Gene expression analysis of cytokines and MMPs in melatonin and rhBMP-2 enhanced bone remodeling

BACKGROUND In the medical and dental fields,there is a need for studies of new therapeutic approaches for the treatment of bone defects that cause extensive bone loss.Melatonin may be an important endogenous biological factor for bone remodeling,and growth factors may enhance the repair process.AIM To evaluate the gene expression of cytokines(IL-1β,IL-6,IL-10 and TNF-α),markers of osteoclastogenesis(RANK,RANKL and OPG)and MMPs(MMP-1,MMP-2,MMP-8 and MMP-13)from the treatment of melatonin associated with an osteogenic membrane and rhBMP-2 on the recovery of a bone injury.METHODS Sixty-four rats were used and divided into 9 experimental groups and were formed according to the treatment carried out in the region of the bone lesion,which varied between the combination of 1,10 and 100μmol/L of melatonin.Gene Expression analysis was performed using real time-PCR by reading the concentration of total RNA and reverse transcription.RESULTS There were differences between groups when compared with clot or scaffold control,and improvement with a higher concentration of melatonin or rhBMP-2.The combination melatonin(1μg)with 5μg of rhBMP-2,using the guided bone regeneration technique,demonstrated some effects,albeit mild,on bone repair of critical bone defects.CONCLUSION This indicates that the approach for administering these substances needs to be reassessed,with the goal of ensuring their direct application to the affected area.Therefore,future research must be carried out,seeking to produce materials with these ideal characteristics.

Genome-Wide Identification, Evolution and Expression Analyses of GA2ox Gene Family in Brassica napus L.

Gibberellin 2-oxidases(GA2ox)are important enzymes that maintain the balance of bioactive GAs in plants.GA2ox genes have been identified and characterized in many plants,but these genes were not investigated in Brassica napus.Here,we identified 31 GA2ox genes in B.napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes.Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm,and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons.Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups,including two C_(19)-GA2ox and two C_(20)-GA2ox clades.Group 4 is a C_(20)-GA2ox Class discovered recently.Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes.BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome.BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development,and most of them were mainly involved in abiotic responses,regulation of phytohormones and growth and development.Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons,as well as an insight into the biological functions of GA2ox family genes in B.napus.

血清IL-2、sST2表达与特发性膜性肾病免疫抑制剂治疗反应性的相关性

目的探讨特发性膜性肾病患者血清白介素-2(IL-2)、可溶性生长刺激表达基因2蛋白(sST2)表达水平与免疫抑制剂治疗反应性的相关性。方法选取2020年1月至2022年10月于医院接受免疫抑制剂治疗的135例特发性膜性肾病患者,于入院时检测患者血清IL-2、sST2,并于治疗完成后测定24 h尿蛋白定量,依据患者治疗反应性分为缓解组与未缓解组。对比两组患者一般资料及入院时血清IL-2、sST2水平,采用点二列相关性分析血清IL-2、sST2水平与特发性膜性肾病免疫抑制剂治疗反应性的关系,并绘制受试者工作特征(ROC)曲线评估血清IL-2、sST2水平预测特发性膜性肾病免疫抑制剂治疗反应性的价值。结果135例特发性膜性肾病患者中共有132例完成规律治疗,经免疫抑制剂治疗6个月后,101例患者疾病缓解,纳入缓解组,其余31例患者纳入未缓解组。未缓解组年龄、入院时肾功能分级、疾病分期、血清IL-2、sST2水平均高于缓解组,差异有统计学意义(P<0.05);点二列相关性分析显示,血清IL-2、sST2水平与特发性膜性肾病免疫抑制剂治疗反应性不良风险呈正相关(r 1=0.428,P 1<0.001;r 2=0.344,P 2<0.001);绘制ROC曲线,结果显示,血清IL-2、sST2预测特发性膜性肾病免疫抑制剂治疗反应性不良的曲线下面积均>0.7,具有一定预测价值,且联合预测价值更高。结论血清IL-2、sST2表达水平与特发性膜性肾病患者免疫抑制剂治疗反应性密切相关,二者表达水平越高,治疗反应性越差,且联合检测可作为预测特发性膜性肾病患者免疫抑制剂治疗反应性的敏感指标。

