Human protein arginine N-methyltransferase 2 (PRMT2, HRMT1L1) is a protein that belongs to the arginine methyltransferase family, and it has diverse roles in transcriptional regulation through different mechanisms depending on its binding partners. In this study, we provide evidences for the negative effect of PRMT2 on breast cancer cell proliferation in vitro and in vivo. Morever, cyclin D1, one of the key modulators of cell cycle, was found to be downregulated by PRMT2, and PRMT2 was further shown to suppress the estrogen receptor α-binding affinity to the activator protein-1 (AP-1) site in cyclin D1 promoter through indirect binding with AP-1 site, resulting in the inhibition of cyclin D1 promoter activity in MCF-7 cells. Furthermore, a positive correlation between the expression of PRMT2 and cyclin D1 was confirmed in the breast cancer tissues by using tissue microarray assay. In addition, PRMT2 was found to show a high absent percentage in breast caner cell nuclei and the nuclear loss ratio of PRMT2 was demonstrated to positively correlate with cyclin D1 expression and the increasing tumor grade of invasive ductal carcinoma. Those results offer an essential insight into the effect of PRMT2 on breast carcinogenesis, and PRMT2 nuclear loss might be an important biological marker for the diagnosis of breast cancer.

AIMS/HYPOTHESIS:Our previous study showed there was a change of microRNA (miRNA) expression profile, and miR-21 was significantly down regulated in insulin-resistant adipocytes (IR-adipocytes). Phosphatase and tensin homologs deleted on chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, was identified to be a target gene of miR-21, which suggested miR-21 might be associated with insulin resistance (IR) or diabetes. However, it is not known whether miR-21 play any role in the development of IR in 3T3-L1 adipocytes.METHODS:Normal adipocytes and adipocytes transfected with pre-miR-21(pmiR-21) or negative control (pNeg) were treated with high glucose and high insulin for 24 h, insulin-stimulated glucose uptake was determined by 2-Deoxyglucose transport assay, miR-21 expression level was measured by using quantitative real-time RT-PCR (qRT-PCR). The protein expression levels of PTEN, Akt, phospho-Akt (Ser473), IRβ, GSK3β, phospho-GSK3β (Ser9) and GLUT4 were detected by western blotting assay.RESULTS:We further confirmed that miR-21 was down regulated in IR-adipocytes by qRT-PCR. Over-expression of miR-21 significantly increased insulin-induced glucose uptake and decreased PTEN protein expression, while it had no significant effect on PTEN mRNA expression in IR-adipocytes. Moreover, over-expressing miR-21 significantly increased insulin-induced phosphorylation of AKT (Ser473), GSK3β (Ser9) and the translocation of glucose transporter 4 (GLUT4) in IR-adipocytes.CONCLUSIONS:In this study, our data demonstrate that miR-21 reverses high glucose and high insulin induced IR in 3T3-L1 adipocytes, possibly through modulating the PTEN-AKT pathway, and miR-21 may be a new therapeutic target for metabolic diseases such as T2DM and obesity.

OBJECTIVE:To explore the protective effect of Schisandrins extract on liver ultrastructure in rats with CCl4 poisoning.METHODS:Twenty five Wistar rats were randomly divided into the control group,the CCl4 poisoned 24 h and 10 d groups,the Schisandrins 24 h and 10 d groups,five groups with 5 rats in each group.The rats of the CCl4 poisoned and the Schisandrins groups were given one peritoneal injection of CCl4(200 ml/L in vegetable oil) at a dosage of 0.2 ml/100 g body weight,the rats of the control group were given the same volume of vegetable oil.Then Schisandrins extract(0.5 ml/100 g body weight)were given orally to rats of the Schisandrins group daily,while saline was given to rats of the poisoned and the control groups.The rats were sacrificed at different time points.The morphologic changes of the hepatocytes were examined by transmission electron microscopy.RESULTS:The ultrastructure of hepatocytes of rats were changed in the CCl4 poisoned 24 h group,the cytoplasm of hepatocytes was loosed and vacuolated,mitochondria were swollen,with cristae breakdown or loss,and the endoplasmic reticulum appeared lamellar.The nuclear membrane was shrunken and ruptured,and the nuclear chromatin condensed and marginated.The hepatocytic nuclei showed obvious atypia in rats of the CCl4 10 d group.By comparing with that of the CCl4 group,the ultrastructure of hepatocytes in rats of the schisandrins group was less severely damaged.CONCLUSION:The use of Schisandrins extract in the treatment of CCl4 poisoned rats exerted a protective effect on liver ultrastructure.

