实时荧光定量PCR 检测胃癌SOX2的 表达及其临床意义

目的： 探讨性别决定相关基因簇2(SOX2)表达与胃癌发生、发展和转移的关系及其临床意义。方法：用Trizol提取52例胃癌组织及对应正常胃黏膜组织的mRNA,并将其逆转录为cDNA。用实时荧光定量PCR方法检测SOX2 mRNA的表达,并分析SOX2基因的表达水平与患者临床病理指标的关系。结果：胃正常黏膜组织中SOX2 mRNA的表达是胃癌组织的2.24倍,两者差异有统计学意义(t=3.22,P=0.002 6)。高分化和中分化的胃癌组织中SOX2 mRNA表达量高于低分化的胃癌组织(t=3.09和2.87,P均<0.05),肿瘤浸润深度为T1+T2的胃癌组织中SOX2 mRNA表达量是T3+T4癌组织的2.57倍(t=2.16,P<0.05),淋巴结无转移组SOX2 mRNA表达量是转移组的2.69倍(t=2.54,P<0.05)。与Ⅰ+Ⅱ期的胃癌组织比较,Ⅲ+Ⅳ期胃癌组织的SOX2 mRNA表达量明显降低(t =2.59,P=0.014)。SOX2 mRNA表达量与患者的性别、年龄无明显关系(P>0.05)。结论：SOX2 mRNA表达水平的降低与胃癌的发生、浸润和转移相关,有望成为胃癌诊断及预后判断的一个指标。     

非小细胞肺癌组织中OCT4和miRNA-155的表达及其与临床病理特征的关系

目的探讨非小细胞肺癌组织中OCT4蛋白、miRNA-155的表达及其与肺癌临床病理特征的关系。方法采用免疫组织化学及RT-qPCR方法,检测60例非小细胞肺癌患者组织中OCT4蛋白和miRNA-155的表达情况,并分析其与临床病理特征相互关系。结果免疫组织化学检测发现OCT4蛋白在肿瘤组织中的阳性表达率为61.67%。OCT4蛋白在高分化、中分化和低分化的肺癌组织中的阳性表达差异有统计学意义(P=0.021),随着癌组织分化程度的升高,OCT4的表达率逐渐下降。OCT4蛋白在有无淋巴结转移两组中的阳性表达差异有统计学意义(P=0.028)。OCT4的表达在临床病理分期中有统计学意义(P=0.049)。miRNA-155在非小细胞肺癌组织中均有不同程度表达,相对Ct值中位数是20.48。miRNA-155与高分化、中分化和低分化的肺癌组织中的表达有统计学意义(P=0.016),随着癌组织分化程度的升高,miRNA-155相对Ct值减小,说明其表达量升高。miRNA-155在OCT4阳性组中的表达量低于OCT4阴性组。结论非小细胞肺癌组织中OCT4蛋白的表达与组织分化程度、淋巴结转移和临床分期相关。miRNA-155在OCT4蛋白阳性组的表达水平低于OCT4蛋白阴性组(P<0.0001)。miRNA-155和OCT4可能是非小细胞肺癌患者在基因和蛋白两个水平的反方向的改变,并且OCT4蛋白的表达受miRNA-155的影响。     

