Objective To investigate the angiogenesis effects of Salvia miltiorrhiza in heterotopically grafted frozen-thawed mouse ovaries. Methods The ovaries thawed after cryopreservation were xenografted into the donated kidney capsules of 8- to 12-week adult male mice. The mice were divided into two groups, saline and Salvia miltiorrhiza groups, the mice either in the saline or in Salvia miltiorrhiza groups were administered I.p. Daily either saline(0.5ml) or Salvia miltiorrhiza(0.5g)respectively, from the day prior transplantation. The two groups were sacrificed 1 day,2 days and 7 days after transplantation respectively, the grafts from thawed,1 day,2 days,7 days were removed for follicle counting, immunohistochemical studying and detecting of the mRNA expression of vascular endothelial growth factor(VEGF). Results The number of follicles and survival rates in grafts after transplantation of Salvia miltiorrhiza group were more than that of saline group (P<0.05);the expression of VEGF increased after transplantation,peaked on day 7,there was no difference between the two groups (P>0.05);the apoptosis index of Salvia miltiorrhiza group was less than that of saline group (P<0.05), the mRNA expression of VEGF188 and VEGF164 of Salvia miltiorrhiza group was more than that of saline group on 48 hours after transplantation(P<0.05). Conclusion Salvia miltiorrhiza may provide benefits for folliculogensis and decreasing the apoptosis index of follicles. Nevertheless,a increase in the VEGF188 and VEGF164 isoform in the Salvia miltiorrhiza group may suggest the positive effect of exogenous Salvia miltiorrhiza therapy in the early stage of angiogenesis.

AIM: To clarify the effect of SC58125 on cell proliferation and apoptosis in HepG-2 cells and explore the molecular mechanisms. METHODS: Cell culture, MTT, TUNEL, DNA ladder, flow cytometry and Western blot analysis were employed in the present study. RESULTS: SC58125 inhibited the growth of HepG-2 cells and induced the apoptosis. Furthermore, it arrested G_0/G_1 phase and inhibited S phase in HepG-2 cells. Depressed expression of P33 cdk2 ,P34 cdc2 ,cyclin B_1,cyclin E ,Mpm-2,Rb ,PCNA proteins were found in HepG-2 cells treated with SC58125. CONCLUSION: SC58125 inhibits cell proliferation and induces apoptosis, which may be related to the altered low protein levels of P33 cdk2 ,P34 cdc2 ,cyclin B_1,cyclin E ,Mpm-2,Rb,PCNA

Objective To summarize the clinical features of 22 probands diagnosed with congenital muscular dystrophy (CMD),and to provide genetic counseling and prenatal diagnosis for 23 fetuses of these pedigrees.Methods Data of 22 CMD patients who were treated in the Pediatric Department of Peking University First Hospital during October 2006 to March 2016 were analyzed.Informed written consents for participation in this study were obtained from the parents or guardians.Prenatal diagnosis was performed using DNA samples extracted from fetal villus cells of 12 cases at 11-13 gestational weeks and amniotic fluid of 11 cases at 18-22 gestational weeks.Direct DNA sequencing by polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) were used to detect CMD-related gene mutations.Linkage analysis of short tandem repeats (STRs) was used to identify maternal blood contamination and biological parents.Results Thirteen out of the 22 probands with CMD were diagnosed with congenital muscular dystrophy type 1 A (MDC1A),and all of them carried compound heterozygous mutations in LAMA2 gene.Prenatal diagnosis of 13 fetuses from these pedigrees found that four fetuses were wild-type,seven were heterozygotes and two carried the same mutations as their proband.Three probands with LMNA-related congenital muscular dystrophy (L-CMD) carried de novo mutations in LMNA gene.In these pedigrees,two fetuses were wild-type and one whose mother was mosaicism carried the same mutations as the proband.One proband with Ullrich congenital muscular dystrophy carried compound heterozygous mutations in COL6A2 gene and the fetus of the same pedigree was wild-type.Five probands were diagnosed with α-dystroglycanopathies.And among them,two cases of muscle-eye-brain disease (MEB) carried compound heterozygous mutations in POMGnT1 gene and the fetuses of the two peidgrees were heterozygotes;one case of congenital muscular dystrophy type 1C (MDC1C) had compound heterozygous mutations in FKRP gene and the fetus carried the same mutations;one patient diagnosed with POMGnT1-related congenital muscular dystrophy with mental retardation (CMD-MR) carried compound heterozygous mutations in POMGnT1 gene,and the fetus was positive for the same mutations;one proband with POMT1-related CMD-MR was positive for compound heterozygous mutations in POMT1 gene and the results of prenatal diagnosis for two fetuses of this pedigree showed that the first fetus had the same mutations as the proband,while the second was heterozygote.Conclusions No effective therapeutic method is available for CMD.Therefore,accurate genetic counseling and prenatal diagnosis are necessary to prevent CMD child from birth.

