Based on the three delivery forms of CRISPR/Cas9 system at the levels of DNA, RNA and protein, this paper mainly approaches the development and new strategies of CRISPR/Cas9 delivery systems, as well as their application in the biomedical field and the clinical treatment of gene-related diseases. By summarizing and elaborating the CRISPR/Cas9 system delivery and gene therapy strategy, new ideas are provided for the discovery of innovative drugs and the development of gene therapy.

Background: In suckling piglets, transmissible gastroenteritis virus (TGEV) causes lethal diarrhea accompanied by high infection and mortality rates, leading to considerable economic losses. This study explored methods of preventing or inhibiting their production.Bovine antimicrobial peptide-13 (APB-13) has antibacterial, antiviral, and immune functions. Objectives: This study analyzed the efficacy of APB-13 against TGEV through in vivo and in vitro experiments. Methods: The effects of APB-13 toxicity and virus inhibition rate on swine testicular (ST) cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT). The impact of APB-13 on virus replication was examined through the 50% tissue culture infective dose (TCID50 ). The mRNA and protein levels were investigated by real-time quantitative polymerase chain reaction and western blot (WB). Tissue sections were used to detect intestinal morphological development. Results: The safe and effective concentration range of APB-13 on ST cells ranged from 0 to 62.5 µg/mL, and the highest viral inhibitory rate of APB-13 was 74.1%. The log10 TCID50 of 62.5 µg/mL APB-13 was 3.63 lower than that of the virus control. The mRNA and protein expression at 62.5 µg/mL APB-13 was significantly lower than that of the virus control at 24 hpi. Piglets in the APB-13 group showed significantly lower viral shedding than that in the virus control group, and the pathological tissue sections of the jejunum morphology revealed significant differences between the groups. Conclusions APB-13 exhibited good antiviral effects on TGEV invivo and in vitro.

Objective To evaluate the nutrional status and malnutrition risks of hospitalized children with acute lymphoblastic leukemia (ALL),provide nutrition support for subsequent treatment,lower nutritional risks and improve therapeutic effects;to complete the statistical analysis of height and weight across age through data normalization.Methods A total of 592 children diagnosed ALL from August 2014 to September 2016 at Beijing Children's Hospital were enrolled,and the directly measured height and weight which reflected the nutritional status of children were collected and normalized.Nutritional risk screening was completed by STAMP screening tool and the nutritional status was evaluated by three Z scores HAZ＜-2,WAZ＜-2 and WHZ＜-2.The effects of nutrition intervention were investigated by weight,hemoglobin and albumin changes between before and after L-asp treatment.Results The Z score test showed that there was no significant difference between the nutritional status of pretreatment ALL patients (0.34％ growth retardation,2.36％ lower body weight,0.17％ emaciation) and normal healthy children (3％ malnutrition);after comparing the STAMP nutritional risk assessment and t test results for children with ALL,it was found that the proportion of children with high malnutrition risk (score≥4) was 9.71％,which had a strong correlation with body mass index;the children with body mass index less than 0.80 (50％ children having malnutrition) or greater than 1.30 (20％ children having malnutrition) had a higher risk of malnutrition,and the body mass indexof the high-risk group (0.852 kg/m2) were significantly lower than that of the low risk group (1.051 kg/m2,score ≤ 3);there were significant differences in height and weight between children with STAMP ≤ 3 and STAMP ≥ 4 (all P ＜0.05) Nutritional interventions made no significant weight change during treatment;For children with STAMP ≤3 and STAMP ≥4 in L-asp treatment,the hemoglobin value changed from (87.46± 19.27) g/L to (95.12±13.51) g/L and (101.55±21.97) g/L to (95.05±11.22) g/L respectively (all P=0.001);The albumin of children with STAMP≤3 changed from (40.63±4.149) g/L to (41.20±5.266) g/L in treatment and that of children with STAMP ≥4 changed from (40.96±8.429) g/L to (42.17±3.574) g/L in treatment (P=0.20,0.05).Conclusion There are no obvious indications of malnutrition in children with ALL,but nearly 10 ％ of them have malnutrition risks.Special dietary guidance is needed during the treatment.Nutrition intervention is effective in the treatment of L-asp.Normalized data is more convenient to use in statistical analysis with more accurate results,which can be used as a supplementary method for cross-age group statistical analysis of children's height,weight and other parameters.