肿瘤干细胞标志物CD133、CD44、SOX2、OCT4、ALDH1在非小细胞肺癌组织中的表达及临床意义北大核心CSCD

目的研究肿瘤干细胞标记物CD133、CD44、SOX2、OCT4、ALDH1在非小细胞肺癌(NSCLC)组织中的表达及临床意义,为探索非小细胞肺癌肿瘤干细胞提供参考。方法采用免疫组织化学方法检测70例NSCLC组织、14例非癌组织中的CD133、CD44、SOX2、OCT4、ALDH1蛋白的表达并对结果进行分析。结果 (1)CD133、CD44、SOX2、OCT4、ALDH1在70例NSCLC组织中的阳性表达率分别为88.57%、98.57%、100%、100%、100%,强阳性表达率分别为48.57%、67.14%、67.14%、31.43%、50%;CD133、C C D 4 4在N S C L C与非癌组织中的表达差异存在统计学意义(P均<0.0001),SOX2、OCT4、ALDH1在NSCLC与非癌组织中的表达差异无统计学意义(P均>0.05)。(2)随着病理级别的升高,CD133、CD44、SOX2、OCT4及ALDH1的表达呈上升趋势,分化越低的NSCLC表达上述指标的概率越高,其中CD133、SOX2、OCT4的表达在高、中、低分化组织中差异存在统计学意义(P值分别为0.001、0.040、<0.0001);CD133的表达在吸烟史、分化程度、淋巴结转移、肿瘤分期四个因素上差异存在统计学意义(P值分别为0.033、0.001、0.033、0.046);CD44与SOX2的表达在年龄上的差异存在统计学意义(P值分别为0.001、0.040)。结论 NSCLC组织中CD133、CD44、SOX2、OCT4、ALDH1阳性率高,CD133、CD44的表达明显高于非癌组织;CD133、SOX2、OCT4与NSCLC的恶性程度有关;CD44与SOX2与年龄因素有关。     

转录因子SOX2在胃癌中的表达及其临床意义

目的 探讨转录因子SOX2在胃癌组织中的表达情况及其与临床病理特征的关系.方法 应用免疫组织化学技术检测67例胃癌组织中SOX2的表达,并选取30例远端切缘胃壁组织作为对照.结果 SOX2在胃癌组织中的阳性表达率为52.24％（35/67）,显著低于对照组胃壁组织的阳性表达率93.33％（28/30）,差异有统计学意义（χ^2=16.326,P＜ 0.01）;在胃癌组织中SOX2的阳性表达率与肿瘤的分化程度、浸润深度、淋巴结转移以及临床分期有关,差异均有统计学意义（均P＜ 0.05）.结论 SOX2表达下调与胃癌的早期发生有关,并与淋巴结转移及TNM分期密切相关,SOX2可作为预测胃癌预后的生物学指标.     

胃癌组织中Survivin、COX-2蛋白表达及其与肿瘤血管生成的关系

目的:探讨Survivin,COX-2蛋白在胃癌组织中的表达及其与临床病理特征和肿瘤血管生成的关系.方法: 用免疫组化方法对65例胃癌组织Survivin,COX-2,CD34的表达进行检测.结果: 胃癌组织中Survivin蛋白阳性表达率为 70.77%(46/65),COX-2蛋白阳性表达率为76.93%(50/65).Survivin表达与分化程度和淋巴结转移有关(P<0.05),而与临床分期无关(P>0.05);COX-2的阳性表达与分化程度、临床分期和淋巴结转移有关(P<0.05).Survivin表达与COX-2的表达密切相关(r=0.235,P<0.05).Survivin阳性表达组织中 的MVD值为103.04±15.06,阴 性组为81.89±12.15,二者有显著差异(P<0.01).COX-2阳性表达胃癌组织中的MVD值为104.82±14.05,阴性组为81.02±13.73,二者有显著差异(P<0.01).结论: Survivin和COX-2对凋亡的抑制的协同作用及其促进血管生成的作用在胃癌的发生发展中起关键作用.     

胃癌组织中mRNA-375基因高甲基化及其表达的研究

目的:探讨胃癌组织中miRNA-375基因表达与基因甲基化调控的相关性.方法:2011年3月至8月在天津医科大学总医院通过胃镜检查收集90例新鲜组织活检标本,分为2组,胃癌组54例,非癌对照组36例.应用实时荧光定量反转录PCR检测miRNA-375基因表达,甲基化特异性PCR检测miRNA-375基因启动子区CpG岛甲基化.结果:胃癌组miRNA-375基因表达下调,与非癌对照组相比差异有统计学意义(P<0.05);胃癌组和非癌对照组 miRNA-375基因启动子区高甲基化阳性率分别为62.96%(34/54)和22.22%(8/36),差异有统计学意义(χ2=14.405, P<0.05).中高分化胃癌组织中miRNA-375基因表达高于低分化组,差异有统计学意义(t=2.634,P=0.011);miRNA-375基因启动子区甲基化阳性率中高分化组与低分化组分别为44.44%(8/18)和72.22%(26/36),差异有统计学意义(χ2=3.971,P=0.046).结论:癌组织中存在miRNA-375基因异常低表达及启动子区的高甲基化,miRNA-375基因高甲基化可能抑制miRNA-375基因表达,在胃癌发生发展中发挥重要作用.     

