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OBJECTIVE： To optimize and improve the quality standard for Citrus reticulata formula granules. METHODS： Totally 13 batches of C. Reticulata formula granules from 4 different manufacturers were used as trial samples， and qualitative identification of hesperidin and nobiletin in the samples were carried out by TLC according to the method of 2015 edition of Chinese Pharmacopoeia （part Ⅳ）. The quantitative analysis of naringin， hesperidin， hesperetin， nobiletin and tangeretin in C. reticulatae formula granules were conducted by UPLC[The determination was performed on Waters Acquity UPLC BEH C18 column with mobile phase consisted of acetonitrile-0.2％ phosphoric acid aqueous solution （gradient elution）. The detection wavelength was set at 283 nm， and sample size was 3 μL]. RESULTS： The results of TLC showed that in the chromatograms of samples， same color spots were shown in the corresponding positions of the chromatogram of reference substance. The results of UPLC showed， that the linear range of naringin， hesperidin， hesperetin， nobiletin and tangeretin were 0.64-6.44， 15.78-157.80， 0.17-1.66， 2.08-20.85 and 2.04-20.43 μg/mL， respectively （all r≥0.999 2）； the limits of detection were 0.03， 0.33， 0.10， 0.20 and 0.06 μg/mL； the limits of quantitation were 0.07， 1.34， 0.20， 0.60 and 0.22 μg/mL. The average recoveries were 99.4％， 99.6％， 99.7％， 99.7％ and 99.7％ （n＝9）； RSDs of precision （n＝6）， stability （n＝7） and reproducibility （n＝6） tests were all≤2.03％； naringin was detected in only 3 batches of samples from one manufacturer （the content ranged from 0.067 3 to 0.069.6 mg/g）， while the other 4 components were detected in 13 batches of samples （the contents of them ranged 0.646 5-1.728 0， 0.102 6-0.290 5， 0.023 1-0.689 8， 0.018 2-0.270 7 mg/g）. CONCLUSIONS： In this study， the quality standard of C. reticulata formula granules was improved by qualitative and quantitative methods， and the contents of hesperidin， hesperetin， nobiletin and tangeretin were not less than 0.60， 0.10， 0.02 and 0.01 mg/g， respectively.
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Objective@#To construct the mmu-miR-155 eukaryotic overexpression vector pmR-155 and to investigate its effect on HBV replication and expression of PTEN in vivo.@*Methods@#The mmu-mir-146a precursor gene fragment pre-mmu-mir-146a was amplified by PCR, then connected to the pmR-mCherry plasmid vector after double enzyme digestion, the accuracy of recombinant vector was verified by colony PCR、double enzyme digestion and sequencing; then the recombinant vector was transfected HBV transgene mice(Experimental Group)with hydrodynamics-based injection via vena caudalis, and pmR-mCherry plasmid、PBS were respectively transfected into the mice as Empty plasmid Group、Blank Group. The concentration of IFN-γ in the serum was detected by ELISA. The expression of SOCS1、PTEN mRNA in the liver was detected by qPCR at 30d post-transfectioned. The Western blot was performed to detect the changes in SOCS1、PTEN、HBX in the liver tissue at 30 d post-transfectioned. The results were analyzed with Student’s t-test, or one-way analysis of variance and the least significant difference test.@*Results@#the colony PCR、double enzyme digestion and sequencing verified that the gene was inserted into the pmR-mCherry vector. Compared with Blank Group, the expression of miR-155 in the Experimental Group was significantly increased(t = 8.90, P < 0.01); the concentration of IFN-γ in the Experimental Group was significantly increased(F = 26.58, P < 0.01); the mRNA(FSOCS1 mRNA = 19.72, P < 0.01; FPTEN mRNA = 7.38, P < 0.05) and protein(FSOCS1 = 50.30, P < 0.01; FPTEN = 129.00, P < 0.01) expression of COCS1、PTEN was significantly decreased in the Experimental group and the protein of HBX was also significantly(FHBX = 77.97, P < 0.01).@*Conclusion@#The pmR-155 eukaryotic overexpression vector is successfully constructed, this recombinant vector can express miR-155 stably; miR-155 can down-regulate cocs1、PTEN gene expression and up-regulate the expression of IFN-γ, it can inhibit the replication of HBV and a potential targets to treating hepatocellular carcinoma.