Cloning and Prokaryotic Expression of Canine IL-2 Gene

[ Objective] To clone and express canine IL-2 gene and thus to provide theoretical support for the development of novel immune enhancers and genetic engineering vaccines. [ Method] Leukocytes separated from canine whole blood were stimulated by concanavalin for 20 h, and then total RNA was extracted. According to the sequence of canine IL-2 gene published in the GenBank, a pair of primers was designed. After PCR am- plificetion, the target fragment was cloned into prokaryotic expression vector pET-28a. The recombinants were transformed into the host bacteria BL21. After IPTG induction, the expression products were analyzed by SDS-PAGE. [ Result] A 500 bp band with the expected size appeared in the RT-PCR products. After the pMD18-T-IL2 was identified by double digestion, an approximately 500 bp fragment was produced, which indicated successful cloning of the gene. After the pET-28a-lL2 was identified by restriction enzyme digestion and PCR, a 500 bp fragment was produced, which indicated successful construction of the expression vector. As revealed by the SDS-PAGE analysis, a protein band with molecular weight of about 20 kDa appeared. [ Conclusion] The canine IL-2 gene was cloned and expressed.

Effects of Light and Temperature on the Expression of the Lhcb2 Gene in Pea

An approximately 800 bp cDNA ( Lhcb 2) encoding light_harvesting chlorophyll a/b_binding protein complex (type Ⅱ) was cloned from the seedling of pea ( Pisum sativum L.) with RT_PCR method. Southern blotting using special probe demonstrated that there existed one copy of Lhcb 2 in pea genome. RT_PCR and Northern blotting revealed the expression of Lhcb 2 which was regulated by light in a time_dependent expression manner. The Lhcb 2 gene didn't express untill 2 h after irradiated with white light. Low temperature (4 ℃) also affected the Lhcb 2 gene by decreasing half of its expression under 25 ℃.

Analysis of Seed-specificity of Silencing fad_2 Gene Expression in Transgenic Rapeseed Line W-4(Brassica napus L.)

This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.

Identification of hub genes associated with Helicobacter pylori infection and type 2 diabetes mellitus:A pilot bioinformatics study

BACKGROUND Helicobacter pylori(H.pylori)infection is related to various extragastric diseases including type 2 diabetes mellitus(T2DM).However,the possible mechanisms connecting H.pylori infection and T2DM remain unknown.AIM To explore potential molecular connections between H.pylori infection and T2DM.METHODS We extracted gene expression arrays from three online datasets(GSE60427,GSE27411 and GSE115601).Differentially expressed genes(DEGs)commonly present in patients with H.pylori infection and T2DM were identified.Hub genes were validated using human gastric biopsy samples.Correlations between hub genes and immune cell infiltration,miRNAs,and transcription factors(TFs)were further analyzed.RESULTS A total of 67 DEGs were commonly presented in patients with H.pylori infection and T2DM.Five significantly upregulated hub genes,including TLR4,ITGAM,C5AR1,FCER1G,and FCGR2A,were finally identified,all of which are closely related to immune cell infiltration.The gene-miRNA analysis detected 13 miRNAs with at least two gene cross-links.TF-gene interaction networks showed that TLR4 was coregulated by 26 TFs,the largest number of TFs among the 5 hub genes.CONCLUSION We identified five hub genes that may have molecular connections between H.pylori infection and T2DM.This study provides new insights into the pathogenesis of H.pylori-induced onset of T2DM.

The Role of Predominant Expression of Th2 Type Cytokines Gene in the Genesis and Development of Human Gliomas

Objective: To explore the expression of Th1/Th2 cytokines gene in human gliomas and its role in the genesis and development of human gliomas.Methods: Using IL-2 and IFNγ as Th1 type cytokines, IL-4, IL-6 and IL-10 as Th2 type cytokines, the biological activity of cytokines in the supernatant of glioma cell lines was assayed by ELISA method, and the gene expression of Th1/Th2 cytokines in human glioma cells, glioma infiltrating lymphocytes and glioma cell lines were detected by RT-PCR.Results: There was predominant expression of Th2 type cytokines in human glioma cells, glioma infiltrating lymphocytes and glioma cell lines, but there was no such expression in normal brain tissues.Conclusion: It suggested that there is a relationship between the Th2 type cytokines expression in human gliomas and the immunosupressive status of human glioma patients. The predominant expression of Th2 type cytokines may play an important role in the genesis and development of human gliomas. Key words glioma - Th1/Th2 - gene expression - RT-PCR This project was supported by a grant from National Natural Sciences foundation of China (No. 30271335).

