Effect of cholesterol on human meniscal fibrochondrocytes / 中华创伤骨科杂志

Objective:To explore the effect of cholesterol on the expression of genes for matrix synthesis and degradation of human meniscal fibrochondrocytes and its mechanism.Methods:Meniscal tissue was taken from patients undergoing arthroscopic surgery to extract fibrochondrocytes. The cells were divided into a control group in which the normal cells were not processed, a positive control group in which interleukin-1 β was used to create a degeneration model, and 2 treatment groups which were subjected to treatment with 15 and 30 μg/mL cholesterol respectively. Safranin O staining, β-galactosidase staining and enzymic kits were used to detect the morphology and total cholesterol (TCH) content of meniscal fibrochondrocytes in the 4 groups. Immunofluorescence and western blot were used to detect the protein expression of type Ⅰcollagen precursor α1 (COL1A1) and type Ⅱ collagen precursor α1 (COL2A1). RT-qPCR was used to detect the mRNA expression of COL1A1, COL2A1, matrix metalloproteinase (MMP) 3, MMP9, MMP13, and genes related to cholesterol efflux pathways [like liver X receptor α (LXR α), ATP binding cassette transporter A1 (ABCA1) and ABCG1]. Results:There was no significant difference between the control and the positive control groups in the TCH content in human meniscal fibrochondrocytes ( P>0.05). The treatments with 15 and 30 μg/mL cholesterol resulted in significantly increased TCH contents in human meniscal fibrochondrocytes in the treatment groups ( P<0.05). Compared with the control group, the mRNA expression of LXR α, ABCA1 and ABCG1 was significantly decreased in the treatment groups ( P<0.05), and the meniscal fibrochondrocytes in the positive group and the treatment groups presented with a lower density, chaotic distribution and obvious signs of degradation. Compared with the control groups, the mRNA expression of matrix synthesis genes (COL1A1 and COL2A1) in the meniscal fibrochondrocytes was significantly inhibited while the mRNA expression of matrix degradation metalloenzymes (MMP3, MMP9 and MMP13) was significantly promoted ( P<0.05). Conclusion:Cholesterol may inhibit the cholesterol efflux pathways of meniscal fibrochondrocytes, and thus cause accumulation of cholesterol in the meniscal fibrochondrocytes, eventually leading to degeneration of meniscus.

Effect of IFN-CSP on gene of JAK-STAT signaling pathway in HBV transgenic mice / 中国生化药物杂志

Objective To investigate the anti-HBV molecular mechanisms of liver targeting interferon ( IFN-CSP ) in Balb/c-HBV transgenic mice.Methods Balb/c-HBV transgenic mice were randomly divided into 3 groups.Control group (treated with physiological saline), IFN α2b group (treated with 103 U/g IFN α2b), IFN-CSP group (treated with 102 U/g IFN-CSP).Another group of the non-transgenic mice were used as the Normal group.Each mouse was intramuscular injected with 50 μL dose once a day for 4 weeks.Total RNA of mice liver were extracted, and STAT1, STAT2, IRF-9, OAS1 gene expression of JAK-STAT signaling pathway were analyzed by real-time PCR.Results IFN α2b and IFN-CSP can significantly up regulate the expression of STAT1, STAT2, IRF-9, OAS1 gene of JAK-STAT signaling pathway (P<0.01).The induce effects of IFN-CSP on STAT1, STAT2, IRF-9, OAS1 were significantly better than that of IFN α2b (P<0.05).Conclusion The anti-HBV molecular mechanisms of liver targeting interferon (IFN-CSP) in Balb/c-HBV transgenic mice maybe related to regulate the expression of STAT1, STAT2, IRF-9, OAS1 gene of JAK-STAT signaling pathway.These results will lay a basis for the application of recombinant liver-targeting interferon.

Preliminary study of liver targeting interferon on the immune toxicity of mice / 中国生化药物杂志

Objective To evaluate the immunotoxicity effect of Liver targeting interferon (IFN -CSP) on mice.Methods Mice were randomly divided into five groups:low, middle and high dose of IFN-CSP, solvent control group(saline) and Positive control group (cyclophosphamide).They were injected subcutaneously for 2 weeks.Delayed hypersensitivity test was used to determine the cell immunefunction and plaque forming cell assay was used to determine the humoral immune function.Results There was no significant difference of the the index of immune organ and the ear swelling degree between IFN-CSP groups and control group.There was also no significant difference on hemolytic plaque test between them.Conclusion IFN-CSP has no significant effect on both cellular immunity function and humoral immune function of mice, this results will provides the basis for further safety evaluation.