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OBJECTIVE:To establish UPLC fingerprint of Glycyrrhiza uralensis. METHODS:UPLC method was adopted. The determination was performed on Waters CORTECS UPLC C18column with mobile phase consisted of acetonitrile-0.1% formic acid(gradient elution)at the flow rate of 0.3 mL/min. The detection wavelength was set at 254 nm,and column temperature was 30 ℃. The sample size was 1 μ L. Using glycyrrhizic acid as control,UPLC chromatograms of 27 batches of sample were determined. Similarity evaluation was conducted by using TCM Chromatogram Fingerprint Similarity Evaluation System(2004 A edition)to determine common peak and conduct cluster analysis of 27 batches of samples. RESULTS:There were 20 common peaks in UPLC chromatograms of 27 batches of samples. The similarity degree of S2,S4,S19,S21,S22,S24 were less than 0.90, the others samples were more than 0.90.After validation,UPLC chromatograms of 21 batches of batches of samples were in good agreement with control fingerprint. 27 batches of samples were clustered into 3 categories,in which S24 was categoryⅠ;S2,S4, S12,S19,S21,S22 were categoryⅡ;other were categoryⅢ. CONCLUSIONS:Established fingerprint can provide reference for quality evaluation of G.uralensis.
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Objective To construct the has-miR 146a eukaryotic overexpression vector pmR 146a and to explore its effect on the expression of c-Myc gene in HepG2.2.15 cells.Methods The has-miR-146a precursor gene fragment pre-has-miR-146a was amplified by PCR,then connected to the pmR-mCherry plasmid vector after double enzyme digestion,the accuracy of recombinant vector was verified by colony PCR,double enzyme digestion and sequencing;then the recombinant vector was transfected into HepG2.2.15 cells as the experimental group,meanwhile the empty vector group (transfecting pmR-mCherry empty plasmid group) and blank group(transfecting reagent lip2000+PBS),then the fluorescent protein expression amount was observed under the fluorescence microscopy at 24,48 h;the expression of has miR-146a was evaluated by qPCR;at 24,48 h after transfection,the expression levels of c-Myc gene mRNA were detected by qPCR,and the c-Myc protein expression level after 48 h was detected by Western blot.Results The colony PCR,double enzyme digestion and sequencing verified that the pre-has-miR-146a gene fragment was inserted into the pmR-mCherry vector;at 24,48 h after transfection in the experimental group and empty vector group,intracellular strong fluorescence was seen by fluorescent microscope,the transfection efficiency was at 50％-60％ contrasting without fluorescence;the has-miR-146a expression level in the experimental group was significantly higher than that in the empty vector group and blank group (P＜0.01);the c-Myc mRNA expression at 24,48 h after tranfection was significantly lower than that in the empty vector group and blank group (P＜0.05);the protein expression amount at 48 h after transfection was lower than that in the empty vector group and blank group (P＜0.01).Conclusion The pmR-146a eukaryotic overexpression vector is successfully constructed,this recombinant vector can express miR-146a stably;miR-146a can down-regulate c-Myc cancer gene expression,which can serve as one of potential targets for treating hepatocellular carcinoma.
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Objective CRISPR/Cas9 genome-editing technique provides an novel method for whole genome editing in eukaryotic cells.Recently,we found that gene subtype library with smaller size and focused pur-pose is more economical and practical. In this study,we aimed to target kinases,a group of pivotal cell signal transducers,to construct a kinase knock-out library using CRISPR/Cas9 technique.The construction strategy wll al-so be discussed. Methods 10 sgRNA was designed for each kinase target.After oligo pool synthesis by semicon-ductor chip,the oligos were eluted from the chip. The oligo templates were amplified and cloned into Cas9 vector and transformed into Stble3 competent cells.Monoclonal colonies were selected for DNA sequencing. Results(1) GO analysis of 507 cell kinases showed that the cell kinases took part in a wide range of cell signaling.(2)The sgRNA pool with about 140 bp in length was successfully amplified by using oligo pool as the template and univer-sal PCR primers.(3)In 40 identified library clones,34 clones were sequenced successfully. Among them,the DNA sequencing results of 25 samples were completely consistent with the designed target sequences.But there are some mutations in the primers of 9 samples.Failure in bacteria shaking,DNA sequencing and other factors were ex-isted in the other clones. Conclusion The CRISPR/Cas9 kinase knock-out library can be widely used for screen-ing the important kinases which may mediate cell proliferation,metastasis,drug resistance and autophagy.This li-brary will play an important role in clarifying the development of disease associated with kinases.
