maspin和bax联合检测对乳腺癌复发的研究

目的：研究乳腺癌组织中mas pin和bax的表达,探讨二者预测乳腺癌复发的价值。方法：采用SP免疫组织化学方法检测30例复发乳腺癌、24例无复发乳腺癌及14例乳腺纤维腺瘤组织中maspin和bax的表达情况。结果：复发乳腺癌（23.3%,20.0%）和无复发乳腺癌组织（54.2%,45.8%）maspin、bax阳性表达率均较乳腺纤维腺瘤组织（85.7%,78.6%）低（P均〈0.05）,复发乳腺癌maspin和bax阳性表达明显低于无复发乳腺癌（P〈0.01）。复发乳腺癌组织maspin表达与组织学分级呈负相关（r=-0.481,P〈0.05）,与激素受体呈正相关（r=0.497,P〈0.05）;bax表达与组织学分级呈负相关（r=-0.522,P〈0.05）;maspin表达与bax表达呈正相关﹙r=0.454,P〈0.05﹚。结论：maspin、bax的异常表达可能在乳腺癌的发生、发展过程中起重要作用,联合检测能更好地判断乳腺癌的复发。     

子宫内膜增生、子宫内膜样腺癌组织中Twist与maspin的表达及相关性分析

目的:研究扭曲蛋白Twist和maspin在典型子宫内膜增生、不典型子宫内膜增生和子宫内膜样腺癌组织中的表达。方法:应用免疫组化检测40例典型子宫内膜增生、40例不典型子宫内膜增生和80例子宫内膜样腺癌组织中Twist、maspin的表达。结果:在典型子宫内膜增生、不典型子宫内膜增生和子宫内膜样腺癌组织中Twist、maspin阳性率分别为10%、37.5%和52.5%,12.5%、42.5%和43.8%。Twist在不典型子宫内膜增生和子宫内膜样腺癌组织中的表达均高于典型子宫内膜增生,差异有统计学意义（P＜0.05）。子宫内膜样腺癌组织中,Twist与组织学分级、浸润深度和淋巴结转移相关,差异有统计学意义（P＜0.05）,maspin与组织学分级、分期和淋巴结转移相关,差异有统计学意义（P＜0.05）。Twist和maspin没有显著相关（r=0.322,P=0.056)。结论:在不典型子宫内膜增生和子宫内膜样腺癌组织中,Twist和maspin蛋白表达不同,表明它们在子宫内膜样腺癌发展中的潜在功能,可能与子宫内膜样腺癌发生有关。     

Gene expression profiles in human gastric cancer: expression of maspin correlates with lymph node metastasis

To seek for a candidate gene that would regulate tumour progression and metastasis in gastric cancer, we investigated gene expression profiles by using DNA microarray. Tumour tissue and adjacent normal tissue were obtained from 21 patients with gastric cancer and then examined for their gene expression profiles by the Gene Chip Human U95Av2 array, which includes 12 000 human genes and EST sequences. A total of 25 genes were upregulated and two genes were downregulated by at least four-fold in the tumour tissue. In a further analysis according to lymph node metastasis, the expressed levels of maspin, as well as carcinoembryonic antigen and nonspecific crossreacting antigen were significantly higher in tumours with lymph node metastasis than in those without it. Maspin expression in 85 gastric cancer patients was further investigated by using immunohistochemistry. Maspin expression was not observed in normal gastric epithelia without intestinal metaplasia. In contrast, maspin was expressed in 74 of 85 tumour tissues. There was a significant correlation between the incidence of maspin-positive tumour staining and lymph node metastasis. These results suggest that maspin has a potential role for tumour metastasis in gastric cancer.     

maspin/pEFIRES-N表达载体的构建及应用

目的构建maspin/pEFIRES-N表达载体,为深入研究maspin基因抑制肝癌生长、转移、浸润作用和机制奠定基础.方法 PCR扩增maspin基因全长,将表达载体pEFIRES-N用EcoRⅠ XbaⅠ进行双酶切后,T4DNA连接酶连接成重组质粒,稳定转染到肝癌高转移细胞株mHCC-97中进行表达,检测稳定转染前后maspin表达的变化.结果重组质粒在大肠杆菌株JM109内扩增.提纯、纯化后用EcoRⅠ、XbaⅠ酶切鉴定及测序鉴定证明maspin基因已完整、正确的插入到pEFIRES-N表达载体,并在肝癌MHCC-97细胞中上调了maspin基因mRNA和蛋白水平表达,抑制了肝癌MHCC-97细胞增殖及浸润.结论成功构建了maspin/pEFIRES-N表达载体,能在真核细胞中表达.     

