西伯利亚鲟免疫球蛋白M重链区mRNA的组织分布和细菌感染后的表达

根据GenBank登录的西伯利亚鲟Acipernser baerii免疫球蛋白M重链区mRNA（IgHM mRNA）设计引物,进行SYBR green实时定量。结果表明：鲟的脑、鳃、性腺、心脏、后肠、肝脏、肌肉、脾脏8种组织中,IgHM mRNA在脾脏中表达最高,在肝脏和心脏中表达较少,在脑、鳃、性腺、后肠、肌肉5种组织中几乎不表达;在水温22.6～24.5℃时,按100 g体质量注射0.1 mL菌液,给体质量为（260.2±37.8）g的西伯利亚鲟注射浓度为2.0×107CFU/mL的维氏气单胞菌Aeromonas veronii,注射后40 h鱼开始死亡,62 h后停止死亡,注射后直至62 h时,处理组脾脏IgHM mRNA表达量一直处于对照组水平,93 h时处理组脾脏IgHM mRNA显著升高（P〈0.05）,为93 h对照组含量的3.7倍,说明机体已经开始特异性免疫应答。

利用原位杂交技术检测免疫球蛋白重链mRNA在T和B淋巴瘤内的表达

应用原位杂交技术和免疫组织化学法对８例Ｂ淋巴瘤和４例Ｔ淋巴瘤进行了ＩｇＨｍＲＮＡ和ＩｇＭ，κ，λ的检测。结果显示，８例Ｂ淋巴瘤ＩｇＨｍＲＮＡ均为阳性，位于胞核周围，有的ｍＲＮＡ阳性反应成点状位于胞质的一侧。７例胞质中ＩｇＭ，κ或λ为阳性。１例胞质内无ＩｇＭ，κ或λ，但胞核内ＩｇＨｍＲＮＡ阳性反应成点状分布，可能是初级ＲＮＡ转录。在Ｔ淋巴瘤中，只有一例胞核内出现初级ＲＮＡ转录，因为早期Ｔ淋巴瘤，也有Ｄ。

基因免疫研究的最新进展

基因免疫是９０年代初开始发展起来的新方法。基因疫苗以其良好持久的免疫反应性，制备简单，易于保存等传统疫苗难以比拟的优点而受到普遍重视，目前已成为分子生物学研究的重点方向之一。不仅对很多病原体的基因疫苗进行了实验研究，而且还有个别疫苗已经进入Ⅰ、Ⅱ期临床试验阶段。同时在实验方法上也有新的探索，如ｍＲＮＡ免疫，表达库免疫，基因免疫毒素的研究，粘膜基因免疫等，使基因疫苗的研究有了一定的深度和广度。本文介绍了这些最新进展的概况。

Expression and Mutation of MAGE-A3 mRNA in Lung Cancer Tissues

Objective： To inwvetigate the expression of MAGE-A3 mRNA in tissue samples derived from lung cancers and to discuss the possibility of using MAGE-A3 antigens as a new peptide vaccine for inunotherapy for lung cancers. Methods： Tumor tissue samples of lung cancers and paired non-tumor tissues of the lung were obtaimed from 31 lung cancer patients. Total RNA was extracted and cDNA was synthesized. Nested polymernse chain reaction amplification using MAGE-A3 specific primer was performed to detect the expression of MAGE-A3. The 10 clones of 5 samples of MAGE-A3 mRNA positive PCR products were DNA sequenced by using DNAs sequencer （PE-377）. Results： Of 31 lung cancers, 26 （83.9%） expressed MACE-A3 mRNA. The expression of MAGE-A3 gene was not detectable in the adjacent lung tissues. The DNA sequencing confirmed that the target gene fragment in all 5 samples of PCR products was MACE-A3 cDNA. Point nmtations occurred in 4 samples （8 clones） detected （C^2773→T^2773; G^2807→A^2807） resulting in alternation of amino acid residue in one position （E^143→K）. Conclusion： （1） The MAGE-A3 gene was expressed exclusively in tumor tissues of the patients with lung cancer in China. This tumor rejection antigen may have potential to be used as a new peptide vaccine for immunotherapy for lung eancers. （2） There are two point mutations of MAGE-A3 gene sequence in some Chinese lung cancer patients.

