恶性肿瘤组织CD8~+/CD28~+细胞表达与细胞凋亡的相关性

目的探讨恶性肿瘤组织中CD8+/CD28+细胞杀伤肿瘤细胞的机制。方法用流式细胞术检测恶性肿瘤组织中CD8+/CD28+细胞表达率及细胞凋亡(APO)水平,并分析两者之间的关系。结果恶性肿瘤组织的CD8+/CD28+细胞检出率为(6.86±4.19)%,显著低于正常对照组织(11.85±3.80)%(P<0.01)。恶性肿瘤组织APO检出率为(1.49±1.24)%,显著低于正常对照组织(4.34±3.28)%,(P<0.01)。在恶性肿瘤组织中,CD8+/CD28+细胞检出率和APO表达水平呈显著正相关(r=0.5995,P<0.01)。结论恶性肿瘤能引起组织中CD8+/CD28+细胞表达率和APO检出率显著下降,且二者检出率呈显著正相关。恶性肿瘤组织中CD8+/CD28+细胞杀伤肿瘤细胞的机制可能与诱导肿瘤细胞凋亡密切相关。     

原发性肝癌患者外周血和癌组织中调节性T细胞数量及其临床意义

目的 探讨原发性肝癌患者外周血中调节性T细胞(Treg)占CD4+T细胞(Treg/CID4+)比值和肝癌组织中Treg数量及其临床意义.方法 63例原发性肝癌患者均行根治性手术切除,采用流式细胞术检测患者手术前外周血中Treg/CD4+比值,用免疫组织化学方法检测肿瘤组织中Treg的表达.结果 原发性肝癌患者外周血中Treg/CD4+比值显著高于乙型肝炎患者(P<0.01)及正常对照组(P<0.01).癌组织中Treg数量为(15.69±13.29)个/mm2,癌旁肝组织、10例乙型肝炎肝组织、10例正常肝组织中未见或极少见Treg.外周血中Treg/CD4+比值与癌组织中Treg数量呈正相关(P=0.024).外周血和癌组织中Treg数量高者,术后5年生存率低(P值分别为0.042、0.019);癌组织中Treg数量高者,术后5年无瘤生存率低(P=0.001).结论 原发性肝癌患者外周血和组织中调节性T细胞水平升高;外周血中Treg/CD4+比值及癌组织中Treg数量可作为预测患者根治术后预后的免疫学指标.     

Use of multiple biomarkers for the localization and characterization of colon cancer stem cells by indirect immunocytochemistry

In this study, we used LGR5, γ-synuclein, p53, KRAS and epiregulin antibodies to localize stem cells by indirect immunocytochemistry in paraffin sections of normal and cancerous colon tissues. In the normal colon tissue, no staining of cells with LGR5, γ-synuclein, p53 and KRAS antibodies was observed, apart from a few scattered cells in between the colon villi that were faintly stained with antibodies to LGR5. Staining of highly differentiated cancer tissue with LGR5 antibodies revealed single cells or clusters of up to 4 cells in the interior space of the carcinoma cell layers. Staining of poorly differentiated cancer tissues (stage I-IV) revealed 9-81 clustered stem cells. The number of clustered stem cells increased significantly with the tumor stage, when comparing stage II to stage IV (p<00048). Occasionally, the clustered stem cells appeared in the interphase between the colon stroma and the tumor tissue. Surprisingly, antibodies to p53 clearly stained the clusters of stem cells both in the nuclei and the cytoplasm. The staining of the nuclei of other cells in the undifferentiated tumors was in general weaker, and no staining was found in the cytoplasm. Antibodies to γ-synuclein heavily stained the endothelial cells of the blood vessels and some other scattered cells in the highly differentiated tumors. Antibodies to γ-synuclein heavily stained the stem cells in both the cytoplasm and the nuclei of poorly differentiated tumors. Antibodies to KRAS stained the cytoplasm and the nuclei of stem cells in poorly differentiated tumors and also stained the cytoplasm of some scattered cells. Antibodies to epiregulin stained the cytoplasm of normal colon tissue cells in the crypt-villus axis. The antibodies weakly stained the highly differentiated tumor cells and moderately stained the moderately differentiated tumor cells. Of note, the antibodies intensively stained the clustered stem cells of the poorly differentiated tumor cells. These antibodies also clearly stained the clustered stem cells of poorly differentiated tumors but were not specific as they clearly stained cells in the crypt-villus axis of the normal colon wall. Our results show that LGR5 antibodies can serve as a reliable marker for colon cancer stem cells. Once the colon stem cells are identified, the targeting of specific drugs to kill these cells should be attempted in the future in order to cure this disease. Moreover, the fact that we did not find any stained cells with antibodies to LGR5 in normal tissues apart from a few scattered cells, suggests that the normal colon stem cells differ from the tumor stem cells at least as regards the expression of this protein. In addition, antibodies to γ-synuclein, p53 and KRAS only stained the tumor stem cells and not the normal tissue. Thus, they can serve as multiple biomarkers for the localization of colon cancer stem cells by indirect immunofluorescence.     

