RNA干扰Mcl-1基因表达对淋巴瘤Raji细胞增殖和凋亡的机制研究

目的:探讨抑制Mcl-1基因表达对淋巴瘤Raji细胞增殖和凋亡的影响及机制。方法:NC-siRNA、Mcl-1-siRNA转染Raji细胞,以不作任何处理的细胞作为空白对照组,48h后Western blot检测各组细胞中Mcl-1的蛋白表达;CCK8实验和流式细胞仪分别检测细胞的增殖和凋亡情况;Western blot检测Cleaved caspase3、Notch1、Hes1蛋白表达。结果:转染Mcl-1-siRNA后Mcl-1的蛋白表达显著降低;与对照组及NC-siRNA组比较,Mcl-1-siRNA组细胞存活率显著降低,细胞凋亡率显著升高,Cleaved caspase3蛋白显著上调表达,Notch1和Hes1蛋白显著下调表达。结论:RNA干扰抑制Mcl-1基因表达可显著降低Raji细胞增殖及诱导细胞凋亡,其机制与抑制Notch1信号通路有关。     

人Hes1-shRNA，Hes5-shRNA慢病毒表达载体的构建、包装及鉴定

目的构建人Hesl-shRNA和Hes5-shRNA慢病毒表达载体,为Notch—Hes信号通路的相关研究奠定基础。方法根据人Hes1,Hes5基因mRNA序列分别设计、合成多对互补的DNA单链寡核苷酸,退火后克隆至pENTR／U6入门载体。通过入门载体瞬时转染神经胶质瘤U251细胞筛选有效干扰序列。将含有效干扰序列的入门载体与pLenti6／BLOCK—iT—DEST载体进行LR重组构建Hesl—shRNA和Hes5-shRNA慢病毒表达载体,经脂质体介导入293FT细胞,包装成慢病毒。用该慢病毒感染U251细胞,Western印迹法分别检测Hes1,Hes5蛋白的表达。结果分别构建了针对Hes1和Hes5基因的特异性shRNA慢病毒表达载体,其包装获得慢病毒可有效感染U251细胞并分别对HeM,Hes5蛋白的表达有显著抑制作用。结论成功构建了Hesl—shRNA和Hes5-shRNA慢病毒表达载体。     

可磷酸化短肽偶联壳聚糖介导IL-1RA与IGF-1共转染对兔关节软骨细胞的作用

探讨可磷酸化短肽偶联壳聚糖(phosphorylatable short peptide coupled chitosan,pSP-CS),介导人白细胞介素 1受体拮抗剂基因(interleukin-1 receptor antagonist protein,IL-1RA)和人胰岛素样生长因子1基因(insulin like growth factor-1,IGF-1) 共转染,对体外培养的兔关节软骨细胞的作用. 将pSP-CS 与共表达质粒pBudCE4.1-IL-1RA+IGF-1、单基因表达质粒pBudCE4.1-IL-1RA、pBudCE4.1-IGF-1和空质粒pBudCE4.1制成pSP-CS/pDNA复合物,转染体外分离培养的正常兔原代关节软骨细胞. ELISA 法检测IL-1RA和IGF-1的表达,以表征pSP CS转染效率;Cell Counting Kit-8 (CCK-8) 法分析软骨细胞的增殖活力;流式细胞仪检测软骨细胞的凋亡;定量PCR检测软骨细胞中基质金属蛋白酶抑制剂-1(matrix metallo proteinase inhibitor-1, Timp-1)、基质金属蛋白酶-3(matrix metalloproteinase-3, Mmp-3)、聚集蛋白聚糖 (Aggrecan) 基因表达. 转基因组IL-1RA和IGF-1有较高的表达水平;各转基因组明显促进细胞增殖、抑制细胞凋亡、下调Mmp-3基因表达、上调Timp 1和Aggrecan基因表达,且双基因组作用明显优于单基因组(P<0.05). 结果表明,pSP-CS可以携带外源基因进入软骨细胞并大量表达, IGF-1与IL-1RA协同作用明显提高体外培养软骨细胞的生物活性, 为今后研究pSP-CS介导多基因体内治疗软骨损伤提供了基础.     