胃癌中Cks1、P27Kip1、Skp2蛋白表达相关性及其意义

目的:探讨Cks1在胃癌发生及Skp2调节P27Kip1降解过程中的作用。方法:应用流式细胞术检测正常胃黏膜、胃不典型增生组织及胃癌组织中Cks1、P27Kip1、Skp2蛋白的表达。结果:由正常胃黏膜、胃不典型增生组织到胃癌中Cks1、Skp2的表达均呈上升趋势(P<0.05),P27Kip1表达则呈下降趋势(P<0.05)。胃癌中Cks1、Skp2表达均与P27Kip1表达呈负相关(r=-0.649,P<0.05;r=-0.732,P<0.05);而Cks1蛋白表达与Skp2蛋白无相关性(P>0.05)。胃癌中Cks1表达与肿瘤分化程度相关(P<0.05),而与肿瘤浸润深度、淋巴结转移及临床分期不相关(P>0.05)。结论:Cks1可能参与胃癌的发生;胃癌中Cks1可能参与Skp2调节P27Kip1泛素化的降解过程。     

自噬因子Beclin 1和LC3表达与胃癌临床病理特征的相关性研究

目的 探讨自噬因子Beclin 1和LC3表达与胃癌临床病理特征的相关性.方法 采用免疫组织化学方法,检测胃癌组织中自噬因子Beclin 1和LC3的表达情况.结果 Beclin 1蛋白在胃癌组织中的表达率为42.5%(51/120),在癌旁组织其表达率为93.3%(112/120),两者比较有显著性差异(χ2=21.3,P<0.05);LC3蛋白在胃癌组织中的表达率为90.0%(108/120),明显高于癌旁组织的表达率(29.3%,34/120),两者有显著性差异(χ2=24.8,P<0.05).Beclin 1和LC3蛋白表达与胃癌肿瘤分化程度和淋巴结转移密切相关,P<0.05.结论 自噬因子Beclin1和LC3表达可能与胃癌的发生发展有关.     

同源盒转录因子CDX2与Y染色体决定因子SOX9在肠上皮化生及胃癌组织中的表达

目的:研究同源盒转录因子CDX2和Y染色体决定因子SOX9在肠上皮化生(intestinal metaplasia,IM)及胃癌组织中的表达,探讨CDX2和SOX9在IM发生、进展及胃癌发生、发展中的作用及相互关系.方法:选取慢性萎缩性胃炎(chronic atrophic gastritis,CAG)伴IM 24例、癌旁IM 17例、胃癌组织50例,用HE染色对IM及胃癌进行组织学分型,免疫组化法观察CDX2、SOX9蛋白在上述各种胃黏膜组织中的表达,研究二者表达与胃癌临床病理学特征之间的关系.结果:CDX2蛋白表达阳性率在CAG伴IM组、癌旁IM组和胃癌组分别为87.50％、76.47％和62.00％,CAG伴IM组显著高于胃癌组(P＜0.05),CAG伴IM组与癌旁IM组、癌旁IM组与胃癌组之间比较无统计学差异(P＞0.05),肠型胃癌显著高于弥漫型胃癌(77.78％ vs 43.48％,P=0.013).CDX2蛋白在胃癌中的表达与胃癌临床分期、分化程度、Lauren分型及是否有浆膜浸润等有关.SOX9蛋白表达阳性率,在CAG伴IM组、癌旁IM组及胃癌组分别为54.17％、70.59％、80.00％,CAG伴IM组显著低于胃癌组(P＜0.05),CAG伴IM组与癌旁IM组、癌旁IM组与胃癌组之间比较无统计学差异(P＞0.05),肠型胃癌显著高于弥漫型胃癌(92.59％ vs 65.22％,P=0.040).SOX9蛋白在胃癌中的表达与胃癌分化程度、临床分期、Lauren分型和是否淋巴结转移等有关联.胃癌组织中CDX2和SOX9的阳性表达存在显著性负相关(r=-0.391,X2=5.778,P=0.016).结论:CDX2、SOX9在IM及肠型胃癌的发生中可能有重要作用,可做为IM及肠型胃癌预测的特异性指标之一.二者可能在胃癌进展及浸润转移过程中起重要作用,联合检测对选择合适治疗方式及预后判断具有重要价值.     

