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Objective:To review the detail data of National Natural Science Foundation of China (NSFC) grant support during past decade and to analyze the tendency of basic and applied science researches in ophthalmology and vision science.Methods:The detail data of grants supported by NSFC in the field of ophthalmology and vision science during 2010—2019 were collected.The project category, project name, principle investigator, amount of grant, supported institute and region distribution were analyzed.Cluster analysis of key words associated with the projects was performed with VOSviewer software.Results:During the past decade, the number of projects and the total amount of funding from NSFC were stable in ophthalmology and vision science, which accounted for about 2% of medicine field.The number of keywords representing advanced researches, hotspots areas and methodologies increased.Retinal and choroidal diseases, corneal and ocular surface diseases, glaucoma and optic nerve diseases ranked in the top three in the granted projects.Granted projects mainly went to the well-developed areas in economy and education.The high-level talent projects primarily were in the institutes and universities with solid and profound scientific research background.Conclusions:The sustained and stable support of NSFC plays a promoting role in the development of ophthalmology and vision science and talent cultivation.The new technology and its integration with interdisciplinary science promote the development of ophthalmology.Successfully granted projects appear to be related to the regional environment of economy, education and scientific research background.
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Objective To study the effects of acupuncture combined with Anchang powder acupoint application in the treatment of irritable bowel syndrome with diarrhea. Methods A total of 100 patients with irritable bowel syndrome with diarrhea in our hospital from July 2014 to July 2016 were enrolled.The subjects were randomly divided into the control group (n=50) and the treatment group (n=50). The control group were treated with trimebutine, while the treatment group were treated with acupuncture combined with Anchang powder acupoint application. The two groups were treated for 4 weeks. The clinical effects of the two groups after treatment were compared. The clinical symptom scale was used to evaluate the clinical symptoms, and the quality of life was evaluated by the quality of life scale (QOL) The recurrence rate after half a year heal of the two groups were compared. Results The total efficacy rate of the treatment group was 92.0% (46/50), significantly higher than 68.0% (34/50) of the control group (x2=9.000, P=0.003). After treatment, the clinical symptom integral(1.89 ± 0.95 vs.4.02 ± 1.13,t=10.202)of the treatment group were significantly lower than the control group(P<0.05).After treatment,the quality of life rating scores(34.15 ± 6.75 vs.28.47 ± 5.01,t=4.803) of the treatment group were significantly higher than the control group (P<0.01). Half a year after treatment, the recurrence rate of the treatment group was 24.1% (7/29), significantly lower than 64.7% (11/17) of the control group (x2=7.405, P=0.007). There was no significant difference of the two groups during the treatment. Conclusions Acupuncture combined with Anchang powder acupoint application showed a good efficacy for the patients with irritable bowel syndrome with diarrhea. And the treatment showed the low incidence of adverse reactions and low recurrence rate, can mprove the clinical symptom and quality of life.
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Objective:To explore the feassibility to edit human p53 and PTEN,two tumor suppressor genes,by CRISPR-Cas9 technology and to evaluate the editing efficiency in vitro,and to provide the experimental study tools for transforming primary healthy cells into malignant tumor cells and establishment of humanized mouse models with human oncogenesis in vivo.Methods:The single-guide RNA(sgRNA)sequences were designed to target the common exon regions of p53 and PTEN mRNA isoforms based on software analysis,that could predict their gene editing efficiency.The sgRNAs with high scores were selected and cloned into Cas9-P2A-GFP plasmid to construct sgRNA-Cas9-P2A-GFP vector that co-expressed sgRNA,Cas9 and GFP.The 293T cells in logarithmic growth phase were transfected with the sgRNA-Cas9-P2A-GFP vector(experimental group)or PBS(control group)by Lipofectamine 2000.After two-week expansion,the GFP-positive 293T cells were purified by flow cytometric sorter,whose genomic DNA was extracted for further analysis.The DNA fragments containing the sgRNA targeting site were amplified from the extracted genomic DNA by PCR and purified by gel extraction.Then they were linked into the pEASY-Blunt Zero cloning vector and transformed into competent E.coli cells.The single colonies formed by pEASY-Blunt Zero vector transformed cells were used to extract the plasmid for DNA sequencing.And the sequencing results of control group and experimental group were compared to judge the gene editing efficiency.Results:Over 82% of the sgRNA-Cas9-P2A-GFP transfected cells were found to express GFP gene after flow sorting in experimental group,which was significantly higher than that of the pre-sorted cells(P<0.05).Genomic DNA was extracted from the sorted cells after expansion and used as PCR template.The length of the amplified fragments containing the p53 mutation site was 612 bp,while the lengths of the amplified fragments containing the PTEN-1/PTEN-2 mutation site were 667 and 947 bp.The results of sequencing showed that the efficiency of editing induced by p53-1,p53-2 and PTEN-2 sgRNA were 54.5%,45.5% and 33.3%,respectively. Conclusion:The sgRNAs p53-1,p53-2 and PTEN-2 designed for p53 and PTEN can successfully guide Cas9-mediated site-specific genome editing with high efficiency at the genome level.
