GENOMIC STRUCTURE OF MOUSE TBXZ AND DETECTION OF EXPRESSION OF TBXZ IN NORMAL AND MALIGNANCE MELANOPHORE BY RT-PCR

T-boxgenesrepresentarecentlyidentifiedfamilyofhighlyevolutionarilyconservedgenesthatencodeproteinsranginginsizefrom--400to>900aminoacids.[],2]Membersofthisgenefamilyshareamotif200aminoacidswhichintheTgeneproducthas.beenshowntoexhibitsequence-specific,DNA-bindingactivity.1']Chapmanandassociates[4]haveshownthatsixmurineT-boxgenesexhibitoverlappingbutuniquepatternsofexpressionduringembryogenesis,indicatingthatexpressionofthesegeneistemporallyandspatiallyregulated.Thissuggeststhat,similartoPaxge…     

人胰腺癌组织中Tbx2蛋白的表达及Wnt/β-catenin信号通路对TBX2基因的调节

Objective:To investigate the expression of Tbx2 protein in pancreatic cancer tissues and its molecular regulation mechanism by Wnt/β-catenin signaling.Methods:49 pancreatic cancer and 13 non-cancer tissue specimens were obtained and examined the expression of Tbx2,and the correlation between the expression of Tbx2 and clinicopathological parameters was analyzed.The immunohistochemistry,immunocytochemistry,RT-PCR and Western blot assay methods were used to detect the changes of expression levels of β-catenin and Tbx2.Results:Tbx2 was amplified in 34 of 49 pancreatic cancers,and in 13 non-cancer tissues,only one sample amplified.The further study demonstrated that Tbx2 had a significant positive correlation with tumor differentiation degree and clinical stage,but it did not relate to the sex,age and the disease region.Inhibition of β-catenin degradation through the treatment of pancreatic cancer cells SW1990 with different concentrations of lithium chloride indicated that accumulation of β-catenin was sufficient to induce TBX2 expression.Conclusion:TBX2 gene plays an important role in the occurrence and development of pancreatic cancer and the accumulation of β-catenin contributes to the expression of TBX2.The Wnt/β-catenin signaling pathway participates the regulation of TBX2 in pancreatic cancer cells.     

荧光定量RT-PCR检测mdr-1基因表达

建立荧光定量ＲＴ－ＰＣＲ检测肿瘤细胞ｍｄｒ－１基因表达的方法,了解肺癌组织中ｍｄｒ的表达水平。方法：建立荧光定量ＲＴ－ＰＣＲ方法,在ＰＥ７７００型检测仪上定量检测Ｋ５６２／ＡＤＭ耐药株和Ｋ５６２不耐药株细胞ｍｄｒ－１有达水平,同时检测４５例初治肺部肿瘤病人组织标本。     

Real-time quantitative RT-PCR assessment of PIM-1 and hK2 mRNA expression in benign prostate hyperplasia and prostate cancer

To investigate the expressions of PIM-1 and hK2 mRNA in normal prostate, benign prostatic glandular hyperplasia (BPH), and prostate cancer (PCa), and to explore the association of PIM-1 and hK2 expressions with PCa progression. The samples were harvested from 37 patients with BPH, 23 patients with PCa, and three with normal prostate tissues. Total RNA was extracted from their prostate tissues and analyzed for PIM-1 and hK2 mRNA levels using SYBR green I-based quantitative real-time RT-PCR (QRT-PCR) assays and Southern blot analysis. The differences of gene expressions were calculated based on standard curve. Quantitative expressions of PIM-1 and hK2 mRNA in normal prostate, BPH, and PCa were 1.05 ± 0.04, 2.57 ± 0.74, 4.45 ± 0.63, and 1.02 ± 0.03, 2.264 ± 0.46, 5.905 ± 0.78, respectively. PIM-1 and hK2 were expressed higher in PCa than those in BPH and normal prostate tissues, the differences among which had statistic significance (P < 0.05). Our results support the hypothesis that PIM-1 and hK2 play a significant role in the growth of PCa and the detection of PIM-1 and hK2 mRNA expressions by QRT-PCR provided more reliable and helpful information on diagnosis, treatment, and prognosis of PCa. Hui-chan He and Xue-cheng Bi contributed equally to this article.     