RNA干扰maspin基因对胃癌细胞株MKN-28凋亡的影响

目的:探讨转染maspin特异性短发夹RNA(short hairpin RNA, shRNA)重组质粒对胃癌细胞株MKN-28凋亡的影响.方法:设计并合成针对maspin基因的shRNA,并将其与真核表达载体pGenesil-1.1连接,构建重组质粒pGenesil-maspin.应用RT-PCR和Western 印迹法检测转染重组质粒后MKN-28细胞中maspin、bcl-2和bax mRNA及蛋白的表达变化,应用FCM法检测转染重组质粒对MKN-28细胞的细胞周期和细胞凋亡的影响.结果:成功构建maspin特异性shRNA重组质粒pGenesil-maspin1、pGenesil-maspin2及pGenesil-HK(阴性对照); pGenesil-maspin成功转染后可明显下调胃癌细胞MKN-28中 maspin和bax mRNA及蛋白的表达水平(P<0.01),并上调bcl-2 mRNA和蛋白的表达水平(P<0.01);重组质粒转染后,MKN-28细胞的细胞周期呈现G0/G1期细胞减少(P<0.01),细胞凋亡率下降(P<0.01).结论:外源性maspin shRNA转染MKN-28细胞后能下调maspin的表达,抑制胃癌细胞的凋亡,且使细胞周期分布发生改变;其分子机制可能与上调bcl-2和下调bax的表达有关.     

Cytoplasmic Maspin Expression Correlates with Poor Prognosis of Patients with Adenocarcinoma of the Uterine Cervix

Background

Maspin is known to be a tumor suppressor protein and its prognostic significance in patients with several types of cancer has been reported. To date, however, no study has focused on the association between maspin expression and the prognosis of patients with adenocarcinoma of the uterine cervix. We explored the prognostic value of maspin expression with particular reference to its subcellular localization in patients with adenocarcinoma of the uterine cervix.

Methods

Paraffin-embedded tissue samples from 46 patients diagnosed as adenocarcinoma of the uterine cervix were immunohistochemically analyzed using an antibody for maspin. The patients were followed up for 3 to 165 months (median: 64.2 months) and the prognostic value was evaluated by the log-rank test and the Cox regression hazard model.

Results

A sample was considered maspin-positive if maspin was expressed in only the cytoplasm; 69.6% (32 cases) of the specimens were maspin-positive, and there was significant correlation between positivity and recurrence (P = 0.022). Maspin-positive patients had both shorter disease free survival and shorter overall survival by the log-rank test (P = 0.023, P = 0.043, respectively). By Cox’s multivariate analysis, the International Federation of Obstetrics and Gynecology (FIGO) status was the only independent prognostic factor for disease free survival and overall survival in patients with adenocarcinoma of the uterine cervix.

Conclusion

This is the first report to reveal an association between cytoplasmic maspin expression and the prognosis of patients with adenocarcinoma of the uterine cervix. Although further studies with a larger series of patients and a longer follow up period are necessary, the present results suggest that cytoplasmic maspin expression could be an indicator of unfavorable prognosis in patients with adenocarcinoma of the uterine cervix.     

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down-regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation is involved in the mechanism of down-regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-beta1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for Western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-beta1 were hypermethylated. Down-regulation of maspin synthesis was also associated with histone H3 dimethylation at lysine 9. Both maspin mRNA and protein were re-expressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down-regulation of maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation.     

Immunohistochemical detection of uPA, uPAR, PAI-1, and maspin in ameloblastic tumors

BACKGROUND: To evaluate the roles of extracellular matrix (ECM)-degrading serine proteinase in progression of odontogenic tumors, expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and maspin was analyzed in ameloblastic tumors as well as in tooth germs. METHODS: Tissue specimens of 10 tooth germs, 45 ameloblastomas, and 5 malignant ameloblastic tumors were examined immunohistochemically with the use of antibodies against uPA, uPAR, PAI-1, and maspin. RESULTS: Immunohistochemical reactivity for uPA, uPAR, PAI-1, and maspin was detected in normal and neoplastic odontogenic tissues: uPA was recognized predominantly in mesenchymal cells, uPAR was evident in epithelial cells, PAI-1 was found in both epithelial and mesenchymal cells, and maspin was expressed only in epithelial cells. The levels of uPA and uPAR immunoreactivity in ameloblastic tumors were slightly higher than the levels in tooth germs, while PAI-1 reactivity in ameloblastomas tended to be lower than that in tooth germs. The level of maspin immunoreactivity in ameloblastomas was significantly higher than that in tooth germs, and ameloblastic carcinoma showed decreased maspin reactivity. CONCLUSION: Expression of uPA, uPAR, PAI-1, and maspin in tooth germs and ameloblastic tumors suggests that interactions among these molecules contribute to ECM degradation and cell migration during tooth development and tumor progression. Altered expression of the serine proteinase and its associated molecules in ameloblastic tumors may be involved in oncogenesis of odontogenic epithelium.