Objective: To study the distribution of natural killer (NK) cells and the CD38 positive subpopulations in different tissues in rheumatoid arthritis (RA) patients and the mechanism of CD38 agonist. Methods: The levels of NK cells, CD38 positive subpopulation and NK cell surface chemokine receptors (CXCR3, CCR5), as well as granzyme B and perforin were detected by flow cytometry in peripheral blood and knee synovial fluid. The isolated cells were then cultured in vitro and divided into 3 groups, namely blank control group, IB4 stimulation group (Anti-CD38 mAb), IL-2 and IB4 co-stimulation group. And the concentration of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in culture supernatants was determined by enzyme linked immunosorbent assay (ELISA). Liposome CD38-siRNA was transfected into peripheral blood lymphocytes of RA patients, and transfection efficiency was detected by reverse transcription-polymerase chain reaction (RT-PCR). The effect of CD38 and CD38-siRNA silencing on the expression of phospholipase C-gamma 1 (PLC-γ1) protein in the peripheral blood lymphocytes of RA was detected by Western blot. Results: The expression of CCR5 and CXCR3 in NK cells in peripercal NK cells of RA was significantly higher than that in normal healthy subjects (P<0.05). The two chemokine receptors (CXCR3, CCR5) were mainly expressed in CD38⁺ NK cell subsets. The levels of granzyme B and perforin in synovial lymphocytes cells were significantly higher than those in peripheral blood lymphocytes (P<0.05). The secretion of IL-6 and TNF-α in synovial and peripheral blood lymphocytes cells existed significant difference only in IL-2 and IB4 co-stimulation group (P<0.05). The levels of PLC-γ1 protein in the peripheral blood lymphocytes of RA patients was significantly decreased than that in the blank control group after the treatment with CD38-siRNA (P<0.05). Conclusions The synovial NK cells are more lethal than the peripheral NK cells in the RA patients. CD38 might mediate the phosphorylation of intracellular proteins via the Protain kinase C (PKC) pathway. Blocking CD38 molecules can reduce the phosphorylation level of intracellular proteins. CD38 maybe involved in the mechanism of chemotaxis and killing capability of NK cells.

Objective To explore the value of the plasma miR-193a-5p level on diagnosis and monitoring the response to treatment in acute myeloid leukemia (AML). Methods Peripheral blood samples were obtained from AML patients enrolled in hematology department of the Second Hospital of Shanxi Medical University from July 2015 to December 2015, including 30 de novo AML patients, 9 patients in complete remission (CR) and 6 patients in relapse. Peripheral blood samples from 15 healthy people were randomly choosed as the health control group. Plasma miR-193a-5p expression levels were detected by using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Results The plasma miR-193a-5p relative expression level of AML patients group [0.465 6 (0.103 1-5.000 2)] was obviously lower than that of health control group [0.766 1 (0.052 1-3.134 4)] (U= 122, P= 0.018 7). The plasma miR-193a-5p relative expression levels of de novo group and relapse AML group were significantly lower than those of CR group and health control group (P<0.05), and there was no significant difference between the CR group and health control group (U= 56, P= 0.511 9). No significant correlation was found between the plasma miR-193a-5p level and age, gender, blast percentage of the bone marrow, peripheral blood leukocyte count, platelet count, CD34+cells'percentage and so on. Conclusion The decreased plasma miR-193a-5p expression level can be served as a new and noninvasive biomarker for the evaluation of diagnosis and treatment in AML.

OBJECTIVETo identify the MPL L391-V392ins12 spliceosome and analyze its frequencies in patients with myeloproliferative neoplasms (MPN).

METHODSMPL aberrant spliceosome was identified through reverse transcription polymerase chain reaction (RT-PCR)combined with cloning sequencing. The mutation of this spliceosome in 248 MPN patients and 200 normal people was determined by allele-specific polymerase chain reaction (AS-PCR).

RESULTSA novel aberrant spliceosome of MPL gene (MPL L391-V392ins12)was identified, i.e. 36 bp intron was retained between exon7 and exon8, and there were 12 amino acids (EGLKLLPADIPV)inserted. MPL L391-V392ins12 mutation was detected in 19 (7.66%)of the 248 patients with MPN, including 1 (1.92%) of 52 patients with PV, 14 (9.66%) of 145 with ET, and 4 (7.84%) of 51 with PMF. And the mutation was not detected in the group of 200 normal people.

CONCLUSIONMPL L391-V392ins12 spliceosome is an aberrant spliceosome present in the MPN. It can be detected in PV, ET and PMF, and more frequently in ET and PMF. This mutation may play an important role in the process of MPN.