抑制素α基因表达下调对鹅卵泡颗粒细胞凋亡和增殖的影响

抑制素α基因是控制家禽排卵率的一个重要候选基因。为研究抑制素α基因对体外培养的鹅卵泡颗粒细胞增殖凋亡的遗传作用,构建psiRNA-INHα1和psiRNA-INHα2两个抑制素α基因干扰载体,以降低抑制素α基因表达。体外培养鹅F1和闭锁卵泡的颗粒细胞,经抑制素α基因干扰48h后,荧光显微镜检测转染效率,同时用流式细胞术检测抑制素α基因表达水平,以及细胞生长周期、细胞凋亡指数和增殖指数的变化,用放免法测定细胞培养上清中雌激素和孕酮水平。结果表明,抑制素α基因干扰后卵泡颗粒细胞抑制素表达水平比对照组降低30%～40%（P〈0.05）;细胞凋亡指数和增值指数显著高于对照组（P〈0.05）;细胞分泌雌激素水平显著下降（P〈0.05）。由此推断,抑制素α基因对颗粒细胞具有显著的抗凋亡效应。     

IQGAP1基因干扰对人食管癌细胞同质粘附能力的影响

目的：研究IQGAP1基因干扰对人食管癌细胞同质粘附能力的影响。方法：体外培养人食管癌KYSE150和 EC9706细胞,利用Western blot方法检测两株细胞IQGAP1蛋白的表达,利用缓慢聚集和细胞分离实验比较两株细胞同质粘附能力的差异;进一步在KYSE150和EC9706细胞中构建IQGAP1基因干扰的稳定细胞系,观察IQGAP1基因干扰后细胞同质粘附能力的改变。结果：KYSE150细胞IQGAP1蛋白表达量低于EC9706细胞,而同质粘附能力高于EC9706细胞;IQGAP1基因干扰后,其蛋白表达量明显降低,而细胞同质粘附能力明显增强。结论：IQGAP1 基因干扰能够显著增强食管癌细胞的同质粘附能力,从而降低肿瘤细胞的恶性表型。     

RNA 干扰沉默缺氧诱导因子1α逆转肺癌细胞耐药性

目的:观察RNA干扰沉默缺氧诱导因子1α(HIF-1α)对肺癌细胞耐药性的影响。方法:构建靶向HIF-1α小干扰RNA基因,并转染到人肺腺癌耐顺铂细胞株A549/DDP细胞中。逆转录聚合酶链反应RT-PCR)检测细胞的HIF-1α、多药耐药基因1(MDR-1)以多药耐药相关蛋白基因(MRP)mRNA变化,免疫细胞化学法观察干扰后HIF-1α、P-糖蛋白以及MRP蛋白的变化。MTT法检测不同浓度的顺铂作用下细胞死亡率。结果:HIF-1αsiRNA组中HIF-1α、MDR-1、MRP mRNA水平显著降低(P<0.05),且蛋白水平也显著下降(P<0.05)。HIF-1αsiRNA组细胞死亡率较未转染组均明显增高(P<0.05),转染siRNA阴性组不影响肿瘤细胞的耐药性。结论:HIF-1αsiRNA可显著降低A549/DDP细胞中HIF-1α、MDR-1、MRP表达,从而起到逆转肺腺癌A549/DDP细胞的耐药作用。     