目的探讨苦瓜蛋白对荷瘤小鼠肿瘤生长的影响和荷瘤小鼠血清基质金属蛋白酶MMP-2、MMP-9的活性及变化以及细胞因子白细胞介素-6(IL-6)及肿瘤坏死因子-α(TNF-α)表达的影响。方法将清洁级ICR雄性小鼠按体重随机分成5组,建立肝癌H22皮下移植瘤模型,分为模型组、苦瓜蛋白高、中、低剂量(100、50、25mg/kg)组及环磷酰胺组(CTX,40mg/kg),各组均ip给药,观察苦瓜蛋白对肿瘤生长的影响;分离血清,明胶酶谱法检测荷瘤小鼠血清基质金属蛋白酶MMP-2、MMP-9的活性及其变化,ELISA检测细胞因子IL-6及TNF-α表达。结果苦瓜蛋白组与模型组相比,肿瘤质量明显减轻(P<0.01),小鼠体重也较模型组小鼠轻(P<0.01);苦瓜蛋白(100、50、25mg/kg)组及CTX组抑瘤率分别为85.81%、66.89%、52.05%、65.10%;各苦瓜蛋白组与模型组相比,MMP-2、MMP-9的活性降低,酶原表达下调(P<0.01);细胞因子IL-6及TNF-α表达明显下调(P<0.01),上述作用与苦瓜蛋白剂量相关。结论苦瓜蛋白具有较强的体内抗肿瘤活性;其抗肿瘤作用可能与苦瓜蛋白抑制MMPs的活性及表达,以及调节IL-6及TNF-α等细胞因子的表达水平有关。

Teachers are principal parts in the innovation and implementation of teaching.Along with the deepening transformation of new medical teaching mode,higher demands have been set for the construction of teachership and for the education and training of teachers.The article gives an analysis on several issues that should be given attention to in the teacher training.

近年来的研究表明,微卫星不稳定性(MI)尤其是高度微卫星不稳定性(MI-H)与许多肿瘤的发生和发展关系密切。本文就MI的发生和致瘤机制、临床病理学意义以及MI相关研究存在问题等方面的研究现状作一综述。

[Purpose] To explore effect of Momordin on expression and activity of matrix metalloprotease in murine transplanted tumor(H22). [Methods] Male mice of ICR species were randomly divided into 5 groups and then constructed the transplantation tumor model of liver cancer H22. The negative control group were treated by normal saline; T1, T2 and T3 group by Momordin with low, middle and high dose respectively and positive control group by cytoxan (CTX group). All the mice were feeded ordinarily with intraperitoneal drugs administration. Tumor inhibitive rate of Momordin was measured. Activity of MMP-2 and MMP-9 in serum was detected by Gelatin Zymography. [Results] Tumor inhibition rate in low, middle and high dose of Momordin and CTX group were 52.05%, 66.89%, 85.81% and 65.10%, respectively. The activity of MMP-2 and MMP-9 significantly decreased in all Momordin treated groups compared to the negative control group (P0.01). The above function correlated to the dose of Momordin. [Conclusion] Momordin plays a role of inhibitory effect on the expression and activity of matrix metalloprotease and its anti-tumor activity may be involved in regulating the expression and activity of matrix metalloprotease.