Research on hepatitis C virus entry inhibitor / 病毒学报

Hepatitis c virus (HCV) infection has become one of the global public health problem,while there is no vaccine to prevent HCV infection, the so-called "cocktail" therapy that use a combination of drugs targeting multiple steps in the HCV infection cycle could achieve better curative effect. the process of HCV entering into host cell is the important step of drug intervention, in which HCV envelope protein El and E2, Host cell factors including Heparan sulfate(HS), CD81, scavenger receptor class B type I (SR-BI), Occludin (OCLD), Claudin (CLDN), low densitity lipoprotein receptor (LDLR), dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), Liver/lymph node specific ICAM-3-grabbing integrin(L-SIGN), trans- ferrin receptor 1 (TfR1) and so on play a important role. The virus and the host factors can be used as targets of hcv entry inhibitors many studies have shown that as novel and promising compounds, HCV entry inhibitors combinating with other drugs can be more effective in the treatment of HCV, this paper have re- viewed targets and inhibitors of HCV enterring into host cell since 1990s.

Optimization of expression conditions of an induction strategy for improving liver targeted interferon (IFN-CSP) production in E. coli / 生物医学工程学杂志

Expression conditions of induction strategies for the cytoplasmic inclusion bodies (IBs) production of liver targeted interferon IFN-CSP by recombinant Escherichia coli (E. coli) BL21 (DE3) were optimized in shake-flask cultures in this study. The factors of the optimized protocol included in the present study were pH, inducer IPTG (isopropyl beta-D-thiogalactoside) concentration, culture growth temperature, incubation time and induction point. The effects of those factors were investigated by 'single variable at a time' method, aimed to analyze characterization of the recombinant strain. Orthogonal experimental design was further used to optimize the above critical factors for IFN-CSP production. According to the expression optimization result, it was confirmed that the main influence factors were cell density and induction temperature. The IFN-CSP gene expression optimized conditions were: pH value of the culture medium was 6.0, culture temperature 37 degrees C, adding IPTG to final concentration 0.4 mmol/L when the recombinant strain growth density OD600 achieved 0.8 and induction time 4 h. At this point, the IBs represented 74.3% of the total cellular protein. Compared with the non-optimized condition, IFN-CSP production obtained in optimized induction strategies were increased by approx. 1.2-fold. The optimized induction strategy yielded 688.8 mg/L of IFN-CSP, providing experimental data to study the biology activity and productive technology of IFN-CSP.

Effects of shuixianzi extracts on lipid regulation and liver protection in hyperlipidemic rats / 中华老年医学杂志

Objective To investigate the preventive and therapeutic effects of shuixianzi extracts on hyperlipidemia rats, and on fatty liver pathology. Methods Shuixianzi was homogenized and filtered. Then the filtrate was freeze-dried after centrifugation. The powder was just the extracts of shuixianzi. During the establishment of rat hyperlipidemic model, the extract was given at the same time. At the end of this experiment, the changes of blood lipid and liver pathology were observed. In therapeutic experiments, after the hyperlipidemia model was established, optimal dose of extract was given, then the changes of blood lipid and liver pathology were also observed and the levels of superoxide dismutase (SOD) and malondialdehyde (MIA) were tested. Results In preventive experiments, high dose of extracts of shuixianzi versus negative control could inhlbit both the increase of TC, TG, LDL-C and the drop of HDL-C. [TC: (3.23±0.01) vs. (6.56±0.01) mmol/L; TG:(2.33±0.01) vs. (4.12±0.02) mmol/L; LDL-C: (2.02±0.01) vs. (3.91±0.02) mmol/L; HDL-C: (0.98±0.01) vs. (0.76±0.01) mmol/L, all P＜0.01]. At the same time, the extracts could inhibit the pathological changes of fatty liver. In therapeutic experiments, extracts versus control could regulate the serum lipid levels [TC: ( 3.67 ± 0.31 ) vs. ( 6.33 ± 0.52 ) mmol/L; TG: ( 1.99 ±0.11) vs. (4.08±0.24) mmol/L; LDL-C: (1.57±0.12) vs. (3.78±0.14) mmol/L; HDL-C:(1.10±0.03) vs. (0.77±0.02) mmol/L, all P＜0.01] and could reverse fatty changes of liver in hyperlipidemic rats. At the same time the extracts versus control could also increase the activity of SOD [(276.3±26.8) vs. (165.4±16.7) U/mg, P＜0.01] and decrease the level of MDA [(3.67±1.23) vs. (7.45±2.33) nmol/mg, P＜0.01]. Conclusions The extracts of shuixianzi could prevent and treat the hyperlipidemia, inhibit the fatty pathological change of liver, and also have the antioxidant function.

Secretion expression of BPI23-haFGF fusion gene in Pichia pastoris X-33 and identification of its biological activity / 生物医学工程学杂志

The BPI23-haFGF fusion gene was subcloned to the yeast expression vector pPICZaA and the recombinant plasmid pPICZaA-BPI23-haFGF was constructed. After linearization by sac I, the construct was introduced into X-33 yeast cells. The efficient engineering strain was obtained by the resistance and phenotype selection and identified by specific PCR. SDS-PAGE and Western blot analysis indicated that a 43 KD protein band coincident with the anticipated fusion protein size expressed in the culture supernatant of the transformed yeast cells, which accounted for above 50% of the total proteins of the culture supernatant. About 90% purity of recombinant BPI23-haFGF fusion protein was obtained by affinity chromatography. The in vitro bioactivity testing showed that the purified fusion protein killed E. coli and promoted proliferation of NIH3T3 cells, suggesting that the recombinant BPI23-haFGF fusion protein possessed both of BPI and FGF functions.

