Objective To investigate the effect of optimized conditions on proliferation and differentiation of the neural stem cells(NSCs) from different age brain of rat in vitro. Methods The brain tissues from embryonic(E12～E15d),newborn(1～2?d) hippocampus and olfactory bulb from adult of rat were isolated.After proliferation in conditional culture medium including growth factor and induced differentiation,the cell type specific antibodies were used for the immune fluorescence staining to identify the cells. Results A lager scale NSCs were obtained from brain tissue and could proliferate in conditional culture in vitro. They could self renew and generate both nerve and glia.Conclusion\ The NSCs got from rat brain can be used for basic and clinical research.\;[

Background and purpose: Thyroid carcinoma cells spread mainly through lymph node metastasis, and lymphangiogenesis plays an important role during the lymph node metastasis, but it is not very clear to understand the formation mechanism. This study was to investigate the correlative expressions of HPSE, VEGF-C, D2-40 and lymphangiogenesis in thyroid carcinoma. Methods: Immunohistochemistry SP method was used to detect the expressions of HPSE, VEGF-C and D2-40 in 77 patients with thyroid carcinoma including papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC),medullary thyroid carcinoma (MTC), 32 of them with lymph node metastasis was enrolled into the study, D2-40 stained the lymphatic vessels, and lymphatic vessel density (LVD) was scanned under the light-microscope, and the correlation among the above indexes in different thyroid carcinoma types were analyzed respectively. Results: The expressions of HPSE, VEGF-C and D2-40 were observed to have a different degree in thyroid carcinoma, and the highest expression of the protein could be seen in the patients with papillary carcinoma (P<0.05),The expression ratios of HPSE,VEGF-C and D2-40 in different carcinoma types were 54.9%, 68.6%, 12.8±5.7 for PTC, 37.5%, 50%, 8.6±1.7 for FTC, 20% and 20%, 4.9±0.8 for MTC, respectively. There were significant different expressions of HPSE, VEGF-C and D2-40 between the patients with lymph node-positive group and node-negative group (P=0.014, P=0.048, respectively). In addition, the expressions of them were positively correlated (P<0.001, r=0.616). Conclusion: HPSE, VEGF-C and D2-40 have a close relationship with lymph node metastasis, HPSE and VEGF-C are related to the lymphangiogenesis.

ObjectiveTo investigate the relationships between traditional Chinese medicine(TCM) syndrome differentiation and serum cystatin C(Cys-C) and homocysteine(Hcy) in patients with chronic heart failure(CHF). Methods 115 cases with CHF admitted into the Department of Cardiology of the First Affiliated Hospital of Zhejiang Chinese Medical University were selected in the CHF group, and 30 cases who had taken health examination in the same period were chosen in the healthy control group. According to the TCM syndrome differentiation, the CHF cases were subdivided into four groups with different types of syndrome: 30 cases of deficiency of both Qi and Yin syndrome, 30 cases of Qi deficiency syndrome and blood stagnation syndrome, 30 cases of heart and kidney Yang deficiency syndrome and 25 casesof flooding due to Yang deficiency syndrome. The serum levels of Cys-C and Hcy in different groups were tested, and the relationships between TCM syndrome differentiation and serum Cys-C and Hcy were analyzed by using Spearman rank correlation analysis.Results The serum levels of Cys-C and Hcy in the patients with CHF were significantly higher than those in the healthy control group〔Cys-C(mg/L):1.24±0.34 vs. 0.77±0.22, Hcy(μmol/L):18.66±4.57 vs. 11.65±3.21,bothP<0.05〕. Compared with the healthy control group, the serum levels of Cys-C and Hcy in the above four groups of different syndromes had a tendency of gradual elevation in the sequence as follows: deficiency of both Qi and Yin, Qi deficiency and blood stagnation, heart and kidney Yang deficiency and flooding due to Yang deficiencygroups〔Cys-C(mg/L):1.02±0.27,1.09±0.31,1.32±0.22, 1.59±0.25; Hcy(μmol/L): 14.94±2.20, 17.66±3.04, 19.79±3.48, 22.96±5.31〕, and the elevation in levels of flooding due to Yang deficiency group was the most prominent compared with that in other groups(P<0.05). The correlation analyses showed that different types of TCM syndrome in patients with CHF were positively correlated with the levels of Cys-C and Hcy(r1=0.73,r2=0.79,bothP<0.05).ConclusionThe changes of serum Cys-C and Hcy levels are consistent with the evolution of regular pattern of TCM syndrome differentiation in patients with CHF, and these two markers can be regarded as the objective indicators of TCM syndrome differentiation of CHF.