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Objective:To construct the eukaryotic expression plasmid carrying hTERT-P2A-EGFP, and to explore its expression and transfection efficiency in the HEK293FT cells.Methods:The recombinant plasmid was constructed by using pBABE-puro-hTERT and pRRLSIN-cPPT-MSCV-EGFP plasmids.The hTERT,P2A,and EGFP genes were obtained using pBABE-puro-hTERT as template by PCR.And the correct hTERT was inserted into pRRLSIN-cPPT-MSCV-EGFP vector.Then the recombinant plasmid containing hTERT-P2A-EGFP gene was obtained and identified.The HEK293FT cells were transfected by the recombinant plasmid, and the expression of green fluorescence protein(GFP) was observed by fluorescence microscope.Results:The PCR results showed that the fragments of hTERT, P2A, and EGFP were 3 400, 110 and 720 bp.And the length of gene fragment(hTERT-P2A-EGFP)was 4 300 bp by enzyme digestion.The results of sequencing showed that the 1 547 site of the target gene was mutated.Using site-directed mutagenesis, the 1 547 site was successfully mutated.And the target gene sequence was completely identical with the sequence published in GenBank.The recombinant plasmid was transfected into the HEK293FT cells, and GFP was observed in the cells.The results of flow cytometry showed that the transfection efficiency of recombinant plasmid was 44.8%.Conclusion:The recombinant plasmid carrying hTERT-P2A-EGFP gene is successfully constructed, and it can be used for cell transfection.
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Myeloid-derived suppressor cells ( MDSC) are a bone marrow-derived heterogeneous cell population with immuno-suppressive activity.Although there is convincing evidence that autoimmune diseases are associated with MDSC expansion ,controversies remained regarding the role of MDSCs in controlling autoimmune responses .Recent studies have shown that the expansion of MDSCs , which are capable of inhibiting effector cell function in vitro ,does not always lead to alleviation of autoimmune diseases ,and in some ca-ses paradoxically exacerbates the disease progression .This review summarizes recent insights into the role of MDSCs in the development of autoimmune responses and the potential of using MDSCs for the treatment of autoimmune diseases .
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Objective To study the effects of cleavage of Bcl-2 by two DNAzymes on apoptosis of human hepatoma cell line (HepZ1).Methods Two “10-23” DNAzymes(DzT and DzTi) targeting Bcl-2 mRNA and their analogues(DzT' and DzTi') were synthesized and used to cleave Bcl-2 mRNA in vitro and in BEL-7402 cells.The RT-PCR was performed to assess the cleaving efficiency.Expression of Bcl-2 protein was determined by immunofluorescent method.Cell apoptosis was detected by flow cytometry.Results The unmodified Enzymes DzT,and its modified form DzTi,which had an added 3'-inverted thymidine,could effectively cleave Bcl-2 mRNA in vitro.After transfected into BEL-7402 cells,DzTi exhibited more powerful cleaving ability than DzT,significantly down-regulated the level of Bcl-2 protein(P ＜0.01) and inhibited the cell growth(P ＜0.05).The results of flow cytometry suggested that the apoptosis rate of DzT and DzTi significantly increased,appeared apoptotic peak.Cell cycle was delayed in DzT and DzTi group,proportion of cells in G0/G1 increased,S phase cells decreased.Conclusion The synthesized DNAzymes could effectively cleave Bcl-2 mRNA,decrease the level of Bcl-2 protein and induce hepatoma cells apoptosis.
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Objective To study the inhibitory effect of hammerhead ribozyme targeting connective tissue growth factor(CTGF) on collagen I synthesis and cell cycle progression of human hepatic stellate cell line(LX-2) cells.Methods Hammerhead ribozyme cDNA targeting CTGF mRNA plus two self-cleaving sequences were inserted into pTriEx2 vector to construct a recombinant vector pTriCTGF-Rz.LX-2 cells were transfected with either pTriEx2 or pTriCTGF-Rz and further stimulated with or without TGF-1.There were five groups in the experiment:control group,pTriEx2 group,pTriCTGF-Rz group,pTriEx2 plus TGF-?1 group,and pTrCTGF-Rz plus TGF?1 group.Semi-quantitative RT-PCR was used to detect the levels of CTGF mRNA and collagen Ⅰ mRNA.ELISA and flow cytometry were used to detect the levels of collagen Ⅰ secretion and cell cycle.Results Transfection of pTriCTGF-Rz into LX-2 cells reduced the CTGF mRNA and collagen Ⅰ mRNA levels as well as collagen Ⅰ protein level compared with pTriEx2 group(P