利用CRISPRi下调MDR1基因表达增强肺腺癌A549/DDP细胞对顺铂敏感性

目的:探讨利用CRISPRi下调MDR1基因表达增强肺腺癌A549/DDP细胞对顺铂敏感性的作用。方法:利用生物信息学技术预测MDR1基因启动子上潜在的CRISPRi干扰位点,设计干扰片段并构建重组载体,采用qRT-PCR、Western blot方法检测各组细胞中MDR1基因mRNA以及蛋白表达水平,筛选干扰效率高的重组载体进行后续实验。将人肺癌A549/DDP细胞分为3组,分别为A549/DDP、Scrambed、sgRNA-MDR1-1,每组设置3个复孔。将各载体转染细胞48 h后,流式细胞术检测各组细胞外排情况,MTT法检测各组细胞的IC₅₀值,激光共聚焦显微镜下观察各组细胞经顺铂处理后的细胞形态。结果:经测序比对,成功构建两种干扰MDR1基因转录的CRISPRi重组载体。转染A549/DDP细胞后,各转染组细胞MDR1基因mRNA以及蛋白水平均显著降低(P<0.01),其中sgRNA-MDR1-1的干扰效率最高,mRNA和蛋白水平干扰效率分别达60%和51%。与Scrambed组相比,转染sgRNA-MDR1-1组细胞的细胞外排能力降低(P<...     

沉默Bmi-1基因表达稳定细胞株的建立及其生物学特性的研究

B细胞特异性莫洛尼鼠白血病病毒插入位点1(B-cell-specific moloney murine leukemia virus insertionsite 1,Bmi-1)基因是多梳基因家族成员,参与细胞增殖调控.研究发现Bmi-1基因可能参与肿瘤的形成,可能成为肿瘤潜在的治疗靶点.用RNA干扰(RNA interference,RNAi)沉默Bmi-1基因表达观察其对乳腺癌细胞株MCF-7侵袭和转移等生物学特性的影响,以探讨Bmi-1在乳腺癌发生发展中的作用.PT67细胞包装质粒后产生的逆转录病毒感染MCF-7细胞,嘌呤霉素筛选建立稳定细胞株,稳定抑制Bmi-1的细胞株命名为MCF-7/Bmi-1si.通过RT-PCR和Western blot分别从mRNA和蛋白水平检测Bmi-1的表达量;平板克隆形成实验检测细胞克隆形成能力;Transwell侵袭小室模型检测细胞体外侵袭和转移能力.MCF-7/Bmi-1si组与MCF-7和MCF-7/GFPsi组相比,Bmi-1 mRNA和蛋白表达量明显减少,克隆形成数及形成率也明显减少(P<0.05).侵袭和转移实验表明:与MCF-7和MCF-7/GFPsi组相比,MCF-7/Bmi-1si组细胞在Transwell侵袭小室中24 h穿膜细胞数明显减少(P<0.05).结果表明沉默Bmi-1基因表达稳定细胞株构建成功,Bmi-1基因表达的沉默能显著降低MCF-7细胞的体外增殖及侵袭转移能力.     

电穿孔介导的人对氧磷酶1基因在小鼠原代骨骼肌细胞中的转移与表达

非病毒载体介导的外源基因在哺乳动物骨骼肌细胞中的表达往往受限于基因转移效率的低下.本文利用电穿孔为基因转移方法,研究了人对氧磷酶基因（PON1）在原代培养的小鼠骨骼肌成肌细胞和成熟肌管中的转移与表达.在上述细胞中加入PON1的真核表达质粒后实施一定条件的电穿孔,通过测定不同时间点培养基与细胞裂解液中芳香酯酶活性的变化以衡量PON1的表达与分泌.结果显示,PON1在成肌细胞中表达的最佳电穿孔条件为800 V/cm, 20 ms and 50 μF;在肌管中为700 V/cm, 20 ms and 50 μF.在此条件下,细胞存活率均达75%以上,且表达的蛋白均可有效分泌.RT PCR分析同样验证了PON1 mRNA在骨骼肌细胞中的高效表达.电穿孔介导的PON1基因表达效率显著高于传统的基因转移方法如磷酸钙法和阳离子脂质体法.因此,以不同分化阶段的骨骼肌细胞为靶细胞,通过电穿孔介导外源基因表达切实可行,并可能在细胞工程与基因治疗等领域均具有潜在的应用前景.     