PTEN、NFκB、Ki-67在胃癌中的检测及意义

目的探讨PTEN、NFκB、Ki-67的表达与胃癌的关系及作用。方法运用免疫组织化学法检测PTEN、NFκB、Ki-67在胃癌中的表达情况。结果PTEN蛋白在胃癌组织中的阳性表达率为24.2%（15/62）,明显低于癌旁正常胃组织为77.3%（17/22）（P<0.001）;而NFκB、Ki-67蛋白在胃癌中的阳性表达率分别为61.3%（38/62）,67.7%（42/62）,明显高于癌旁正常胃组织的18.2%（4/22）和13.6%（3/22）（P<0.01,P<0.001）。PTEN、NFκB、Ki-67的阳性表达率与临床病理分期、组织分化程度及5年生存期有关,而与性别、年龄无关（P>0.05）;临床病理分期低、组织分化好的患者生存时间往往较长,差异有统计学意义（P<0.05）。此外,胃癌中PTEN与NFκB呈负相关（χ²=6.519,P<0.05）,PTEN与Ki-67呈负相关（χ²=4.022,P<0.05）,NFκB则与Ki-67呈正相关（χ²=5.641,P<0.05）。结论PTEN基因的失活、NFκB基因的活化可能促进了胃癌的发生、发展和浸润转移的进程; Ki-67基因的表达情况不仅能客观反眏胃癌的增殖活性,还能估计肿瘤细胞发生转移的潜在危险性,可作为临床评价肿瘤恶性度及预后的指标。     

Radiation sensitivity of merkel cell carcinoma cell lines

: Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions.     

桥接整合因子1通过AKT-mTOR通路诱导非小细胞肺癌H1975细胞周期阻滞

目的：研究桥接整合因子1（bridging intergrator 1,Bin1）基因过表达后对非小细胞肺癌细胞株H1975细胞周期的影响及其作用机制。方法：构建携带Bin1基因的CMV-MCS-GFP-SV40-Neomycin-Bin1质粒,并转染H1975细胞（Bin1+组）,另设置空白质粒转染组（Bin1-组）及空白对照组（Ctrl组）,利用RT-PCR和Western blotting分别检测3组细胞中Bin1在mRNA和蛋白质水平的表达情况。流式细胞术检测不同处理组H1975细胞周期的变化,Western boltting分别检测各组中AKT、mTOR磷酸化水平及细胞周期相关蛋白（周期蛋白D1、CDK4、Rb）的表达情况。结果：与Bin1-组、Ctrl组比较,Bin1+组H1975细胞中Bin1在mRNA、蛋白水平表达明显上调（均P<0.05）; H1975细胞阻滞在G1期\[（60.53±1.89）% vs（46.14±1.56）%、（47.33±2.07）%,均P<0.05\]; Bin1+组H1975细胞内p-AKT、p-mTOR表达下调（均P<0.05）,AKT、mTOR表达变化无统计学差异（P>0.05）;周期蛋白D1、CDK4的表达量均明显下调(P<0.05),Rb表达量明显增加（P<0.05）。结论：Bin1基因在H1975细胞株过表达后明显诱导细胞周期阻滞,其机制可能是通过抑制AKT-mTOR通路及其细胞周期相关蛋白实现的。     