AIM: To establish a method for simultaneously determining chlorogenic acid,puerarin and paeoniflorin in Kangganning Mixture(Flos Lonicerae japonicae,Radix Puerariae Lobatae,Radix Paeoniale rubra,Fructus Forsythiae,etc.). METHODS: A multiple wavelength HPLC method was devoloped.The analysis was performed on an Agilent Zorbax SB C_(18) column(250 mm?4.6 mm,5 ?m) with acetonitrile-0.1%H_3PO_4(11∶89) as the mobile phase.The detection wavelengthes were monitored at 324 nm,254 nm and 230 nm for chlorogenic acid,puerarin and paeoniflorin,respectively.The flow rate was 1.0 mL/min and the column temperature was at 30 ℃.(RESULTS:) The calibration curves of chlorogenic acid,puerarin and paeoniflorin showed good linearity at the ranges of 0.179-2.864 ?g,0.071 55-1.144 8 ?g and 0.372 5-5.96 ?g,respectively.The average recoveries were(100.7%,) 103.3%,102.6% with RSD of 2.2%,2.4%,1.9%,respectively. CONCLUSION: The method is simple,accurate and reproducible for determining chlorogenic acid,puerarin and paeoniflorin in Kangganning Mixture.

ObjectiveTo explore the role of the lymphocyte subsets in the peripheral blood in diagnosis, treatment and prognosis of hemophagocytic lymphohistiocytosis (HLH) in children.MethodA total of 30 children with HLH were enrolled in this study and treated according to the HLH-2004 diagnostic guidelines. 20 children with HLH entered complete remission (CR) and 10 children with HLH died. Thirty age-matched healthy children were selected as normal controls. T cell subsets in the pe-ripheral blood were measured by lfow cytometry.ResultsCompared with control group, CD3+T and CD8+T cells were signiif-cantly increased, CD4+T and CD3-CDl6+CD56+ NK cells were signiifcantly decreased, and CD4+/CD8+ cell ratio was signiifcantly decreased in 20 CR children and 10 died children with HLH in acute phase (P<0.05). CD19+B cells was not statistically different in 20 CR children and 10 died children with HLH in acute phase from control group (P>0.05). In acute phase, the lymphocyte subsets were not statistically different between 20 CR children and 10 died children (P>0.05). In 20 CR children, the proportion of CD3-CD16+CD56+NK in CR phase was statistically different than that in acute phase (P<0.05).ConclusionsChildren with HLH have obvious changes in peripheral blood lymphocyte subsets and have cellular immunity disorders. Dynamic detection of the changes may help determine the therapeutic effect and prognosis of HLH.

Objective To determine the cellular localization and expression pattern of AR and FSHR in adult rat testis must be helpful to understand the action site and mechanisms that T and FSH regulate spermatogenesis. Methods We applied in situ Hybridization to detect the expression of AR and FSHR on adult testis, in which Dig labeled cRNA probe was used to carry out the experiment on frozen sections; at the same time, following the technique of transillumination assisted microdissection we separated seminiferous epithelium into four stages(Ⅱ Ⅵ,Ⅶ Ⅷ,Ⅸ Ⅻ and ⅩⅢ Ⅰ), extracted total RNA and carried out dot hybridization, using ? 32 P labeled cDNA probe, in order to test qualitatively and quantitatively the location of AR and FSHR mRNA and their expression pattern in adult rat testis. Results Our results showed that the positive signal of AR mRNA was located in Sertoli cells and Leydig cells. The signal in Sertoli cells began to appear in Ⅱ Ⅵ stages, strongest in Ⅶ Ⅷ stages and weakest in Ⅸ Ⅰ stages ( P

Objective To study the gene mutation of fibroblast growth factor receptor 3 (FGFR3) and p53 in bladder cancer tissue and to explore their relationship with tumor recurrence. Methods DHPLC and PCR direct sequence were used to detect the mutation of FGFR3 and p53 in BTCC (n=98) and normal bladder mucosa (n=10). Genomic DNA of 98 BTCC was extracted. The exon 5-8 of P53 and the exon 7, 10, 15 were amplification by PCR. The products of PCR was screened by DHPLC to detect the mutation of the production. The results of the FGFR3 and p53 mutation were analyzed by Kaplan-Meier method and no recurrence survival rate was tested by log rank test. All the analysis were aim to explore the clinical biological value of the mutation of FGFR3 and p53. Results Mutation of FGFR3 in BTCC (44. 9%) was higher than normal bladder mucosa(0, P<0.01). Mutation in T_a-T_1 was 75. 6%(33/45) ,T_2 -T_4 was 26. 6%C10/53). Mutation in G_1 was84. 6%(11/13),inG_2 was 61. 4% (27/44), in G_3 was 14. 6% (6/41), (P<0. 05). The mutation rate was lower with the higher of stage and grade. Mutation of p53 in BTCC (34. 6%) was higher than normal bladder mucosa (0%) (P<0. 01). Mutation in T_a - T_1 was 20. 0% (9/45), T_2 - T_4 was 47. 2%(25/53). Mutation in G_1 was G_1 7. 7%(1/13), in G_2 18. 2%(8/44),in G_3 58. 1%(25/41) , (P<0. 05). The mutation rate was higher in the higher stage and grade. Kaplan-Meier method results revealed that mutation of FGFR3 indicating a favorable prognosis while mutation of p53 indicating a poor prognosis. As to the analysis of genotype, the type of FGFR3mut/p53wt had a relative longer recurrent interval (P<0. 01). Conclusions Mutation of FGFR3 indicated a relative longer recurrent interval, which revealed a favorable prognosis of BTCC. Mutation of p53 indicated a relative shorter recurrent interval, which revealed a poor prognosis.