Objective To study the diagnostic value of Spyglass visual impression and Spybite targeted biopsies for biliary strictures of unknown reasons. Methods Several Chinese and English databases were electronically searched for studies on biliary strictures diagnosed with Spyglass visual impression and Spybite targeted biopsies compared with golden standard ( pathological biopsy, autopsy and long-term clinical follow-up) . The methodological quality of the included studies was assessed according to QUADAS-2 items. The software Meta-DiSc ( version 1. 4 ) was used to conduct pooling on sensitivity, specificity, positive likelihood radio, negative likelihood radio and diagnostic odds ratio ( DOR ) . Heterogeneity test was performed and the summary receiver operating characteristic curve ( SROC ) was drawn for area under the curve ( AUC) . Results A total of 12 studies met the inclusion criteria, involving 532 patients who received Spyglass visual impression and 525 who received Spybite targeted biopsies. The combined specificity, sensitivity, positive likelihood ratio, negative likelihood ratio and DOR were 0. 90 (95％CI:0. 85-0. 94), 0. 89 (95％CI:0. 85-0. 93), 7. 12 (95％CI: 4. 36-11. 64), 0. 12 (95％CI:0. 07-0. 22)and 82. 40 (95％CI:33. 73-201. 28)for Spyglass visual impression, and 0. 98 (95％CI:0. 96-1. 00), 0. 66 (95％CI:0. 60-0. 71), 13. 29 (95％CI:6. 92-25. 53), 0. 37 (95％CI:0. 28-0. 47)and 51. 05 (95％CI:23. 58-110. 53) for Spybite targeted biopsies, respectively. The AUC on the SROC of Spyglass visual impression and Spybite targeted biopsies were 0. 9574 and 0. 9398, respectively. Conclusion Spyglass visual impression is useful for detecting malignant lesion, whereas Spybite targeted biopsies is better at confirming malignant diagnosis, which indicates combination of the two methods have good diagnostic value for indeterminate biliary strictures, but their negative results are not perfect in excluding biliary cancer.

Objective To observe whether transcutaneous electrical acupoint stimulation (TEAS) could improve the emergence and recovery of patients undergoing robotic gynecologic surgery,and to explore the mechanism behind it.Methods Patients (aged 18-65 years,BMI 18-30 kg/m2,ASA grade Ⅰ or Ⅱ) scheduled for elective robotic gynecologic surgery were screened and randomized into three groups:group TEAS (groups T),no acupoint group (group N) and control group (group C),receiving TEAS (ST-36,SP6,BL59,BL60),stimulation at bilateral hips and no-stimulation respectively.Stimulations were given from 30 min before anesthesia induction to the end of surgery.Recovery measurements during emergence,PACU stay and 24 h after surgery were recorded.Levels of serum AQP4,MMP9 and S100β were analyzed.Results Time to response to verbal command and time to extubation in group T [(18.3± 6.7) min and (19.4 ± 6.6) min respectively] were significantly shorter than those in group C [(21.9±7.3) min and (23.1±7.3) min respectively] (P ＜0.05).Maximum VAS scores during PACU stay were significantly lower in group T than that in groups C and N (P＜0.05).Postoperative AQP4 level in group T significantly decreased compared with baseline (P＜0.05).However,postoperative MMP9 and S100β level in group C significantly in creased compared with the baseline (P＜0.05 or P＜0.01).Conclusion TEAS could fasten emergence of patients after robotic gynecologic surgery and improve postoperative analgesia.Mechanisms involving AQP4,MMP9 and S100β may be involved.