[Objective]To explore the inhibitory action and mechanisms of ribosome inactive protein(RIP)from Momordica Charantia seeds on transplanted H22 hepatoma.[Methods] Sixty depurative grade made of ICR species were used to establish hepatoma model through peritoneal injection with H22 cells and divided randomly into three groups: the normal saline treated group(con group,12 mice);Momordin treated mice (RIP group,36 mice) positive control group(CTX group,12 mice).All mice were ordinary fed with basic food for 7 days,Con group were treated with normal saline;RIP groups including three sub-groups(12 mice)were treated with high middle and low dose momordin(100,50 and 25 mg/kg respectively);CTX group were also treated with cytoxan(40 mg/kg),the way of administration was peritoneal injection(once a day,12 days).Condition of tumor growth in each group was investigated and serums were also isolated from each group mice,the expression of interleukin-6 and tumor necrosis factor-α were assayed by enzyme linked immunosorbent assay(ELISA) too.[Results] Tumor weight of RIP and CTX group was more lowver than that of con group(P<0.01) and there was a positive correlation between tumorostatic rate and momordin dose in RIP sub-groups(P<0.01);the serum levels of interleukin-6 and tumor necrosis factor-α in RIP and CTX groups were markedly down-regulated compared to con group(P<0.01) and there was a negative correlation among RIP sub-groups groups(P<0.05).[Conclusion] RIP from Momordica Charantia seeds shows a remarkable inhibitory effect on the transplanted H22 hepatoma;its mechanism may involve regulating the level of cytokines such as interleukin-6 and tumor necrosis factor-α.

Objective:To explore the anti-tumor activity and effects of Momordin from Momordica Charantia Seeds on Murine Transplanted Tumor H22 and immune organ such as spleen and liver.Methods:Mice of ICR species which had got the transplanted tumor H22 were divided randomly into four groups:the normal saline treated control group,low,middle and high dose Momordin treated mouse and labeled with t1 and t2 and t3 group,All of the mouse received the medicines such as normal saline and gradient dose of Momordin through peritoneal injection on the based of ordinary feeding.Anti-tumor activities,as well as the changes of animal weights and their immune organ such as liver and spleen index were also investigated.Results:Low,middle and high dose of Momordin treated group had a tumorostatic rate 52.05 %and 66.8% and 85.81% respectively,and meanwhile an evident inhibitory effect on body weigh was also seen(P0.05),but not on the liver and spleen index(P0.05).Conclusion:Momordin in a certain dosage show a marked inhibitory effect on the murine transplanted tumor H22,no harm to their immune organs such as spleen and liver.

Objective:To explore the feasibility of astrong cation exchange chromatographic column(SP Sepharose High Performance) in purifying ribosome inhibiting protein(RIP)from momordica charantia seeds and observe the cytotoxicity and anti-tumor activity of RIP in erythroleukemic K562.Methods:Using gradient acetone precipitation,strong cation exchange chromatographic column(SP Sepharose High Performance) to isolate the RIPfrom momordica charantia seeds,applying sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and isoelectric focusing electrophoresis(IFE)to detect the purityof RIP.Erythroleukemic K562cells were treated by gradient concentrationof RIP for 72 hours and detected its cytostatic activity by(CCK-8) kit;as well as FACScan(FITC-Annexin V staining),Light and electron microscopy were used to detect its activity of apoptosis.Results:After using the SP Sepharose High Performance chromatographic column,we obtained the RIPfrommomordica charantia seeds with high purityand convenience;Not only the RIP had strong cytostatic activity but also could induce K562 cells to apoptosis understand a appropriated concentrationof RIP(9.1μg/mL).Conclusion:The methods presented in this paper are feasible to extract and purify the RIP and the RIP extracted from momordica charantia seeds may exert its anti-tumor activity through induction of apoptosis.

BACKGROUND AIM: To explore the effects of resveratrol in suppressing growth and inducing apoptosis in human esophageal carcinoma EC109 cells. MATERIALS AND METHODS: EC109 cells were treated with resveratrol at different concentrations,methyl thiazolyltetrazolium (MTT)assay was used to examine the effect of resveratrol on growth of EC109 cells. Hoechst 33258 staining and phase contrast microscope were used to examine the apoptosis status of EC109 cells. The cell cycle arrest and cell apoptosis were analyzed by flow cytometry. RESULTS: Resveratrol(15.62-500 μmol/L)could inhibit the growth of EC109 cells in time-and dose-dependent manner. 500 μmol/L resveratrol had 87.43% inhibitory rate on the growth of EC109 cells at 72 h, 48 h after treatment with resveratrol, typical apoptosis was seen under phase contrast microscope and fluorescence microscope in 500 μmol/L treatment groups. Resveratrol could arrest EC109 cells growth in S phase, inhibit DNA synthesis and induce cell apoptosis and the apoptotic ratio was 62.3%. CONCLUSION: Resveratrol could inhibit proliferation, cause S phase arrest and induce apoptosis of EC109 cells.

