增强子结合蛋白C/EBPα对白血病BALB/c裸鼠的肿瘤抑制作用

目的 探讨增强子结合蛋白C/EBPα在白血病裸鼠体内的肿瘤抑制作用.方法 将30只BALB/c裸鼠随机分转染组(10只)、空载组(10只)、对照组(10只),建立皮下瘤模型,并将30只BALB/c裸鼠如上分组建立白血病模型,将C/EBPα稳定表达细胞株pEGFP-C/EBPα-K562、空载细胞株pEGFP-K562及白血病细胞株K562分别经皮下和尾静脉注射到相应组裸鼠体内,形成皮下瘤和血液病模型.观测皮下肿瘤的变化,应用TUNEL检测细胞的凋亡,瑞特-吉姆萨染色观察血液病模型裸鼠外周血和骨髓中白血病细胞增殖能力,RT-PCR检测增殖相关基因的表达.结果 皮下瘤模型中pEGFP-C/EBPα-K562组肿瘤质量及最大直径为(24±0.1)g和(11±2)mm,空载组和对照组分别为(5.1±0.3)g、(19±3)mm和(5.7±0.4)g、(23±3)mm(均P＜0.05),TUNEL检测发现pEGFP-C/EBPα-K562组肿瘤细胞凋亡明显增加(P＜0.05);血液病模型鼠外周血可见白血病细胞,pEGFP-C/EBPα-K562组白血病细胞增殖能力明显低于空载组和对照组,可见明显细胞分化现象,RT-PCR检测发现p53基因上调和c-myc基因下调.结论 增强子结合蛋白C/EBPα在白血病小鼠体内具有促进肿瘤细胞凋亡和抑制白血病细胞增殖的能力,并能促进白血病细胞分化.C/EBPα的白血病抑制作用可能是通过对相应基因的调控来实现.     

急性白血病组蛋白乙酰化修饰及其对错配修复基因表达的调控作用

目的 探讨急性白血病患者组蛋白乙酰化修饰规律,并探索组蛋白乙酰化对错配修复基因hMSH2和hMLH1差异表达的调控作用.方法 用反转录-聚合酶链反应(RT-PCR)方法检测56例急性白血病患者和30名健康志愿者单个核细胞(MNC)的错配修复基因hMSH2和hMLH1 mRNA的表达,用Western blot法检测组蛋白H3、H4、去乙酰化酶(HDAC1)、hMSH2和hMLH1基因的蛋白表达情况.用组蛋白去乙酰转移酶抑制剂(TSA)诱导30例白血病患者MNC乙酰化,并检测处理后MNC的组蛋白H3、H4、HDAC1、hMSH2和hMLH1的表达状态变化.结果 急性白血病组的hMSH2和hMLH1、组蛋白H3、H4的蛋白表达量分别为0.4610±0.1211、0.4013±0.1143、0.4103±0.1241和0.4251±0.1081,均明显低于健康志愿者组的蛋白表达量(0.9461±0.1841、0.9960±0.2021、0.8971±0.1194、0.9513±0.1953),差异均有统计学意义(t值分别为3.341、3.935、2.843、3.575,P＜0.05);而急性白血病患者组的HDAC1表达(0.8841±0.2018)高于健康志愿者组的表达量(0.5142±0.1340),差异有统计学意义(t=2.634,P＜0.05);TSA作用于白血病单个核细胞后,组蛋白H3、H4、hMSH2和hMLH1的表达上调,分别比阴性对照组表达上调2.9倍、3.4倍、1.5倍和1.6倍,而HDAC1的蛋白表达出现明显的抑制,表达下调为阴性对照组的40%.结论 急性白血病患者的组蛋白乙酰化呈低表达现象,组蛋白乙酰化在急性白血病患者中对错配修复基因差异表达具有调控作用.     

