Objective To construct miRNA-29b1 gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29b1 sequence in Genbank .sgRNA and Cas9 were transcribed to RNA in vitro, these RNA were then microinjected into zygotes of C 57BL/6 mice.After mouse birth, the genome DNA was extracted and sequenced to identify its genotype; meanwhile , real-time PCR was used to assay the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney of mutated mice .Result A 20 bp sgRNA targeted on miRNA-29b1 was synthesized and transcribed to RNA with Cas 9.After microinjection, miRNA-29b1 gene-mutated mice were obtained.The sequencing results showed that there were two types of genotype for the mutated mice , one was 10 bp deletion, and another was 23 bp deletion accompanied with a 3 bp insertion.Compared with the wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney was reduced significantly .Conclusions miRNA-29b1 gene knockout mice are constructed successfully by using CRISPR /Cas9 technology.

Objective To knockout Rag2 and IL2rg genes and construct severe combined immunodeficiency mice based on CRISPR/Cas9 technology. Method Design and synthesis of 25 bp sgRNA were made according to the Rag2 and IL2rg sequences in Genbank. After annealing, sgRNA was cloned into pX330 vector. Recombination plasmid Rag2?sgRNA, IL2rg?sgRN and Cas9 were then transcribed into RNA, these RNA were microinjected into zygotes and the zygotes were transplanted into recipient ICR mice. F0 founders were born and mutated F0 founders mated with wild type mice to obtain F1 generation heterozygous mice. Mutated F1 mice were crossed and got F2 generation homozygous mice. Genotype and phenotype of the knockout mice were identified by sequencing, flow cytometry and xenograft model. Results Rag2?sgRNA and IL2rg?sgRNA recombination plasmids were constructed and transcribed into RNA. After microinjection and mat? ing, F0 founders were born and F2 homozygous mice were obtained. The results of sequencing showed that there were two types of genotype in IL2rg gene, 10 bp or 11 bp deletion;however, there was only one genotype in Rag2 gene, which was 8 bp deletion. Compared with wild?type BALB/c mice, the number of CD3 +, B220 + and NKp46 + cells in peripheral blood of the knockout mice was reduced significantly. After inoculation of human breast cancer cell line SKBR?2HL cells, tumor size in the xenograft mouse model was increased gradually along with time extension. Conclusion CRISPR/Cas9 is an efficient way to mutate Rag2 and IL2rg gene in mice in vivo, leading to aberrant T cells, B cells and NK cells.

Objective To investigate the effect of NS-398 combined with docosahexaenoic acid (DHA) on apoptosis of cholangiocarcinoma QBC939 cells and its mechanism.Methods In vitro,cultured cholangiocarcinoma QBC939 cells were exposed to 0,25,50,100,150 and 200 μmol/L NS-398 with 0,15,30,45,60 and 75 μg/ml DHA respectively.The absorbance of the QBC939 cells were measured by CCK8 and its growth inhibition ratio were calculated.Flow cytometry was applied to detect cell apoptosis.The level of β-catenin and c-myc mRNA and protein were measured by real-time PCR,Western blot and enzyme-linked immunoabsordent assay respectively.Results Exposure to NS-398 combined with DHA suppressed the growth of QBC939 cells.When NS-398 was at 100 μmol/L and DHA at 45 pμg/ml,the relative growth inhibition rate of QBC939 cells was 90％ (F =5.85,P ＜ 0.05).NS-398 combined with DHA promoted QBC939 cells apoptosis at the early stage (F =8.16,P ＜ 0.01).Real-time PCR could detect low β-catenin and c-myc expression in QBC939 cells disposed by NS-398 combined with DHA (F =7.61,P ＜0.01),(F =7.92,P ＜0.01).NS-398 combined with DHA decreased β-catenin (F =7.75,P ＜ 0.01),(F=8.17,P＜0.01) and c-myc (F=8.76,P＜0.01),(F=8.12,P＜0.01) protein expression in QBC939 cells.Conclusion NS-398 combined with DHA promoted apoptosis and inhibit proliferation of cholangiocarcinoma cells QBC939 in vitro possibly through downregulated mRNA and protein expression of β-catenin and c-myc.

Objective To establish a set of operational status assessment indicators to meet the needs of informationized hospital management.Methods Assessment indicators were selected and weights were set respectively through literature review,field interview,and questionnaire survey.Six target dimensions were key performance indicators medical business,operational performance,cost control,medical insurance,balance and risk management,and development capability.Thus a set of operational status evaluation indicators was established in IT means,and based on the informationization level of a tertiary A general hospital in Zhejiang province.Results In the principle of public welfare,objectivity,effectiveness and prospectiveness,we analyzed and sorted out relevant data in the current hospital informationization,identifying six quantitative indicators,15 level-1 indicators,and 86 level-2 indicators.Conclusions It is feasible to build a set of assessment indicators for hospital operation and management in view of both technology and methodology.