目的:探讨淫羊藿苷(ICA)对乳腺癌细胞MCF-7的作用,以及ICA对表皮生长因子(EGF)诱导的MCF-7细胞上皮间质转化(EMT)的作用及机制.方法:CCK-8法检测ICA对乳腺癌细胞MCF-7存活能力的影响并确定后续试验的干预浓度;设立空白对照组、EGF刺激组、EGF+ICA组;划痕实验、Transwell迁移及侵袭实验检测EGF及ICA+EGF对MCF-7细胞迁移及侵袭能力的影响;Western Blot检测AKT、p-AKT、N-cadherin、E-cadherin、Vimentin蛋白表达水平.结果:ICA对乳腺癌细胞MCF-7有杀伤作用,且呈浓度依赖性;各组MCF-7细胞24h愈合率由高到低依次为:EGF刺激组＞EGF+ICA组＞空白对照组(P＜0.01);各组穿过膜的细胞数由高到低依次为:EGF刺激组＞EGF+ICA组＞空白对照组.与空白对照组比较,EGF刺激组p-AKT、N-cadherin、Vimentin蛋白表达量显著增加(P＜0.01),E-cadherin蛋白表达量显著减少(P＜0.01).与EGF刺激组比较,EGF+ICA组p-AKT、N-cadherin、Vimentin蛋白表达量显著减少(P＜0.01),E-cadherin蛋白表达量显著增加(P＜0.01).结论:ICA能够抑制乳腺癌细胞MCF-7的增殖.同时ICA还可以抑制EGF所诱导的EMT过程,可能是通过下调PI3K/AKT信号通路实现的.

在新发传染病不断增加的背景下,传统的突发公共卫生事件教学方式已无法满足培养创新、实践能力强的应用型预防医学人才的需求.虚拟仿真实验教学作为传统实验教学的有效补充,在医学教育领域的应用已日益广泛.本文对虚拟仿真实验技术在新发传染病暴发疫情处置教学中的优势进行了初步分析,为培养合格的公共卫生高素质创新应用型人才和实现学校精英教育的要求提供理论依据.

探讨胸苷激酶TK1和热休克蛋白HSP90α联合CEA、CA199检测在结直肠癌患者临床诊断中的意义.选取该院接收符合手术治疗的直肠癌患者147例,另选取30例健康体检者为正常对照组,采用酶联免疫吸附法测定血清TK1和血浆HSP90α,以电化学发光法检测血清CEA、CA199.联合临床病理分析通过联合检测分析两者与结直肠癌(CRC)临床病理特征之间的关系,绘制ROC曲线分析联合检测方案对结直肠癌的诊断效能.与正常对照组比较,CRC血清TK1、HSP90α、CEA、CA199水平均有所升高,差异均有统计学意义,P＜0.05.CRC组TK1、HSP90α水平与患者性别、年龄无关,P＞0.05,但与治疗效果相关,P＜0.05.TNM分期Ⅰ+Ⅱ期患者TK1、HSP90α水平低于Ⅲ+Ⅳ期患者,差异有统计学意义,P＜0.05.TK1表达与HSP90α(r=0.108,P＜0.05)、CEA(r=0.100,P＜0.05)、CA199(r=0.308,P＜0.05)表达相关;HSP90α表达与CEA(r =0.144,P＜0.05)、CA199(r =0.216,P＜0.05)表达相关.单项检测诊断效能分析显示,ROC曲线下面积AUC:TK1 (0.904)＞HSP90α(0.862)＞ CA199(0.849)＞ CEA(0.798),其中TK1单项检测的诊断效能最高.TK1、HSP90α联合CEA、CA199检测提高了CRC临床诊断的敏感度和检出率,同时对CRC治疗效果具有一定的指导意义.

随着市场经济与医疗卫生事业的发展,医院定将不负人民重托、无愧历史选择,努力奋斗与拼搏,高度重视和加强廉政文化建设,营造崇廉、尚廉的良好社会氛围,激励全体医务工作者不断奋进、凝聚同心、共筑中国梦的磅礴力量,筑牢医院发展基石,共谋医院持续、和谐及健康发展.