Expression of cytokine mRNA during immuno-modulation of murine suppressor macrophages

In order to analyze the mechanism of immunomodulation by LPS on murine peritoneal suppressor macrophages, we have, using RNase protection assay,checked the changes of mRNA expression pattern of several cytokine genes during the immuno-modulation.It has been found that, after treating peritoneal suppressor macrophages with LPS, mRNAs of IL-12 p35, IL-12 p40,IL-6 and IFN-γ are newly appeared, while those of IL-1α, IL-1β and IL-1Ra are increased and those of other cytokines, like TGF-β1 and MIF are not changed at all.It seems certain that those cytokines, whose expression is increased by LPS stimulation, may be responsible for the functional changes of suppressor macrophages during immuno-modulation. Among these changes, the appearance of IL-12 mRNA may play a critical role, and, in this regard, the synergetic effect between IFN-γ and LPS on the increase of IL-12 p35 and IL-12 p40 mRNA expression is an interesting finding.

血清Tim-3 mRNA、MCP-1、IL-22、sCD30对慢性心力衰竭并发肺部感染患者的诊断价值

目的探讨血清人T细胞免疫球蛋白黏蛋白分子3(T cell immunoglobulin mucin molecule 3,Tim-3)mRNA、人单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)、白细胞介素-22(interleukin-22,IL-22)、人可溶性CD30(soluble CD30,sCD30)对慢性心力衰竭并发肺部感染患者的诊断价值。方法选取2016年10月到2019年1月北京市社会福利医院收治的老年慢性心力衰竭患者144例作为研究对象,依据是否发生肺部感染将患者分为对照组72例和肺部感染组72例,另选72名健康体检者为健康组。抽血检测3组人群血清中Tim-3mRNA、MCP-1、IL-22、sCD30浓度并进行比较。结果肺部感染组患者血清Tim-3 mRNA、MCP-1、IL-22、sCD30浓度高于对照组,对照组高于健康组,差异有统计学意义(P<0.05)。慢性心力衰竭并发肺部感染患者血清MCP-1、IL-22、sCD30、Tim-3 mRNA之间互为正相关,并且随着肺感染程度分级和纽约心脏协会心功能分级的升高而升高,不同分级间比较差异具有统计学意义(P<0.05)。Tim-3 mRNA、MCP-1、IL-22、sCD30诊断肺部感染的受试者工作特征曲线(receiver operating characteristic curve,ROC)的曲线下面积(area under curve,AUC)均接近于0.9,说明诊断准确性较高。结论Tim-3mRNA、MCP-1、IL-22、sCD30可能是慢性心力衰竭患者是否肺部感染的诊断标志物,监测其浓度变化对患者病情评估具有重要临床意义。

Cloning and Phylogenetic Analysis of NS1 Genes from Different Isolates of H9N2 Subtype Duck Influenza Virus

[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.

α-catenin expression is decreased in patients with gastric carcinoma

AIM: To assess the expression of α-catenin in gastric carcinoma and to determine the role of α-catenin expression in gastric carcinogenesis.METHODS: α-catenin expression was assessed by semi quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical staining in 49 gastric carcinomas,26 adjacent non-cancerous mucosae, and gastric biopsy specimens from 11 healthy controls.RESULTS: mRNA levels of α-catenin were reduced or absent in 34 of 49 (69%) gastric carcinoma tissues and in 5 of 26 (19%) tumor-free gastric mucosae of carcinomapatients, respectively. Of the carcinoma samples with altered α-catenin mRNA levels, α-catenin expression was negative in 20 and decreased in 14 cases. Up to 69% of tumors were stained abnormally for α-catenin. Of the 34 cases whose mRNA expression of α-catenin was reduced, 32 (94%) showed abnormal immunostaining patterns, while only 2 showed a normal α-catenin expression. The frequency of reduced expression of α-catenin mRNA was 14% in well-differentiated carcinomas, higher than that in poorlydifferentiated carcinomas (86%). A significant correlation was not shown between α-catenin expression and bothdepth of invasion and lymph node metastasis. Moreover, there was no statistical difference between loss or down-regulation of α-catenin mRNA and Helicobacter pylori ( H pylori) infection.CONCLUSION: Downregulation of α-catenin expressionis common in gastric carcinoma, and α-catenin expression may be used as a differentiation marker. Downregulation of α-catenin expression may be an early event in tumorigenesis. Reduced α-catenin expression is not correlated with H pylori infection.