SNP Identification in α_(2A)-Adrenergic Receptor Gene in Chinese and the Effect on Gene Expression

Objective: To scan single nucleotide polymorphism ( SNP ) in Chinese alpha-2Aadrenergic receptor (α_(2A)-AR) gene and study the effects of the SNP on the gene expression.Methods: The complete sequence of α_(2A)-AR gene was analyzed with automated DNA sequencer to scanSNPs. Genomic DNA was extracted from whole blood and a 239 bp fragment containing the G/Cpolymorphism was amplified with PCR using a pair of. specific primers. PCR-RFLP was used to performthe genotyping of the SNP at the site-1 296 bp of the people in the North of China. Electrophoresismobility shift assay ( EMSA ) was used to study the binding of the 390 bp fragments (- 1 414-1 025bp) with G or C at the site-1 296 bp and nuclear extracts . Results: In our study, two SNPs werefound in α_(2A)-AR gene. Allele frequencies of the SNP at the site-1 296 bp were 0.61 and 0.39 forG and C , and the genotype frequencies were 0.34 , 0.54 and 0.13 for GG, GC and CC respectively fromthe people in the North of China. In the EMSA, a specific binding appeared in the complex ofnuclear extracts and DNA with C at-1 296 bp . Conclusion: Two SNPs exist in α_(2A)-AR gene from thepeople in the North of China , and DNA fragment with allele C of the SNP at the site-1 296 bp couldbind with a specific protein, which could influence the gene expression.

急性心肌梗死患者血清sST2、Hcy、IL-33水平与冠脉狭窄程度及MACE的关系研究

目的探讨急性心肌梗死(AMI)患者血清可溶性生长刺激表达因子2(sST2)、同型半胱氨酸(Hcy)、白细胞介素(IL)-33水平与冠脉狭窄程度及主要不良心血管事件(MACE)的关系。方法回顾性选取该院于2021年5月至2023年5月收治的139例AMI患者,按照是否发生MACE,分为预后良好组54例,预后不良组85例。采用Spearman相关性分析AMI患者血清sST2、Hcy、IL-33水平与冠脉狭窄程度的关系。采用多因素Logistic回归分析患者发生MACE的影响因素,以及绘制受试者工作特征(ROC)曲线评价血清sST2、Hcy、IL-33水平对AMI患者发生MACE的预测价值。结果预后不良组血清sST2、Hcy、IL-33水平显著高于预后良好组,差异有统计学意义(P<0.05)。重度狭窄、中度狭窄患者血清sST2、Hcy、IL-33水平均明显高于轻度狭窄患者,差异有统计学意义(P<0.05)。Spearman相关性分析显示,血清sST2、Hcy、IL-33水平与冠脉狭窄程度呈正相关(P<0.05)。血清Hcy、sST2、IL-33均是影响AMI患者发生MACE的危险因素。ROC曲线分析显示,血清sST2、Hcy、IL-33水平对于AMI患者发生MACE的预测价值的曲线下面积(AUC)分别为0.838、0.726和0.800,灵敏度分别为87.1%、80.0%和84.7%,特异度分别为77.8%、68.5%和77.8%,三者联合对AMI患者发生MACE的预测价值的AUC为0.906,灵敏度和特异度分别为88.2%、90.7%。结论血清sST2、Hcy、IL-33水平对于AMI患者发生MACE具有一定的预测价值。

