AIM: To study the application of superfine comminution in Renzhujianwei Granule. METHODS: After being pretreated, Rhizoma Altractylodis macroleplea and coix seed were crushed into ultra-fine powder by TC20 medical superfine crusher. Then the ultra-fine powder was made into the granule. RESULTS: Detected by scanning electron microscope and EDS X-ray detector, the mean diameter of the particle was ≤5 ?m; The detection of the normal distribution was 87.93%≤20 ?m and 95.08%≤20 ?m. CONCLUSION: The application of ultra-fine powder in Renzhujianwei Granule develops a new method of producing granule, that can save the pharmaceatic adjuvants and keep the effective components, and is propitious to the molding of the preparation, and also can improve the stability.

OBJECTIVE:To establish a method for simultaneous determination of gamabufotalin,arenobufagin,telocinobufa-gin,desacetylcinobufotalin,bufotalin,cinobufotalin,bufalin,cinobufagin and resibufogenin in Liushen pills. METHODS:HPLC method was adopted. The determination was performed on ODS-2 C18 column with mobile phase consisted of acetonitrile-0.15%phosphoric acid(gradient elution)at the flow rate of 1.0 mL/min. The detection wavelength was set at 296 nm,and column temper-ature was 40 ℃. The sample size was 10 μL. RESULTS:The linear ranges of gamabufotalin,arenobufagin,telocinobufagin,de-sacetylcinobufotalin, bufotalin, cinobufotalin,bufalin, cinobufagin and resibufogenin were 1.10-70.39 μg/mL(r=0.9996), 4.03-257.78 μg/mL(r=0.9999),4.09-261.89 μg/mL(r=0.9999),0.67-42.96 μg/mL(r=0.9999),3.36-214.73 μg/mL(r=0.9999), 5.73-366.44 μg/mL(r=0.9999),3.77-241.56 μg/mL(r=0.9999),7.31-468.11 μg/mL(r=0.9999),5.18-331.56 μg/mL(r=0.9999). The limits of quantitation were 1.10,0.85,1.02,0.34,0.84,1.43,0.94,3.66,2.59 μg/mL;the limits of detection were 0.27, 0.21,0.51,0.17,0.42,0.72,0.47,0.91,1.30 μg/mL,respectively. RSDs of precision,stability and reproducibility tests were all lower than 3.0%. The recoveries were 96.35%-103.10%(RSD=2.72%,n=6), 96.76%-103.24%(RSD=2.49%,n=6), 97.01%-101.39%(RSD=1.64%,n=6),97.32%-104.01%(RSD=2.61%,n=6),95.76%-103.60%(RSD=2.92%,n=6), 95.07%-102.59%(RSD=2.92%,n=6),95.77%-101.43%(RSD=2.03%,n=6),95.11%-103.72%(RSD=3.19%,n=6), 95.23%-103.34%(RSD=3.24%,n=6),respectively. CONCLUSIONS:The method is simple,rapid,accurate,reliable and can be used for the determination of bufadienolide in Liushen pills .

Objective To investigate the effects of intervertebral bridging ossifications in patients of osteoporotic vertebral compression fracture (OVCF) on bone fracture healing.Methods A total of 170 patients of thoracolumbar vertebral endplate fracture who were admitted into our hospital were selected.Divided these patients into the observation group,namely 60 patients with nonunion of vertebral endplate after 3 months of conservative treatment,and the control group, including 110 patients with well healed vertebra after 3 months of conservative treatment.Compared the distribution of intervertebral bridging ossifications of the two groups 3 weeks after injury.Results The incidence of bridging ossification at levels of T9 to T10,T10to T11,T11to T12 in the observation group were significantly higher than that in the control group.And it showed a significantly higher incidence of bridging ossification at the second proximal intervertebral segment in the observation group than that of the control group.There was a significantly greater sagittal wedge angle in the observation group compared with the control group.Conclusion Conservative treatment may increase the risk of nonunion of osteoporotic vertebral compression fractures when there is a bridging ossification at the second proximal intervertebral level or the sagittal wedge angle was greater than 14.2°in a fresh osteoporotic vertebral compression fracture.It should be a careful choose whether to take conservative treatment or surgical intervention.