miR-125b靶向MCL1抑制胃癌 MGC-803细胞增殖

探讨mi R-125b对胃癌MGC-803细胞增殖的影响及机制,为阐明胃癌发病的分子机制提供实验依据.采用q RT-PCR和原位杂交,检测mi R-125b在正常胃黏膜(NGM)和胃癌(GAC)组织中的表达.将mi R-125b导入胃癌MGC-803细胞,观察mi R-125b高表达对MGC-803细胞增殖的影响.利用Targetscan 6.2软件及荧光素酶报告基因检测,分析mi R-125b对MCL1基因的靶向性作用.构建MCL1干扰载体,观察干扰MCL1基因表达对MGC-803细胞增殖的影响.结果发现,mi R-125b在胃癌组织中低表达,其表达与胃癌的分化程度及患者预后呈正相关,与TNM分期、淋巴结转移呈负相关(P0.01).mi R-125b高表达后MGC-803细胞的增殖降低、凋亡率增加、裂解caspase-3与裂解PARP表达增加(P0.01);mi R-125b与MCL1基因的3′UTR(2 613~2 620)结合,抑制MCL1的m RNA及蛋白质表达(P0.01);沉默MCL1基因表达后MGC-803细胞的增殖降低、凋亡率增加、裂解caspase-3与裂解PARP表达增加(P0.01).从而得出结论,mi R-125b在胃癌组织中低表达,其表达与胃癌组织分化程度、TNM分期、淋巴结转移及患者预后密切相关;mi R-125b靶向抑制MCL1基因表达,活化caspase-3信号通路,抑制MGC-803细胞增殖.     

Persistent and high levels of Hes1 expression regulate boundary formation in the developing central nervous system

The developing central nervous system is partitioned into compartments by boundary cells, which have different properties than compartment cells, such as forming neuron-free zones, proliferating more slowly and acting as organizing centers. We now report that in mice the bHLH factor Hes1 is persistently expressed at high levels by boundary cells but at variable levels by non-boundary cells. Expression levels of Hes1 display an inverse correlation to those of the proneural bHLH factor Mash1, suggesting that downregulation of Hes1 leads to upregulation of Mash1 in non-boundary regions, whereas persistent and high Hes1 expression constitutively represses Mash1 in boundary regions. In agreement with this notion, in the absence of Hes1 and its related genes Hes3 and Hes5, proneural bHLH genes are ectopically expressed in boundaries, resulting in ectopic neurogenesis and disruption of the organizing centers. Conversely, persistent Hes1 expression in neural progenitors prepared from compartment regions blocks neurogenesis and reduces cell proliferation rates. These results indicate that the mode of Hes1 expression is different between boundary and non-boundary cells, and that persistent and high levels of Hes1 expression constitutively repress proneural bHLH gene expression and reduce cell proliferation rates, thereby forming boundaries that act as the organizing centers.     

Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium

To clarify the mechanisms that regulate neuroendocrine differentiation of fetal lung epithelia, we have studied the expression of the mammalian homologs of achaete-scute complex (Mash1) (Ascl1 - Mouse Genome Informatics); hairy and enhancer of split1 (Hes1); and the expression of Notch/Notch-ligand system in the fetal and adult mouse lungs, and in the lungs of Mash1- or Hes1-deficient mice. Immunohistochemical studies revealed that Mash1-positive cells seemed to belong to pulmonary neuroendocrine cells (PNEC) and their precursors. In mice deficient for Mash1, no PNEC were detected. Hes1-positive cells belong to non-neuroendocrine cells. In the mice deficient in Hes1, in which Mash1 mRNA was upregulated, PNEC appeared precociously, and the number of PNEC was markedly increased. NeuroD (Neurod1 - Mouse Genome Informatics) expression in the lung was detected in the adult, and was enhanced in the fetal lungs of Hes1-null mice. Expression of Notch1, Notch2, Notch3 and Notch4 mRNAs in the mouse lung increased with age, and Notch1 mRNA was expressed in a Hes1-dependent manner. Notch1, Notch2 and Notch3 were immunohistochemically detected in non-neuroendocrine cells. Moreover, analyses of the lungs from the gene-targeted mice suggested that expression of Delta-like 1 (Dll1 - Mouse Genome Informatics) mRNA depends on Mash1. Thus, the neuroendocrine differentiation depends on basic helix-loop-helix factors, and Notch/Notch-ligand pathways may be involved in determining the cell differentiation fate in fetal airway epithelium.     