Expression of BCRP gene in the normal lung tissue and lung cancer

The development of multidrug resistance during chemotherapy was found to be correlated with over-expression of two transmembrane xenobiotic transporter protein, P-glycoprotein (Pgp) and the multidrug resistance protein (MRP).[1(6] Because the discordance between drug efflux and P-glycoprotein expression in human leukemic cells and unclear drug resistance exhibited during chemotherapy in advanced breast cancer patients, a alternative multidrug resistance transporter might be existed and has b…     

An immunohistochemical and ultrastructural study of the sinusoids of hepatocellular carcinoma.

The sinusoids of 30 human hepatocellular carcinomas of various types were examined by electron microscopy and histochemically for binding to the Ulex europaeus lectin (UEA1). A population of sinusoidal macrophages was identified with an antibody to lysozyme (muramidase). The UEA1 binding was negative in normal sinusoids but positive in the tumor vessels. Macrophages resembling Kupffer cells were found within the tumor vessels but in smaller numbers than in either normal or cirrhotic liver tissue. Fibrolamellar and sclerosing carcinomas contained the smallest numbers. Ultrastructurally, endothelial cells of tumor vessels were thicker than normal, with fewer fenestrations. They contained bundles of microfilaments and showed basement membrane formation. Subendothelial myoid cells were found. These findings indicate that the sinusoidal vessels of hepatocellular carcinomas show features of true capillaries and precapillary blood vessels. The degree of this difference from normal hepatic sinusoids may reflect the relative immaturity of the cancer cells.     

Differential expression and activity status of MMP-1, MMP-2 and MMP-9 in tumor and stromal cells of squamous cell carcinomas of the lung.

Matrix metalloproteinases (MMPs) play a key role in cancer progression. Interstitial collagenase (MMP-1) and type IV collagenases (MMP-2, MMP-9) are involved in the initial breakdown of collagen and basement membrane components during tumor growth and invasion. Besides tumor cells, fibroblasts are especially involved in MMP production. The aim of this study was to quantify MMP-1, MMP-2 and MMP-9 within tumor cells and tumor-surrounding fibroblasts compared to normal lung epithelial cells to gain an insight into the function of these MMPs in squamous cell carcinomas of the lung. The expression and activity of MMP-1, MMP-2 and MMP-9 were analyzed in 30 squamous cell carcinomas and in normal lung tissue from the same patients by immunohistology and gelatin zymography. The majority of tumor cells were positive for MMP-1 (mean +/- SD: 67.3 +/- 26.7%) and MMP-9 (64.7 +/- 22.8%), whereas a significantly lower percentage of normal bronchoepithelial cells (47.3 +/- 25.4 and 40.3 +/- 24.2%, respectively; p < 0.01) and fibroblasts located in the tumor-surrounding tissue (39.7 +/- 14.3 and 38.1 +/- 24.1%, respectively; p < 0.01) expressed these MMPs. Only a few tumor cells showed any immunoreactivity for MMP-2 (4.4 +/- 6.7%), whereas a higher percentage of fibroblasts tested positive for this enzyme (8.6 +/- 13.1%; p < 0.01). Using gelatin zymography, we could demonstrate that MMP-2 is activated in the tumor only, not in normal lung tissue. The coordinated expression of MMP-1, MMP-2 and MMP-9 in tumor cells and/or their induction in tumor-surrounding fibroblasts and further activation in the tumor tissue may be involved in the high invasive and metastatic potential of squamous cell carcinomas of the lung. Comparing the results from immunohistology and zymography can give indications for distribution and activity of proteinases, especially certain MMPs such as MMP-2.     