颅脑外伤患者血清IL-33、sST2、TNF-α、HMGB1水平与病情严重程度及短期预后的关系

目的探讨颅脑外伤患者血清白细胞介素(IL)-33、可溶性生长刺激表达基因2蛋白(sST2)、肿瘤坏死因子-α(TNF-α)、高迁移率族蛋白B1(HMGB1)与病情严重程度及短期预后的关系。方法回顾性分析2022年1月至2024年1月渭南市中心医院收治的142例颅脑外伤患者的临床资料。根据格拉斯哥昏迷量表(GCS)评估病情严重程度分为轻度组(13~15分)(n=47)、中度组(9~12分)(n=59)、重度(3~8分)(n=36)。根据格拉斯哥预后量表(GOS)评估28 d预后分为预后良好组(4~5分)(n=115)、预后不良组(1~3分)(n=27)。收集患者资料,包括性别、年龄、体重指数、吸烟、饮酒、高血压、糖尿病、外伤原因、IL-33、sST2、TNF-α、HMGB1。比较不同病情严重程度、不同预后患者的临床资料;采用Spearman秩相关分析检验颅脑外伤患者病情严重程度与IL-33、sST2、TNF-α、HMGB1水平的相关性;通过受试者操作特征(ROC)分析IL-33、sST2、TNF-α、HMGB1预测颅脑外伤患者预后不良的价值。结果重度组IL-33、sST2、TNF-α、HMGB1水平分别为(40.69±9.64)pg/mL、(7.75±2.14)ng/mL、(252.43±41.37)ng/L、(11.42±3.27)μg/L,中度组IL-33、sST2、TNF-α、HMGB1水平分别为(30.47±7.22)pg/mL、(6.45±1.37)ng/mL、(181.29±22.75)ng/L、(8.05±2.13)μg/L,均高于轻度组[(21.53±5.84)pg/mL、(5.61±1.05)ng/mL、(140.36±16.41)ng/L、(4.87±1.42)μg/L],差异均有统计学意义(P<0.05)。IL-33、sST2、TNF-α、HMGB1水平与颅脑外伤患者病情严重程度呈正相关(r=0.561、0.518、0.593、0.537,P<0.05)。预后不良组IL-33、sST2、TNF-α、HMGB1水平分别为(74.08±20.37)pg/mL、(10.56±2.54)ng/mL、(286.31±42.81)ng/L、(13.45±3.07)μg/L,均高于预后良好组[(22.64±6.19)pg/mL、(6.95±1.73)ng/mL、(153.76±31.45)ng/L、(5.86±1.49)μg/L],差异均有统计学意义(P<0.05)。ROC曲线分析结果显示,IL-33、sST2、TNF-α、HMGB1预测颅脑外伤患者预后不良的曲线下面积为0.891、0.846、0.905、0.928,均有P<0.05。结论IL-33、sST2、TNF-α、HMGB1表达水平随颅脑外伤患者病情严重程度升高而升高,且与其短期不良预后有关。

Identification and Expression Analysis of Abscisic Acid Signal Transduction Genes in Hemp Seeds

Abscisic acid(ABA)is involved in regulating diverse biological processes,but its signal transduction genes and roles in hemp seed germination are not well known.Here,the ABA signaling pathway members,PYL,PP2C and SnRK2 gene families,were identified from the hemp reference genome,including 7 CsPYL(pyrab-actin resistance1-like,ABA receptor),8 CsPP2CA(group A protein phosphatase 2c),and 7 CsSnRK2(sucrose nonfermenting1-related protein kinase 2).The content of ABA in hemp seeds in germination stage is lower than that in non-germination stage.Exogenous ABA(1 or 10μM)treatment had a significant regulatory effect on the selected PYL,PP2C,SnRK2 gene families.CsAHG3 and CsHAI1 were most significantly affected by exogenous ABA treatment.Yeast two-hybrid experiments were performed to reveal that CsPYL5,CsSnRK2.2,and CsSnRK2.3 could interact with CsPP2CA7 and demonstrate that this interaction was ABA-independent.Our results indicated that CsPYL5,CsSnRK2.2,CsSnRK2.3 and CsPP2CA7 might involve in the ABA signaling transduction pathway of hemp seeds during the hemp seed germination stages.This study suggested that novel genetic views can be brought into investigation of ABA signaling pathway in hemp seeds and lay the foundation for further exploration of the mechanism of hemp seed germination.

Effect of small interference RNA on expression of the Skp2 in human chondrocytes cell

Objective:To study the inhibitory effect of siRNA mediated by expression vectors on Skp2 expression in human chondrocytes.Method:Three Skp2 sequences siRNA-59,siRNA-318 and siRNA-504 were designed as target sites using online siRNA design tools.Skp2 siRNA expression vectors were successfully constructed in vitro by gene recombination technology,and the influence of recombinant plasmid transfection on Skp2 mRNA expression was detected.DNA electrophoresis was used to verify the results.Results:The sequence of Skp2 interference was correct by sequence analysis.The expression of Skp2 mRNA in siRNA-59,siRNA-318,siRNA-504 transfection group was significantly lower than that in no-load group and NC group(P<0.05),the inhibition rates of Skp2 mRNA in siRNA-59,siRNA-318 and siRNA-504 were respectively 60%,41%and 64%,and the siRNA-504 transfection group had the highest inhibition rate.Conclusion:The siRNA eukaryotic expression vector of Skp2 gene was constructed successfully which effectively inhibit Skp2 mRNA expression in human chondrocytes cell,and can provide strong evidence for the treatment of osteoarthritis.