Tyrosine1248-phosphorylated HER2 expression and HER2 gene amplification in female invasive ductal carcinomas

Background  Phosphorylated HER2 (pHER2) may more accurately reflect the signaling and functional activity of the HER2 protein than detection of HER2 itself. The detection of HER2 gene amplification using fluorescence in situ hybridization (FISH) provides superior prognostic information for the diagnosis of breast cancer. However, the relationship between pHER2 expression in tissue samples and HER2 gene amplification remains unclear. Methods  A total of 210 cases were recruited. The expression of HER2 and tyrosine (Tyr)1248-pHER2 was investigated by immunohistochemistry, and HER2 gene amplification was analyzed by FISH. Spearman’s rank correlation test was employed to confirm correlation between HER2 and Tyr1248-pHER2. Chi-square and Student’s t test were used to determine a significant difference between the baseline characteristics of tumors and the FISH, HER2 and Tyr1248-pHER2 results. The phosphorylation rate of HER2 was calculated using a digital-analysis system. Results  HER2 expression was significantly (P < 0.001) associated with Tyr1248-pHER2 expression. HER2 gene amplification could be detected in 55 (26.2%) of the 210 tumors; 40 were HER2 positive and 32 were Tyr1248-pHER2 positive. The sensitivity and specificity of HER2 and Tyr1248-pHER2 for HER2 gene amplification were 72.7 and 58.2%, and 91.6 and 95.5%, respectively. In cases with an HER2 score of 2, and a phosphorylation score of 2 or 3, gene amplification was observed in 4 (80.0%) out of 5 tumors. Conclusions  Tyr1248-pHER2 expression is highly specific for HER2 gene amplification. The phosphorylation status might provide an adjunct to the assessment of gene amplification in patients with an HER2 score of 2.     

INT2 and ERBB2 amplification and ERBB2 expression in breast tumors from patients with different outcomes

Summary The relationships of INT2 and ERBB2 amplification and of ERBB2 overexpression in primary breast tumors to prognostic factors, recurrence, and survival have generated considerable controversy. The rationale for this study is that long-term, recurrence-free survival is a more direct criterion for testing the validity of a tumor marker than correlation either with prognostic factors or with short-term recurrence and survival. We examined the association of recurrence with INT2 and ERBB2 amplification and ERBB2 expression by comparing primary breast tumors from patients surviving without recurrence for 8.5 years after diagnosis. the LTS group, to tumors from patients recurring within two years, the RR group. The RR (N = 63) and LTS (N = 61) samples were coded and examined for amplification by Southern blotting and for expression by immunohistochemistry. Comparison between the RR and LTS groups demonstrated that INT2 amplification was associated with a significantly (P = 0.018) higher (5.6-fold) risk of recurrence, an association that remained significant after controlling for lymph node (LN), tumor size (TS), and histograde (HG) status. ERBB2 amplification and expression were not associated with a higher recurrence risk. Survival analyses within the RR group, however, demonstrated significantly shorter survival time among cases with than without ERBB2 amplification (P = 0.018, median survival 16 vs 25 months), or ERBB2 expression (P = 0.019, median survival 15 vs 25 months), but not INT2 amplification. Univariate Cox proportional hazards regression models also demonstrated significantly shorter survival among cases with ERBB2 amplification (P = 0.016) or expression (P = 0.049), that remained significant in multivariate analyses (P = 0.022) for ERBB2 amplification. These results indicate a significant positive association between INT2 amplification and risk for tumor recurrence in the RR as compared to the LTS group. The relationship of ERBB2 amplification or overexpression to patient outcome is more complex. ERBB2 amplification and expression have a significant relationship with shorter survival among patients recurrent within two years, but their occurrence in tumors from women surviving without recurrence for 8.5 years suggests that ERBB2 status is not predictive of shorter survival for all breast cancers.     

ALK基因蛋白在肿瘤细胞和正常细胞中表达的意义

背景与目的：以往的研究认为CD30是间变性大细胞淋巴瘤（amaplastic large cell lymphoma,(ALCL)的诊断指标,但是该指标特异性并不强,需要寻找更特异指标。已有研究表明,在ALCL的三种染色体移位产生的融合基因中,均涉及ALK,设想ALK比CD30和P80在诊断ALCL时更特异。本文的目的是探讨间变性淋巴瘤激酶（anaplastic lymphoma kinase,ALK)基因蛋白在多种肿瘤细胞和正常细胞中表达的意义,以此作业进一步研究的基础。方法：应用免疫组化链霉素－过氧化酶染色法检测了14例ALCL、10例霍奇金淋巴瘤／病（Hodgkin‘s lymphoma/disease,HL or HD)、46例B细胞淋巴瘤（B cell lymphoma,BCL）、13例T细胞淋巴瘤（T cell lymphoma,TCL)、21例恶性星形胶质细胞瘤、5例髓母细胞瘤、5例室管膜瘤、75例各种上皮来源的癌、19例不同间叶组织来源的肉瘤以及多种正常组织中ALK基因蛋白的表达。结果：（1）ALCL中ALK蛋白表达率为64．3％,显著高于HD、BCL和其它TCL（P＜0．01）。（2）ALK在肝细胞癌、恶性星形胶质细胞瘤和髓母细胞瘤中有较强的表达。（3）ALK在胎盘组织和胶质细胞神经元中有少量表达。结论：（1）ALK蛋白是ALCL的重要分子标志,对ALCL的诊断和鉴别诊断具有很高价值。（2）ALK基因可能参与了肝细胞癌、恶性胶质细胞瘤和髓母细胞瘤的发生和发展,ALK基因可能是一个重要的肿瘤相关基因。     