Embryonic hepatocyte transplantation for hepatic cirrhosis:Efficacy and mechanism of action

AIM： To investigate the efficacy and mechanism of action of allogeneic embryonic hepatocyte transplantation for the treatment of hepatic cirrhosis. METHODS： Rat embryonic hepatocytes were characterized by examining cell markers. Wistar rats with CCl4-induced cirrhosis were randomly divided into two groups： a model group receiving continuous CCl4, and a cell transplantation group receiving continuous CCh and transplanted with embryonic fluorescent-labeled hepatocytes. In addition, a normal control group was composed of healthy rats. All rats were sacrificed after 2 wk following the initiation of the cell transplant. Ul- trasound, pathological analyses and serum biochemical tests were used to evaluate the efficacy of embryonic hepatocyte transplantation. To analyze the recovery status of cirrhotic hepatocytes and the signaling pathways influenced by embryonic hepatocyte transplantation, real-time polymerase chain reaction was performed to examine the mRNA expression of stellate activation-associated protein （STAP）, c-myb, ~ smooth muscle actin （^-SMA） and endothelin-1 （ET-1）. West- ern blotting and immunohistochemistry were employed to detect ^-SMA and ET-1 protein expression in hepatic tissues. RESULTS： Gross morphological, ultrasound and his- topathological examinations, serum biochemical tests and radioimmunoassays demonstrated that hepatic cir- rhosis was successfully established in the Wistar rats. Stem cell factor receptor （c-kit）, hepatocyte growth factor receptor （c-Met）, Nestin, ~ fetal protein, albu- min and cytokeratin19 markers were observed in the rat embryonic hepatocytes. Following embryonic hepa- tocyte transplantation, there was a significant reversal in the gross appearance, ultrasound findings, histo- pathological properties, and serum biochemical param- eters of the rat liver. In addition, after the activation of hepatic stellate cells and STAP signaling, ^-SMA, c-myb and ET-1 mRNA levels became significantly lower than in the untreated cirrhotic group （P 〈 0.05）. These levels, however, were not statistically different from those of the normal healthy group. Immunohisto- chemical staining and Western blot analyses revealed that ^-SMA and ET-1 protein expression levels in the transplantation group were significantly lower than in the untreated cirrhotic group, but being not statistically different from the normal group. CONCLUSION： Transplantation of embryonic hepatocytes in rats has therapeutic effects on cirrhosis. The described treatment may significantly reduce the expression of STAP and ET-1.

Vascular endothelial growth factor 165b expression in stromal cells and colorectal cancer