Ras-p21,p53在阴茎癌腹股沟淋巴结中的表达及临床意义

Objective: The aim of this study was to investigate the expression and clinical significance of ras-p21 and p53proteins in inguinal lymph nodes with penis carcinoma. Methods: The clinical data of 44 patients of penis (squamous) carcinoma and 40 non-tumor patients from 1990 to 2002 in our hospital were added to our research, 84 inguinal lymph nodes were got by lymph node biopsy from each patient at random. Pathological examination showed that 18 cases of cancer group were metastatic carcinoma as group A, the other 26 cases were inflammatory affection as group B. 20 cases of non-tumor group were nonspecific inflammatory inguinal lymph nodes as group C and the other 20 cases were normal lymph nodes as group D, all the 84 cases in our research were investigated by immunohistochemistry to detect the expression of ras-p21 and p53protein. Results: Immunohistochemistry demonstrated that the expression of as-p21 and p53 protein were significantly higher in cancer group A (88.89%, 72.22%) and B (30.77%, 23.08%) than in control group C (5%, 0%) and group D (0%, 0%.). The expression of two proteins showed significant differences between group A and group B (P ＜ 0.01), and no significant differences between group B and group C (P ＞ 0.05). The expression of two proteins showed significant difference between group A and control group (C + D) (P ＜ 0.01). The expression of two proteins showed significant differences between cases of cancer groups (A + B) and control groups (C + D) (P ＜ 0.01). Significant differences were showed between group (A + B) and group D with the expression of ras-p21 and p53 (P ＜ 0.01). The expression of ras-p21 and p53 in three different differentiated groups were G1 (well-differentiated) group: (22.73%, 13.64%), G2 (moderate-differentiated) group: (81.25%, 68.75%), G3 (poorly differentiated) group: (100%, 83.33%). There was significant differences between G1 group and G3 group (P ＜ 0.05), and no significant differences between G1 group and G2 group, G2 group and G3 group (P ＞ 0.05). There was significant differences between three clinical stages with the expression of ras-p21 and p53 (P ＜ 0.05). Conclusion: Ras-p21 and p53 protein werehighly expressed in cancer groups in this study, while, two proteins hardly detected from control groups. If the inflammatory lymph nodes of penile cancer patients show the positive expression of p21 and p53 protein, the inguinal lymph nodes also need dissection, which is important to improve the diagnosis of inguinal lymph node metastasis rate and patient survival of penile cancer. Ras-p21 and p53 protein detection can act an objective indicator of tumor metastasis and prognosis, and also for our treatment of penile cancer in the inguinal lymph node dissection surgery provides determine indicators.     

淋巴细胞功能相关抗原-1/细胞间黏附分子-1介导的细胞因子诱导的杀伤细胞体外抑瘤机制

目的 探讨淋巴细胞功能相关抗原-1(LFA-1)/细胞间黏附分子-1(ICAM-1)介导的细胞因子诱导杀伤细胞(CIK)的体外抑瘤机制.方法 从白血病患儿外周血分离淋巴细胞,经过干扰素-γ(IFN-γ)、抗CD3单克隆抗体(CD3McAb)、白细胞介素-2(IL-2)诱导并与树突状细胞(DC)共培养,获得大量的DC-CIK.在经10、20μg/ml等不同质量浓度小鼠抗人LFA-1单克隆抗体处理后,采用MTT法研究DC-CIK细胞对多种白血病细胞株的杀伤活性,RT-PCR与Western blotting方法检测GATA-3和T-bet基因表达水平的变化.ELISA方法测定DC-CIK细胞释放细胞因子IL-12、IFN-γ、肿瘤坏死因子-α(TNF-α)的表达水平.结果 诱导后的DC-CIK细胞形态规则,经不同浓度的LFA-1单克隆抗体处理后,MTT结果:20μg/ml LFA-1单克隆抗体封闭组DC-CIK细胞对B95细胞杀伤作用下降最为明显(t=10.138,P＜0.05);RT-PCR与Western blotting结果:20μg/ml LFA-1单克隆抗体封闭的B95细胞组,GATA-3基因mRNA水平和蛋白水平表达增加最为明显(t=16.386,P＜0.05;t=22.652,P＜0.05);同时T-bet基因mRNA水平和蛋白水平表达降低最为明显(t=17.728,P＜0.05;t=17.452,P＜0.05);ELISA结果:20μg/ml LFA-1单克隆抗体封闭的B95细胞组中细胞因子IL-12、IFN-γ、TNF-α分泌水平下降最为明显(t=21.621,P＜0.05;t=13.739,P＜0.05;t=15.278,P＜0.05).结论 GATA-3和T-bet基因参与了LFA-1/ICAM-1介导的DC-CIK抑瘤途径,并且通过分泌Th1型细胞因子IL-12、IFN-γ、TNF-α等发挥抑瘤作用.     

