[Application of exosomes in oral regenerative medicine].

In recent years, exosomes had been recognized as a potential cell-free therapeutic tool in the field of regenerative medicine due to their easy accessibility, low immunogenicity, and good therapeutic effect. As paracrine products of cells, exosomes not only have the functions of their donor cells but also involve fewer ethical disputes. They are more stable than traditional stem cell therapy, and easy to store, transport and sterilize, which are gradually applied in the field of oral regenerative medicine. This article provides a review of the general properties of exosomes and their application in the field of oral regeneration, with a view to exploring new pathways for functional regeneration of the stomatognathic system.

[Evaluation of the outcomes of cochlear implant in children with auditory neuropathy].

Objective: To investigate the auditory and speech abilities of children with congenital auditory neuropathy (AN) after cochlear implant (CI), and to analyze the role of genetic testing in predicting the postoperative outcomes of CI in AN patients. Methods: Fourteen children diagnosed with AN by audiological battery test and underwent CI surgery in Xijing Hospital of the Air Force Medical University from 2002 to 2021 were included in this study (9 males and 5 females), with an implantation age of (3.1±1.7) years (mean±standard deviation, the same as follows). The preoperative audiological results and deafness gene results were analyzed. Another 52 children with ordinary sensorineural hearing loss (SNHL) were selected as the control group (30 males and 22 females), with an implantation age of (2.2±0.9) years. The demographic factors such as age and gender were matched with those of the AN group. The modified Category Auditory Performance (CAP-Ⅱ) and Speech Intelligence Rate (SIR) were used to evaluate the development of postoperative auditory and speech abilities in two groups. The Mandarin Speech Test System was used to test the speech recognition rate of monosyllabic and disyllabic words and sentences. Matlab 2022 software was used to analyze the data. Results: The results of gene in 14 children with AN showed that 6 cases had OTOF gene mutations, 2 cases (siblings) were confirmed to have TNN gene mutations through whole exome sequencing, and the remaining 6 cases were not find any clear pathogenic gene mutations. All subjects underwent CI surgery with electrodes implanted into the cochlea smoothly, and there were no postoperative complications. After surgery, all AN children had improved auditory and speech abilities, but only 64% (9/14) of AN children with CI had auditory ability scores comparable to the control group of SNHL children (including 2 children with TNN gene mutations), and 36% (5/14) of AN children had lower scores than the control group of SNHL children.The average speech recognition rate of two children with TNN gene mutations was 86.5%, and of two children with OTOF gene mutations was 83.2%. Conclusions: AN children achieved varying degrees of auditory and speech abilities after CI, but the postoperative effects varied greatly. Some children achieved similar results as ordinary SNHL children, but there were still some children whose effects were worse than those of ordinary SNHL children. The postoperative efficacy of CI in two children with AN caused by TNN pathogenic genes were comparable to that of ordinary SNHL in children. Genetic testing had certain reference value for predicting the postoperative effect of CI in AN children.

[The regulatory function of tumor-infiltrating Th9 cells to anti-tumor activity of CD8(+) T cells in patients with gastric cancer].