Jmjd3 activates Mash1 gene in RA‐induced neuronal differentiation of P19 cells

Covalent modifications of histone tails have fundamental roles in chromatin structure and function. Tri‐methyl modification on lysine 27 of histone H3 (H3K27me3) usually correlates with gene repression that plays important roles in cell lineage commitment and development. Mash1 is a basic helix‐loop‐helix regulatory protein that plays a critical role in neurogenesis, where it expresses as an early marker. In this study, we have shown a decreased H3K27me3 accompanying with an increased demethylase of H3K27me3 (Jmjd3) at the promoter of Mash1 can elicit a dramatically efficient expression of Mash1 in RA‐treated P19 cells. Over‐expression of Jmjd3 in P19 cells also significantly enhances the RA‐induced expression and promoter activity of Mash1. By contrast, the mRNA expression and promoter activity of Mash1 are significantly reduced, when Jmjd3 siRNA or dominant negative mutant of Jmjd3 is introduced into the P19 cells. Chromatin immunoprecipitation assays show that Jmjd3 is efficiently recruited to a proximal upstream region of Mash1 promoter that is overlapped with the specific binding site of Hes1 in RA‐induced cells. Moreover, the association between Jmjd3 and Hes1 is shown in a co‐Immunoprecipitation assay. It is thus likely that Jmjd3 is recruited to the Mash1 promoter via Hes1. Our results suggest that the demethylase activity of Jmjd3 and its mediator Hes1 for Mash1 promoter binding are both required for Jmjd3 enhanced efficient expression of Mash1 gene in the early stage of RA‐induced neuronal differentiation of P19 cells. J. Cell. Biochem. 110: 1457–1463, 2010. © 2010 Wiley‐Liss, Inc.     

Basic helix-loop-helix proteins and the timing of oligodendrocyte differentiation

An intracellular timer in oligodendrocyte precursor cells is thought to help control the timing of their differentiation. We show here that the expression of the Hes5 and Mash1 genes, which encode neural-specific bHLH proteins, decrease and increase, respectively, in these cells with a time course expected if the proteins are part of the timer. We show that enforced expression of Hes5 in purified precursor cells strongly inhibits the normal increase in the thyroid hormone receptor protein TR(&bgr;)1, which is thought to be part of the timing mechanism; it also strongly inhibits the differentiation induced by either mitogen withdrawal or thyroid hormone treatment. Enforced expression of Mash1, by contrast, somewhat accelerates the increase in TR(beta)1 protein. These findings suggest that Hes5 and Mash1 may be part of the cell-intrinsic timer in the precursor cells.     

Premature differentiation and aberrant movement of pituitary cells lacking both Hes1 and Prop1

In the pituitary, the transition from proliferating progenitor cell into differentiated hormone producing cell is carefully regulated in a time-dependent and spatially-restricted manner. We report that two targets of Notch signaling, Hes1 and Prop1, are needed to maintain progenitors within Rathke's pouch and for the restriction of differentiated cells to the ventral pituitary. We observed ACTH and αGSU producing cells that had prematurely differentiated within Rathke's pouch along with correlated ectopic expression of Mash1 only when both Prop1 and Hes1 were lost. We also discovered that downregulation of N-cadherin expression in cells as they transition from Rathke's pouch to the anterior lobe appears to be essential for their movement. In the Prop1 mutant, cells are trapped in Rathke's pouch and N-cadherin expression remains high. Also, Slug, a marker of epithelial-to-mesenchymal transition, is absent in the dorsal anterior lobe. When Hes1 is lost in the Prop1 mutant, N-cadherin is downregulated and cells are able to exit Rathke's pouch but have lost their migrational cues and form ectopic foci surrounding Rathke's pouch. Our data reveal important overlapping functions of Hes1 and Prop1 in cell differentiation and movement that are critical for pituitary organogenesis.