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.

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.

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.

The productivity of cells per unit area determines the scale-up potential of the cell culture process,and the largescale application of the microcarrier system provides space for the high-yield culture of anchorage-dependent animal cells. The microcarrier animal cell culture technology is suitable for efficient production process development and optimized amplification. In recent years,microcarrier-based culture technology has been widely used in various types of animal cell culture to produce many important biological products such as vaccines,enzymes,hormones,antibodies,interferons and other probiotics. In this paper,the research progress of domestic and foreign microcarrier cell culture technology,the comparison of new microcarriers and traditional microcarriers and their applications were reviewed,so as to provide reference for the in-depth research and application of large-scale cell culture technology based on new microcarriers.

ObjectiveTo study the effect and underlying mechanism of Stemona tuberosa alkaloids on the proliferation and apoptosis of human non-small cell lung cancer NCI-H460 cells. MethodNon-small cell lung cancer NCI-H460 cells were divided into a blank group and S. tuberosa alkaloids groups （50， 100， 150， 200， and 250 mg·L^-1）. The effect of S. tuberosa alkaloids on the proliferation of human NCI-H460 cells was observed by thiazolyl blue tetrazolium bromide （MTT） assay and colony formation assay. Cell apoptosis was observed by Hoechst 33258 staining and flow cytometry. Real-time fluorescence-based polymerase chain reaction （Real-time PCR） was used to detect the effect of S. tuberosa alkaloids on the mRNA expression of cysteinyl aspartate-specific protease 3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and epidermal growth factor receptor （EGFR）. The protein expression levels of Caspase-3， Bax， Bcl-2， protein kinase B （Akt）， phosphorylated （p-）Akt， EGFR， c-Jun N-terminal kinase （JNK）， p-JNK， p38 mitogen-activated protein kinase （p38 MAPK）， and p-p38 MAPK were measured by Western blot. ResultCompared with the blank group， the S. tuberosa alkaloids groups showed increased inhibition rate on cell proliferation （P<0.01）， reduced number of cell clones formed and the rate of cell clonal formation （P<0.05， P<0.01）， and increased karyopyknosis， cytoplasmic aggregation， and cell apoptosis rate （P<0.01）. The S. tuberosa alkaloids groups at 100， 150， 200， and 250 mg·L^-1 showed increased Caspase-3 mRNA expression （P<0.05）， decreased EGFR mRNA expression （P<0.05， P<0.01）， up-regulated protein expression of Caspase-3 and p-JNK （P<0.01）， and down-regulated protein expression of EGFR and p-Akt （P<0.05， P<0.01）. Additionally， compared with the blank group， the S. tuberosa alkaloids groups showed increased expression of Bax mRNA （P<0.01）， decreased expression of Bcl-2 mRNA （P<0.01）， up-regulated protein expression of Bax and p-p38 MAPK （P<0.01）， and down-regulated protein expression of Bcl-2 （P<0.01）. ConclusionsS. tuberosa alkaloids can inhibit proliferation and induce apoptosis of human non-small cell lung cancer NCI-H460 cells， and the mechanism may be related to the inhibition of EGFR protein expression and phosphorylation of Akt protein， as well as the activation of the JNK/p38 MAPK signaling pathway.

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.

OBJECTIVE： To establish HPLC fingerprint of Zhuang and Yao medicine Lespedeza formosa， and to provide reference for quality control of L. formosa from different producing areas. METHODS： Totally 10 batches of samples were collected from 5 producing areas as Guangxi Nanning， Guilin， Wuzhou and so on. HPLC fingerprints was established and similarity analysis was carried out by using “Similarity evaluation system of TCM chromatographic fingerprint” （2012 edition） software. The determination was performed on Inertsil ODS-3 column with mobile phase consisted of acetonitrile-0.2％ phosphoric acid solution （gradient elution） at the flow rate of 0.8 mL/min. The detection wavelength was set at 350 nm， and the column temperature was 35 ℃. The sample size was 10 μL. Common peaks were identified by comparing substance control. Cluster analysis and principal compoent analysis were performed by using IBM SPSS 21.0 statistical software. RESULTS： HPLC fingerprint was established， and 12 common peaks were calibrated. 3 common peaks were identified （common peak 8， 10， 11 were chafotalin， vitexin and isovitexin）. The similarity of 10 batches of samples were all higher than 0.9. Through cluster analysis， 10 batches of medicinal materials could be clutered into 2 groups. According to the principal component analysis， the cumulative variance contribution rate of the two principal component factors was 86.108％ （contribution rates of first principal components and second principal components were 66.891％ and 19.217％）. CONCLUSIONS： HPLC fingerprint of L. formosa is established successfully. The method is simple and easy to use， provides a reliable evalution method for quality control of L. formosa.

