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.

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: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.

Objective:By establishing a rat model of experimental autoimmune thyroiditis(EAT), to investigate the effects of different iodine intake on the hippocampal morphology, monoamine neurotransmitters and ethology of the offspring of EAT rats.Methods:A total of 60 female and 20 male Lewis rats with a body weight of 50 - 60 g were selected. Female rats were divided into 4 groups (15 rats in each group) with random number table method according to their body weight: control group (NI group), thyroglobulin group (Tg group), Tg + high iodine Ⅰ group (Tg + HⅠ group), and Tg + high iodine Ⅱ group (Tg + HⅡ group), and the latter three groups were model groups. The contents of iodine in drinking water of the 4 groups were 100 μg/L, 100 μg/L, 20 mg/L and 200 mg/L, respectively. Rats in the model groups were immunized with porcine thyroglobulin (PTg) subcutaneously at multiple sites, and the NI group was injected with normal saline, once every 2 weeks, 3 times in total. The rats in each group were mated in cages according to the ratio of 3 : 1 between female and male. After experiment of the offspring, the urine samples of mother rats were collected within the previous week, urinary iodine concentration was determined by As 3+-Ce 4+ catalytic spectrophotometry; then the mother rats were killed, HE staining was used to observe the changes of thyroid histomorphology and the infiltration of inflammatory cells; serum thyroglobulin antibody (TgAb) and thyroid peroxidase antibody (TPOAb) of mother rats were determined by radioimmunoassay. Brain tissues were collected from 7 days old offspring, hippocampal morphology of 7 days old offspring was observed by toluidine blue staining; the contents of norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) in brain tissues of 7 days old offspring were measured by enzyme-linked immunosorbent assay (ELISA); 30 and 60 days old offspring were used for water maze-location navigation test and open field test. Results:The levels of urinary iodine increased significantly of mother rats in Tg + HⅠ and Tg + HⅡ groups than that in NI group (median, μg/L: 35 380.18, 236 847.16 vs 221.43, P < 0.05). HE staining showed that the thyroid tissue of mother rats in Tg, Tg + HⅠ and Tg + HⅡ groups had different degrees of destruction and inflammatory cells infiltration, and the degree of destruction and infiltration increased with the increase of iodine intake. Compared with NI group, the contents of TgAb and TPOAb in serum of mother rats in Tg, Tg + HⅠ and Tg + HⅡ groups were significantly increased(2.118 4 ± 0.675 1, 2.103 0 ± 0.714 1, 2.783 6 ± 1.084 3 vs 0.790 1 ± 0.101 0, P < 0.05; 1.015 8 ± 0.252 8, 1.019 5 ± 0.202 0, 0.936 6 ± 0.183 4 vs 0.692 2 ± 0.111 9, P < 0.05), and the content of TgAb in Tg + HⅡ group was significantly higher than that in Tg and Tg + HⅠ groups ( P < 0.05). Compared with NI group, the number of hippocampal neurons decreased and relative damage occurred in Tg, Tg + HⅠ and Tg + HⅡ groups of the offspring. Compared with NI group, the NE contents in brain tissues of the offspring in Tg, Tg + HⅠ and Tg + HⅡ groups decreased (pg/ml: 1 232.01 ± 253.45, 1 197.64 ± 222.46, 1 074.40 ± 366.38 vs 1 733.67 ± 158.12, P < 0.05); there were no significant differences in DA and 5-HT contents in brain tissues of offspring in each group ( P > 0.05). In the water maze-location navigation test, the latency of the Tg + HⅡ group on the 4th day of the 30 days old offspring reaching the platform was significantly longer than that of the NI and Tg groups ( P < 0.05). In the open field test, there was no significant difference in 30 and 60 days old offspring in the latency of moving the original quadrant ( P > 0.05). Conclusions:With the increase of iodine intake, the degrees of thyroid tissue destruction and inflammatory cells infiltration in EAT rats increase, and the levels of TgAb in serum increase significantly. Iodine has certain effects on the hippocampal morphology and the level of monoamine neurotransmitters in the brains of the offspring of EAT rats. The effects of different iodine-induced EAT rats on their offspring's learning, memory and spatial exploration are mainly shown in childhood.

Dental hard tissues lack the ability to self-heal. In dentin and cementum, hydroxyapatite (HA) can exist outside and/or inside collagen fibers. It is difficult to repair or regenerate HA with a highly ordered orientation in the presence of collagen fibers. At present, the biomimetic mineralization of dentin and cementum, mainly carried out by imitating its biological formation process and its physiological structure, can be divided into those originating from the fiber mineralization mechanism and those with HA as the main component. The materials used include natural materials such as demineralized dentin matrix (DDM) and calcined bovine hydroxyapatite (BHA), and synthetic materials such as polymer-induced liquid precursor (PILP) and synthetic HA. In the future, natural materials and synthetic materials should be combined for the restoration and regeneration of dentin and cementum by means of biomimetic mineralization of calcium phosphate released by remineralization solution-HA.