Objective: To explore the heterogeneity and correlation of clinical phenotypes and genotypes in children with disorders of sex development (DSD). Methods: A retrospective study of 1 235 patients with clinically proposed DSD in 36 pediatric medical institutions across the country from January 2017 to May 2021. After capturing 277 DSD-related candidate genes, second-generation sequencing was performed to analyzed the heterogeneity and correlation combined with clinical phenotypes. Results: Among 1 235 children with clinically proposed DSD, 980 were males and 255 were females of social gender at the time of initial diagnosis with the age ranged from 1 day of age to 17.92 years. A total of 443 children with pathogenic variants were detected through molecular genetic studies, with a positive detection rate of 35.9%. The most common clinical phenotypes were micropenis (455 cases), hypospadias (321 cases), and cryptorchidism (172 cases) and common mutations detected were in SRD5A2 gene (80 cases), AR gene (53 cases) and CYP21A2 gene (44 cases). Among them, the SRD5A2 mutation is the most common in children with simple micropenis and simple hypospadias, while the AMH mutation is the most common in children with simple cryptorchidism. Conclusions: The SRD5A2 mutation is the most common genetic variant in Chinese children with DSD, and micropenis, cryptorchidism, and hypospadias are the most common clinical phenotypes. Molecular diagnosis can provide clues about the biological basis of DSD, and can also guide clinicians to perform specific clinical examinations. Target sequence capture probes and next-generation sequencing technology can provide effective and economical genetic diagnosis for children with DSD.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics* ; Child ; China/epidemiology* ; Cryptorchidism/genetics* ; Disorders of Sex Development/genetics* ; Female ; Genital Diseases, Male ; Genotype ; Humans ; Hypospadias/genetics* ; Male ; Membrane Proteins/genetics* ; Penis/abnormalities* ; Phenotype ; Retrospective Studies ; Steroid 21-Hydroxylase/genetics*

The main factors which affected the isolation, purification and cultivation of Pinellia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW + 1.0% Cellulose +0.1% Pectolase, at pH 6.0, temperature (25-28 degrees C ) for 4 h. The sucrose density gradient centrifugation was adopted to purificate the protoplasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA + 13% mannitol at the density of 2.5 x 104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular calli appeared for 30 days. Calli was proliferated on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.
Cell Separation ; methods ; Culture Media ; Pinellia ; physiology ; Protoplasts ; physiology ; Regeneration

Objective To evaluate the therapeutic effects of 2% minocycline hydrochloride liposome controlled-release gel on the periodontitis in an established rat periodontitis model. Methods Biocompatibility was tested by oral perfusion sample solution for long-term observation. Minocycline hydrochloride liposome controlled-release gel was utilized to treat the established rat periodontitis model. The rats were selected randomly and divided into three groups: group A (PERIO-treated group), group B (minocycline hydrochloride liposome controlled-release gel treated group), and group C (negative control group). The gingival index (GI) and probing depth (PD) were detected, and the number of mononuclear and broken bone cells were examined after 7, 14, 28, and 56 d. Results The minocycline hydrochloride liposome controlled-release gel exhibited excellent biocompatibility based on weight measure and tissue section evaluation. The rats with periodontitis demonstrated that GI, PD, and the number of mononuclear and broken bone cells of group B decreased in 14, 28, and 56 d. Pathological observation showed that new bones and fibers were formed in group B. Conclusion Minocycline hydrochloride liposome controlled-release gel improves rat periodontitis, thereby providing valuable evidence for clinical application.

Objective To investigate the effects of enamel matrix proteins (EMP) on proliferation and gene expression of cementum related proteins in human periodontal ligament cells (PDLC).Methods Human PDLC were treated with EMP at concentrations of 0 (control),50,100,200,300 mg/L respectively.At day 1,3,5,7,methyl thiazolyl tetrazolium(MTT) assay was used to measure cell proliferation.At day 7,real-time reverse transcriptase polymerase chain reaction(RT-PCR) was used to detect mRNA expression of cementum attachment protein (CAP) and cementum protein-23 (CP-23).Results EMP significantly increased cell proliferation at concentrations of 50-200 mg/L,but inhibited cell proliferation at concentration of 300 mg/L.The mRNA expression of both CAP and CP-23 at EMP concentrations of 100,200 mg/L (CAP:4.661 ± 0.154,5.923 ± 0.788 ; CP-23:1.222 ± 0.089,3.795 ± 0.640) was significantly higher thanthat of the control (CAP:1.006±0.062,CP-23:1.012±0.163),P＜0.05.Conclusions EMP can promote cell proliferation and increase the gene expression of CAP and CP-23.It suggests that EMP may enhance cementum regeneration through promoting human PDLC's proliferation and cementum related protein production.

OBJECTIVETo establish a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA load in subgingival specimens from the patients with aggressive and chronic periodontitis, and to investigate the relationship between HCMV infection and the periodontal status.

METHODSA total of 114 subgingival plaque specimens were taken from 18 subjects with aggressive priodontiti (AgP), 24 subjects with chronic periodontitis (CP) and 15 healthy control subjects. Standard quantification was performed with recombinant plasmid containing a conserved fragment of HCMV. The SYBR Green I fluorescent quantitative real-time PCR assay was established based on positive plasmid. HCMV DNA load in the specimens were detected with quantitative real-time PCR based on SYBR Green I fluorescence.