AIM:To characterize the implications of vascular endothelial growth factor(VEGF)-A in stromal cells and colorectal cancer and the expression of VEGF-A splice variants.METHODS:VEGF-A expression in tumor and stromal cells from 165 consecutive patients with colorectal cancer was examined by immunohistochemistry.The association between VEGF-A expression status and clinicopathological factors was investigated.Twenty freshfrozen samples were obtained for laser capture microdissection to analyze the splice variants of VEGF-A.RESULTS:VEGF-A was expressed in 53.9% and 42.4% of tumor and stromal cells,respectively.VEGF-A expression in tumor cells(t-VEGF-A) was associated with advanced clinical stage(stage 0,1/9;stage 1,2/16;stage 2,32/55;stage 3,38/66;stage 4,16/19,P < 0.0001).VEGF-A expression in stromal cells(s-VEGF-A) increased in the earlier clinical stage(stage 0,7/9;stage 1,6/16;stage 2,33/55;stage 3,22/66;stage 4,5/19;P = 0.004).Multivariate analyses for risk factors of recurrence showed that only s-VEGF-A expression was an independent risk factor for recurrence(relative risk 0.309,95% confidence interval 0.141-0.676,P = 0.0033).The five-year disease-free survival(DFS) rates of t-VEGF-A-positive and-negative cases were 51.4% and 62.9%,respectively.There was no significant difference in t-VEGF-A expression status.The five-year DFS rates of s-VEGF-A-positive and-negative cases were 73.8% and 39.9%,respectively.s-VEGFA-positive cases had significantly better survival than s-VEGF-A-negative cases(P = 0.0005).Splice variant analysis revealed that t-VEGF-A was mainly composed of VEGF165 and that s-VEGF-A included both VEGF165 and VEGF165b.In cases with no venous invasion(v0),the level of VEGF165b mRNA was significantly higher(v0 204.5 ± 122.7,v1 32.5 ± 36.7,v2 2.1 ± 1.7,P = 0.03).The microvessel density tended to be lower in cases with higher VEGF165b mRNA levels.CONCLUSION:s-VEGF-A appears be a good prognostic factor for colorectal cancer and includes VEGF165 and VEGF165b.

Prunella vulgaris L. extract improves cellular immunity in MDR-TB challenged rats

Objective： To study the effect of the extract of Prunella vulgaris L. on multiple drugs resistant bacillus tuberculosis （MDR-TB）. Methods： Experimental animal model in rats was induced by MDR-TB. Normal group model group and Prunella vulgaris L. group were set up. The contents of IFN-7, IL-4, IL-10 and IL-12 were examined by ELISA. Their genome mRNAs were extracted, the target genes were amplified by PCR. RT-PCR was used to detect the mRNA levels of them. Results： The content of IFN-q, of the extract of Prunella vulgaris L. group was 1.98±0.67 pg/ml, IL-4 was 6.47±1.46 pg/ml, IL-10 was 12.13±3.43 pg/ml and IL-12 was 3.02±0.86 pg/ml. Compared with the model group, Prunella vulgaris L. group was notable difference in serum IFN-γ, IL-12 and IL-10 （P〈0.05）. The mRNA levels of IFN-γ, IL-12 increased and IL-10 decreased obviously, the differences were quite significant （P〈0.05）, but IL-4 had no obvious change. Conclusion： The extract of Prunella vulgaris L. can enhance the cellar immunological function in rats from up-regulation of the level of genetic transcription, accordingly provide the theory basis of healing of tuberculosis with it.

Reduction of natural regulatory T cells in thymomas accompanying myasthenia gravis and its possible association with Foxp3 and thymic stromal lymphopoietin

Objective: To investigate the expression of both thymic regulatory T cells (CD4+CD25+Foxp3+cells, Treg) and thymic stromal lymphopoietin (TSLP) in thymomas accompanying myasthenia gravis. Methods: We used immunohistochemistry and real-time reverse trancription polymerase chain reaction (real-time RT-PCR) techniques to determine Foxp3+ Treg counts and the expression levels of Foxp3 mRNA and TSLP mRNA in thymomas of 23 MG patients and thymuses of 4 healthy controls. Results: The CD4+ Foxp3+ nTreg (natural regulatory T cells) counts in thymomas were significantly lower than those in normal thymuses (P<0.01), and the expression levels of Foxp3 mRNA and TSLP mRNA were also lower in thymomas(P<0.01). Among the thymoma types, type B1 thymoma had the highest Foxp3+ nTreg count and standard values of Foxp3 mRNA and TSLP mRNA. There was a strong positive correlation between the mRNA transcriptional levels of Foxp3 and TSLP. Conclusion: The insufficient expression of Foxp3 in thymoma, which may be caused by decreased transcription of TSLP, may result in the reduction of Tregs and cause autoimmune disorders.

