目的 研究DNA聚合酶δ催化亚基基因1(POLD1)反义RNA对人肝癌细胞SMMC-7721的抑制作用及相应机制.方法 将SMMC-7721细胞分为3组:实验组、阴性对照组、空白组.实验组及阴性对照组分别将POLD1基因反义RNA表达质粒及空质粒分别转染至SMMC-7721细胞,未经处理的SMMC-7721细胞为空白组.用聚合酶链反应法检测各组细胞POLD1基因表达情况;用细胞计数-8(CCK-8)法检测细胞增殖情况;用流式细胞术分析细胞凋亡率;用蛋白质印迹(Western blot)法检测p53、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)等凋亡相关蛋白表达量.结果 实验组、阴性对照组和空白组肝癌细胞POLD1基因相对表达量分别为0.18±0.03,1.03±0.18和1.00±0.00;转染72 h增殖情况(OD450值)分别为0.57±0.06,0.73±0.09和0.77±0.12;凋亡率分别为(23.43±4.12)％,(9.12±1.03)％,(1.24±0.14)％,实验组的上述指标与阴性对照组和空白组比较,差异均有统计学意义(均P<0.05).结论 POLD1基因反义RNA可显著抑制人肝癌细胞SMMC-7721的增殖,且能诱导人肝癌细胞SMMC-7721凋亡.

目的 观察氧化苦参碱(oxymatrine,OMT)联合全反式维甲酸(all-trans retinoic acid,ATRA)抑制二乙基亚硝胺(DEN)诱导的大鼠肝癌形成效果,并探讨其作用机制.方法 采用DEN诱导建立肝癌大鼠模型,造模18周,将Wistar大鼠随机分为空白组、模型组、干预组,分别在诱癌后6、12、18周处死大鼠,通过HE染色观察大鼠肝组织病理变化,检测大鼠肝功能相关指标,ELISA检测大鼠IL-6含量;采用RT-qPCR法检测大鼠let-7a、NF-κB-p65、IL-6,STAT3 mRNA表达变化;采用Western blot检测大鼠STAT3、p-STAT3的蛋白变化.结果 HE染色病理学观察显示,干预组大鼠肝癌结节数明显少于模型组,肝细胞病变坏死减轻;与模型组比较,干预组大鼠血清IL-6及肝功能指标ALT、GGT、AST、ALP水平均明显降低(P<0.05);RT-qPCR检测结果:与模型组比较,干预组大鼠肝组织中let-7a mRNA表达水平明显上升,而NF-κB-p65、IL-6和STAT3 mRNA表达量均明显降低(P<0.05).实验第18周,Western blot检测结果干预组STAT3、p-STAT3蛋白表达均明显低于模型组(P<0.05).结论 氧化苦参碱联合全反式维甲酸能够明显抑制DEN诱发的大鼠肝癌形成,在一定程度上延缓肿瘤生长.

Objective To investigate the feasibility of RNA interfering ( RNAi ) on liver cancer by using this technique to inhibit the expression of POLD 1 gene in human liver cancer cells SMMC-7721.Methods Four expression plasmids with shRNAs targeting POLD 1 gene were constructed ,and then the plasmids were transfected to SMMC-7721 for 48 hours.The expression of POLD1 gene was detected by real-time RT-PCR.CCK-8 assay was used to analyze the change of cell proliferation .Results The four expression plasmids were constructed successfully , confirmed by sequencing .After the plasmids were transfected to SMMC-7721 , the cell proliferation was significantly inhibited in NEO-POLD1-1 and NEO-POLD1-4.The mRNA expression of POLD1 gene in NEO-POLD1-1 and NEO-POLD1-4 were significantly inhibited as (0.1425 ±0.0205) and (0.209 ±0.009).Conclusion shRNAs targeting the POLD1 gene can inhibit the mRNA expression of POLD1 gene and liver cancer cell proliferation in vitro ,and the RNA interfering is sequencing alternative .

Nitidine chloride(Nitidine chloride,NC) is isolated from the foot of Zanthox-ylum nitidum(Roxb.)DC.It has good anti-tumor activity,and can inhibit cell proliferation of nasopharyngeal carcinoma,lung cancer in vitro.Studies have shown that NC can result in blocking the cell cycle G2/M period,and the induction of apoptosis.E2F/RB played an important role in cell cycle regulation,with CycLinD1,CDK4,CyclinE,and CDK2 complex feedback controlling the network.Arious chromatin regulatory complexes have been linked to RB/E2F proteins adjusting the cell from G1 to S phase transition.In addition,E2F is also an important transcription factor,which has traditionally been viewed in the context of cell-cycle control and involved in transcriptional regulation of the genes.The molecular changes of E2F/RB pathway in SMMC-7721 cells treated with Nitidine chloride(NC) were studied and the role of E2F/RB pathway in NC inhibiting the proliferation and participating the apoptosis of SMMC-7721 cells was illustrated.The effects of nitidine chloride on human Hepatoma SMMC-7721 cell in vitro were assayed by cell counting Kit-8.CCK8 was used to detect the cell survival level effects of NC on SMMC-7721 cells.Tumor cell apoptosis and necrosis were studied by Annexin-V and propidium iodide(PI).E2F and RB mRNA levels were quantified by real-time fluorescence quantitative PCR after detecting E2F protein expression levels by Western Blot.Results showed that NC was obviously inhibiting the proliferation of human hepatoma cells,reducing the cell survival rate,and promoting the cell apoptosis significantly.The mRNA and protein expression levels of E2F,RB were up-reduced after SMMC-7721 cells were exposed to NC.NC plays an important role on the E2F/RB regulatory pathway,inhibits the expression of E2F/RB and participates in the inhibition of apoptosis of cell growth.