BACKGROUND AIM: To study the correlation between EGFR gene fragments and nuclear matrix proteins (NMPs) in human bladder transitional cell carcinoma (hBTCC). MATERIALS AND METHODS: The nuclear matrix proteins were prepared from fresh tissues and T24 culture cells, and total cell proteins was also prepared from fresh tissues of hBTCC. Genomic DNA and nuclear matrix DNA (high concentration sail and gradient DNaseⅠ treated) were separated from hBTCC tissues. Two pair primers were synthesized according to the cDNA sequence of EGFR gene.Two EGFR fragments were amplified by PCR using the two pair primers in genomic DNA and different nuclear matrix DNA.The positive fragments with primersⅠ were also sequenced. The binding capacity of EGFR gene to nuclear matrix in bladder cancer was also measured by Southwestern Blot assay using single stranded end labeled probes prepared by PCR product Ⅱ . RESULTS: Both genomic DNA and nuclear matrix DNA (NM DNA0, NM DNA25 ,NM DNA50 ,NM DNA100) of human bladder carcinoma could amplify 110 bp positive fragment with primersⅡ . However both genomic DNA and nuclear matrix DNA showed a 940 bp positive fragment with primersⅠ except NM DNA100. The sequence of 940 bp was identical to the DNA sequence of EGFR gene. Southwestern blot analysis demonstrated that EGFR gene fragment (3901-4010nt) was bound to an about 60 kD nuclear matrix protein in both NMPs (tissues and T24 cells) and total cell protein of bladder carcinoma tissues, but not in the cytoplasm. CONCLUSION: Active EGFR gene is strongly bound to the nuclear matrix and nuclear matrix proteins.Nuclear matrix proteins may regulate the high expression of EGFR gene including the course of transcription and post-transcription and/or translation in the human bladder transitional cell carcinoma.

[Objective] To develop a method for determination of Schisandrin B by LC-MS and to observe the pharmacological action of Schisandrin B in Wistar rat. [Methods] The efficacious components was extracted from the fruit of Schisandra chinensis (Turcz.) Baill. The extract was orally administrated to Wistar rat. Blood samples were obtained at different time points after the extract was administrated. The plasma concentration of Schisandrin B was analyzed by LC-MS, and the plasma concentration-time data of Schisandrin B was also analyzed. The parameters of pharmacokinetics of Schisandrin B in the rats were calculated with 3p97 pharmacokinetics program. [Results] The method was simple and rapid for determination of Schisandrin B in different samples. The retention time of the Schisandrin B was 2.96 min. The maximum mean plasma level of Schisandrin B was 1.01 μg/mL which was reached at 5.65 hr after the oral administration. The mean value of area under concentration-time curve (AUC) was be successfully applied to analysis of Schisandrin B in different samples and to investigating the pharmacokinetics action of Schisandrin B in experimental animals.

Objective To investigate the change of laminB1 expression in normal esophageal epithelia and esophageal squamous cell carcinoma(ESCC).Methods We prepared frozen section nuclear matrix in situ,detected the expression of laminB1 proteins before and after preparing nuclear matrix in normal epithelia and ESCC tissues using immunohistochemistry.We prepared nuclear matrix protein and detected the expression of laminB1 proteins in normal mucosa and ESCC using Western blot.Results The positive rate of normal epithelia,nuclear matrix of normal epithelia,carcinoma,and nuclear matrix of carcinoma were 93.3%,86.7%,96.7% and 86.7% respectively.In normal epithelia,the expression level of laminB1 diminished from basal cells to granular cells.In nuclear matrix of normal epithelia,the expression level decreased and most positive cells were basal cells.Most positive cells showed whole nuclear staining.In ESCC,laminB1 expression showed no regular staining patterns.In nuclear matrix of carcinoma tissue,the expression level of laminB1 was reduced and showed a rim in the nuclear periphery.The expression level in nuclear matrix of carcinoma was higher than in nuclear matrix of normal epithelia,and the difference between them was significant(x2=5.042,P<0.05).Western blot analysis showed that the bands of laminB1 in normal mucosa nuclear matrix protein were weaker staining than in carcinoma nuclear matrix protein.Conclusion LaminB1 proteins are expressed in most normal esophageal epithelia and esophageal squamous cell carcinoma.The expression level of laminB1 protein in nuclear matrix of carcinama is higher than in the nuclear matrix of normal epithelia.The distribution of laminB1 protein in nuclear matrix of carcinoma was different from that in the nuclear matrix of normal epithelia.