Effects of anesthetic methods on preserving anti-tumor T-helper polarization following hepatectomy

AIM:To investigate the impact of different anesthetic techniques on T-helper(Th) cell subsets in hepatocellular carcinoma(HCC) patients undergoing hepatectomy.METHODS:Sixty-one HCC patients who received hepatectomies were randomized into an epidural combined general anesthesia(G + E;n = 31) or a general anesthesia(G;n = 30) group.Blood samples were obtained the morning before the operation(d0),and on the second(d2) and seventh(d7) day after the operation.Th cell contents were evaluated using flow cytometry,realtime reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay.RESULTS:In all 61 patients,Th1 and Th2 cell frequencies,and interferon-(IFN-) mRNA expression markedly increased on d2,compared to d0.They recovered slightly on d7,and the Th1/Th2 ratio increased markedly on d7,compared with d2.In contrast,Th17,regulatory T cell(Treg),and interleukin-17(IL-17) levels and FOXP3 mRNA expression showed no significant change on d2,and then markedly decreased on d7.Similarly,plasma IFN-concentration on d2 was much higher than that on d0,and then partly recovered on d7.As compared with the G group,in the G + E group,Th1 cell frequencies and the Th1/Th2 ratio were slightly higher on d2 and significantly higher on d7,while Th2,Th17,and Treg cell frequencies were slightly lower on d2,and significantly lower on d7.Consistently,on d7,IFN-mRNA and protein levels and the IFN-/IL-4 ratio in the G + E group were higher than those in the G group.In contrast,the IL-17 mRNA level,and IL-17 and transforming growth factor-1 concentrations in the G + E group were lower than those in the G group.CONCLUSION:G + E is superior to G in shifting the Th1/Th2 balance towards Th1,while decreasing Th17 and Treg,potentially benefiting HCC patients by promoting anti-tumor Th polarization.

Lipopolysaccharide induces and activates the Nalp3 inflammasome in the liver

AIM:To examine the activation of the Nalp3 inflammasome and its downstream targets following lipopolysaccharide(LPS) -induced stimulation in the liver. METHODS:Six-to-eight-week-old C57BL/6 chow fed mice were injected intraperitoneally with 0.5μg/g bodyweight LPS and sacrificed 2,4,6,18 or 24 h later. LPS-induced liver damage was confirmed by a biochemical assay to detect alanine aminotransferase(ALT) levels.To determine if LPS stimulation in the liver led to activation of the inflammasome,real-time quantitative polymerase chain reaction was used to evaluate the mRNA expression of components of the Nalp3 inflammasome.Enzyme-linked immunosorbent assays were used to determine the protein expression levels of several downstream targets of the Nalp3 inflammasome,including caspase-1 and two cytokine targets of caspase-1,interleukin(IL) -1βand IL-18. RESULTS:We found that LPS injection resulted in liver damage as indicated by elevated ALT levels.This was associated with a significant increase in both mRNA and protein levels of the proinflammatory cy-tokine tumor necrosis factor(TNF) -αin the liver,as well as increased levels of TNFs in serum.We showed that LPS stimulation led to upregulation of mRNA levels in the liver for all the receptor components of the inflammasome,including Nalp3,Nalp1,pannexin-1 and the adaptor molecule apoptosis-associated specklike,caspase recruitment domain-domain containing protein.We also found increased levels of mRNA and protein for caspase-1,a downstream target of the inflammasome.In addition,LPS challenge led to increased levels of both mRNA and protein in the liver for two cytokine targets of caspase-1,IL-1βand IL-18. Interestingly,substantial baseline expression of pre-IL1βand pre-IL-18 was found in the liver.Inflammasome and caspase-1 activation was indicated by the significant increase in the active forms of IL-1βand IL-18 after LPS stimulation. CONCLUSION:Our results show that the Nalp3 inflammasome is upregulated and activated in the liver in response to LPS stimulation.