OBJECTIVE: To carry out genetic testing for an abortus suspected with Cornelia de Lange syndrome (CdLS). METHODS: History of gestation and the family was taken. Combined with prenatal ultrasonography and the phenotype of the abortus, a diagnosis was made for the proband. Fetal tissue and peripheral blood samples of its parents were collected for the extraction of genomic DNA. Whole exome sequencing was carried out to detect mutations related to the phenotype. Suspected mutations were verified in the parents through Sanger sequencing. RESULTS: Prenatal ultrasound found that the forearms and hands of the fetus were anomalous, in addition with poorly formed vermis cerebellum, slight micrognathia, and increased echo of bilateral renal parenchyma. Examination of the abortus has noted upper limb and facial malformations. Whole exome sequencing revealed that the fetus carried a heterozygous c.2118delG (p.Lys706fs) frameshift mutation of the NIPBL gene. The same mutation was not found in either parent. CONCLUSION The heterozygous c.2118delG (p.Lys706fs) frameshift mutation of the NIPBL gene probably underlies the CdLS in the fetus. Above finding has provided a basis for the genetic counseling for the family.
Cell Cycle Proteins/genetics* ; DNA Mutational Analysis ; De Lange Syndrome/pathology* ; Female ; Fetus ; Humans ; Male ; Mutation ; Phenotype ; Pregnancy ; Whole Exome Sequencing

Objective To explore the changes of cell proliferation and differentiation of neural stem cells induced by fasudil treatment,and to primarily study the mechanism in C17.2 mice.Methods C17.2 cells were cultured in vitro; 5,25,50 and 100 μmol/L fasudil were given to the cells,respectively,for 24 h,and cells in the blank control group were given the same volume of culture medium.The changes of cell morphology were observed under a phase-contrast microscope; cell viability and cell necrosis rate were determined by MTT assay and lactate dehydrogenase (LDH) assay,respectively.Western blotting was applied to detect the expression levels of neural markers (nestin,glial fibrillary acidic protein [GFAP],double cortisol [DCX],microtubule-associated protein-2 [MAP-2]),and Notch Ⅰ and Hes 1 proteins in the notch signaling in cells from the 100 μmol/L fasudil treatment group and blank control group.Immunofluorescence staining was used to detect the nestin and GFAP expressions in the C 17.2 cells.Results As compared with that in the blank control group,the cell viability in the 50 and 100 μmol/L fasudil treatment groups was significantly decreased; that in the 100 μmol/L fasudil treatment group was significantly lower than that in 50 μmol/L fasudil treatment group (P＜0.05); LDH assay showed no significant difference of cell necrosis among the five groups (P＜0.05).Western blotting indicated that 100 μmol/L fasudil treatment group had significantly decreased nestin expression,significantly elevated DCX,MAP-2 and GFAP expressions,and statistically decreased expression levels of Notch 1 and Hes 1 as compared with blank control group (P＜0.05).Immunofluorescence staining indicated that the percentage of nestin positive cells was markedly decreased,the percentage of GFAP positive cells was significantly increased in the 100 μmol/L fasudil treatment group as compared with those in the blank control group (P＜0.05).Conclusion Fasudil treatment could inhibit the proliferation of C17.2 cells and promote them differentiate into neuronal and glial cells via decreasing the expression level of Notch signaling.