Fluid abnormalities occur in the chronically cannulated mid-gestation but not late gestation ovine fetus

Indomethacin, an inhibitor of cyclo-oxygenase given orally, reduced the tumorigenicity of cancer cells in a non-immunogenic murine model of mammary adenocarcinoma (4T1). In the presence of indomethacin, a dose-dependent immune protection could be induced most effectively by immunizing mice with 1 to 3 doses of irradiated tumor cells inoculated at intervals of 7 days prior to challenge with a tumorigenic cell dose. Three immunizations given without indomethacin resulted in tumor growth in 88% of the recipients, and indomethacin treatment started 28 days prior to the challenge dose and given without immunizations led to tumor onset in 83% of mice. In contrast, tumor was documented only in 12% of mice vaccinated with 3 immunization doses and given concomitantly indomethacin. Moreover, 53% of disease-free survivors resisted a second challenge with a high tumorigenic dose. Induction of an anti-tumor immunity in indomethacin-treated mice was further studied as a therapy for tumor-bearing mice. Complete cure was induced in 50% of mice, and a significant reduction in tumor size as well as prolonged survival time were observed in the remaining animals. Immunostimulation by tumor cell vaccination given in the presence of a tolerable dose of indomethacin, therefore, may be incorporated into immunotherapy protocols to activate an anti-tumor response against residual tumor cells that escaped surgery and/or high-dose chemo/radiotherapy.     

Bax is an important determinant of chemosensitivity in pediatric tumor cell lines independent of Bcl-2 expression and p53 status

Susceptibility of a tumor cell to undergo chemotherapy-induced apoptosis appears to be dependent upon the balance of proapoptotic and survival factors that are expressed within any given cell. We have chosen to evaluate how expression of several of these proteins influences chemosensitivity of a panel of 10 pediatric tumor cell lines chosen from three tumor histiotypes: neuroblastoma, rhabdomyosarcoma, and pediatric glial tumors. The proteins evaluated were p53 and six members of the Bax/Bcl-2 family: three proapoptotic proteins (Bax, Bak, and Bcl-xS) and three survival factors (Bcl-2, Bcl-xL, and Mcl-1). We investigated whether there was any relationship between endogenous expression of these proteins and chemosensitivity (or resistance) to three chemotherapeutic agents that directly damage DNA (doxorubicin, actinomycin D, and topotecan) and a mitotic spindle poison (vincristine). Even though exogenous overexpression of wild-type p53 has been associated with a chemosensitive phenotype in several model systems we demonstrated no such relationship in these studies. In addition, expression levels of Bcl-2, Bcl-xL, Bcl-xS, Bak, or Mcl-1 did not correlate with sensitivity or resistance to the four drugs. However, there was a statistically significant correlation between endogenous levels of Bax protein and sensitivity to both doxorubicin and actinomycin D. We conclude that even though many proteins such as p53 and Bcl-2 have been shown to influence drug response when exogenously overexpressed in model systems, in unmodified cell lines endogenous protein levels may not generate the same results. We have demonstrated that endogenous Bax expression was the only protein found to be associated with chemosensitivity across the three different tumor histiotypes and propose that analysis of Bax may be a more useful prognostic indicator for tumor response to therapy than either p53 or Bcl-2.     

A meta-analysis of 61 sperm count studies revisited

The effect of radioimmunotherapy (RIT) on target antigen expression was studied in breast carcinomas transplanted in immunodeficient mice. In nine separate experiments, a single dose of 1500 microCi of 131I-labeled monoclonal antibody (MAb) Mc5 was given to groups of mice carrying well-established, vascularized, transplantable breast tumors (MX-1). Mc5 recognizes an epitope on the tandem repeat of the breast epithelial MUC-1 mucin. This dose suppressed tumor growth for at least 20 days, after which the tumors began to regrow. At various times thereafter, tumors were removed and analyzed for target antigen expression by flow cytometry and immunohistochemistry. In no case was there any significant decrease in antigen content/cell in the tumors of treated mice compared to tumors in control untreated mice. Similar results were obtained with four other breast carcinomas (MCF-7, MDA-MB-331, MDA-MB-435, and MX-2A). To assess the effect of repeated RIT doses on target antigen expression, groups of mice with MX-1 tumors were given 2, 3, and 4 consecutive doses of 1200 microCi of 131I-labeled Mc5. One mouse each at 2, 3, and 4 doses (3 of 18) was cured of its tumor. Control mice were sacrificed after 50 days due to the excessive size of their tumors. Tumors from four mice from each group (2, 3, and 4 doses), after they began to regrow, were excised and analyzed for mucin content and compared to tumors from untreated mice with similar-size tumors transplanted at later dates. In none of the treated groups was there any decrease in mucin content. These results demonstrate that RIT with an anti-breast mucin MAb does not result in the appearance of antigen-negative tumor cells, thus indicating that repeated fractionated doses, which will most likely be necessary for an eventual cure of breast cancer with MAb therapy, are possible.     