Study on the effect of doxorubicin on expressions of genes encoding myocardial sarcoplasmic reticulum Ca^2＋ transport proteins and the effect of taurine on myocardial protection in rabbits

To investigate the effect of doxorubicin(DOX) on gene expression of the myocardial sarcoplasmic reticulum (SR)Ca 2+ transport proteins and the mechanism of taurine(Tau) protecting cardiac muscle cells, 9 rabbits were injected with DOX , 8 rabbits with DOX and Tau, and 9 rabbits with normal saline. Cardiac function , concentration of calcium in cardiomyocytes (Myo[Ca 2+ ] \%i\%), activity of SR Ca 2+ ATPase(SERCA2a), level of SERCA2a mRNA and Ca 2+ released channels(RYR2)mRNA were detected. The left ventricle tissues were observed by electron microscopy. The results showed that cardiac index, left ventricular systolic pressure, activity of SR Ca 2+ ATPase and level of SERCA2a mRNA decreased , while Myo[Ca 2+ ] \%i\% increased in DOX treated rabbits. DOX could not affect the level of RYR2 mRNA. Tau intervention could alleviate the increase of left ventricular diastolic pressure, Myo[Ca 2+ ] \%i\% and the decrease of SERCA2a mRNA induced by doxorubicin. The results suggested that downregulation of SERCA2a gene expression was an important mechanism of DOX induced cardiomyopathy and that Tau could partially improve the heart function by reducing calcium overload and alleviating downregulation of SERCA2a mRNA.

Expression of MTA2 Gene in Ovarian Epithelial Cancer and Its Clinical Implication

In order to investigate the roles of MTA2 in the pathogenesis of ovarian epithelial cancer, the expression of MTA2 in 4 ovarian cell lines were detected by semi-quantitative RT-PCR and Western-blot assays. MTA2 expression in normal, borderline, benign and malignant epithelial ovarian tissues was immunohistochemically examined. The expression of MTA2 mRNA and protein was detected in all of 4 cell lines of ovarian epithelial cancer. The expression of MTA2 mRNA and protein was higher in strong migration cell lines than in weak migration ones. In borderline and ma lignant ovarian tissues tested, MTA2 staining was dramatically stronger than in normal and benign tissues （P〈0. 01）. The expression levels in malignant ovarian tissues were significantly higher than that in borderline epithelial ovarian tissues （P〈0.01）. The expression of MTA2 was correlated with clinical stage, histopathological grade and lymph node metastasis. It was concluded that the high expression of MTA2 was associated with more aggressive behaviors of epithelial ovarian cancer. MTA2 provides a novel indicator of ovarian cancer.

Gene expression and MR diffusion-weighted imaging after chemoembolization in rabbit liver VX-2 tumor model

AIM: To investigate the dynamic characteristics and the correlation between PCNA, Bax, nm23, E-cadherin expression and apparent diffusion coefficient (ADC) on MR diffusion-weighted imaging (DWI) after chemoembolization in rabbit liver VX-2 tumor model. METHODS: Forty New Zealand rabbit liver VX-2 tumor models were included in the study. DWI was carried out periodically after chemoembolization. All VX-2 tumor samples in each group were examined by histopathology and Strept Avidin-Biotin Complex (SABC) immunohistochemical staining. RESULTS: The PCNA expression index in VX-2 tumors was higher than in the normal parenchyma around the tumor (P < 0.001). Nm23, Bax or E-caderin expression index in VX-2 tumors were lower than in the normal parenchyma around the tumor (all P < 0.001). PCNAand nm23 expression in the VX-2 tumor periphery first increased and then decreased (P < 0.001 and P = 0.03, respectively), while the expression of Bax and E-cadherin before and after chemoembolization was insignificant. When b-value was 100 s/mm2, there was a linear correlation between PCNA expression and ADC in the area of VX-2 tumor periphery (P < 0.001), and PCNA expression in VX-2 tumor periphery influenced the ADC. CONCLUSION: The potential of VX-2 tumor infiltrating and metastasizing decreases, while its ability to proliferate increases for a short time after chemoembolization. To some degree, the ADC value indirectly reflects the proliferation of VX-2 tumor cells.