Endothelial plasticity governs the site‐specific leukocyte recruitment in hepatocellular cancer

The correct programming of the endothelial cell phenotype is crucial for efficient leukocyte recruitment to tumor tissue. It has been previously described that T cells infiltrated hepatocellular cancer (HCC) tissue mainly in peritumoral, stromal and tumor border areas. In the current study, phenotype features of tumor endothelial cells and their potential impact on leukocyte recruitment were analyzed in murine tissue of HCC. In the murine model, proinflammatory stimulation with IL‐1β induced leukocyte recruitment in the blood vessels of peripheral tumor areas and in nonmalignant liver tissue, but not in deeper tumor blood vessels. Furthermore, peripheral tumor endothelium, but not deeper tumor blood vessels exhibited a “normalized” hepatic sinusoidal endothelial cell (HSEC)‐like phenotype with regard to the expression of adhesion molecules and liver sinusoidal endothelial markers. When tumor endothelial cells were isolated and incubated in vitro, their phenotype rapidly changed and became almost identical to normal hepatic endothelial cells. Interestingly, cytokine production in HCC was strongly dysregulated as compared to normal liver, with IL‐1RN exhibiting the most prominent elevation. Experiments with isolated hepatic endothelial cells showed that IL‐1RN effectively antagonized the activating action of IL‐1β on the expression of adhesion molecules and T cell attachment. These novel insights indicate that tumor endothelium of HCC represents a plastic system that is susceptible to microenvironmental changes. The peritumoral and tumor border areas have distinct endothelial cell phenotype, which promotes leukocyte recruitment to HCC tissue.     

Expression of Tiam1 in lung cancer and its clinical significance

The aim of this study was to analyze T-cell lymphoma invasion and metastasis-inducing factor 1 (Tiam1) expression in lung cancer patients. A total of 204 patients with lung cancer tissue lesions were enrolled in the present study, along with 40 cases of normal lung tissue and 40 of normal fetal lung tissue. Tiam1 protein expression level was determined using intensity quantitative analysis, for comparison in lung cancer, metastatic, normal lung, and fetal lung tissue. The positive unit (PU) of Tiam1 was 13.5 ± 5.42 in lung cancer, 5.67 ± 1.56 in normal epithelial cells, and 5.89 ± 1.45 in fetal lung epithelial cells. The value in the lung cancer tissue was significantly higher than that in the normal lung tissue and the fetal lung tissue (P<0.01). The Tiam1 PU values with lymph node metastasis and without lymph node metastasis were 15.2 ± 4.34 and 12.5 ± 4.23, respectively, and the difference was statistically significant (P<0.05). The Tiam1 PU values in different tumor, nodes, metastasis (TNM) stages, III-IV period, and I-II phase were 14.7 ± 4.14 and 11.0 ± 5.34 (P <0.05). A correlation was found between Tiam1 expression and the age of patient, tumor size, tumor type, and tumor differentiation. Tiam1 protein expression in the lung tumor tissue is significantly higher than that in the normal lung tissue and fetal lung tissue. Tiam1 expression may be closely related to lung cancer development and metastasis.     