结肠癌细胞系SW480对不同化疗药物化疗敏感性的研究

Objective:The aim of this study was to investigate the sensitivity of chemotherapeutic agents 5-FU, cisplatin (DDP) or TAXOL on colon cancer cell line SW480 with different methods, to find out the best examine time period for further study of chemotherapeutic sensitivities. Methods:The SW480 cell was treated with 5-FU (200 μg/mL), DDP(150 μg/mL) or TAXOL (50 μg/mL) respectively for 4 h, 8 h or 12 h. MTT assay was used to examine the cell survival rate, Annexin V/PI assay was used to analysis the apoptosis rate, Western Blot assay was applied to examine the expression of apoptotic protein.Results:(1) Results of MTT assay showed that the survival rate of SW480 cells at 4h, 8 h or 12 h was:5-FU (200 μg/mL)96.0% ± 8.2%, 85.4% ± 7.8%, 74.4% ± 10.2% (P < 0.05); DDP (150 μg/mL) 99.0% ± 6.4%, 88.7% ± 4.7% (P < 0.05), 46.9%± 2.6% (P < 0.01); TAXOL (50 μg/mL) 51.5% ± 4.2% (P < 0.01), 31.9% ± 3.1%, 17.6% ± 2.3%, or blank group 97.2% ± 5.8%,98.7% ± 7.2%, 97.5% ± 7.5% respectively. (2) The apoptosis rate of cancer cells at 4 h, 8 h or 12 h was:control group:3.4%± 0.2%, 6.2% ± 0.4%, 7.0% ± 0.5%; 5-FU (200 μg/mL) 4.0% ± 0.3%, 4.8% ± 0.4%, 7.7% ± 0.7%; DDP (150 μg/mL) 8.5%± 0.9%, 18.6% ± 1.6% (P < 0.05), 67.0% ± 6.2% (P < 0.01); or TAXOL (50 μg/mL) 32.0% ± 5.2% (P < 0.01), 76.6% ± 8.5%,94.0% ± 8.2%, respectively. (3) Western Blot assay showed that the expression of apoptosis associated protein. PARP, Xlinked inhibitor of apoptasis (XIAP), Caspase-9 and Bcl-xL were changed. Conclusion:The sensitivity of chemotherapy could be assessed by MTT assay, Annexin V/PI assay and Western Blot. The best examine time of the three chemotherapuc agents was 5-FU (200 μg/mL):> 12 h, DDP(150 μg/mL):8-12h, or TAXOL (50 μg/mL):< 4 h.     

华蟾素注射液对人肝癌HepG-2细胞增殖及凋亡的影响

Objective: The aim of our study was to investigate the effect of Cinobufacini injection on the proliferation and apoptosis of human hepatocarcinoma HepG-2 cells. Methods: Cells proliferation was assessed by MTT assay, cells morphologic was observed by the inverted microscopy, Annexin V/PI stein was used to detect the apoptosis and necrosis of the tumor cells. The expression of TOPOⅠ mRNA and TOPO Ⅱ mRNAwere examined by RT-PCR. Results: Cinobufacini injection significantly inhibited HepG-2 cells proliferation in dose- and time-dependent ways. After Cinobufacini injection intervention,HepG-2 cells showed typical morphological changes: cells changed from polygon into round, chromatin looseness and karyolysis were observed. The percentages of apoptosis were 88.49%, 76.02%, 61.73% corresponding to the 48 h interference of 0.42 μg/mL, 0.21 μg/mL, 0.105 μg/mL Cinobufacini injection, perspectively. RT-PCR assay showed that Cinobufacini injection down-regulated TOPOⅠ and TOPO Ⅱ expression at mRNA level. Conclusion: Cinobufacini can inhibit human hepatocarcinoma HepG-2 cells growth and induce tumor cells apoptosis, the mechanism of which might partly relate to the down-regulation of TOPOⅠ mRNA and TOPO Ⅱ mRNA induced by Cinobufacini injection.