EXPRESSION AND SWITCHING OF TH1/TH2 TYPE CYTOKINESGENE IN HUMAN GLIOMAS

Objective To study the expression and switching of Thl/Th2 cytokines gene in human gliomas and its effects on occurring and developing of human gliomas. Methods Interleukin（IL）-2 and interferon-3, represent Thl type cytokines. IL-4, IL-6, IL-10, and IL-13 represent Th2 type cytokines. The gene expressions of Th1/Th2 cytokines in human glioma cells, glioma infiltrating lymphocytes, and glioma cell lines were detected by reverse transcription polymerase chain reaction （RT-PCR）. The biological activity of cytokines in the supematant of glioma cell lines was assayed by enzyme-linked immunosorbent assay （ELISA） method. Results The total positive rates of Th1 and Th2 type cytokines gene in human glioma cells were 14.77% and 75%. The total positive rates of Th1 and Th2 type cytokines gene in glioma infiltrating lymphocytes were 22.73% and 68.17%. There was obviously predominant expression of Th2 type cytokines in human glioma tissues, glioma infiltrating lymphocytes, and glioma cell lines. There was no unbalanced expression of Th1/Th2 cytokines in normal brain tissues. Conclusion There is a predominant expression of Th2 type cytokines in human glioma cells. The switching of Th1/Th2 cytokines gene may play an important role in the occurring and developing of human gliomas.

Expression Pattern of Testis-specific Expressed Gene 2 in Cryptorchidism Model and Its Role in Apoptosis of Spermatogenic Cells

In our previous study, we identified a novel testis-specific expressed gene 2 （TSEG-2） from mouse testis. To further investigate its functions, 35 male Balb/c mice （8 weeks old） were divided into cryptorchidism group （n=20）, sham group （n=10）, and control group （n=5）. In cryptorchidism group, the right testes were anchored to the inner lateral abdominal wall. In situ hybridization （ISH） was applied to measure the localization of TSEG-2 in mouse testis. Real-time quantitative PCR was performed to detect the expression of TSEG-2 gene. Meanwhile, under the mediation of polyethylenimine （PEI）, the recombinant vector pEGFP-TSEG-2 （n=5） or empty vector （mock, n=5） was transfected into the testis of male mice. The transfection efficiencies were measured under a fluorescence microscope. The apoptosis of spermatogenic cells was detected by terminal deoxynuleotidyl-mediated nick end labeling （TUNEL）. The results showed that TSEG-2 was expressed in convoluted seminiferous tubules, more precisely, in spermatogonia and spermatocytes. As compared with sham and control groups, the TSEG-2 transcription was significantly enhanced （P〈0.05） and was correlated with apoptosis of spermatogenic cells in cryptorchid testes （P〈0.05）. PEI was efficient in mediating transfeetion of TSEG-2 into seminiferous tubules of testis. One week post-transfection, intratesticular injection of TSEG-2 resulted in increased apoptosis of spermatogenic cells in vivo （P〈0.05）. These results indicate that TSEG-2 may participate in the apoptosis of spermatogenic cells and the pathogenesis of cryptorchidism.

Expression and Chromosomal Mapping of Mouse Gpx2 Gene Encoding the Gastrointestinal Form of Glutathione Peroxidase, GPX-GI

GPX-GI is a cytosolic tetrameric Se-dependent glutathione peroxidase, similar in properties to GPX-1. Unlike the almost ubiquitous GPX-1, GPX-GI is mainly expressed in the epithelium of gastrointestinal tract. GPX-GI contributes to at least fifty percent of GPX activity in rodent small intestmal epithelium. The total GPX activity consists of at least 70% of selenium-dependent GPX activity in this compartment.By analyzing a panel of mouse mterspecies DNA from the Jackson Laboratory's backcross resource,we mapped Gpx2 gene to mouse chromosome 12 between D12Mit4 and D12Mit5, near the Ccs1 locus which contains a colon cancer susceptibility gene. A pseudogene, Gpx2-ps is mapped to mouse chromosome 7.Comparison of Gpx2 gene expression in three pairs of C57BL/6Ha and ICR/Ha mice which are respectively resistant and sensitive to dimethylhydrazine-induced colon cancer, we found a higher Gpx2 mRNA level in C57BL/6Ha colon than ICR/Ha colon. Interestingly, a lower level of GPX activity is found in the resistant strain of mice. Because GPX-1 has three times higher specific activity than GPX GI, our data suggest that the decreased GPX activity may result from a higher level of Gpx2 gene expression in those cells co-express GPx1 gene

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.