Objectives:To discuss the inhibitory effect of Lactobacillus reuteri on the replication of rotavirus(RV)strain SA11 in vivo and in vitro,and to clarify its effect on the expression of related immune factors.Methods:For in vitro experiments,Lactobacillus reuteri was cultured and identified,and the standard curve and growth curve were plotted to screen the optimal time and concentration for Lactobacillus reuteri cultivation.The cells were infected with Lactobacillus reuteri at the concentrations of 5×108,10×108,50×108,100×108,200×108,and 500×108 CFU·mL-1,and the surival rates of Caco-2 cells were detected by trypan blue staining method.Various concentrations of Lactobacillus reuteri were co-incubated with RV in vitro and applied to the Caco-2 cells.The cells were divided into negative control group(NC group),positive control group(PC group),and 107,108,109,and 1010 CFU·mL-1 Lactobacillus reuteri groups.Immunofluorescence focus method was used to detect the viral titers in the Caco-2 cells after treated with Lactobacillus reuteri and real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the copy numbers of RV VP6 gene in the Caco-2 cells after treated with various concentrations of Lactobacillus reuteri.In in vivo experiments,25 litters of SPF suckling mice were divided into control group,RV group(infected with SA11 strain),Ab-NC group(treated with antibiotic to deplete gut microbiota),Ab-RV group(depleting gut microbiota and then infected with SA11 strain),and Ab-Lac-RV group(depleting gut microbiota,treated with Lactobacillus reuteri,and then infected with SA11 strain).The fecal samples were collected on days 2,4,6,8,and 10 gavage,colon tissue sample were collected on day 4 of and RT-qPCR method was used to detect the copy numbers of RV VP6 gene in feces and the mRNA expression levels of interleukin(IL)-1β,IL-8,IL-10,interferon-γ(IFN-γ),and tumor necrosis factor-α(TNF-α)in colon tissue of the suckling mice in vartious groups.Results:The Lactobacillus reuteri grew well,with round,smooth,and milky white convex colonies and neat edges.After Gram staining,the bacteria appeared purple,irregular,and square-shaped rods.16SrDNA sequencing showed 99%sequence homology,indicating successful activation of Lactobacillus reuteri.The number of live Lactobacillus reuteri was linearly related to the absorbance(A)value,and the standard curve for regression analysis was Y=0.437 5X+0.000 6,R2=0.999 4.During the 0-2 h cultivation period,the bacteria were at the logarithmic growth phase with slow growth;from 2-14 h,the bacteria grew rapidly and stabilized at 14-16 h,reaching the growth rate peak at 16 h,after which they entered the decline phase.Infection with Lactobacillus reuteri at concentrations of 5×108,10×108,50×108,100×108,and 200×108 CFU·mL-1 resulted in the survival rates of Caco-2 cells were all>90%,so these concentrations were selected for the further experiments.Compared with PC group,the copy numbers of RV VP6 gene in the Caco-2 cells in 5×108,10×108,50×108,100×108,and 200×108 CFU·mL-1 Lactobacillus reuteri groups were significantly decreased(P<0.01).Compared with PC group,the viral titers in the Caco-2 cells in 107,108,109,and 1010 CFU·mL-1 Lactobacillus reuteri groups were significantly decreased(P<0.01).Compared with control group,the numbers of gut microbiota colonies in Ab-NC,Ab-RV,and Ab-Lac-RV groups were significantly decreased,indicating successful depletion of gut microbiota in the suckling mice.On days 2 and 4 after gavage,the RV VP6 gene copy number in the feces in Ab-RV group was significantly lower than that in RV group(P<0.05).On days 4,6,8,and 10 after gavage,the RV VP6 gene copy number in the feces in Ab-Lac-RV group was significantly lower than that in Ab-RV group(P<0.05).Compared with control group,the expression levels of IL-1β,IL-10,IFN-γ,and TNF-α mRNA in colon tissue in Ab-RV and Ab-Lac-RV groups were significantly increased(P<0.05 or P<0.01),while the expression level of IL-8 mRNA was significantly decreased(P<0.05),and the expression level of IL-10 mRNA in colon tissue in Ab-LAC-RV group was significantly increased(P<0.01).Conclusion:Lactobacillus reuteri may inhibit the RV replication by upregulating the expressions of IL-1β,IL-10,IFN-γ,and TNF-α mRNA and downregulating the expression of IL-8 mRNA.

OBJECTIVE: To diagnose a fetus with Papillorenal syndrome by prenatal ultrasonography and genetic testing, and to correlate its genotype with phenotype. METHODS: Ultrasound finding of the fetus was reviewed. Muscle sample of the abortus was taken, and genetic variant related to the clinical phenotype was screened by whole exome sequencing (WES). Suspected pathogenic variant was verified by Sanger sequencing. RESULTS: Prenatal ultrasound revealed severe dysplasia of the fetal kidneys and oligohydramnios. WES revealed that the fetus has carried a c.736G>T (p.Glu246Ter) nonsense variant of the PAX2 gene, which was unreported previously. The result of Sanger sequencing was consistent with that of WES. Both parents of the fetus were of the wild-type, suggesting a de novo origin of the fetal variant. CONCLUSION The novel heterozygous c.736G>T (p.Glu246Ter) variant of the PAX2 gene probably underlay the Papillorenal syndrome in the fetus. Above finding has provided a basis for genetic counseling and clinical decision-making.

OBJECTIVE: To explore the genetic basis for fetus with short limbs detected by prenatal ultrasonography. METHODS: Results of clinical imaging of the fetus was collected. Amniotic fluid sample was collected through amniocentesis for the extraction of fetal DNA. Whole exome sequencing was carried out to detect variants related to the clinical phenotypes. Candidate variant was verified by Sanger sequencing. RESULTS: Prenatal ultrasound showed that the fetus had short limbs but no other abnormality. Whole exome sequencing has identified that the fetus carried two heterozygous pathogenic variants c.484G>T and c.1436dupA of the SLC26A2 gene, for which its mother and father were heterozygous carriers, respectively. CONCLUSION The fetus was diagnosed with atelosteogenesis type 2 by combined prenatal ultrasonography and whole exome sequencing, which may be attributed to the compound heterozygous variants of the SLC26A2 gene. Above findings provided evidence for the diagnosis of the fetus and genetic counseling.

