Objective To investigate the effects of DNA demethylation drugs combined with histone deacetylase in-hibitors on fragile X mental retardation 1 neighbor protein (FMR1NB) expression and its promoter methylation in human oral cancer cells and try to find a strategy of weakening the heterogeneity of FMR1NB expression.Methods Human oral cancer cell lines Cal27 and SCC-9 were treated with decitabine (DAC) , an inhibitor of DNA meth-yltransferase, combined with trichostatin A (TSA) and valproic acid (VPA), inhibitors of histone deacetylase.Then reverse transcription-polymerase chain reaction (RT-PCR) , quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression of FMR1 NB and pyrosequencing was used to detect the methylation of FMR1NB promoter.Results Compared with the blank control group, DAC and its combination with TSA and VPA significantly induced the expression of FMR1NB mRNA and protein in Cal27 and SCC-9 cells.Compared with DAC alone group, FMR1NB mRNA expression of each DAC-combined drug groups significantly increased, but FMR1NB protein did not significantly change in Cal27 cells; for SCC-9 cells, except for DAC+TSA group, the mRNA and protein levels of FMR1NB significantly increased in all other groups.In addition, there was no signifi-cant difference in the expression of FMR1 NB mRNA and protein between the three-combined drugs group and two-combined drugs groups.Further methylation assay showed that the methylation level of the overall FMR1NB promot-er and its each CpG site measured were reduced to varying degrees in all treatment groups except for three-combina-tion drug group of SCC-9.Conclusion DAC and its combination with TSA and VPA can enhance the expression of FMR1NB by mediating the demethylation of FMR1NB promoter, wherein the enhanced expression effect of the com-bination of the two drugs is stronger, suggesting that they have the potential to weaken the heterogeneity of FMR1NB expression and improve the immunotherapy effect of oral cancer.

ObjectiveTo investigate the effect of Stemona tuberosa alkaloids （STA） on apoptosis and phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） and c-Jun N-terminal kinase/p38 mitogen-activated protein kinase （JNK/p38 MAPK） signaling pathways in human lung cancer A549 cells. MethodA549 cells were classified into blank group and STA groups （100， 150， 200， 250， 300 mg⋅L^-1）. Thiazole blue （MTT） assay and colony formation assay were used to evaluate the proliferation of A549 cells. Apoptosis was observed based on Hoechst 33258 staining， flow cytometry， and Annexin V-FITC/PI staining. Western blot was employed to detect the expression of apoptosis-related proteins cysteine-aspartic acid protease-3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）， and Bcl-2， and the expression of PI3K， phosphorylated （p）-PI3K， Akt， p-Akt， JNK， p-JNK， p38 MAPK， and p-p38 MAPK. ResultCompared with the blank group， STA groups （150， 200， 250， 300 mg⋅L^-1） demonstrated the increase in inhibition rate of cell proliferation （P<0.01） and cell clone inhibition rate， and decrease in cell clone formation rate （P<0.01）. In comparison with the blank group， STA groups showed typical characteristics of apoptosis， such as chromatin condensation and enhanced fluorescence reaction. The apoptosis rate of STA groups was significantly higher than that of the blank group （P<0.01）. Compared with the blank group， STA （150， 200， 250， 300 mg⋅L^-1） significantly up-regulated the protein expression of Caspase-3 and Bax （P<0.05， P<0.01） and down-regulated the expression of Bcl-2 protein （P<0.01）. Compared with the blank group， STA had no significant influence on the total protein expression of PI3K， Akt， JNK， and p38 MAPK. However， STA （150， 200， 250， 300 mg⋅L^-1） significantly decreased the levels of p-PI3K and p-Akt （P<0.05， P<0.01） and increased the level of p-p38 MAPK （P<0.05， P<0.01）. Compared with the blank group， STA （200， 250， 300 mg⋅L^-1） significantly raised the level of p-JNK （P<0.05， P<0.01）. ConclusionSTA can inhibit the proliferation and induce the apoptosis of A549 cells by inhibiting PI3K/Akt signaling pathway and activating JNK/p38 MAPK signaling pathway.