RESULTSHCMV were detected in 58.3% of AgP sites and 41.7% of CP sites, however, only 6.7% of periodontally-healthy sites were HCMV positive. The detection rate of HCMV in periodontitis lesions was significantly higher than in periodontal health (P < 0.01). High copy-counts more than 10(4) of HCMV were detected in 33.3% of AgP sites, which were significantly higher than in CP sites (10.4%) (P < 0.05).

CONCLUSIONSubgingival infection with HCMV is closely associated with periodontitis. Active HCMV infection may be related to the rapid tissue destruction of AgP.

Adult ; Chronic Periodontitis ; Cytomegalovirus ; Cytomegalovirus Infections ; Dental Plaque ; Female ; Humans ; Male ; Periodontitis ; Polymerase Chain Reaction

OBJECTIVETo examine the expression of periodontal ligament-associated protein-1 (PLAP-1) in the periodontal tissues and periodontal ligament cells (PDLC).

METHODSThe PLAP-1 expression in normal periodontal tissue was examined by immunohistochemistry. The protein expression and mRNA transcription of PLAP-1 in PDLC were investigated by immunocytochemistry and reverse transcription-polymerase chain reaction.

RESULTSPLAP-1 was expressed in periodontium but not in cementum, alveolar bone and gingival tissues. PLAP-1 expression was observed in cell plasma, but not in nuclei. There was a 350 bp electrophoresis band representing PLAP-1 mRNA.

CONCLUSIONSPLAP-1 may play a role in physiology of periodontal tissues and cells in normal adult rats.

Animals ; Extracellular Matrix Proteins ; genetics ; metabolism ; Immunohistochemistry ; Male ; Periodontal Ligament ; cytology ; metabolism ; Periodontium ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the expression of interleukin-10 (IL-10) mRNA in gingival tissue of active and stable stage in patients with adult periodontitis.

METHODS12 patients with acute abscesses of the periodontium, 12 patients after periodontal initial treatment and 6 periodontal healthy patients having extraction of impacted wisdom tooth were randomly divided into group A (active stage group), group B (stable stage group) and the control group. Biopsies of gingival tissues were collected from every subject of three groups. Technique of in situ hybridization was applied to observe the expression of IL-10 mRNA in the biopsies from three groups semi-quantitatively.

RESULTSIL-10 mRNA was positively expressed in lymphocytes, macrophages and fibroblasts. The quantity of IL-10 mRNA of group A was the lowest in the three groups and was significantly lower than that of control group and group B respectively (P < 0.01). The quantity of IL-10 mRNA of group B was the highest in the three groups and was significantly higher compared with the control group and group A (P < 0.01).

CONCLUSIONThe quantities of IL-10 mRNA expression are closely related with various clinical states of periodontitis.

Case-Control Studies ; Chronic Periodontitis ; metabolism ; Gingiva ; metabolism ; Humans ; Interleukin-10 ; metabolism ; RNA, Messenger ; metabolism

OBJECTIVETo investigate the role of p75 neurotrophin receptor (p75NTR) in the regeneration of facial nerve crush injury.

METHODSIn p75NTR knockout mice and wild type mice, the regenerating fibres in the facial nerve were also labelled by an anterograde tracer cholera toxin B (CTB). The next day after injury of facial nerve, CTB was injected into the trunk of the nerve in the proximal side of the crush, and then anterograde tracing and immunohistochemistry were used to examine the regeneration of axons after facial nerve crush injury. In p75NTR knockout mice and wild type mice, the facial nerves on one side were crushed and regenerating neurons in the facial nerve nucleus were labelled by Fast Blue. The facial nerve trunk was cut in the bifurcated region in the 4th day after injury and the stump was inserted into a small polymer tube containing Fast Blue. Retrograde tracing and labling motoneuron counting were used to examine the survival of motoneurons in the facial nerve nucleus after facial nerve crush injury.

RESULTSThe results showed that the axonal growth of injured axons in the facial nerve of p75NTR knockout mice was significantly retarded. The number of regenerated neurons in the facial nerve nucleus in p75NTR knockout mice was significantly reduced (P < 0.05). Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR knockout mice (P < 0.01).

CONCLUSIONp75NTR plays an important role in the regeneration of injured peripheral nerves after injury.

Animals ; Axons ; Facial Nerve ; Mice ; Motor Neurons ; Nerve Regeneration ; Neurons ; Receptor, Nerve Growth Factor ; Receptors, Nerve Growth Factor

OBJECTIVETo investigate the influence of high mobility group box 1 (HMGB1) on the expression of interleukin 6 (IL-6), receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) on periodontal ligament fibroblasts.

METHODSHuman periodontal ligament fibroblasts were stimulated with HMGB1 at concentrations of 10, 30, and 100 ng x mL(-1) for 24 h. RT-PCR and Western blot analysis were performed to check mRNA and protein expression of IL-6, RANKL and OPG on the cells.

RESULTSThe ratio of RANKL/OPG was increased at both mRNA and protein level after HMGB1 stimulation at 10, 30, 100 ng x mL(-1). Inflammatory cytokine IL-6 was upregulated by HMGB1 at the concentration of 100 ng x mL(-1).

CONCLUSIONIncreased ratio of RANKL/OPG and IL-6 on periodontal ligament fibroblasts suggests that HMGB1 might play a role in the pathogenesis and progression of periodontal disease.

Cells, Cultured ; Fibroblasts ; metabolism ; HMGB1 Protein ; metabolism ; Humans ; Interleukin-6 ; metabolism ; Osteoprotegerin ; metabolism ; Periodontal Ligament ; cytology ; RANK Ligand ; metabolism