DNA甲基化对miR-133a表达及胶质瘤细胞增殖及凋亡的影响

目的:探讨miR-133a在胶质瘤细胞中的表达及DNA甲基化对其的调控机制并初步探究miR-133a对胶质瘤细胞增殖及凋亡的影响。方法:通过RT-PCR检测胶质瘤组织与正常对照脑组织中miR-133a的表达差异;MSP实验检测胶质瘤组织与正常对照脑组织中miR-133a基因甲基化程度;BSP实验检测正常胶质细胞株HEB与胶质瘤细胞株A172、U87、U251中miR-133a基因的甲基化水平;去甲基化试剂AZA与胶质瘤细胞株A172、U87、U251共培养72 h后,RT-PCR检测上述3株胶质瘤细胞中的miR-133a表达变化;MTT及Annexin V-FITC凋亡实验分别检测AZA处理后的3株胶质瘤细胞的增殖与凋亡能力变化。结果:RT-PCR实验表明与正常对照脑组织相比,胶质瘤组织中miR-133a表达显著下降;MSP实验结果显示,与正常对照脑组织相比,胶质瘤组织中miR-133a基因的甲基化水平明显增高;BSP实验结果显示与正常对照胶质细胞株HEB相比,胶质瘤细胞A172、U87、U251中miR-133a基因的甲基化水平显著增加;RT-PCR实验显示与去甲基化试剂AZA共培养72 h后,胶质瘤细胞株A172、U87、U251细胞中miR-133a的表达显著增加。MTT与Annexin V-FITC凋亡实验结果表明,去甲基化试剂AZA处理后,胶质瘤细胞A172、U87、U251的增殖能力明显下降,细胞的凋亡发生率显著增加。结论:胶质瘤细胞中DNA甲基化通过调控miR-133a的表达而沉默后者发挥抑制肿瘤细胞增殖,促进肿瘤细胞凋亡的功能。     

5-FU concentration in tumor tissue and the antitumor effect in patients with gastric cancer after oral administration of UFT

Serum and tumor tissue concentration of FT, 5-FU and uracil were measured in 23 patients with gastric cancer who were administered UFT preoperatively. The degeneration of cancer cells was evaluated histologically and the correlation between 5-FU concentration in the tumor tissue and the antitumor effect was assessed. The average concentration of 5-FU in tumor tissue (0.122 microgram/g) was significantly higher than that in normal gastric tissues. Serum 5-FU concentration was very low, suggesting no accumulation of 5-FU in blood. A positive correlation between the concentration of 5-FU and uracil in tumor tissues was found. A 5-FU level higher than 0.05 microgram/g was frequently demonstrated in tumor tissue, resulting in moderate degeneration of cancer cells in many cases. When tumors were classified according to histological type, intratumoral 5-FU concentration was not always correlated with degeneration of cancer cells. The above results suggested that UFT was a very effective drug for stomach cancer because of high tumor affinity for 5-FU, but that it is necessary to consider the sensitivity of tumor cells to antitumor drugs in order to obtain an excellent antitumor effect.     

Intracellular distribution of macrophage migration inhibitory factor predicts the prognosis of patients with adenocarcinoma of the lung

BACKGROUND: Macrophage migration inhibitory factor (MIF) is known to be a proinflammatory cytokine and glucocorticoid-induced immunomodulator as well as a regulator of tumor growth. Although positive and negative effects of MIF on tumor cell growth have been reported, to the authors' knowledge the precise role of MIF in tumorigenesis remains unclear. In the current study the authors assessed expression of MIF protein and mRNA in lung adenocarcinomas with regard to patient prognosis. METHODS: Immunohistochemical analysis was performed on tissue specimens surgically obtained from 74 patients with primary lung adenocarcinoma (American Joint Committee on Cancer pathologic Stages I, II, and IIIa). In addition, expression of MIF mRNA in the cancerous tissue was investigated using in situ hybridization. Patient prognosis was evaluated with regard to MIF expression levels and its distribution was analyzed with the Kaplan-Meier method. RESULTS: MIF mRNA and MIF protein were observed in the bronchial epithelium, alveolar epithelium, vascular smooth muscle, and alveolar macrophages in the normal lung tissue. In tumor tissue from lung adenocarcinoma specimens, both MIF mRNA and protein were observed at much higher levels than in the normal alveolar epithelium. MIF protein was observed diffusely in the cytoplasm of tumor cells in all tumor specimens examined. MIF protein also was observed in the nuclei of tumor cells from 59 patients (79.7%), whereas it was not observed in the nuclei of tumor cells from 15 patients (20.3%). The patients without nuclear MIF expression had a worse prognosis compared with those patients with MIF expression in the nuclei (P = 0.04). CONCLUSIONS: The results of the current study suggest that intracellular MIF distribution predicts patient prognosis in individuals with adenocarcinoma of the lung.     