探讨氯化两面针碱(Nitidine chloride,NC)通过影响POLD1基因启动子甲基化率及调节转录调控因子p53和E2F的特异性结合对肝癌SMMC-7721细胞增殖和凋亡的影响.该研究采用CCK8,焦磷酸测序,RT-PCR,Western blot等技术方法论证了NC通过影响POLD1基因启动子甲基化率,间接调控POLD1基因转录活性,从而降低了DNA聚合酶ε的合成效率,抑制肝癌SMMC-7721细胞的增殖.不同剂量的NC(0.5,1.0,2.0 mg/mL)处理肝癌细胞SMMC-7721 48 h后,细胞增殖抑制率逐渐增大,分别为21.1％,35.2％,92.1％,呈明显的剂量依赖性.POLD1基因启动子的甲基化水平整体上升.经方差分析,与对照组相比,1.0,2.0 mg/mL实验组的E2F结合位点甲基化率随浓度增加而增高(F=10.21,P＜0.05),差异有统计学意义,0.5 mg/mL实验组与对照组相比无明显差异(P =0.694).而p53结合位点甲基化率随NC浓度增加呈下降趋势,实验组与对照组有明显差异(F =9.76,P ＜0.05),其中2.0 mg/mL实验组有显著差异(P＜0.01).实验组POLD1 mRNA和蛋白p125随NC浓度增加而下降,采用秩和检验,x2分别为40.19,36.65,P＜0.05,1.0、2.0 mg/mL实验组与对照组相比,差异有统计学意义.结果显示氯化两面针碱可能通过调节SMMC-7721细胞POLD1基因的甲基化水平,影响其启动子转录调控因子的作用,从而抑制了POLD1基因表达,降低DNA聚合酶ε活性,抑制肝癌细胞增殖的作用.

目的：分析和探讨肺癌患者外周血 T 淋巴细胞亚群及调节性 T 细胞的变化。方法选取我院2013年1月至2014年1月收治的70例肺癌患者作为研究对象，对照组为同时期30例健康体检查者，采用流式细胞术对其进行外周血 T淋巴细胞亚群及调节性 T 细胞的检测。结果肺癌患者 T 细胞百分数较高，C D3+、CD3+ CD 4+及 CD16+CD56+ 细胞较健康人明显下降，早中期 CD3+CD8+变化小，晚期细胞明显下降（P<0.05），差异具有统计学意义。结论肺癌患者外周血 T 淋巴细胞亚群及调节性 T 细胞发生了一定的变化，为患者细胞免疫功能的评估提供了科学的参考依据。

Objective To analyze the effect of POLD1 gene antisense RNA on the proliferation of human liver cancer cell SMMC-7721 and human normal liver cell HL-7702 so as to explore the feasibility of POLD1 gene antisense RNA for the treatment of human liver cancer. Methods Expression plasmid with human POLD1 gene antisense RNA was constructed.The experiment consisted of negative control group( HL-7702 cells and SMMC-7721 cells transfected with empty plasmid ) , blank control group ( untransfected HL-7702 cells and SMMC-7721 cells) and experimental group( HL-7702 cells and SMMC-7721 cells transfected with human POLD1 gene antisense RNA) . Cell counting kit-8(CCK-8) assay was used to analyze the cell growth conditions.Results There was no significant difference in the absorbance value(A450 nm) of liver HL-7702 cells 0,24,48 or 72 hours after transfection among three groups(P>0.05).Compared with blank control group and negative control group, experimental group obtained lower absorbance values ( A450 nm ) of liver cancer cells SMMC-7721 24,48 and 72 hours after transfection(P<0.05).Conclusion POLD1 gene antisense RNA can inhibit the proliferation of liver cancer cells while the growth of normal hepatocytes is not affected.