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Objective To construct the miRNA‐21 eukaryotic overexpression vector pmR‐21 and to explore its regulation effect on the expression of c‐myc gene in HepG2 .2 .15 cells .Methods The miRNA‐21 precursor gene fragment pre‐miRNA‐21 was amplified by PCR ,then connected to the pmR‐mCherry plasmid vector after double enzyme digestion ,the accuracy of the recombi‐nant vector was verified by double enzyme digestion and sequencing ;then the recombinant vector was transfected into HepG2 .2 .15 cells ,the fluorescent protein expression was observed under the fluorescence microscopy at 24 h and the transfection efficiency was detected by flow cytometry ;the expression of miRNA‐21 was evaluated by real‐time quantitative PCR;at 72 h after transfection ,the expression levels of c‐myc gene were detected by RT‐PCR and Western blot ;CCK‐8 was used to detect the cell proliferation in each group .Results The double enzyme digestion and Western blot verified that the target gene fragment was inserted into the pmR‐mCherry vector;at 24 h after transfection ,intracellular strong fluorescence was seen ,the transfection efficiency was higher than 50% ;miRNA‐21 expression level of the pmR‐21 recombinant vector group was significantly increased;c‐myc gene expression was increased in the pmR‐21 recombinant vector group at 72 h after transfection ,the cell proliferation in the pmR‐21 recombinant group was faster than that in the control group(P<0 .05) .Conclusion The pmR‐21 eukaryotic overexpression vector is successfully con‐structed ,this recombinant vector can express miRNA‐21 stably ;miRNA‐21 can up‐regulate c‐myc gene expression ,c‐myc gene is one of miR‐21′s targets for playing a cancer‐promoting action .
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Objective:To investigation of the long-term-siRNA treatment with HBV transgene mice on inhibit replication of hepatitis B virus .Methods:The constructed siRNA expressed vectors was transfected HBV transgene mice by hydrodynamics -based in-jection via vena caudalis .Different groups were set including:specificity siRNA groups ( pSilencer5.1/C2,pSilencer4.1/C2,pSilenc-er3.1/C2),PBS group and negative vector group (n=10).The effect was observed in different periods (6 d,21 d,1 months,3 months, 6 months and 9 months after injection ) .HBsAg was analyzed by Chemiluminescence method , HBV-DNA was analyzed by real time quantitative PCR ( RQ-PCR) .Results:Compared with the PBS group , specificity siRNA groups showed decreased levels of HBsAg and HBV-DNA (P0.05).Conclu-sion:The siRNA based on the expression vector can suppress the expression and replication of HBV in HBV transgene mice .The inhi-bition effects of long-term-siRNA treatment was specific .
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Aim: To preliminarily investigate the antioxidant activity of the acidic exopolysaccharide(EPS-A) from cultivated Cordyceps sisnensis in vitro and in vivo. Methods: PC12 cells were injured by H_2O_2 after the cells were treated with different doses of EPS-A for 6 h, and then the levels of malondialdelyde( MDA) and superoxide dis-mutase (SOD) in PC12 cells were determined. H22 cells were inoculated into the right oxter of each mouse after ICR mice were treated with EPS-A for 7 days, and tumor-bearing mice were treated with EPS-A for 7 days. MDA level and SOD activity in liver, brain and serum were assayed, and tumor weight of each mouse was measured as well. Results: EPS-A significantly decreased the production of MDA and increased SOD activity in H_2O_2-injured PC12 cells. Moreover, EPS-A inhibited H22 tumor growth in mice, in which the level of MDA in liver and brain was reduced, and SOD activity in liver, brain and serum was enhanced. Conclusion: EPS-A exhibited antioxidant activity in cell cultures and mice.
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To accommodate the requirement of quality education and achieve the goal of the 7-year-schooling clinical medicine,experimental training of cell immunology based on pathogen-induced immunology was established in combination with traditional experiment teaching method.In this course,inductive teaching method was used.The independent study and innovation ability of student were trained,which had a better teaching effect.
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Objective: To investigate the antitumor actions of polysaccharide extracts from the fruiting body of coriolus versicolor (CVE). Methods:Hepatoma HepA cells were injected into mice subcutaneously. Different doses of CVE were given by gavage. On the 7 th and 14 th day, tumor inhibitive rates were calculated. ELISA was performed to measure the serum IgG level; MTT was used to examine CVE′s effects on the proliferation of T lymphocytes of thymus. Immunohistochemistry was used to determine CVE′s influence on the expression of tumor related genes P53 and VEGF in liver. Results: CVE may evidently inhibit the growth of the transplanted HepA tumors. Its effects on the serum IgG level and on the proliferation of T lymphocytes of thymus were also significantly. Also, CVE markedly decreased the expression of P53, VEGF genes in liver. Conclustion: CVE had significant antitumor effects in vivo . The mechanisms may involve immune modulation effects and antimetastasis actions.
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Object To extract and purify a novel fibrinolytic enzyme from Perinereis aibuhitensis Grube Methods The enzyme was precipitated from the extract by ammonium sulfate, dialyzed, chromatographed on Sephadex G 100 column, and then purified by polyacrylamide gel electrophoresis Its fibrinolytic activity was assessed with fibrin plate method Results The purified enzyme showed an isoelectric point (PI) around 4 5 as tested by gel isoelectric focusing It consisted of two polypeptide chains with molecular weights around 33 000 u and 14 400 u, respectively Conclusion This was a novel fibrinolytic enzyme discovered from P aibuhitensis for the first time