Objective To analyze mutations in the protein-coding region of the POLD1 gene in the human hepatocarcinoma cell line SMMC-7721;this gene encodes an enzyme critical for DNA replication. Methods Total RNA was isolated from SMMC-7721 cultures,and the POLD1 gene coding region was amplified by RT-PCR,purified and A-tailed, and then cloned into a T-vector. Mutations were detected by sequencing. Results The protein-coding region of the POLD1 gene was successfully amplified and found to contain 12 mutations,11 of which were nucleotide changes and 1 was a nucleotide deletion. Conclusions SMMC-7721 en-codes a mutated POLD1 gene, and the mutations lead to changes in the protein sequence.

OBJECTIVE:To inhibit the expression of POLD1 gene by antisense RNA in the cells of human liver cancer SMMC-7721,and explore the feasibility of intervention for liver cancer.METHODS: Expression plasmid with human POLD1 antisense RNA was constructed.The blank control group and negative control group were established in the same time.SMMC-7721 cells were divided into three groups:blank control group(untransfected cells),negative control group(cells transfected with blank vector),and experimental group(cells transfected with antisense RNA).MTT assay,RT-PCR and western blot were used to analyze the data.RESULTS:MTT assay showed that cell proliferation was significantly lower in transfected group.Compared with negative control group and blank control group,the A490 value of experimental group was 0.306 7±0.015 4 in 24 h,0.459 2±0.033 2 in 48 h,0.567 1±0.061 1 in 72 h afer transfacting(P0.05).The mRNA expression of POLD1 gene was down-regulated by real-time RT-PCR,which were 0.142 5±0.020 5 in experimental group,1.017±0.188 in negative control group and 1.00±0.00 in blank control group(F=26.5,P0.05).It meant antisense RNA could inhibit the gene expression.The protein expression of POLD1 gene was down-regulating by Western Blot,which were 0.237 9±0.005 9 in negative control group,0.222 3±0.009 7 in experimental group and 0.235 4±0.003 4 in blank control group(F=136 5.754,P0.05).Similar results were also obtained for the protein expression levels of p125.It means antisense RNA can inhibit the protein expression.CONCLUSION:Antisense RNA could inhibit the proliferation of human liver cancer cell SMMC-7721 possibly by down-regulating POLD1 expression.

Objective To construct the eukaryotic expression plasmid BFP-cyclin D1 and investigate the effects of cyclin D1 on the proliferation and migration of MCF-7 cells.Methods The cyclin D1 gene was amplified by RT-PCR from total RNA of MCF-7 cells,digested with EcoR I and Sal I,and inserted into the eukaryotic florescence expression vector pEBFP-N1.The resulting recombinant plasmid BFP-cyclin D1 was transfected into MCF-7 cells.In parallel,one negative control group was transfected with pEBFP-N1 and another with transfection reagent without any DNA.Following transfection,cell proliferation was detected by MTT assay,cell migration by cell scratch assay,and mRNA expression by quantitative real-time PCR.Results The eukaryotic expression plasmid BFP-cyclin D1 was successfully constructed and high expression of cyclin D1 was detected in MCF-7 cells.Cell proliferation and migration were significantly higher in the cells transfected with BFP-cyclin D1 than in the two control groups(P0.01).Conclusions High expression of cyclin D1 can accelerate proliferation and migration of MCF-7 cells,perhaps by shortening the cell cycle and promoting DNA replication.

目的:构建包含突变型CDK4基因的真核绿色荧光表达载体,作为POLD1基因依赖的细胞周期复制调控的模型,研究POLD1基因相关的癌性增殖的机制,为干预细胞恶性增殖提供新的思路.方法:设计人突变型CDK4基因全长特异性引物进行PCR扩增人肝癌细胞系SMMC-7721总cDNA,以pEGFP-Cl质粒为模板连接,得到重组质粒GFP-CDK4后进行测序和生物信息学比对分析；转染细胞分3组:实验组(转染突变型CDK4重组真核表达质粒GFP-CDK4),阴性对照组(转染空载体pEGFP-Cl组)和空白对照组(SMMC-7702).通过MTT试验分析细胞增殖变化:实时荧光定量PCR技术检测CDK4、POLDl及细胞周期相关因子的表达量,Western blot检测蛋白表达的差异.结果:成功构建了人突变型CDK4基因真核表达质粒GFP-CDK4,转染到肝细胞SMMC-7702后使细胞表达融合绿色荧光的CDK4蛋白；SMMC-7721细胞中突变型的CDK4存在5个碱基突变,4个碱基插入,2个碱基缺失,这使得5个氨基酸序列发生了改变；与空白对照组及阴性对照组相比,实验组细胞增殖明显升高(0.826±0.08 vs 0.596±0.06,0.609±0.10,F=7.033,均P＜0.05)；实验组CDK4 mRNA表达水平差异明显(1.94±0.11 vs 1.01±0.00,1.05±0.12,F=54.046,P＜0.01),POLD1 mRNA相应地升高(2.47±0.25 vs 1.16±0.00,1.26±0.23,F=135.496,P＜0.01)；稳定转染细胞的蛋白水平变化趋势与基因相同,其中实验组CDK4(0.65±0.03 vs 0.41±0.03,0.39±0.05,F=14.665,均P＜0.05),P125(0.54±0.04 vs 0.30±0.07,0.25±0.06,F=11.788,均P＜0.05).结论:人突变型CDK4基因的真核表达载体GFP-CDK4显著促进肝细胞的增殖能力,这与POLD1基因及P125蛋白的高表达相关.