Objective:To investigate the cytopathic effect of amino acids 86-175 of rotavirus non-structural protein 4 (NSP4 86-175) on rat cardiomyocytes and the possible mechanism. Methods:Rat H9C2 cardiomyocytes were treated with NSP4 86-175 protein. Changes in the growth and morphology of the cells were observed. The activity of LDH in the cell culture medium was detected. Fluo-3AM was used to label intracellular free calcium ions and the concentrations of calcium ions in rat cardiomyocytes with and without NSP4 86-175 treatment were detected by confocal laser microscopy. The expression of Bax, Bcl-2, caspase-3, 78 kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP) and caspase-12 at mRNA level was detected by real-time PCR. The expression of caspase-3, caspase-8, caspase-9, GRP78, CHOP and caspase-12 at protein level was detected by Western blot. Results:Normal cardiomyocytes showed a typical myoblast-like morphology, presenting as spindle-shaped cells with clear boundaries. Obvious cytopathic effect, vacuolar degeneration, shriveled and rounded cells, and cell fragmentation were observed after the treatment with purified NSP4 86-175 protein. The activity of LDH in cell culture medium was enhanced by NSP4 86-175 protein. NSP4 86-175 protein also enhanced the fluorescence of the calcium ions in rat cardiomyocytes, promoted cell apoptosis, up-regulated the expression of apoptotic factors including caspase-3, caspase-8, caspase-9 and Bax-2, and increased the expression of classical markers of endoplasmic reticulum stress such as GRP78, CHOP and caspase-12. Conclusions:NSP4 86-175 had a cytopathic effect on rat cardiomyocytes, which might be related to the induced intracellular calcium overload, endoplasmic reticulum stress, apoptosis and necrosis. These results might be used as theoretical reference for further study on rotavirus infection and myocardial injury.

Objective To evaluate the role of hippocampal CD200 receptor 1(CD200R1)in peri-operative neurocognitive disorders(PND)in mice.Methods Sixty clean-grade male C57BL/6 mice,aged 9-10 months,weighing 32-38 g,were used in the study.The experiment was performed in two parts.Ex-periment Ⅰ Thirty-six mice were divided into 2 groups(n＝18 each)using a random number table meth-od: control group(group C)and PND group.Group C only received isoflurane anesthesia.Partial left lo-bectomy of the liver was performed under isoflurane anesthesia in group PND.Contextual fear conditioning test was performed at 1,3 and 7 days after surgery,and the freezing time was recorded.The mice were then sacrificed,and the hippocampus was isolated for determination of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)contents(by enzyme-linked immunosorbent assay)and CD200 and CD200R1 expression(by Western blot).ExperimentⅡ Twenty-four mice were divided into 2 groups(n＝12 each)using a random number table method: CD200-Fc group and IgG1-Fc group.Recombinant proteins CD200-Fc and human IgG1-Fc were injected into the lateral cerebral ventricle in CD200-Fc group and IgG1-Fc group,respectively.Partial left lobectomy of the liver was performed after the end of injection.Contextual fear conditioning test was performed at 1 and 3 days after surgery,and the freezing time was recorded.Re-sults Experiment Ⅰ Compared with group C,the freezing time in the contextual fear conditioning test was significantly shortened,and the contents of IL-1β were increased at 1 and 3 days after surgery,the contents of TNF-α were increased at 3 and 7 days after surgery,and the expression of CD200 and CD200R1 was up-regulated at 1 day after surgery in group PND(P＜0.05).ExperimentⅡ Compared with IgG1-Fc group,the freezing time in the contextual fear conditioning test was significantly prolonged at 1 day after surgery in CD200-Fc group(P＜0.05).Conclusion Up-regulated expression of hippocampal CD200R1 is the endogenous protective mechanism of PND in mice.

Objective:To analyze the clinical features and mutation of myeloid differentiation factor 88 (MYD88) L265P in patients with diffuse large B-cell lymphoma (DLBCL) of central nervous system (CNS).Methods:The clinicopathological materials of 45 cases of DLBCL of CNS were retrospectively collected in Xuanwu Hospital, Capital Medical University from September 2014 to February 2017. The clinicopathological data were retrospectively analyzed, combined with immunohistochemistry, EB virus in situ hybridization, imaging and medical history. The mutation of MYD88 L265P gene was detected by pyrosequencing and its clinical significance was analyzed. Results:The age of the patients ranged from 42 to 82 years [(57.6±8.8) years], including 24 males and 21 females. Totally 93.3% (42/45) of the patients had supratentorial tumours, which were single or multiple. The cerebral hemisphere (31/45, 68.9%) was the most common involved site, and 21 cases (21/45, 46.7%) had multiple lesions. Histologically, DLBCL in the CNS showed diffuse infiltration of tumor tissue, some of which grew around blood vessels in a "sleeve" arrangement. CD 20 and CD 79a were diffusely and strongly positive. Thirty-nine cases (39/45, 86.7%) were non-germinal center B cell (non-GCB) subtype and 6 cases (6/45, 13.3%) were germinal center B cell (GCB) subtype. MYD88 L265P mutation was found in 64.4% (29/45) patients. There was statistically significant difference between non-GCB type (71.8%, 28/39) and GCB type DLBCL (1/6, P=0.017). Compared with the operation/biopsy group without chemotherapy, operation+chemotherapy, biopsy+chemotherapy, operation/biopsy+chemotherapy+stem cell transplantation can improve the survival and prognosis ( HR=0.05, 95% CI 0.01-0.33 , P=0.002; HR=0.04, 95% CI 0.01-0.36 , P=0.004; HR=0.01, 95% CI 0.00-0.17 , P=0.001; respectively). Conclusions:DLBCL of the CNS is aggressive tumor with poor prognosis, the clinical manifestations are complex and diverse, and the diagnosis is challenging. MYD88 L265P is a common and specific gene mutation in primary CNS lymphoma(PCNSL), which is of great significance in the diagnosis and treatment of lymphoma. The MYD88 L265P mutation was more frequently detected in non-GCB than GCB subtype. Chemotherapy can improve the survival rate of PCNSL patients. If chemotherapy achieves complete remission and autologous hematopoietic stem cell transplantation is performed, there may be a chance of long-term survival.