Inhibition of S-adenosylhomocysteine hydrolase decreases cell mobility and cell proliferation through cell cycle arrest

S-adenosylhomocysteine hydrolase (AHCY) hydrolyzes S-adenosylhomocysteine to adenosine and l-homocysteine, and it is already known that inhibition of AHCY decreased cell proliferation by G2/M arrest in MCF7 cells. However, the previous study has not indicated what mechanism the cell cycle arrest is induced by. In this study, we aimed to investigate the different cell cycle mechanisms in both p53 wild-typed MCF7 and p53 mutant-typed MCF7-ADR by suppressing AHCY. We extensively proved that AHCY knockdown has an anti-proliferative effect by using the WST-1 assay, BrdU assay, and cell cytometry analysis and an anti-invasive, migration effect by wound-healing assay and trans-well analysis. Our study showed that down-regulation of AHCY effectively suppressed cell proliferation by regulating the MEK/ERK signaling pathway and through cell cycle arrests. The cell cycle arrest occurred at the G2/M checkpoint by inhibiting degradation of cyclinB1 and phosphorylation of CDC2 in MCF7 cells and at the G1 phase by inhibiting cyclinD1 and CDK6 in MCF7-ADR cells. Finally, we determined that AHCY regulates the expression of ATM kinase that phosphorylates p53 and affects to arrest of G2/M phase in MCF7 cells. The findings of this study significantly suggest that AHCY is an important regulator of cell proliferation through different mechanism in between MCF7 and MCF7-ADR cells as p53 status.     

Mechanisms of kaposis-sarcoma cell supernatant-induced vascular cell invasion

Kaposi's sarcoma (KS) is a highly angiogenic lesion frequently associated with acquired immune deficiency syndrome. Histologically the lesions appear to contain proliferative 'spindle shaped' cells with a mixed smooth muscle-endothelial-fibroblastic histotype and a conspicuous neovascularization, derived from host cell recruitment. Media conditioned by cultured KS cells (KS-CM) have angiogenic properties. KS-CM is able to promote endothelial and smooth muscle cell migration and invasion. The mechanisms of this KS-CM activity are still unknown. We hypothesize that KS-CM contains numerous factors with different roles in inducing the neo angiogenic process. We show that AIDS-IST-KS cell supernatants induce gelatinase A production and plasminogen activator (PA) up-regulation in vascular cells. KS-CM activity in vivo is heparin dependent. Also bFGF alone, a heparin dependent factor, alone can induce endothelial and smooth muscle cell invasion, MMP-2 production and PA activity. However, antibodies to bFGF do not block KS-CM activity and do not reduce the effect on PA up-regulation. This evidence suggests that heparin-binding factors other than bFGF may be present. Chromatography of KS-CM on heparin-sepharose demonstrates the presence of two heparin-binding fractions with chemotactic and gelatinase A inducing activity. The flow through was also active. KS-CM absorption on heparin-sepharose beads did not modify its induction of PA activity, further evidence for the presence of non heparin-binding factors as well.     

Landscape of immune cell infiltration in clear cell renal cell carcinoma to aid immunotherapy

The tumor microenvironment, comprised of tumor cells and tumor-infiltrating immune cells, is closely associated with the clinical outcome of clear cell renal cell carcinoma (ccRCC) patients. However, the landscape of immune infiltration in ccRCC has not been fully elucidated. Herein, we applied multiple computational methods and various datasets to reveal the immune infiltrative landscape of ccRCC patients. The tumor immune infiltration (TII) levels of 525 ccRCC patients using a single-sample gene were examined and further categorized into immune infiltration subgroups. The TII score was characterized by distinct clinical traits and showed a significant divergence based on gender, grade, and stage. A high TII score was associated with the ERBB signaling pathway, the TGF-β signaling pathway, and the MTOR signaling pathway, as well as a better prognosis. Furthermore, patients with high TII scores exhibited greater sensitivity to pazopanib. The low TII score was characterized by a high immune infiltration level of CD8⁺ T cells, T follicular helper cells, and regulatory T cells (Tregs). Moreover, the immune check point genes, including CTLA-4, LAG3, PD-1, and IDO1, presented a high expression level in the low TII score group. Patients in the high TII score group demonstrated significant therapeutic advantages and clinical benefits. The findings in this study have the potential to assist in the strategic design of immunotherapeutic treatments for ccRCC.     