Objective: To study the expression of long non-coding RNA LINC01152 in glioma and its influence on the malignant biological behavior of glioma cells． Methods: LINC01152 expression in glioma was analyzed by LncSpA V2．0 and GEPIA database．qRT-PCR was applied to detect the expression of LINC01152 mRNA in 10 samples of human normal brain tissues，40 samples of glioma tissues and 5 glioma cell lines．GO and KEGG enrichment analysis of LINC01152 co-expressed genes were performed using the DAVID database to predict the related functions． The AnoLnc2，TargetScan，LinkedOmics and miRDB databases were used to predict the LINC01152 related miRNAs and target genes to construct a ceRNA regulatory network．LINC01152 expression was knocked down in glioma cell lines by small interfering RNA (siRNA) transfection．The CCK-8 test，scratch healing experiments，Transwell，flow cytometry and Western blot experiments were used to measure the influence of LINC01152 on the proliferation，migra- tion，invasion and apoptosis of glioma cells. Results : Database analysis showed that compared with other tumor types，LINC01152 was highly expressed in glioblastoma (GBM) and low grade glioma (LGG) ，and was higher than normal brain tissue．qRT-PCR showed that the expression of LINC01152 mRNA in glioma tissues was significantly higher than that in normal brain tissues (P＜0. 01)．The expression of LINC01152 was correlated with Ki-67 (P < 0. 05) ，but not with clinical parameters such as gender，age，tumor size，P53 protein，glial fibrillary acidic protein (GFAP) ，O-6-methylguanine-DNAmethyltransferase ( MGMT) and WHO grade of glioma patients． Functional enrichment analysis of co-expressed genes indicated that the LINC01152 was mainly involved in biological processes such as cell adhesion and synaptic signaling．LINC01152-miRNA-mRNA regulatory network was constructed according to predicted target genes．After down-regulation of LINC01152 expression，the proliferation，migration and invasion abilities of A172 and U87 cells decreased(P＜0. 01) ，while the apoptosis of glioma cells significantly increased (P＜0. 001) ． Conclusion LINC01152 is highly expressed in glioma，which can promote the malignant biological behavior of glioma cells by enhancing proliferation，migration as well as invasion and inhibition of apoptosis.

OBJECTIVE To establish the fingerprint of Zhuang medicine Jinmu granules and carry out chemometric analysis ， and determine the contents of three components . METHODS High performance liquid chromatography （HPLC） method was adopted. Using rutin as the reference ，HPLC fingerprints of 10 batches of Jinmu granules were drawn and similarity evaluation was performed by Similarity Evaluation of Chromatographic Fingerprint of Traditional Chinese Medicine （2012 edition）；the common peak was determined by comparing with mixed control ；SPSS 21.0 software was used for cluster analysis ，and SIMCA 14.1 software was used for principal component analysis and orthogonal partial least squares-discriminant analysis. The differential components affecting the quality of Jinmu granules were screened by taking the variable importance in projection （VIP）value ＞1 as the standard ；the HPLC method was used to determine the contents of astilbin ，polydatin and berberine hydrochloride in Jinmu granules. RESULTS There were 22 common peaks in 10 batches of Jinmu granules ，and the similarities were 0.962-0.997；five common peaks were identified ，namely gallic acid （peak 2），polydatin（peak 9），rutin（peak 11），astilbin（peak 13）and kaempferol （peak 20）. The results of cluster analysis showed that 10 batches of Jinmu granules could be clustered into 3 categories：S1 and S 3-S4 were grouped into one category ；S5-S6 and S 9 were grouped into one category ；S2，S7-S8 and S 10 were grouped into one category. The results of principal component analysis showed that the parameter of model interpretation was 0.951 and that of prediction ability was 0.723. The classification results were basically consistent with cluster analysis. The classifica tion results of orthogonal partial least squares- com discriminant analysis were also ba sically consistent with clus- ter analysis. The common peaks with VIP value ＞1 in the order were peak 7＞peak 11（rutin）＞peak 17＞peak 13（astilbin）＞ peak 3＞peak 8＞peak 6＞peak 16 respectively. The linear ranges of astilbin ，polydatin and berberine hydrochloride were 0.012 6- 1.225 0，0.010 8-1.052 5 and 0.020 0-1.562 5 mg/mL，respectively（all R 2＝0.999 9）. RSDs of precision ，stability（24 h）and repeatability tests were all less than 3％. The average recoveries were 99.48％（RSD＝2.67％，n＝9），98.57％（RSD＝1.77％，n＝ 9）and 100.84％（RSD＝2.49％，n＝9）. The contents were 1.221 0-7.011 6，2.251 1-4.462 9，1.252 4-3.328 7 mg/g，respectively. CONCLUSIONS Established fingerprint and the method of content determination are accurate ，stable and simple. Combined with chemometric analysis ，it can be used for the quality control and evaluation of Zhuang medicine Jinmu granules.