目的 探讨鼻咽癌患者外周血淋巴细胞亚群和NK细胞活性与治疗前后EB病毒IgA/VCA抗体水平高低、是否转移及不同临床分期的关系及其意义.方法 采用流式细胞术(FCM)检测58例鼻咽癌患者治疗前后外周血淋巴细胞亚群和NK细胞的百分含量.结果 ①鼻咽癌患者治疗后和治疗前比较,CD8+细胞水平显著升高,CD4+和CD19+细胞水平和CD4+/CD8+的比值显著下降,差异有统计学意义(P＜0.05),CD3+的差异无统计学意义(P＞0.05);②EB病毒IgA/VCA抗体高滴度和低滴度的鼻咽癌患者比较,在治疗前,NK细胞和CD19+细胞水平显著升高,差异有统计学意义(P＜0.05),其余指标差异无统计学意义(P＞0.05);在治疗后,各项指标差异无统计学意义(P＞0.05);③有淋巴结转移和无淋巴结转移的鼻咽癌患者比较,在治疗前,NK细胞和CD19+细胞水平显著升高,差异有统计学意义(P＜0.05),其余指标差异无统计学意义(P＞0.05);在治疗后,各项指标差异无统计学意义(P＞0.05);④Ⅲ～Ⅳ期和Ⅰ～Ⅱ期的鼻咽癌患者比较,在治疗前,CD19+细胞水平显著升高,差异有统计学意义(P＜0.05),其余指标差异无统计学意义(P＞0.05);在治疗后,各项指标差异无统计学意义(P＞0.05).结论 鼻咽癌患者机体细胞免疫状态与机体是否存在局部淋巴结和远处转移无明显相关性,但在进行治疗后机体细胞免疫抑制状态较治疗前明显改善.

目的探讨氯化两面针碱（nitidine chloride,NC）对肝癌SMMC-7721细胞POLD1基因甲基化及细胞增殖的影响。方法不同剂量的NC（0.5、1.0和2.0mg/mL）作用于人肝癌SMMC-7721细胞48h后,采用焦磷酸技术、RT-PCR和CCK8方法分别测定不同剂量的NC对SMMC-7721细胞中POLD1基因甲基化率,mRNA表达及细胞增殖抑制率的影响。结果不同剂量的NC处理肝癌细胞SMMC-7721 48h后,实验组POLD1基因的甲基化水平整体上升,对照组与实验组甲基化率分别为4.26±0.49、4.14±0.75、4.90±0.64和5.67±0.77。采用方差分析,组间两两比较,1.0、2.0mg/mL实验组与对照组相比较,甲基化率有明显的上升,差异有统计学意义,P值分别为0.041、0.001;0.5mg/mL实验组与对照组相比差异无统计学意义,P＞0.05。实验组间两两比较,差异有统计学意义,P值＜0.05。对照组和实验组的POLD1基因mRNA相对表达量分别为1.02±0.81、0.83±0.19、0.44±0.09和0.49±0.23,呈下降趋势。秩和检验分析,组间表达差异明显;1.0、2.0mg/mL实验组与对照组相比,差异有统计学意义,P＜0.001;0.5mg/mL实验组与对照组相比差异无统计学意义,P＞0.05。实验组间两两比较,差异有统计学意义,P＜0.05。随着NC浓度的增加,细胞增殖抑制率逐渐增大,分别为21.1%、35.2%和92.1%,呈明显的剂量依赖性。结论 NC可能通过调节SMMC-7721细胞POLD1基因的甲基化水平,增强其启动子的甲基化,抑制POLD1基因表达,从而降低DNA聚合酶δ活性,达到抑制肝癌增殖的作用。

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 .

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.

DNA聚合酶δ是参与真核生物DNA合成、损伤与修复、细胞周期调控等过程的重要聚合酶之一.近期有研究发现原发性肝癌组织中DNA聚合酶δ催化亚基p125表达水平较癌旁组织高,也有研究发现肺癌组织中DNA聚合酶δ第4个亚基p12低表达,且在结肠癌组织及细胞系中发现DNA聚合酶δ催化亚基p125的编码基因POLD1发生突变.这些均说明DNA聚合酶δ的异常与肿瘤的发生、发展有着非常重要的关系.

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.