贲门癌mdm2 基因扩增及蛋白表达的研究

 目的 检测贲门癌组织中mdm2基因扩增及蛋白表达，探讨其在贲门癌发生发展中的作用，分析其临床病理意义。方法 用dPCR技术检测贲门癌组织mdm2基因扩增；用免疫组织化学技术及流式细胞术检测mdm2蛋白表达水平。结果 该组标本中18．42％有mdm2基因扩增，64．86％有mdm2蛋白过表达，其mdm2表达FJ值（1．45±0．34）明显高于正常黏膜组织的FJ值（1．00±0．13，P〈0．05）。高中分化的贲门癌组织，mdm2基因扩增率及蛋白表达水平均较低分化者高（33．30％Vs5．00，80．00％VS47．06％，P〈0．05）。结合该组标本此前已检测的p53基因突变和蛋白表达结果发现，两种基因及蛋白表达异常在本组标本中的分布有不重叠趋势。结论 dm2基因扩增和过表达是贲门癌较常见的分子改变，并可能通过抑制野生型p53基因功能参与贲门癌形成；有mdm2扩增及表达的贲门癌组织分化程度较高，提示预后较好。     

Pim-2基因在成人急性白血病中的表达

目的探讨Pim蛳2基因在成人急性白血病患者中的表达。方法应用半定量RT蛳PCR方法分别检测38例健康对照者和194例急性白血病患者Pim蛳2基因的表达。结果正常人Pim蛳2基因的表达最高,Pim蛳2/G3PDH的半定量比值分别为(1.188±0.471);急性淋巴细胞白血病初治及复发患者Pim蛳2基因的表达最低,分别为(0.682±0.590)与(0.800±0.538),与正常组差异有显著性(P<0.05),急性髓系白血病初治和复发患者与正常人Pim蛳2基因的表达无明显差异,其半定量比值为(0.903±0.590)及(0.861±0.544)。结论急性淋巴细胞白血病初治及复发患者Pim蛳2基因的表达低于正常人。     

乳腺癌C—erbB—2和nm23基因表达的研究

目的：探讨Ｃ－ｅｒｂＢ－２和ｎｍ２３基因表达与乳腺癌组织学类型、临床分期以及对乳腺癌淋巴结转移的影响。方法：９８例乳腺癌术后石腊包埋标本行免疫组化ＳＰ法。结果：Ｃ－ｅｒｂＢ－２在淋巴结转移组表达率为６０．２９％，在非淋巴结转移组表达率为３３．３３％，差别有显著意义；Ｃ－ｅｒｂＢ－２在乳腺癌不同临床分期表达率各不相同，Ⅳ期为１１．７８％，Ⅱｂ＋Ⅲ期为５６．６０％，Ⅰ＋Ⅱａ期为２５．９２％，差别有显著     

小鼠Doc-1R基因的克隆及其表达

背景与目的：Doc-1R基因是1999年克隆的一种新的基因,已有的研究表明Doc-1R基因可能是一种潜在的抑癌基因。为了进一步研究此基因的功能,我们首先克隆了小鼠的Doc-1R基因,并对此基因的表达进行了初步的研究。方法;根据小鼠的Doc-1R基因cDNA序列,设计合成了基因组特异引物,应用巢式PCR对小鼠基因组步移文库进行扩增。对测序结果进行序列分析及剪接位点的鉴定。另外应用RT－PCR的方法研究了Doc-1R基因在小鼠肝,脾,胰,肾,肺,肠,心,脑,骨,肌肉,膀胱,卵巢,睾丸等13种组织的表达。结果：经二次基因组步移获得了小鼠Doc-1R基因组全长序列。基因组全长2787bp,含4个外显子和3个内含子,外显子与内含子接头符合GT／AG法则。RT－PCR结果表明,Doc-1R基因在小鼠13种组织和器官中均有表达。结论：成功克隆了小鼠Doc-1R基因,为进一步研究此基因的功能奠定了基础。RT－PCR表达结果提示此基因可能是对维持组织器官的功能具有重要的作用的管家基因。     

