Effect and Mechanism of Cuscutae Semen Total Flavonoids Combined with Tripterygium wilfordii Polyglycoside Tablets on Ovarian Germline Stem Cells of Physiological Mice by Notch Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Cuscutae Semen total flavonoids combined with Tripterygium wilfordii polyglycoside tablets （TWPT） on ovarian germline stem cells of female physiological mice through neurogenic locus notch homolog （Notch） signaling pathway. MethodSixty female Kunming mice （5 weeks old） were randomly divided into normal group， Tripterygium wilfordii polyglycoside tablets low-， high-dose groups （13.65 mg·kg-1·d-1 and 27.3 mg·kg-1·d-1， 1 and 2 times clinical equivalent dose）， Cuscutae Semen total flavonoids low- and high-dose groups （150 mg·kg-1·d-1 and 300 mg·kg-1·d-1）， and combination group （13.65 mg·kg-1·d-1 TWPT and 150 mg·kg-1·d-1 Cuscutae Semen total flavonoids）， with 10 in each group. After 3 weeks of continuous administration， the uterus/brain and ovarian/brain indexes were calculated， and the pathological changes of ovarian tissue were observed under light microscope. The content of estradiol in serum was determined by enzyme linked immunosorbent assay （ELISA）. Immunofluorescence assay was performed to observe the expressions of germline stem cell markers in ovarian epithelium， including mouse vasa homologue （Mvh）， octamer-binding transcription factor 4 （Oct4）， tyrosine-protein kinase receptor （c-kit）， Nanog， Notch signaling pathway molecules， neurogenic locus notch homolog protein 1 （Notch1）， hes family BHLH transcription factor 1（Hes1）， and jagged canonical Notch ligand 1 （JAG1）. ResultCompared with the normal group， low and high doses of TWPT had no significant effect on the uterus/brain and ovary/brain indexes and the uterus and ovary morphologies of mice， while only the number of atretic follicles was increased （P<0.01）. The expressions of ovarian germline stem cell markers and Notch signaling pathway molecules had a decreasing trend in TWPT low-dose group， while the expressions of Mvh， c-kit， and Nanog were down-regulated （P<0.05， P<0.01） and the expressions of Notch1 and Hes1 were also reduced （P<0.01） in TWPT high-dose group. However， the above indexes were increased in Cuscutae Semen total flavonoids low-dose group （P<0.05， P<0.01）. Compared with the low does of TWPT group， the combination group had a decrease in the increased number of atretic follicles （P<0.01）， an improvement in the down-regulated expressions of Mvh and Nanog （P<0.01）， and an increase in the expressions of Notch1 and Hes1 （P<0.05， P<0.01）. ConclusionOvarian germline stem cells are the source target of the reproductive toxicity of TWPT. Cuscutae Semen total flavonoids participate in the regulation of the germline stem cell pathways to alleviate the reproductive toxicity caused by TWPT， and its mechanism of action may be related to the Notch signaling pathway.

Role of karyotyping combined with chromosomal microarray analysis in prenatal diagnosis of balanced translocation or inversion carriers / 中华围产医学杂志

Objective:To explore the value of karyotyping and chromosomal microarray analysis (CMA) in the prenatal diagnosis of balanced translocation/inversion carriers.Methods:This was a retrospective study involving 117 balanced translocation/inversion carrier couples. Among them, 90 women had a history of spontaneous abortion(≥2 times), stillbirth, fetal multiple malformations, or giving birth to children with chromosome abnormality disease and the peripheral blood karyotyping and fluorescence in situ hybridization testing confirmed that one partner was balanced translocation/inversion carrier. The present pregnancies of these cases were spontaneous and lasted until 18-25 weeks. The other 27 cases were confirmed by chromosome examination at the present pregnancy after the indication of fetal structural abnormalities by fetal karyotyping due to advanced maternal age and abnormal ultrasound and prenatal screening results. The results of karyotyping and CMA by amniocentesis during 18 to 25 gestational weeks were all summarized and described. Results:The successful rate of both methods was 100.0% (117/117). Unbalanced and balanced translocation/inversion were detected in seven (6.0%) and 39 (33.3%) fetuses by karyotyping, respectively. CMA revealed 14 fetuses with pathogenic copy number variation (CNV) and one with variants of uncertain significance(VUS), with an anomaly detection rate of 12.8% (15/117). Among the 15 cases with CNV, 13 were related to the parental translocation/inversion, one with de novo mutation (22q11.2 microdeletion syndrome), and one Duchenne muscular dystrophy mutation carrier. Based on the results of karyotype and CMA, there were 12 fetuses with unbalanced chromosomal fragments (10.3%), 37 fetuses with balanced translocation/inversion (31.6%), and 68 fetuses with normal chromosomes (58.1%). Conclusions:The combination of karyotyping and CMA can provide more accurate prenatal genetic diagnosis when one of a couple carries balanced chromosomal translocations/inversion.

Genetic analysis and prenatal diagnosis of Duchenne or Becker muscular dystrophy / 中华妇产科杂志

