cells

Plasma low-density lipoprotein-cholesterol (LDL-C) is mainly taken up and cleared by the hepatocellular LDL receptor (LDL-R). LDL-R gene expression is regulated by the sterol regulatory element binding proteins (SREBPs). Previous studies have shown that curcumin reduces plasma LDL-C and has hypolipidemic and anti-atherosclerotic effects. Herein, we investigated the effect of curcumin on LDL-R expression and its molecular mechanism in HepG2 cells. Curcumin increased LDL-R expression (mRNA and protein) and the resultant uptake of DiI-LDL in a dose- and time-dependent manner. Using a GFP reporter system in a transfected HepG2/SRE-GFP cell line, we found that curcumin activated the sterol regulatory element of the LDL-R promoter. In HepG2/Insig2 cells, curcumin reversed the inhibition of LDL-R expression induced by Insig2 overexpression. These data demonstrate that curcumin increases LDL-R protein expression and uptake activity via the SREBPs pathway. These findings contribute to our further understanding of the cholesterol-lowering and anti-atherosclerotic effects of curcumin.