The association of altered RARβ gene expression with the cervical lesion pathogenesis

Objective:Previous reports have shown that the gene promoter region of retinoic acid receptor β(RARβ) was hypermethylated in cervical carcinoma,implying the inhibition of gene transcription.The aim of this study was to investigate the association of cervical cancer development with the RARβ gene expression at the mRNA and protein level to assess the impact of RARβ as a marker for early detection of the cancer.Methods:We collected 126 cases of formalin fixed and paraffin embedded cervical tissue specimens as well as 37 cases of fresh tissue samples from women with cervicitis,cervical intraepithelial neoplasia(CIN) and cervical squamous cell carcinoma(CSCC).The RARβ mRNA and protein expression was detected by quantitative RT-PCR and immunohistochemistry,respectively.Results:(1) The mRNA expression of RARβ in CIN and cervical cancer was markedly decreased compared to cervicitis with a statistically very significant difference,but no difference was found between CIN and cervical cancer.(2) RARβ protein was normally expressed in the epithelial cells of cervicitis and partially lost in a few cases,but with the development of cervical lesion pathogenesis and cancer,a significant loss of protein expression was detected in CIN(38%) and CSCC(57%) compared to cervicitis(P < 0.01).Conclusion:The downregulation of RARβ transcription or loss of protein expression is an important indicator of cervical cancer and its precursur lesions.The detection of RARβ expression coupled with aberrant methylation of the gene may become a biomarker for the early prognosis or diagnosis of the cancer.

Inhibition of SIRT1 Increases EZH2 Protein Level and Enhances the Repression of EZH2 on Target Gene Expression

Objective To study the regulatory roles of SIRT1 on EZH2 expression and the further ef-fects on EZH2's repression of target gene expression. Methods The stable SIRT1 RNAi and Control RNAi HeLa cells were established by in-fection with retroviruses expressing shSIRT1 and shLuc respectively followed by puromycin selection. EZH2 protein level was detected by Western blot in either whole cell lysate or the fractional cell extract. Reverse transcription-polymerase chain reaction was performed to detect the mRNA level of EZH2. Cycloheximide was used to treat SIRT1 RNAi and Control RNAi cells for protein stability assay. Chromatin immunoprecipitation (ChIP) assay was applied to measure enrichment of SIRT1, EZH2, and trimethylated H3K27 (H3K27me3) at SATB1 promoter in SIRT1 RNAi and Control RNAi cells. Results Western blot results showed that EZH2 protein level increased upon SIRT1 de-pletion. Fractional extraction results showed unchanged cytoplasmic fraction and increased chromatin fraction of EZH2 protein in SIRT1 RNAi cells. The mRNA level of EZH2 was not affected by knockdown of SIRT1. SIRT1 recruitment was not detected at the promoter region of EZH2 gene locus. The protein stability assay showed that the protein stability of EZH2 increases upon SIRT1 knockdown. Upon SIRT1 depletion, EZH2 and H3K27me3 recruitment at SATB1 promoter increases and the mRNA level of SATB1 decreases. Conclusions Depletion of SIRT1 increases the protein stability of EZH2. The regulation of EZH2 protein level by SIRT1 affects the repressive effects of EZH2 on the target gene expres-sion.

Upregulated CD133 expression in tumorigenesis of colon cancer cells

AIM: To analyze the upregulated CD133 expression in tumorigenesis of primary colon cancer cells. METHODS: Upregulated CD133 expression in tumorigenesis of colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) was analyzed by flow cytometry. Human colon cancer tissue samples were stained with anti-human CD133. SW620 cells were sorted according to the CD133 expression level measured by fluorescence-activated cell sorting. Spheroids of colorectal cancer cells were cultured with the hanging drop. Expression of CD133 and Lgr5 in spheroids of colorectal cancer cells and monolayer culture was detected by RT-qPCR. Spheroids of colorectal cancer cells were analyzed using anti-human CD133 with immunohistochemical staining. RESULTS: CD133 antigen was expressed in colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) as well as in primary and metastatic human colon cancer tissues. However, the CD133 was differently expressed in these cell lines and tissues. The expression levels of CD133 and Lgr5 were significantly higher in spheroids of parental, CD133hi and CD133-cells than in their monolayer culture at the mRNA level (P < 0.05). Immunohistochemical staining of spheroids of CD133-cells showed that CD133 was highly expressed in colorectal cancer cell lines. CONCLUSION: Upregulated CD133 expression plays a role in tumorigenesis colorectal cancer cells, which may promote the expression of other critical genes that can drive tumorigenesis.