NDRG2 suppresses the proliferation of clear cell renal cell carcinoma cell A-498

Background

Recently, the anti-tumor activity of N-myc downstream-regulated gene 2 (NDRG2) was shown decreased expression in clear cell renal cell carcinoma (CCRCC), but the role of the down-expression of NDRG2 has not been described.

Methods

The NDRG2 recombinant adenovirus plasmid was constructed. The proliferation rate and NDRG2 expression of cell infected with recombinant plasmid were mesured by MTT, Flow cytometry analysis and western blot.

Results

The CCRCC cell A-498 re-expressed NDRG2 when infected by NDRG2 recombinant adenovirus and significantly decreased the proliferation rate. Fluorescence activated cell sorter analysis showed that 25.00% of cells expressed NDRG2 were in S-phase compared to 40.67% of control cells, whereas 62.08% of cells expressed NDRG2 were in G1-phase compared to 54.39% of control cells (P < 0.05). In addition, there were much more apoptotic cells in NDRG2-expressing cells than in the controls (P < 0.05). Moreover, upregulation of NDRG2 protein was associated with a reduction in cyclin D1, cyclin E, whereas cyclinD2, cyclinD3 and cdk2 were not affected examined by western blot. Furthermore, we found that p53 could upregulate NDRG2 expression in A-498 cell.

Conclusions

We found that NDRG2 can inhibit the proliferation of the renal carcinoma cells and induce arrest at G1 phase. p53 can up-regulate the expression of NDRG2. Our results showed that NDRG2 may function as a tumor suppressor in CCRCC.     

肿瘤细胞自噬的诱导及其细胞周期分析

背景与目的:自噬作为Ⅱ型程序性死亡——自噬性死亡过程中的主要现象,与细胞的自噬性死亡有着密切的关系。研究者们对凋亡与细胞周期的关系进行了深入而细致的研究,但对自噬性细胞死亡与细胞周期的关系却知之甚少。本研究的目的是探讨不同方法诱导的细胞自噬与细胞周期之间的关系。方法:用Hanks’液替代培养基的饥饿诱导和用长春新碱诱导两种方法分别处理对数生长期的HeLa细胞、SW480细胞以及经过和未经过植物血凝素(phytohemagglutinin,PHA)刺激的健康人外周血淋巴细胞;应用激光共聚焦显微镜和透射电镜检测细胞自噬的发生,兔抗人微管相关蛋白1轻链3Ⅱ(microtubule-associatedprotein1lightchain3,MAP1-LC3-Ⅱ)/DNA双参数流式细胞术分析自噬细胞的细胞周期。结果:HeLa细胞和SW480细胞用饥饿和长春新碱两种方法诱导的细胞自噬在G1、S、G2/M期均可以发生,且自噬发生率随诱导时间的延长逐渐增加。处于静止期(未经PHA刺激)的外周血淋巴细胞没有自噬的发生,48h时LC3-Ⅱ表达率<2.62%(HanksL液饥饿诱导)或<6.16%(长春新碱诱导);经PHA刺激48h进入细胞周期的外周血淋巴细胞,2h时已有明显的自噬发生。结论:MAP1-LC3-Ⅱ/DNA双参数流式细胞术是对细胞自噬与细胞周期进行同步分析的一种新的简便可靠的方法;细胞自噬只发生在细胞进入周期后,而静止期细胞对自噬诱导因素不敏感。