Objective:To construct BimS lentivirus RNA interference(RNAi) vector and to study its infection efficiency by using RNAi technique.Methods:Three interference targets were designed according to the BimS sequence.The single chain primer was annealed into double-stranded oligo sequences,and then connected with vector linearized with Age Ⅰ and EcoR Ⅰ enzyme.The recombinant plasmid was packaged,and the infection efficiency was observed by infecting ACC-2 cells.Results:After amplification,a 337 bp band was appeared in the electrophoresis results of positive clones.Sequence of inserted fragments were identical with the result of DNA sequencing.Restructuring lentivirus was packed in 293T cells,the virus titer was 2 × 108 TU/ml,MOI =20,and the transfection efficiency was 85％.The BmS mRNA relative expression of pFU-GV-BmS-1,pFU-GV-BMS-2 and control group was 0.743 ±0.025,0.466 ±0.023 and 1.266 ±0.042 respectively(between each 2 groups,P ＜0.05).Conclusion:BimS lentivirus virus RNA interference vectors can be constructed,and can efficiently infect ACC-2 cells.

Objective To consecutively investigate the quality of auto-antibody testing of the whole country and to improve quality.Methods A nation-wide investigation was carried out and hospitals or departments participating were notified by letter or telephone communication.Autoantibodies tested for quality control survey included anti-nuclear antibody (anti-ANA),anti-double-stranded DNA (anti-dsDNA)antibody,anti-extractable nuclear antigens (anti-ENA) antibody,anti-mitochondria antibody (AMA),anti-smooth muscle antibody (ASMA) and anti-citrulline antibody (anti-CCP).There were 15 samples in total for testing,including 3 control samples for each test.Same sample was used for both AMA and ASMA test.Sample distribution and data analysis were carried out double-blindly.A total of 114 hospitals or departments participated in the survey.Multiple testing methods were adopted including indirect immumo-fluo-rescence (IIF),immuno-blot (IB),dot-blot (DB),double immuno-diffusion (DID),enzyme linked immuno-sorbent assay (ELISA),chemiluminescent assay,dot-immunogold filtration assay.Results The accurate rates for this survey were 98％,96.6％,89.5％,98.1％,92.1％,96.4％ respectively for ANA,anti-dsDNA,anti-ENA,AMA,ASMA and anti-CCP.Anti-ENAs were further divided into anti-RNP,anti-Sm,anti-SSA,anti-SSB and anti-Scl-70 subgroups,and the accurate rates were 88.4％,96.8％,100％,100％ and 95.8％,respectively.Titers of ANA varied greatly among different labs,so did quantitative analysis of anti-CCP,AMA and antidsDNA by ELISA.However,the accuracy of ANA types determined by IIF was greatly improved.Detection rate of AMA and AMSAwas still low.Conclusion Among detected antibodies,ANA,anti-dsDNA and antiCCP are the most prominently improved.Accurate rate of anti-ENA antibody is slightly increased.

Objective To investigate the current national situation of autoantibody test in order to improve the quality of autoantibodies test.Methods Hospitals or departments in the whole country participated voluntarily or on invitation.Fifteen samples in total were distributed double-blindly,and autoantibodies including anti-nuclear antibody (ANA),anti-double-stranded DNA (anti-dsDNA) antibody,anti-extractable nuclear antigens (anti-ENA),anti-citrulline antibody (anti-CCP),anti-mitochondria antibody (AMA) and anti-smooth muscle antibody (ASMA) were tested in 6 samples.The samples were used for AMA and ASMA tests.Results A total of 148 hospitals or departments participated and multiple testing methods were adopted.The accurate rate of ANA(97.3％),AMA (96.1％),ASMA (92.1％) and anti-CCP (97.4％) were higher than that of anti-dsDNA (81.9％) and anti-ENA (77.2％).Anti-RNP and anti-Scl70 in anti-ENAs had the lower accurate rate,90.9％ and 80.3％ respectively.Taking data of the past 10 years together,the accuracy of antiSSA,anti-SSB,anti-Sm had been stable since 2009,while that of anti-RNP and anti-Scl70 decreased slightly.For methodology,indirect immunofluorescence was mainly adopted in the testing of ANA,anti-dsDNA,AMA and ASMA,immunoblotting was mainly adopted in anti-ENA detection and enzyme linked immunosorbent serologic assay was used for anti-CCP test.Conclusion No major variation of primary testing method is found in recent 10 years.Although diverged greatly among different methods,the accuracy of antibody detection has improved year by year.

Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between miRNAs and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qRT-PCR method. Two microRNA genes, osaMIR156 and osaMIR397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice miRNA target in vivo, and may be suitable for functional analysis of miRNAs of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice.
Gene Targeting ; Green Fluorescent Proteins ; genetics ; MicroRNAs ; genetics ; Oryza ; genetics ; Plasmids ; Protoplasts ; metabolism ; RNA, Plant ; genetics ; Transfection

Objective To evaluate the effect of endemic disease prevention in Shaanxi province caused by earthquake and supply a scientific basis for making decision regarding restoration and reconstruction after the earthquake.Methods Forty different drinking water samples were collected respectively from Mianxian county and Chencang district the earthquakes'most severely hit areas in Shaanxi province with drinking-water-borne fluorosis.The contents of fluorine and arsenic in the water was detected by Fluoride ion-selective electrode and hydride generation atomic fluorescence spectrometry,respectively.Two hundreds iodized salt and urine samples were collected from affected household women aged 18-45 in severely hit Ningqiang and Lveyang countie with iodine deficiency disorders.The contents of iodine in the salt was detected by semi-quantitative and of urinary iodine of household women was determined by AsⅢ-Ce4+ catalytic spectrophotometry.Two hundred children aged 7-12 were detected by clinical and X-ray examination from Ningqiang and Lveyang counties of Kashin-Beck disease prevailing areas.Hair and food samples from 20 children were collected.The contents of selenium were determined by 2,3-Diamino naphthalene fluorescence spectrophotometry and of T-2 toxin in the food was detected by ELISA.Results No incidents of serious hydrologieM or geological damage were found.Fluoride content in one of eighty water samples was beyond 1.5 mg/L,accounting for 1.25%;five samples were between 1.0 mg/L and 1.5 mg/L,accounting for 6.25%:the arsenic content of all samples were below 0.01 mg/L;all the salt samples from the home of household aged 18-45 were iodine salt,the median of urine iodine of household wonlen Was 303.03μg/L (21.40-1133.54 μg/L) and 325.21 μg/L(27.61 - 1191.46 μg,/L) in Ningqiang and Lveyang counties, respectively.No clinical and X-ray patients were found in the children aged 7 - 12, the average content of selenium in hair was (0.2638 ± 0.0875)mg/kg; the content of T-2 toxin in the forty food samples was below 100 μg/kg. Conclusions The prevention of the endemic disease in the four counties has been moderately affected by the earthquake, but the situation of endemic disease is stable and there is no evidence of the endemic disease.

Objective:To study the indications and clinical efficacy of video assisted small incision in treatment of infected pancreatic necrosis.Methods:A retrospective study was conducted on 27 patients with infected pancreatic necrosis treated by video assisted small incision at the Department of Pancreatic and Splenic Surgery, Hunan Provincial People's Hospital, from January 2018 to December 2019. The surgical approach, operation time, intraoperative blood loss, postoperative hospital stay and complications were analysed. Postoperative follow-up was carried out at outpatients’ clinic, and the patient's time to full recovery and long-term complications were studied.Results:The 27 patients successfully underwent the operations. There were 22 males and 5 females, aged (50.6±6.2) years. The treatment results were analyzed according to the different surgical approaches: the retroperitoneal approach group ( n=4); the omental sac approach group (n=14); the intercostal space approach group ( n=2); and the combined approach group ( n=7). The operation time was (85.3±31.6)min. The intraoperative blood loss was 65.0(45.2, 121.4)ml. The postoperative hospital stay was 23.0(12.5, 36.1)days. The incidence of complications (Clavien-Dindo grade Ⅲ and above) was 14.8%(4/27). There were 2 patients in this study who were admitted to the intensive care unit due to postoperative hemorrhage: 1 patient responded well to conservative treatment and the remaining patient required interventional treatment. Another patient because of poor results, underwent debridement by laparotomy 2 weeks after the operation. There was 1 patient who developed grade C pancreatic fistula which was cured by surgical treatment 6 months later. On follow-up, 2 patients developed colonic fistula 2 weeks after surgery and 2 patients gastric fistula 1 week and 3 weeks after surgery. These patients responded to conservative treatment. Conclusion:With proper case selection, video assisted small incision could safely and effectively be used to treat infected pancreatic necrosis.