Objective To investigate the ability of trans-acting HDV(hepatitis delta virus) ribozyme to suppress the replication of hepatitis B virus(HBV) in vivo.Methods 1) Selection of potential HDV ribozyme target sites within the pregenome of HBV in vitro: an HBV genome full sequence was cloned from HepG2.2.15 cell's genomic DNA,and aligned with 202 HBV full sequences of various genotypes which were retrieved from GenBank database,potential target sites which conserved in most of the genotypes(homology 90%) were identified.Several sites located in C region were examined by RNase H hydrolysis with oligonucleotides(18nt) and one site that may be accessible for the ribozyme was selected.Cleavage activity of the trans-acting HDV ribozyme constructed in our lab for this site was tested in vitro;2) Construction of HDV riboxyme expression vectors: the cDNA of HDV ribozyme was recombined with three eukaryotic promoters,tRNAVal,hMCV and U6,and three vectors ptVHRz,pSURz and pcDHRz were constructed respectively.Then they were transfected respectively into HepG2.2.15 cells by LipofectomineTM2000(Invitrogene);3) Assay: the expression of HDV ribozymes in cells was assayed by Northern blot dot hybridization;HBsAg,HBeAg and HBV in the cultural supernatant were assayed by ELISA and real time quantity PCR respectively.Results The selected site can be effectively cleaved by the constructed ribozyme in vitro.When transfected into HepG2.2.15 cells after 48 hours,three vectors were all expressed highly and two of them,ptVHRz and pcDHRz,have significantly inhibited the expression of HBeAg but not for HBsAg.However no any suppression for the replication of HBV was observed in the study.Conclusion The ability of trans-acting HDV ribozyme specially suppressing the expression of HBeAg in vivo shows that it is a new potential tool for HBV gene therapy.But the fact that the replication of HBV was not suppressed by the HDV ribozyme is remained to elucidate and further work needs to be done.

The discussion method adequately applied to the teaching module of new medical teaching mode may promote the study of medical professional knowledge and cultivate the medical students' moral and cultural quality in the course of medical education,thus further optimizing the new medical teaching mode on a certain extent.

Aims and Background It was documented that nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) and that EBV-encoded latent membrane protein-1 expression (LMP1) plays an important role in the pathogenesis of NPC. In preclinical studies, arsenic trioxide (As2O3) has been identified as a promising anticancer agent for treatment of NPC. The purpose of this study is to investigate if this agent can inhibit the expression of LMP1 and therefore lead to growth inhibition of NPC cells in vitro. Methods LMP1-positive NPC cells, HNE1-LMP1, were treated with 3 umol/L of As2O3 for 96 hours. The LMP1 protein expression and mRNA level in HNE1-LMP1 cells were determined by western blot, confocal immunofluorescence staining and semiquantitative reverse transcriptase reaction (RT-PCR). Apoptosis was determined by light microscopy and the TUNEL method. Alterations in the cell cycle distribution were also investigated by flow cytometry. MTT assay and colony formation assay were used to detect the proliferation of the cells. The LMP1-negative parental cell lines HNE1 and HNE2 were used as control in an attempt to elucidate the role of LMP1 in the anticancer effect of As2O3 on NPC cells. Results The expression of LMP1 at the protein and mRNA level was reduced after exposure to 3 umol/L As2O3. This dose of As2O3 significantly induced apoptosis and growth retardation of HNE1-LMP1 cells. In addition, more HNE1-LMP1 cells were induced to G0/G1 and G2/M arrest. The same dose of As2O3 had a moderate effect on HNE1 and HNE2 cells. Conclusion Arsenic trioxide can inhibit LMP1 expression and dictate apoptosis and alterations of cell cycle distribution as well as growth retardation. LMP1-positive NPC cells are more sensitive to As2O3 treatment than LMP1-negative NPC cells.