Objective:To construct a mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 with hfq gene deletion and to analyze its biological characteristics. Methods:The hfq gene of NTUH-K2044 was knocked out by homologous recombination technology to construct △ hfq mutant strain. Its biological characteristics including growth rate, environmental stress tolerance, biofilm formation, capsular polysaccharide synthesis, resistance to neutrophil phagocytosis and lethality to Galleria mellonella larvae were analyzed by comparing with the wild-type strain using phenotypic experiments. Results:The △ hfq mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 was successfully constructed. Phenotypic experiments showed that the △ hfq mutant strain had significantly slower growth rate, smaller colonies and decreased hypermucoviscosity. Its growth was significantly inhibited under different environmental stress conditions such as pH9, pH5.5, 0.7 mmol/L SDS, 5% NaCl, 0.1% H 2O 2 and high temperature of 50℃. In terms of virulence and pathogenicity, the △ hfq mutant strain showed decreased ability to form biofilm and capsule, significantly down-regulated expression of magA and rmpA genes required for capsule synthesis, lower survival rate in the neutrophil bactericidal test and obviously reduced lethality to Galleria mellonella larvae. Conclusions:As a RNA chaperone, Hfq protein could participate in post-transcriptional regulation and play an important role in regulating the physiology, environmental adaptability and virulence of hypervirulent Klebsiella pneumoniae. This study provided reference for further study on hypervirulent Klebsiella pneumoniae.

Objective:To explore the accuracy and clinical application value of a Multi-Agent Reinforcement Learning framework (MARL framework) in three-dimensional ultrasound to automatically locate the coronal plane of the uterus.Methods:A total of 144 female patients who underwent routine gynecological examinations in Luohu People′s Hospital during May 2020 were selected as the experimental subjects. The three-dimensional volume data of the uterus of all the experimental subjects were collected by using the Resona-8 high-end color Doppler ultrasound system. A sonographer with more than 5 years of clinical experience manually locate the coronal plane of the uterus in all collected data, and at the same time automatically locate the coronal plane of the uterus MARL framework. The coronal plane images of the uterus obtained by the two methods were saved, and the operation time of the two methods was recorded. The coronal plane uterine images obtained by the two methods were mixed together, and the images were scored 0-1 by two senior ultrasound experts in a double-blind manner. The average score greater than or equal to 0.6 points was considered qualified.Results:①In 144 volunteers, among the coronal planes of the uterus located by the two methods, 131 were qualified by the manual method, and 137 were qualified by the automatic method.There was no statistical difference between the manual and automatic coronal plane images of the uterus (χ 2=1.934, P=0.164) by the chi-square test. ②Using interquartile range analysis, the median and interquartile range of the image score of the automatic group was 0.80(0.75, 0.90), while the median and interquartile range of the image score of the manual group was 0.80(0.75, 0.90). The Wilcoxon signed rank test was used to analyze the quality of the coronal plane images obtained by manual and automatic methods, and the difference was not statistically significant ( Z=1.241, P=0.215). ③The paired t test was used to compare the time required to locate the coronal surface of the uterus, by manual method (63.65±10.182)s, by automatic method (3.25±0.294)s, the difference between the two methods was statistically significant ( t=19.52, P<0.001). Conclusions:The method based on MARL framework has a high correlation with the manual locating of the coronal plane of uterus in three-dimensional ultrasound, and greatly reduces the operation time. It can be effectively applied in clinical practice and lays a foundation for the automatic diagnosis of uterine related diseases.