携带绿色荧光报告基因EBV-LMP2A真核表达载体构建及转染鼻咽癌细胞

目的 构建携带绿色荧光基因的真核表达载体pIRES2-Zs-Green1-LMP2A,并转染至鼻咽癌CNE2细胞。方法 从EB病毒阳性的狨猴淋巴瘤细胞B95-8中克隆EB病毒编码的ＥＢＶ潜伏膜蛋白2A（latent membrane protein 2A,LMP2A）序列,并定向克隆入pIRES2-Zs-Green1载体,双酶切及测序鉴定重组的真核表达载体pIRES2-Zs-Green1-LMP2A;通过脂质体转染将重组的真核表达载体pIRES2-Zs-Green1-LMP2A转染至鼻咽癌CNE2细胞（实验组）,同时另设转染pIRES2-Zs-Green1载体的阴性对照组及未转染的空白对照组。利用质粒所携带的绿色荧光蛋白表达计算细胞转染效率,RT-PCR检测目的基因LMP2A在鼻咽癌CNE2细胞中的表达。结果 双酶切及测序鉴定证实真核表达载体pIRES2-Zs-Green1-LMP2A构建成功,荧光显微镜下发现实验组和阴性对照组细胞均发出绿色荧光,实验组细胞转染率约为75%;RT-PCR检测发现实验组细胞中有目的基因LMP2A表达,但阴性对照组和空白对照组均未检测到目的基因表达。结论 成功构建了pIRES2-Zs-Green1- LMP2A真核表达载体并转染鼻咽癌CNE2细胞,目的基因LMP2A可在转染的鼻咽癌CNE2细胞中稳定表达。     

Genomic gain of 5p15 leads to over-expression of Misu (NSUN2) in breast cancer

We have examined expression of the Myc target gene Misu (NSUN2) in breast cancer. There was extensive copy number gain, and increased mRNA and protein levels, of Misu in approximately one third of breast cancer cell lines and primary tumours examined, irrespective of tumour subtype. Genes on 5p15.31-33, where Misu is located, showed evolutionary synteny. siRNA-mediated knockdown of Misu reduced cell number in over half of the cell lines tested, irrespective of estrogen receptor status. We conclude that Misu is up-regulated in a substantial proportion of breast cancers and has therapeutic potential as a drug target.     

干扰素对小鼠乳腺癌细胞侵袭力及转移相关基因表达的调变

目的研究不同干扰素对肿瘤细胞侵袭力及侵袭转移相关基因表达的调变作用。方法根据细胞穿过铺有Ｍａｔｒｉｇｅｌ胶多孔膜的数量检测细胞侵袭力,用流式细胞分析和Ｎｏｒｔｈｅｒｎ杂交检测细胞表面抗原和ｍＲＮＡ表达水平。结果ＭＡ－８９１细胞经ＩＦＮ－γ处理体外侵袭力明显增强（Ｐ＜０．０１）,经ＩＦＮ－α处理侵袭力下降。ＩＦＮ－γ显著增加细胞表面ＩＣＡＭ－１和７２０００ＩＶ型胶原酶表达水平,ＩＦＮ－α不增加细胞ＩＣＡＭ－１的表达但显著上调ｎｍ２３基因的表达水平。结论ＩＦＮ－γ和ＩＦＮ－α对ＭＡ－８９１细胞侵袭转移相关的基因和基因产物表达有不同的作用,而对其转移潜能产生不同的调变作用。     

鼻咽癌患者HER-2/neu基因扩增和表达及其临床意义

目的：研究鼻咽癌HER-2／neu基因扩增、表达及其临床意义。方法：采用原位荧光杂交、逆转录多聚链式反应和免疫组化技术检测鼻咽癌组织HER-2／neu基因扩增、表达。结果：HER-2／neu基因在鼻咽癌中无扩增，但有过表达，其原因是由于mRNA高表达所致；这种过表达与鼻咽癌预后之间未显示有相关性。结论：HER-2／neu基因在鼻咽癌无扩增，有过表达，这种过表达未显示预后意义。     

大肠癌组织中Bcl-2表达与临床病理参数相关性的研究

目的：探讨抑制凋亡基因Bcl-2在大肠癌及癌旁组织中的表达及意义。方法：应用免疫组化S-P法检测72例大肠癌组织和48例癌旁组织中Bcl-2的表达情况。结果：Bcl-2在癌组织与癌旁组织的表达率分别是54．17％（39／72）和20．83％（10／48）,二者差异有统计学意义,P〈0.001。Bcl-2在高、中和低分化腺癌组织中表达分别是66．67％、47．06％和35．71％,统计学分析高、低分化腺癌之间差异有统计学意义,P=0．016。大肠癌组织Bcl-2表达与年龄、性别和瘤体大体分型等临床病理参数无关,P〉0．05。在伴有淋巴结转移癌组织中Bel-2表达率为34．78％,无淋巴结转移者为63．27％,统计学分析差异有统计学意义,P-0．024。DukeD期Bcl-2表达率显著低于A、B、C期,P〈0．05。结论：Bcl-2蛋白参与了大肠癌的发生,其表达高低与肿瘤细胞的病理分级、Duke临床分期和淋巴结转移密切相关。