Objective:To explore the application effect of five-in-one anti-cancer support model in patients with breast neoplasms.Methods:A total of 70 patients who were admitted to the Fourth Ward of Department of Breast Surgery in Harbin Medical University Cancer Hospital from May to September 2019 were selected as research objects by using the convenient sampling method. According to the random number table method, they were randomly divided into the study group and the control group, with 35 cases in each group.The control group was given routine nursing, while the study group was given five-in-one anti-cancer support model nursing. The depression, anxiety, social isolation, social support and quality of life scores of patients of the two groups were evaluated before and after the intervention.Results:After intervention, the depression scores of patients in the study group were lower than those in the control group. The total score of quality of life, score of social and family status and score of functional status in the study group were lower than those in the control group. The differences were statistically significant ( P<0.05) . Conclusions:The five-in-one anti-cancer support model is adopted to design body, mind, spirit, and social relaxation courses that meet the characteristics of breast cancer patients to meet their psychological and social needs, which has certain clinical significance.

Objective:Experimental autoimmune thyroiditis (EAT) rat model was establish to observe the effects of iodine excess on thyroid function, antibody and thyrotropin receptor (TSHR) gene expression in EAT rats, and to explore the role of TSHR gene in autoimmune thyroiditis.Methods:According to body weight (80 - 180 g), 48 rats (4-week-old female Lewis) were randomly divided into control group, thyroglobulin (TG) group, TG + high iodine Ⅰ(TG + HⅠ) group, and TG + high iodine Ⅱ (TG + HⅡ) group, 12 rats per group. The iodine concentration in drinking water given to each group was 50 μg/L, 50 μg/L, 20 mg/L and 200 mg/L, respectively. At the same time, rats in TG, TG + HⅠ and TG + HⅡ groups were immunized once every two weeks for three times using pTg and CFA as immunoreagent. Paraffin embedded sections of thyroid tissues were used to observe the pathological changes of rats. The serum levels of thyroglobulin antibody (TgAb), thyroperoxidase autoantibody (TPOAb), free triiodothyronine (FT 3) and free thyroxine (FT 4) in rats were determined by radioimmunoassay. Serum TSHR content in rats was determined by enzyme linked immunosorbent assay (ELISA). The expression of TSHR mRNA in whole blood and thyroid tissue of rats was determined by RT-PCR. The expression of TSHR protein in thyroid tissue of rats was determined by immunohistochemistry (IHC). Results:Hematoxylin-eosin (HE) showed that the thyroid follicles in control group were complete in structure and regular in shape, and no lymphocyte infiltration was observed. A small number of lymphocytes were observed in TG group and scattered in distribution. Follicular structure destruction, fusion and interfollicular infiltration were observed in TG + HⅠ group and TG + HⅡ group. There were significant differences in serum TgAb, TPOAb, FT 3 and FT 4 levels among all groups ( H = 30.28, 21.99, 12.87, 26.69, P < 0.05). Compared to the control group [6.89 (6.32, 7.27), 11.02 (7.60, 12.53), 5.05 (2.71, 7.99), 7.51 (6.50, 9.24) pmol/L], the levels of TgAb [34.99 (25.39, 41.35), 37.70 (29.06, 43.99), 46.41 (38.52, 55.26)], TPOAb [22.87 (13.65, 31.82), 22.22 (14.82, 28.33), 14.61 (12.95, 19.34)], FT 3 [57.74 (24.56, 64.27), 43.64 (5.69, 80.03), 38.56 (17.73, 47.59) pmol/L], and FT 4 [62.16 (41.22, 91.57), 60.61 (35.52, 103.31), 47.96 (31.84, 112.71) pmol/L] were significantly higher in TG group, TG + HⅠ group, and TG + HⅡ group ( P < 0.05). Compared with the control group [(249.37 ± 38.12) μU/L], TG group [(225.33 ± 41.28) μU/L], and TG + HⅠ group [(218.15 ± 65.51) μU/L], TSHR expression level in TG + HⅡ group [(154.26 ± 25.95) μU/L] were significantly decreased ( P < 0.05). The mRNA expression levels of TSHR gene in the whole blood (0.89 ± 0.19, 0.89 ± 0.30, 0.85 ± 0.24) and thyroid tissue(0.63 ± 0.25, 0.46 ± 0.16, 0.51 ± 0.25) of TG group, TG + HⅠ group and TG + HⅡ group were significantly lower than that of control group (1.00 ± 0.05, 1.13 ± 0.21, P < 0.05). IHC showed that the positive intensity of TSHR protein in control group was significantly higher than that in TG group, TG + HⅠ group and TG + HⅡ group. Conclusions:Long-term exposure to high iodine will eventually lead to the damage of iodine-uptake function in thyroid gland and thyroid diseases. Abnormal expression of TSHR gene may lead to antigenicity of thyrotropin binding site in extracellular receptor region and autoimmune thyroid disease.