F-asparaginask(F-LSP)is onk of thk cke agknts in thk long-tkrm chkmothkrape of lemphoid malignanciks in childrkn with acutk lemphoblastic lkuckmia(LFF)and non Hodgcin＇s lemphoma(NHF). If F-LSP rksistanck occurs in patiknts,it is highle suggkstivk of poor prognosis. Thkrkfork,thk mkchanism of F-LSP rksistanck is a major stude in thk fikld of diagnosis and trkatmknt of lkuckmia in childrkn. Ovkr thk ekars,rklatkd studiks havk shown that thk bask lkvkl of asparagink senthktask in tumor cklls,bonk marrow hkmatopoiktic support cklls,somk advkrsk mkta-bolic changks aftkr F-LSP chkmothkrape,and thk F-LSP silkncing inactivation induckd be sklf nkutralizing antibode can lkad to F-LSP rksistanck. In this papkr,according to thk litkraturk rklatkd rkports in rkcknt ekars,providk rkfkrknck for domkstic collkaguks,in ordkr to carre out thk rklkvant basic and clinical rkskarch,to providk thkorktical basis for thk rkalization of individualization of chkmothkrape.

Objective To analyze the efficacy of different doses of intravenous immunoglobulin (IVIG) in the treatment of acquired severe aplastic anemia (AA) in children. Methods The clinical data of hospitalized children with severe AA who received adjuvant immunosuppressive therapy of IVIG from January 2000 to December 2015 were retrospectively analyzed. According to different doses of treatment, the children were divided into low dose group ( IVIG 200-400 mg/ (kg·d) once every 4 weeks for 6 times), high dose group (IVIG 1 g/ (kg·d ) x 2 days once every 4 weeks for 6 times). Results All the children were followed up until December 31, 2015. Among the 61 children, it was effective in 41 children and total effective rate was 67.2%. The effective rate of anti thymocyte globulin (ATG) treatment in high dose group was higher after 3 months than that of low dose group, and there was statistical difference (P=0.020). The interval between first dose of IVIG and first dose of ATG in 20 cases of ineffectiveness was 2.0 (2.0-5.0) d, while that in 41 cases of effectiveness was 8.0 (7.0-9.0) d, and the difference is statistically significant (P<0.001); Among the 20 ineffective children, 18 children had the interval <7 day. The survival rates of the two groups were 80% and 87.1%, respectively, and there was no difference between two groups (P>0.05). The incidence of severe infections in the high-dose group was lower than that in the low-dose group after the use of ATG for 6 months, and there was statistical difference (P=0.008). Conclusions High dose of IVIG therapy can increase the early response rate in children with acquired severe AA, but it does not increase the long-term effectiveness, cure rate and 5 year survival rate. In addition, it can reduce the severe infection rate, but cannot reduce the total infection rate and infection related mortality rate.