Objective:To evaluate the clinical efficacy of Buyang-Huanwu Decoction combined with modified Tingli-Dazao-Xiefei Decoction on the basis of conventional western medicine therapy in the treatment of chronic heart failure (CHF) with syndrome of qi deficiency and blood stasis. Methods:Seventy patients who met the inclusion criteria from November 2017 to November 2019 in Shijingshan District Hospital of Traditional Chinese Medicine were randomly divided into two groups, 35 in each group. The control group was treated with western medicine for chronic heart failure, and the treatment group was treated with Buyang-Huanwu Decoction combined with Tingli-Dazao-Xiefei Decoction on the basis of the control group. Both groups were treated for 2 weeks. The TCM syndrome scores were observed and compared before and after treatment. Minnesota Living with Heart Failure Questionnaire (MLHFQ) was used to evaluate the quality of life of patients. Lee’s Heart Failure Score was used to evaluate the severity of symptoms. The N-terminal pro-B type natriuretic peptide(NT-proBNP) was detected by ELISA. The adverse reactions during treatment were observed and the clinical efficacy was evaluated. Results:The total effective rate was 91.4% (32/35) in the treatment group and 77.1 % (27/35) in the control group, and the difference was statistically significant ( χ2=7.050, P=0.014). After treatment, the serum NT-proBNP in the treatment group [(1 725.3 ± 1 473.8) ng/L vs. (2 485.7 ± 2 164.4) ng/L; U=341.200, P=0.031] was significantly lower than that of the control group. The NT-proBNP [(54.3 ± 26.7) % vs. (35.5 ± 19.8)%; U=4.310, P=0.003] was significantly higher than that of the control group. After treatment, the TCM syndrome scores and MLHFQ scores in the treatment group were significantly lower than those in the control group ( t=3.785, 9.925, P=0.031, 0.001). During the treatment, no obvious adverse reactions were observed in both groups. Conclusion:On the basis of standardized treatment of Western medicine, Buyang-Huanwu Decoction and Tingli-Dazao-Xiefei Decoction can improve the clinical efficacy of CHF patients with qi deficiency and blood stasis syndrome, promote the repair of damaged myocardium (reduce NT-proBNP), and improve the quality of life of patients.

Objective To observe the gastric changes in adult male Sprague-Dawly (SD) rats irradiated by the single large dose electron beam,providing animal experimental evidence for intraoperative radiotherapy for gastric cancer.Methods Thirty-eight SD rats were randomly divided into the control and experimental groups.The stomach of the rats in the experimental group were subject to single 6 MeV 20 Gy irridiation by using the patent technology of Accurate Irradiation Experiment Table for Small Animal Radiation.The general conditions,gastric injury and body weight change were observed at different days following irradiation.Results The most severe gastric damage of rats was observed on the 14th d after irradiation.The gastric injury was gradually repaired accompanied with glandular atrophy at 28 d postirradiation,and the gastric injury was manifested as cellulose fibrinous repair on the 56th d after irradiation.Within 1 week post-irradiation,weight loss was noted in the experimental group,which significantly differed from the rats in the control group (P＜0.05).During the 2nd week,the body weight was increased in the experimental group,significantly lower compared with the rats in the control group (P＜ 0.05).The body weight of rats did not significantly differ between two groups at 6 weeks after irradiation (P＞ 0.05).Conclusions The most severe gastric injury is observed at 2 weeks after the single-dose 6 MeV electron beam 20 Gy irradiation,whereas no gastric perforation occurs.The gastric injury can be restored to normal status within 8 weeks following irradiation.

To summarize the clinical manifestations of a case with 46, XY sex development disorder caused by myelin regulatory factor(MYRF) gene mutation and review the literature to deepen the specialists′ understanding of the clinical disease spectrum resulting from MYRF gene variations. The child had a female phenotype with mild masculinity, chromosome 46, XY, sex-determining region of Y gene(SRY gene) positive, laboratory tests were consistent with primary hypogonadism, ultrasound did not detect the gonads, but the residual reproductive tract was visible, and echocardiography suggested coarctation of the aorta, MYRF gene c. 2518C>T(p.R840*) heterozygous variant. The father did not carry this variant. The mother was untraceable, and genetic testing had not been completed. It was analyzed as pathogenic variation according to American College of Medical Genetics and Genomics(ACMG) guidelines. Sixteen cases of disorders of sex development caused by MYRF gene variation reported from 2018 to 2021 were reviewed, MYRF gene variants, 46, XY, and 46, XX individuals can be pathogenic, can affect the gonad and reproductive tract at the same time, and can also affect multiple systems. In this case, the patient presents with 46, XY sex development disorder due to MYRF gene mutation, accompanied by rare cardiovascular complications. When encountering 46, XY primary hypogonadism without well-developed Müllerian duct structures, this condition should be considered. Following confirmation, a comprehensive assessment of multi-organ function is necessary.