Objective To explore the mutation characteristics of DMD gene in patients with Duchenne or Becker muscular dystrophy and female carriers, to provide effective prenatal diagnosis. Methods Samples were collected from 94 male patients clinically diagnosed with Duchenne or Becker muscular dystrophy and 121 corresponding female relatives from Qingdao Women and Children′s Hospital from June 2011 to October 2018. Multiplex ligation-dependent probe amplification (MLPA) was used to detect their DMD gene, and 23 high risk pregnants were performed prenatal diagnosis. Any candidate of DMD gene single-exon deletion was validated by further PCR amplification. The sample with whole DMD gene deletion was confirmed by chromosomal microarray analysis (CMA) to detect copy number variations and break site. Results Among 94 clinical Duchenne or Becker muscular dystrophy patients, 66(70.2%, 66/94) were detected gene mutation; 56 cases were exon deletion mutation and 10 cases were duplication mutation. In 121 female relatives, 48 cases (39.7%, 48/121) were diagnosed as carriers. The mutation carrying rate, was 64.5% (40/62) identified in 62 mothers of Duchenne or Becker muscular dystrophy patients. Five Duchenne or Becker muscular dystrophy fetuses and 5 carrier fetuses were prenatally diagnosed in 23 high risk pregnants. Two children with the entire DMD gene deletion were identified more deletions at Xp21, with deletions of 6.66 Mb and 10.64 Mb respectively. Conclusions MLPA may be an important method to detect DMD gene mutation of deletion and duplication. Therefore, the diagnosis of probands, female carriers and making an effective prenatal diagnosis are essential to reduce the birth of children with Duchenne or Becker muscular dystrophy.

Application of chromosomal microarray analysis and next-generation sequencing for the analysis of abortic tissues / 中华医学遗传学杂志

OBJECTIVE@#To assess the value of chromosomal microarray analysis (CMA) and next-generation sequencing (NGS) for the analysis of abortic tissues.@*METHODS@#A total of 242 samples of spontaneous abortion were collected and tested by CMA or NGS.@*RESULTS@#The detection was successfully in 238 cases (98.35%). In total 143 cases of chromosomal abnormalities were detected, which accounted for 60.08% of all cases. Numerical chromosomal abnormalities were found in 133 cases(93.01%), structural abnormalities were found in 9 cases (6.29%), and uniparental disomy was found in 1 case(0.70%).@*CONCLUSION@#Both CMA and NGS have the advantages of high-throughput, good coverage, high resolution and rapid analysis. They can be used for the detection of the causes of spontaneous abortions. CMA is more useful for the detection of aneuploidies and uniparental disomy, while NGS has advantages in its throughput, capacity in detecting low percentage chimerism and cost, which can provide more options for clinicians.

Expression profile of long non-coding RNA in gingival tissues from the patients with aggressive periodontitis / 中华口腔医学杂志

Objective@#To detect and analyze the differential expression profile of long non-coding RNA (lncRNA) in aggressive periodontitis (AgP) and healthy gingival tissues, in order to explore the role of lncRNA in AgP.@*Methods@#After the informed consents were obtained, gingival tissues from AgP patients (n=40) and healthy volunteers (n=40) were collected in Department of Periodontology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine (from Mar. 2012 to Aug. 2012) and Department of Periodontology, Hospital of Stomatology, Tianjin Medical University (from Oct. 2016 to Apr. 2017). The differential expression of lncRNA of tissues from AgP patients (n=20) and healthy volunteers (n=20) were examined via microarray assay. Bioinformatics was applied to analyze the expression data of lncRNA and correlative mRNA. Two lncRNAs (lncRNA-TNFRSF13C and lncRNA-API5) were chosen to verify the microarray results by using real time quantitative polymerase chain reaction (PCR) in the other gingival tissues.@*Results@#Compared with the result of healthy gingival tissues, totally 8 632 lncRNAs were differentially expressed in tissues from AgP patients. From these data, 1 986 lncRNAs were significantly upregulated while 6 646 lncRNAs were downregulated, amongst which 48 lncRNAs were upregulated (>10 times) (P<0.05), 14 lncRNAs were downregulated (>10 times) (P<0.05). Furthermore, totally 5 519 correlative mRNAs were differentially expressed, amongst which 1 676 mRNAs were upregulated (≥2 times, P<0.05) and 3 843 mRNAs were downregulated≤0.5 (P<0.05). The selected lncRNA-TNFRSF13C and lncRNA-API5 were up-regulated in AgP (P<0.05), which confirmed the results of microarray. From bioinformatics, differential expression lncRNAs were in association with many signal pathways including toll-like receptor signaling pathway, cell cycle and apoptosis pathway, and tumor necrosis factor receptor superfamily pathway.@*Conclusions@#LncRNA may be involved in the pathogenesis of AgP through various pathways, which need to be further explored.

Correlation between the expression of activator protein-1 (c-Fos/c-Jun) mRNA and gingival inflammation / 国际生物医学工程杂志

Objective To study the correlation between the expression of activator protein-1 (c-Fos/c-Jun) mRNA and gingival inflammation,so as to discuss the pathogenesis of periodontitis.Methods The gingival tissues were divided into three groups according to the gingival index (GI),including GI=0 group (control group,14 cases),GI=1 group (15 cases) and GI=2 group (11 cases).The total RNA in each gingival tissue was extracted,and cDNA was synthesized by reverse transcription synthesis.The expressions of c-Fos and c-Jun mRNA in healthy gingival tissue (GI=0 group) were detected by reverse transcription-polymerase chain reaction.The levels of c-Fos and c-Jun mRNA in all the groups were detected by real-time quantitative PCR.Results Both c-Fos and c-Jun mRNA was expressed in healthy gingival tissues.The levels of c-Fos and c-Jun mRNA in GI=1 group was 15.58±9.19 and 3.47± 1.77,respectively,which was significantly higher than 1.31±1.03 and 1.32±0.94 in GI=0 group,and the differences were statistically significant (all P＜0.05).The level of c-Fos mRNA in GI=2 group was 3.01±1.48,which was lower than that in GI=1 group (P＜0.05) and higher than that in GI=0 group (P＜0.05).The level of c-Jun mRNA in GI=2 group was 1.48±0.65,which was lower than that in GI=1 group,and had no significant difference with GI=0 group (P＞ 0.05).Conclusions Activator protein-1 (c-Fos/c-Jun) is associated with the degree of gingival inflammation,suggesting that it is involved in the occurrence and development of gingival inflammation.