Objective This study was designed to compare the effects of the anti-human T lymphocyte globulin (Fresenius,ATG-F)and rabbit anti-human thymocyte immunoglobulin (Genzyme,R-ATG) in the treatment of childhood aplastic anemia (AA) and their effects.Method A total of 59 children with aplastic anemia were analyzed in the present study,including 34 cases of severe aplastic anemia (SAA),12 cases of very severe aplastic anemia (VSAA) and 13 cases of transfusion-dependent non-severe aplastic anemia (NSAA).While receiving immunosuppressive therapy (IST),30 and 29 patients,with long-term oral supplement with cyclosporin A (CSA),androgen and Chinese traditional medicines,were treated with ATG-F and R-ATG,respectively.When it was necessary,some supportive cares such as component transfusion and infection control were also employed.Absolute counts of peripheral blood lymphocyte (ALC) at various time points were dynamically detected after ATG therapy.Result According to the International Aplastic Anemia Treatment and Effect standards.There were no statistically significant differences in the overall response rate (67％ (20/30) vs.69％ (20/29),x2 =0.036,P =0.676) and the survival rate (87％ (26/30)vs.83％ (24/29),x2 =0.173,P =0.676) between the ATG-F and R-ATG groups.There were significant and long-term ALC decrease after ATG therapy,the rate of ALC decrease in ATG-F and R-ATG group,the ALC only recovered to 47.8％ (ATG-F group) and 47.4％ (RATG group) of the pre-treatment level respectively.Conclusion ATG-F 5 mag/(kg · d) and R-ATG 3.75mg/(kg · d) could achieve similar effects in the treatment of childhood AA,through similar significant clearance of T cells.Therefore,all of these suggest that ATG-F and R-ATG might serve as the drugs of frontline choice for IST in childhood AA patients who do not have an available human leukocyte antigen identical related donor.

Objective To analyze the percentage and functional changes of natural killer T (NKT) cells in peripheral blood and bone marrow of severe aplastic anemia (SAA) children before immunosuppressive therapy (IST) comparing to that of healthy children.Methods Ten children with severe aplastic anemia were included in the study and ten healthy children at the same age were selected as the control group. By lfow cytometry, the percentage of CD3+CD1d tetramer+ NKT cell in peripheral blood and bone marrow were detected from March 2014 to December 2014 in our hospital. Immune magnetic bead separation was used to isolate and purify iNKT cells .The puriifed iNKT cells were cultured in the OCH(50 ng/ml,100 ng/ml or 200 ng/ml)+rhIL-2+rhG-CSF culture systems. The ampliifcation of iNKT cells after cultured in different systems were calculated. Elispot method was used to analyze the spotting form cells (SFCs) of IFN-γ or IL-4 expressed by activated iNKT cells.Results The percentage of CD3+CD1d tetramer+ NKT cells in peripheral blood of SAA group(0.72±0.03)% was signiifcantly lower than that of the control group(0.92±0.02)%(P=0.000). The percentage of CD3+CD1d tetramer+ NKT cells in bone marrow of SAA group(0.82±0.02)% was signiifcantly lower than that of the control group(1.05±0.05)%(P=0.000).In vitro iNKT cell ampliif-cation ability of bone marrow in SAA group was signiifcantly lower than the control group, and in medium concentration(50±6) and high concentration OCH group(52±6), the ampliifcation ability was higher than that in low concentration OCH group(30±5) (P<0.05). The secretion of IFN-γ in the iNKT cells of SAA bone marrow was signiifcantly lower in medium concentration(33±3) and high concentration(35±3)OCH group than that of the low concentration(50±3)OCH group(P<0.01). The secretion of IL-4 in the iNKT cells of SAA bone marrow was signiifcantly higher in medium concentration(50±3)and high concentration(75±3) OCH group than that of the low concentration(33±3) OCH group(P<0.01).Conclusions The quantity and function of NKT cells from children with SAA are lower than that of the healthy children.In vitro, they had better ampliifcation ability and could improve IL-4/IFN-γ imbalance in medium concentration and high concentration OCH group than in low concentration OCH group.

Objective:To investigate the quantitative expression of Melanoma-associated antigen MAGE-E1 mRNA in glioma, and explore its potential for immunotherapy in glioma.Methods:To establish a quantitative real-time polymerase chain reaction ( qRT-PCR) method to quantitatively determine MAGE-E1 mRNA in glioma.A total of 47 human glioma and 14 normal brain tissue specimens were analyzed.MAGE-E1 mRNA was normalized to HPRT1, a housekeeping gene.MAGE-E1/HPRT1 was used to evaluate the expression level of MAGE-E1 mRNA.Results:High-level expression of MAGE-E1 was observed in 7.1%of normal brain and 66.0%glioma tissues,which shown significant difference with P<0.05.There was irrelevant between the expression of MAGE-E1 mRNA and clinicopathogical parameters ,such as gender ,age,histological subtype and grade.Conclusion:With higher expression of MAGE-E1 in glioma tissues,it might be a potential target for immunotherapy of glioma.

10.3969/j.issn.1000-3606.2013.06.007