Epidermal growth factor receptors in spontaneous ovarian granulosa cell tumors of SWR-derived mice

Epidermal growth factor (EGF) receptor binding properties were examined in spontaneous ovarian granulosa cell (GC) tumors from SWR and SWR-derived strains of mice. EGF binding was measured at room temperature in tissue homogenates from GC tumors and normal ovaries from adult randomly cycling mice. GC tumor tissue displayed significantly increased EGF binding and 2 receptor populations (R1 and R2). Normal ovarian tissue appeared to have only one receptor population with a dissociation constant (KD) similar to the R1 (high-affinity) receptor in GC tumors. In subsequent experiments, GC tumor and normal granulosa cells from immature mice were analyzed in primary cultures for EGF binding, immunofluorescence microscopy for receptors, and cell proliferation. After 24 hr in culture, the GC tumors bound 10-fold more EGF/micrograms protein than did normal granulosa cells. GC tumor cells, but not normal granulosa cells, showed specific immunofluorescence when reacted with a polyclonal antibody to mouse EGFR. During 96 hr in culture, GC tumor cells, but not normal cells, showed a significant proliferative response to EGF. In conclusion, the EGF binding capacity is markedly increased in GC tumor cells and the proliferation data suggest that this growth factor supports tumor growth in the SWR model system.     

弥漫大B细胞淋巴瘤中B7-H1临床表达水平分析

目的 对弥漫大B细胞淋巴瘤(DLBCL)中B7-H1的表达水平进行分析.方法 收集DLBCL患者病理活检或手术切除的肿瘤组织标本50例,另收集瘤周正常淋巴组织标本50例作为对照.采用Western Blot法检测B7-H1在瘤周正常淋巴组织与DLBCL肿瘤组织中的表达水平;采用流式细胞仪检测瘤周正常淋巴组织与DLBCL肿瘤组织中B7-H1和调节性T细胞(Treg)的表达水平.结果 Western Blot检测结果显示,B7-H1在DLBCL肿瘤组织与瘤周正常淋巴组织中均有表达,与瘤周正常淋巴组织比较,DLBCL肿瘤组织中B7-H1的表达水平较高.HLA-DR+CD14+巨噬细胞在DLBCL肿瘤组织中表达B7-H1的比例较瘤周正常淋巴组织高(P﹤0.05).与瘤周正常淋巴组织相比较,DLBCL肿瘤组织中(包括IPI值≥2分与IPI值﹤2分)pDC、Treg、mDC与调节性T细胞(CD4+/CD8+)的比例较高(P﹤0.05).结论 B7-H1在DLBCL患者的淋巴组织中呈高表达,提示B7-H1与淋巴瘤细胞的免疫逃逸有关,从而造成了DLBCL细胞免疫平衡的失调.     

Some characteristic features of the biotransport of the cytostatic agent 1,6-dibromo-1,6-dideoxy-dulcitol

Aspects of the biological transport of cytostatically active 1,6-dibromo-1,6-dideoxydulcitol (DBD) were studied using ⁸²Br-labelled material on dogs and Yoshida tumorbearing rats. Thirty-five per cent of the DBD dose was excreted in the urine within 12 hours. Besides unchanged DBD, metabolites containing C-Br bonds could also be detected in the urine. In vivo, the 50% decomposition time of the C-Br bonds in DBD is about 5 to 6 hours. One hour after administration of DBD, more than 50% of the persisting dose had entered the cells. Accumulation of the drug in the tumor tissue could not be demonstrated. A fraction of the dose enters the enterohepatic circulation.     

流式细胞术检测肺癌瘤组织和外周血角蛋白阳性细胞表达

[目的]探讨肺癌患者瘤组织和外周血角蛋白(cytokeratin,CK)阳性细胞的表达及其临床意义.[方法]采用流式细胞术检测和分析了43例肺癌患者瘤组织和外周血CK 细胞的表达情况.[结果]肺癌患者瘤组织和外周血CK 细胞检出率均显著高于相应对照组(P＜0.01或P＜0.05),且两者呈良好的正相关(r=0.325,P＜0.05).肿瘤患者CK 细胞检出率与肿瘤患者性别、年龄无关(P＞0.05);而与肿瘤临床分期、病理分级、瘤灶数、肿瘤大小、转移情况以及癌细胞倍体类型、增殖活性和凋亡水平密切相关(P＜0.05或P＜0.01).[结论]CK 细胞表达水平与患者临床生物学行为及瘤细胞生物学特性关系十分密切;CK 细胞可以作为肺癌的诊断、恶性度判断、微转移及患者预后估计的标志物.