Association between Bmi1 and clinicopathological status of esophageal squamous cell carcinoma

AIM： To investigate the clinicopathological roles of Bmil in esophageal squamous cell carcinoma （ESCC）.METHODS： Quantitative real-time polymerase chain reaction and immunohistochemical staining for Broil were performed in cancerous and adjacent non-cancerous paraffin-embedded esophageal specimens.RESULTS： The Bmil expression level was unaffected by gender and age. The level of Broil mRNA in ESCC was significantly higher than that in the adjacent non-cancerous tissues （2.181 ± 2.158 vs 0.931 ± 0.894, P = 0.0152）, and its over-expression was aggressively associated with lymph node metastasis （3.580 ± 2.487 vs 1.703 ± 0.758, P = 0.0003）, poorer cell differentiation （P = 0.0000） and advanced pathological stage （3.827± 2.673 vs 1.590 ± 0.735, P = 0.0001）. The patients were divided into high-expression and low-expression groups based on the median expression level of Bmi1 mRNA, and a shorter overall survival time in the former group was observed. Immunohistochemistry for Bmi1 oncoprotein showed diffusely positive, focally positive and negative expression in 44, 16 and 10 of 70 ESCC cases, respectively, compared with three, two and five of 10 adjacent non-cancerous cases （P = 0.027）. The positive rate of the oncoprotein in samples of histological grade Ⅲ was higher than that of grade Ⅱ（P = 0.031）, but its expression had no relation to the lymph node metastasis and pathological staging. In 70 ESCC samples, Bmi1 showed high intense expression in the cytoplasm and less or even no expression in the nucleus.CONCLUSION： Bmi1 was over-expressed in ESCC. Increased Bmi1 mRNA expression was significantly associated with ESCC progression, and the oncoprotein was largely distributed in the cytoplasm of tumor cells.

Aberrant methylation of the 3q25 tumor suppressor gene PTX3 in human esophageal squamous cell carcinoma

AIM:To identify the novel methylation-silenced gene pentraxin 3(PTX3) in esophageal squamous cell carcinoma(ESCC).METHODS:PTX3 mRNA expression was examined in six human ESCC cell lines,one human immortalized normal esophageal epithelial cell line,primary ESCC tumor tissue,and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction(RTPCR).Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels.Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene.RESULTS:In the majority of ESCC cell lines,we found that PTX3 expression was down-regulated due to gene promoter hypermethylation,which was further confirmed by bisulphite genomic sequencing.Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines.Methylation was more common in tumor tissues(85%) than in adjacent nontumor tissues(25%)(P < 0.01).CONCLUSION:PTX3 is down-regulated through promoter hypermethylation in ESCC,and could potentially serve as a biomarker of ESCC.

Expression and localization of paxillin in rat pancreas during development

AIM:To investigate the expression and localization of paxillin in rat pancreas during development. METHODS:Pancreata from Sprague Dawley rat fetuses, embryos, young animals, and adult animals were used in this study. Expression levels of paxillin in pancreata of different development stages were detected by reverse transcription polymerase chain reaction and Western blotting. To identify the cell location of paxillin in the developing rat pancreas, immunohistochemistry and double-immunofluorescent staining were performed using antibodies for specific cell markers and paxillin, respectively. RESULTS:The highest paxillin mRNA level was detected at E15.5 (embryo day 15.5) following a decrease in the later developmental periods (P < 0.05 vs E18.5, P0 and adult, respectively), and a progressively increased paxillin protein expression through the transition from E15.5 to adult was detected. The paxillin positive staining was mainly localized in rat islets of Langerhans at each stage tested during pancreas development. CONCLUSION:The dynamic expression of paxillin in rat pancreas from different stages indicates that paxillin might be involved in some aspects of pancreatic development.