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.

Objective To investigate the different biological characteristics of high-and low-expression of ATP-binding cassette transporter(ABCG2)in human multi-drug resistant nasopharyngeal carcinoma CNE2/DDP cell line(abbreviated as ABCG2High cell and ABCG2Low cell),and to analyze drug-resistant characteristics of the two cell lines.Methods ABCG2High and ABCG2Low cells were isolated by flow cytometry,while their cloning efficiency,cell cycle distribution,and the expression of ABCG2 before and after the 5th passage were examined as well.mRNA expressions of drug-resistant genes,such as ABCG2,Bcl-2,MDR1,MRP and MGMT,were detected by reversed transcriptive polymerase chain reaction(RT-PCR).MTT assay was utilized to detect the light absorbance(A)from day 1 to 14 to determine growth kinetics.Moreover,the drug sensitivity of the two cell lines to fluorouracil,cisplatin,vincristine,carboplatin,epirubicin,daunorubicin,paclitaxel and mitomycin were detected by MTT assay.Results The cloning efficiency of ABCG2High and ABCG2Low cells was 25.24%?1.18% and 16.28%?1.11%,respectively,on day 7.ABCG2High cells in S phase accounted for 41.5%,while the ABCG2Low cells in the G1/M0 phase went up to as high as 63.9%.The expression of ABCG2 in original ABCG2HighCNE2/DDP cells was 98%,while in ABCG2Low CNE2/DDP cells was 2%.However,at the 5th passage,the expression of ABCG2 decreased to 75% in the former cells and increased to 20% in the latter ones.The expression of ABCG2 mRNA in ABCG2High cells was obviously higher than that in ABCG2Low cells,while there was no difference in expression of other drug resistance genes between two cell lines.The growth velocity of ABCG2High cells exceeded that of ABCG2Low cells in early stage,but it slowed down 7 days later.MTT assay showed that IC50 of ABCG2High cells to 8 kinds of anti-tumor drugs were twofold higher than that of ABCG2Low cells,with statistically significant difference between them(P

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 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.

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.

AIM: To evaluate the role of human angiotensin Ⅱ(AngⅡ) type Ⅰ receptor (AT1R) antisense cDNA (ahAT1) on migration of cultured artery smooth muscle cells (VSMCs). METHODS: Two recombinant adenoviral vectors, Ad/CMV. ahAT1 containing full length antisense cDNA targeting to human AT1R mRNA, and Ad/CMV.LacZ containing LacZ called report gene, were constructed by orientation clone technology and homologous recombination, and then were used to transfect VSMCs in vitro. AT1R expression detected by RT-PCR and immunohistochemistry, and migration of VSMCs measured by Boyden's Chamer methods, were compared between transfected and non- transfected VSMCs. RESULTS: Forty-eight hours after Ad/CMV. ahAT1 transfection, the level of AT1R mRNA decreased markedly (50% of control group), and AT1R protein expression was significantly less (P

Objective To evaluate the effect of different kind of intravenous immunoglobulin (IVIG) therapy in treating Kawasaki disease (KD) and preventing cardiac consequences (coronary artery lesion, CAL). Methods A questionnaire form and guideline for KD diagnosis were sent to 50 hospitals providing pediatric medical care in Shanghai. The data from a total of 1 682 KD patients were collected. It included 1 064 males and 618 females from 1998 through 2008 in Shanghai. The average age of the KD patients was (2.57±2.33) years old (0.1-18.8 years).The patients had been divided into 6 groups for different IVIG therapy, which included 1 g/kg once, 2 g/kg once, 0.4-0.5 g/kg five times, 1 g/kg twice, 2 g/kg twice and others. SAS 6.12 software was used for statistical analysis. Results In all KD patients, the patients treated with IV1G in 5th-10th day of illness has the least cardiac complication and CAL incidence and the group with IVIG therapy of 1 g/kg twice also has the least cardiac complication and CAL incidence. Conclusions The best doses of IVIG in treating KD is 1 g/kg twice and the IVIG therapy should be used in 5th-10th day of KD illness.

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.

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.

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.