Adult ; Bacteremia ; Hematopoietic Stem Cell Transplantation ; Humans ; Male ; Nontuberculous Mycobacteria ; Postoperative Complications

Objective To study the relationship between urinary albumin excretion rate(UAER)and cardiac structure and function in type 2 diabetes mellitus(T2DM).Methods Five hundred and twenty patients with T2DM were divided into three groups by 8 h UAER:normoalbuminuria group(424 cases),microalbuminuria group(75 cases)and macroalbuminuria group(21 cases).At the same time,50 healthy people were selected as control group.The cardiac structure and function in four groups were evaluated by echocardiographic measurements.Results Left atrial dimensio[(34.39±3.46)mm],left ventricular posterior wall thickness[(10.34±1.05)mm],relative wall thickness(0.46±0.05),interventricular septal thickness[(10.49±1.07)mm],left ventricular mass[(167.37±32.12)g],left ventricular mass index[(87.62±12.16)g/m2]in macroalbuminuria group were significantly higher than those in control group[(32.59±2.71)mm,(9.30±0.77)mm,0.42±0.04,(9.75±0.81)mm,(147.33±27.23)g,(80.14±13.81)g/m2](P＜0.05),mean while the ratio of E/A(0.82±0.19)in macroalbuminuria group was significantly lower than that in control group(1.21±0.16)(P＜0.05).Conclusions In T2DM patients with albuminuria,the cardiac structure and function have changed.With the increase of UAER,the impairments are more prominent.And it may predict diabetes cardiomyopathy.

Objective To study nano-structured porous polycaprolactone combined with bone marrow stromal cells on CTGF expression in rabbit impaired cartilage. Methods 50 rabbits were randomly divided into normal group, model group, NSP-PCL group, BMSCs group, or NSP-PCL + BMSCs group. A model of rabbit impaired cartilage was surgically established. Then NSP-PCL, BMSCs, and NSP-PCL + BMSCs were separately administered to the latter three groups once a week from the 2nd week to the 5th week after the procedure. The impaired cartilage tissues were collected 24 h after the last administration. The cartilage tissues were pathologically examined by H & E staining. Number of surviving BMSCs was detected by Hoeehst33342 Markers 1 week and 3 weeks after transplantation. Levels of CTGF protein in cartilage were determined by immunohistochemistry and Western blotting. Results In the model group, cartilage layer became thinner, with proliferation of fibroblast cells and a obvious cartilage surface hollow; New cartilage cells appeared in the surface hollow of the impaired cartilage in each treatemnt group, with a thicker layer. The number of transplanted BMSCs after 3 weeks was significantly increased in BMSCs group and NSP-PCL + BMSCs group. As compared with the model group, levels of CTGF protein were increased in each treatment group, with significant differences (P < 0.05). Conclusions Nano-porous polycaprolactone combined with bone marrow mesenchymal stem cells can repair cartilage damage by enhancing the expression of CTGF protein.

Objective To explore the mechanism of the low-frequency electrical stimulate on pedunculopontine nu-cleus to treat the Parkinson (PD) through observinge the low-frequency electrical stimulation of Pedunculopontine Nucleus (PPN) in PD rat model and the effects of neurotransmitters (GPi) neurons discharge in the medial part of the globus pallidus. Methods Thirty SD rats were randomly assigned to the control group and the PD model group, with 15 in each group. PD model was established through injecting 6-OHDA into Substantia nigra compact (SNc) of black rat. Effect of low frequency electrical stimulation, micro-electrophoresis glutamate (Glu) and its receptor blocking breaking agent MK-801,γ-aminobu-tyric acid (GABA) and its receptor antagonist bicuculline (BIC) on discharge of rat neuron GPi was examined using extracel-lular unit recording methods through seven glass microelectrode recording. Results When stimulated by low frequency electrical stimulation of PPN, reactions from the control group and neuronal response GPi in PD rats were inhibited. The aver-age discharge frequency was reduced compared to pre-stimulation (P < 0.01). Micro-electrophoresis and BIC Glu excite neurons while microiontophoresis MK-801 and GABA restrain neurons. In the background of micro-electrophoresis BIC’s excitatory effects on neuron, low-frequency electrical stimulation on PPN reduced neuronal firing frequency. And in the background of inhibition effect of micro-electrophoresis MK-801, low-frequency stimulation PPN further restrain neuronal discharge frequency. Conclusion Low frequency electrical stimulation inhibits GPi PPN neuronal activity probably though regulating neurons projecting to the Glu and GABA nerve pathways in GPi neuron.