Humans ; Mutation ; Myeloproliferative Disorders ; genetics ; Neoplasms ; genetics ; Polymerase Chain Reaction ; Receptors, Thrombopoietin ; genetics ; Spliceosomes

Objective To study the relationship among iron metabolism indicators in the patients with hepatitis B and to analyze their clinical significance.Methods Serum ferritin,iron and hepcidin were detected in 96 cases of hepatitis B and 58 negative control subjects.The above indicators detected results and their relationship were analyzed.Results Compared with the control group,ser-um ferritin and serum iron concentration in the hepatitis B group were increased,he difference was statistically significant (P <0.05);serum ferritin and serum iron concentration in the hepatitis B group demonstrated the positive correlation (P <0.05);serum ferritin in the patients with hepatitis B was negatively correlated with hepcidin (P =0.018).Conclusion The patient with hepatitis B often have the iron metabolism abnormality,and the iron metabolism indicators is expected to be an assessment indicator of treat-ment and prognosis in the patients with hepatitis B.

Objective To explore the effect of a novel colloidal gold immunochromatographic test strip for detection of group B streptococci (GBS).Methods A total of 202 cases of swab of vagina or neck of uterus were collected,and they were detec-ted by novel strip and control strip to evaluate their clinical applications.Results Sensitivity of novel strip was about 105 CFU/ml and the detection time was about 5 to 8 minutes,and it showed better sensitivity and shorter detection time com-pared with control strip.In the 202 cases of clinical samples,the detection results of 197 cases were in consistent with the control strip,however,the detection results of 5 cases were not in consistent.The positive coincidence rate and negative coin-cidence rate were 97.5% and 97.54% respectively,and the total coincidence rate and Kappa value were 97.52% and 0.948 respectively.The consistency test showed no significant difference between this strip and control strip.Conclusion This method was a effective technology for diagnosing of infection caused by GBS,and had high value in clinical application.

Objective To investigate the expression of nuclear factor erythroid-2 related factor 2 (Nrf2) gene in chronic myeloid leukemia (CML) and explore its relationship with clinical characteristics and thioredoxin reductase (TrxR).Methods The expressions of Nrf2 and TrxR genes in bone marrow cells and K562 cells were analyzed in 30 bone marrow preparations of CML patients in different phases,including 20 in chronic phase,3 in accelerated phase,7 in blastic phase by real-time quantitative polymerase chain reaction.Ten health subjects were served as normal controls.Results The relative quantitation expression of Nrf2 and TrxR mRNA were 5.601±1.069 and 9.017±2.398 in chronic phase,1.698±0.349 and 5.590±1.015 in accelerated phase,1.252±0.807 and 5.050±1.469 in blastic phase,1.377± 0.344 and 1.867±0.977 in normal controls.The expressions of both Nrf2 and TrxR mRNA in CML had significant differences from those of the normal controls (x2 =14.680,P =0.002,x2 =8.271,P =0.041).The expression of Nrf2 mRNA in accelerated phase,blastic phase group showed no significant difference (Z =0.011,P =0.496),but lower than that in chronic phase group (Z =2.155,P =0.016,Z =2.534,P =0.006).The difference between the first visit and post-treated group was significant (Z =2.015,P =0.022).The expression in K562 cells and normal controls had significant difference (Z =1.898,P =0.029).In CML patients,the expression of Nrf2 was positively correlated with that of TrxR (r =0.738,P =0.037).Conclusion The expression of Nrf2 gene is higher in the first visit group of CML than that in the other groups,and is decreased after therapy,which may be the molecular marker predicting the progress of CML.Nrf2 mRNA expression level is correlated with TrxR.

Objective To investigate epidermal growth factor receptor(EGFR)expression and tumor residual to the prognostic value in patients with nasopharyngeal cancer(NPC).Methods 200 patients with NPC were examined for EGFR expression by immunohistochemistry analysis,neck cancer and nasopharyngeal residual.Results In 200 cases with NPC,expression of EGFR of positive and negative were 160 cases(80.0%) and 40 cases (20.0%);the rate of overall survival(OS),disease-free survival (DFS),loeoregional relapse-free survival (LRFS) and distant metastasis-free survival(DMFS)in patients with positive EGFR were 72.3%,63.6%,72.2%,63.8% and negative EGFR were 90.0%,90.0%,90.0%,90.0%,respectively in 3-year(X2=3.95,X2=4.12,X2=3.98,X2=4.15,P＜0.05),the rate of local recurrence,distant metastasis rate in residual were 26.6%,32.5% which is significantly higher than 8.1%,18.1% in without residual(X2=4.75,X2=4.94,P＜0.05);the hish expression of EGFR with DFS,OS were significantly correlated(r=6.457,P＜0.05).Conclusion The overexpress of EGFR had the tendency of poor prognosis.tumor residual after radiotherpy can be a prognostic indicator for patients with NPC.