Application of microarrays in screening the antibacterial peptide associated genes of Musca domestica / 生物工程学报

To screen the candidate genes associated with Musca domestica antibacterial peptides using DNA microarray technique, the hybrid probes were designed from the conservative domains of the encoded area of the insect antibacterial peptide genes in GenBank with biology software Designer 2.0, and were synthesized by a chemical process, with the assistance of the automated Gen III Microarray Spotter, those oligo probes were printed on a special ready-made glass, and a cDNA microarray was constructed. The total RNA was extracted from the fat body of Musca domestic third-instar larve induced after 24 hours by Escherichia coli and Staphylococcus aureus, the strands of cDNA were labled with fluoresceine Cy3 using the method of reverse transcription PCR, after prehybridization, hybridization and washing procedure, the results of hybridization were scanned using computer system, and the data were analyzed using the software of MIDAS, fifteen valid hybridization signals were detected through two times of hybridization and scanning (the positive samples as a control were excluded). DNA microarray technique can be successfully applied screen the candidate genes associated with Musca domestica antibacterial peptides, and further provide significant evidence to discover its antibacterial peptide new genes.

Cloning of the antibacterial peptide cecropin gene of Musca domestica larvae and its fusion expression in Escherichia coli / 中国人兽共患病学报

In the present study, the total RNA was extracted from three instar larvae of Musca domestica, the cDNA sequence encoding the ORF of cecropins was amplified by RT-PCR, and the target fragment was further sequenced after being cloned into T vector pUCm-T. Then, the cDNA sequence of the mature cecropins was amplified by PCR with recombinant plasmid pUCm-T/cecropin as template, the N-terminal rare codon GGA of E. coli was changed to the favorable codon GGC,and a Asn codon AAC was added in front of the stop coden TAA in the C- terminus. This mutant gene designated as mCecropin was then ligated with the fusion expression vector pGEX-4T-1. After restriction analysis and DNA sequencing, the positive recombinant plasmid pGEX-4T-1/mCecropin was transformed to different strains of E. coli cells and the fusion protein was expressed after IPTG induction. The fusion protein was assayed by SDS-PAGE and the E. coli BL21(DE3) cell was chosen as the host cell for the expression of the fusion protein. The expressed fusion protein GST-mCecropin was purified by GSTrap affinity coloum and the GST marker was then cleaved by thrombin. In this way, the fusion protein mCecropin with antibacterial activity was obtained after purification with HiTrap benzamidine column.

Exploring ways of practical and innovative talents cultivation in local medical schools / 中华医学教育探索杂志

Based on the analysis of current condition of local medical schools and medi-cal talents demand at the grassroots level,this paper indicates that,taking students’personality development as a breakthrough point,with practice platform construction as main part,supple-mented by multi-knowledge hierarchy and scientific and cultural exposure,backed by workable policy.

DIAGNOSTIC VALUE AND FACTORS EFFECTING OF IMMUNOENZYMATIC STAINING TEST(IEST) FOR SCHISTOSOMIASIS JAPONICA / 中国血吸虫病防治杂志

The most suitable antigenic frozen sections are the sections of infected mice liver and adult worm of Schistosoma japonicum at 7th week and 6th week after infection respectively; the most suitable thickness of section is 7?m. The antigenicity of liver tissue-egg frozen section(TEFS) is markedly higher than that of adult worm frozen sectin(AWFS) and of liver tissue-egg paraffin embedded section.TEFS, as antigen, is considered to be of high sensitivity(96.7%), high specificity (98.7%) and lower cross reaction rates(0~3.3%) in IEST for diagnosis of schistosomiasis. The sensitivity of TEFS-IEST ig markedly higher than that of AWFS-IEST and that of COPT. The main advantages of TEFS-IEST are lower cost of the antigen, high sensitivity and rapid yield of results.

COMPARISON OF IMMUNOENZYMATIC STAINING TECHNIQUE (IEST), DOUBLE GLUING STRIP CIRCUMOVAL PRECIPITIN TEST(DGS-COPT) AND CONVENTIONAL CIRCUMOVAL PRECIPITIN TEST(CV-COPT) FOR DIAGNOSIS OF SCHISTOSOMIASIS JAPONICA / 中国寄生虫学与寄生虫病杂志

IEST, DGS-COPT and CV-COPT using lyophilized ova of schistosoma japonicum were performed on sera from 120 cases of schistosomiasis japonica, 120 cases of schis-tosomiasis japonica 3-8 years after being cured with praziquantel and 120 healthy individuals by single-blind method. The sensitivity and specificity of IEST was 91.7% and 95.8% respectively which were significantly higher than that of both DGS-COPT and CV-COPT. The negative conversion rate of cured patients was 70.8% with IEST, 80.8% with DGS-COPT and 81.7% with CV-COPT. The results showed that IEST has higher diagnostic value for schistosomiasis than both COPT. DGS-COPT has the same diagnostic value as CV-COPT, however, it was easy to perform and time-saving, thus it might be applied in the fields for practical purposes.