Objective To investigate the inhibitory effect of epidermal growth factor receptor inhibitor AG1487 on the expression of extracellular signal-regulated kinase(ERK) of Tca8113 cells. Methods Immunocytochemistry and Western blot were employed to detect the expression of active ERK and total ERK by using different concentrations of AG1487. Results The results of immunicytochemistry showed that the expression of active ERK in nucleus was inhibited by AG1487,but the expression of total ERK in cytoplasm was not affected.The results of western blot further demonstrated that AG1487 could inhibit the expression of active ERK and the inhibitor effect showed a dose-dependent pattern. Conclusion Epidermal growth factor receptor inhibitor AG1487 could inhibit the MAPK signaling pathway of Tca8113 cells.These inhibitory effects may impact the proliferation of tumor cells.

Objective To explore the protecting effects of schisandrins on the nuclear matrix proteins and structure of hepatocyte of CCl_4-intoxicated rat. Methods The Wistar rats were randomly divided into the control group, the intoxicated group, and the treating group. The rats of intoxicated and treating group were given one-time peritoneal injection of CCl_4 (200 mL/L in vegetable oil) at a dosage of 0.2 mL/100 g body mass. The CCl_4-intoxicated rats in the treating group were treated with schisandrins extract by oral administration [0.5 mL/ (100 g·d)], while saline was given to rats in the intoxicated group. The rats were sacrificed at different time pionts. The morphologic changes of the hepatocytes were examined under the light and electron microscope. The expression of lamin B and the metabolism of nuclear matrix proteins were analyzed by immunohistochemical method and SDS-PAGE, respectively. Results The application of schisandrins extract attenuated the pathological changes of the hepatocyte, increased the expression of lamin B protein, and modulated the metabolism of nuclear matrix proteins. Conclusion Schisandrins may promote cytothesis of damaged hepatocytes by increasing the expression of lamin B protein and modulating the metabolism of nuclear matrix proteins in CCl_4-intoxicated rat, thus, schisandrins play a role of protecting the nuclear matrix proteins and the structure of hepatocyte in CCl_4-intoxicated rat.

Nasopharyngeal carcinoma (NPC) is notorious for the metastases, which are in close association with Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Arsenic trioxide (As2O3) has been shown to induce apoptosis and differentiation in NPC xenografts. Then, can it repress the cancer cells' metastasis potential? To elucidate this issue, the present study was performed. LMP1-negative cell line HNE1 and LMP1-positive cell line HNE1-LMP1 were used as in vitro model. Cells (1 x 10(5)/mL) were cultured with or without 3 microM As2O3 for 48 h. Then the survival cells were collected to investigate their potential of colony formation, attachment, invasion, and migration. Both confocal immunofluorescence staining and Western blot were used to detect the changes of LMP1 expression. The changes of MMP-9 were examined by RT-PCR assay and Western blot. The results were as follow: i) the colony formation inhibition rate (75.41 +/- 3.9% in HNE1-LMP1 cells vs 37.89 +/- 4.9% in HNE1 cells), the rate of attachment (HNE1-LMP1 vs HNE1: 56.40 +/- 3.5 vs 65.87 +/- 5.9%), the invasion inhibitory rate (HNE1-LMP1 vs HNE1: 56.50 +/- 3.7 and 27.91 +/- 2.1%), and the migration inhibitory rate (HNE1-LMP1 vs HNE1: 48.70 +/- 3.9 vs 29.19 +/- 6.27%) were all significantly different between the two cell lines (P < 0.01). ii) LMP1 was down-regulated in As2O3-treated HNE1-LMP1 cells. iii) The reduction of MMP-9 was found in As2O3-treated groups, more evident in HNE1-LMP1 cells. Thus, we conclude that As2O3 can reduce metastasis potential of NPC cells, involving inhibition of MMP-9 expression. LMP1 were also reduced in this process and seemed to enhance anti-metastasis activity of As2O3.