OBJECTIVE To investigate the effect of arctigenin(ATG) on liver fibrosis in rats induced by carbon tetrachloride(CCl4)and to explore its underlying mechanism. METHODS Sprague-Dawley rats were randomly divided into six groups:vehicle,ATG 3.0 mg · kg-1 group,CCl4 model group,CCl4+ATG 1.0 and 3.0 mg·kg-1 groups,and CCl4+colchicine(COL)0.1 mg·kg-1(toxicity)group. Liver fibrosis was induced by subcutaneous injection of CCl4 in rats for 8 weeks. ATG and colchicine were administrated ig once a day starting from the fifth week after the CCl4 treatment for 4 weeks subsequent. At the end of the study,glutamic pyruvic transaminase (GPT),glutamic oxaloacetic transaminase(GOT),albumin(ALB),and total bilirubin (TBIL) as well as the contents of hydroxyproline (HYP) in liver tissues were measured. Histopathological changes were observed in the liver tissues using hematoxyline-eosin(HE)and Masson’s trichrome staining. The proliferation of hepatic stellate cells (HSC) and expression of cell cycle-related proteins were assayed by indirect immunofluores?cence staining and Western blotting,respectively. RESULTS Compared with CCl4 model group,ATG 1.0 and 3.0 mg · kg-1 improved the liver function by decreasing serum contents of GPT,GOT and TBIL (P<0.05),and increasing serum content of albumin(P<0.05). Histological results indicated that ATG 1.0 and 3.0 mg · kg-1 alleviated liver damage and reduced the formation of fibrous septa. Moreover, ATG 1.0 and 3.0 mg · kg-1 significantly decreased liver HYP when compared with CCl4 model group(P<0.05). In addition,CCl4-induced proliferation of activated HSC was inhibited by ATG 1.0 and 3.0 mg·kg-1, and this was accompanied by down-regulation of cyclin D1,cyclin-dependent kinase(CDK)2,CDK4, and proliferating cell nuclear antigen (PCNA)(P<0.05),and up-regulation of p27kip1 in activated HSC (P<0.05). CONCLUSION ATG can alleviate hepatic injury and fibrosis induced by CCl4,which is probably associated with suppression of the proliferation of activated HSC.

OBJECTIVE:To compare genetic diversity of Spatolobi caulis from different areas of Guangxi by random amplified polymorphic DNA(RAPD)and inter-simple sequence repeat(ISSR). METHODS:Through using POPGENE 32 software,Ntsys software and SPSS 17.0 software,RAPD and ISSR methods were used to study genetic diversity of 9 samples of S. caulis from dif-ferent areas of Guangxi. RESULTS:After amplification of screened 3 RAPD primers and 4 ISSR primers,and there were 198 and 315 locus,and 37 and 80 polymorphism locus. Rates of polymorphism locus were 18.7% and 25.4%;the number of effective al-leles were 1.416 8 and 1.584 0;genetic diversity index were 0.269 4 and 0.351 3;Shannon diversity index were 0.431 6 and 0.529 9. All the values of ISSR marker were higher than RAPD marker. The average genetic similarity coefficient of ISSR and RAPD were 0.757 64 and 0.683 80,indicating ISSR was more sensitive for the detection of genetic diversity. The clustering result of them was close to each other. The correlation coefficient of them were 0.847,indicating very significant positive correlation at the level of 0.001. CONCLUSIONS:ISSR could reflect more information of genetic diversity than RAPD,and is more suitable for research of genetic diversity of S. caulis from different areas of Guangxi.

Objective To study the effect of Du meridian electroacupuncture on growth associated protein-43 (GAP-43) expression in rats after spinal cord injury (SCI). Methods The SCI models were established with MASCIS Impactor at T11 segment in Sprague-Dawley rats. They were equally divided into control group (group A, n=18) and Du meridian electroacupuncture group (group B, n=18). Group B received electroacupuncture once a day since 1 week after SCI. They were evaluated with the Basso-Beattie-Bresnahan (BBB) score 1, 2, 4, 8 weeks after SCI. The mRNA and protein of GAP-43 was detected with RT-PCR and immunohistochemistry 2, 4, 8 weeks after SCI. Results Compared with group A, the BBB score, the expression of GAP-43 mRNA and protein increased in group B after SCI (P<0.01). Conclusion Du meridian electroacupuncture can promote the expression of GAP-43 after spinal cord injury.