Objective: To investigate the levels of Th9 cells and interleukin-9 (IL-9), and to assess the regulatory activity of Th9/IL-9 to anti-tumor immune response in patients with gastric cancer. Methods: Thirty-four patients with gastric cancer who received operation in the First Affiliated Hospital of Xinxiang Medical University between October 2018 and August 2019 were included. Twenty individuals who received physical examination in the same period were also enrolled. Peripheral blood was collected, and then plasma and peripheral blood mononuclear cells (PBMCs) were isolated. Tumor-infiltrating lymphocytes (TILs) and autologous gastric cancer cells were isolated from resected gastric cancer tissues. CD4(+) T cells, CD8(+) T cells, and CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells were purified from PBMCs and TILs. Plasma IL-9 level was measured by enzyme linked immunosorbent assay (ELISA). The percentage of CD3(+) CD4(+) IL-9(+) Th9 cells in PBMCs and TILSs was assessed by flow cytometry. The mRNA levels of IL-9 and transcriptional factors purine-rich nucleic acid binding protein 1 (PU.1) were semi-quantified by real-time quantitative polymerase chain reaction (RT-qPCR). PBMCs and TILs from gastric cancer patients were stimulated with recombinant human IL-9. Cellular proliferation was measured by cell counting kit-8. The phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and STAT6 were investigated by western blot. Cytokine production was measured by ELISA. Purified CD8(+) T cells from TILs of gastric cancer patients were stimulated with recombinant human IL-9. CD8(+) T cells and autologous gastric cancer cells were cocultured in direct contact and indirect contact manner. The percentage of target cell death was calculated by measuring the lactate dehydrogenase (LDH) level. These cretion of γ-Interferon (γ-IFN) and tumor necrosis factor-α (TNF-α) was measured by ELISA. CD4(+) CCR4(-)CCR6(-)CXCR3(-)cells, CD8(+) T cells, and autologous gastric cancer cells were directly cocultured, and anti-IL-9 neutralizing antibody was added. The target cell death was measured. Results: The percentages of CD3(+) CD4(+) IL-9(+) Th9 cells in PBMCs of control group and PBMCs of gastric cancer group were (1.21±0.25)% and (1.14±0.19)%, respectively. The difference was not statistically significant (P=0.280). The percentage of CD3(+) CD4(+) IL-9(+) Th9 cells in TILs of gastric cancer group was (2.30±0.55)%, which was higher than those in PBMCs of control group and PBMCs of gastric cancer group (P<0.001). The plasma IL-9 level in control group and gastric cancer group were (5.04±1.51) and (4.93±1.25) ng/ml. The difference was not statistically significant (P=0.787). The relative levels of IL-9 mRNA in PBMCs of control group and PBMCs of gastric cancer group were 1.33±0.39 and 1.36±0.27. The difference was not statistically significant (P=0.691). The relative level of IL-9 mRNA in TILs of gastric cancer group was 2.90±0.75, which was higher than those in PBMCs of control group (P<0.001) and PBMCs of gastric cancer group (P<0.001). The relative levels of PU.1 mRNA in PBMCs of control group and PBMCs of gastric cancer group were 1.21±0.12 and 1.20±0.11. The difference was not statistically significant (t=0.21, P=0.833). PU.1 mRNA relative level in TILs of gastric cancer group was 2.81±0.65, which was higher than those in PBMCs of control group (P<0.001) and PBMCs of gastric cancer group (P<0.001). Recombinant human IL-9 stimulation did not affect the proliferation of PBMCs and TILs of gastric cancer patients (P>0.05), but elevated the phosphorylation level of STAT6 and induced the secretions of γ-IFN, IL-17, and IL-22 by TILs (P<0.05). In direct contact culture system, IL-9 stimulation promoted tumor-infiltrating CD8(+) T cells-induced autologous gastric cancer cell death [(20.62±2.27)% vs. (16.08±2.61)%, P<0.01)]. In indirect contact culture system, IL-9 stimulation did not increase CD8(+) T cell-induced autologous gastric cancer cell death [(5.21±0.70)% vs. (5.31±1.22)%, P=0.998)]. However, the secretion levels of γ-IFN were elevated in response to IL-9 stimulation in both culture systems [direct contact culture system: (100.40±12.05) pg/ml vs. (76.45±8.56) pg/ml; indirect contact culture system: (78.00±9.98) pg/ml vs. (42.09±10.71) pg/ml; P<0.01]. The TNF-α secretion level did not significantly changed (P>0.05). In direct contact culture system, the percentage of target cells was (22.01±3.05) % and γ-IFN secretion level was (104.5±12.84) pg/ml in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells group, which was higher than (16.08±2.61)% and (76.45±8.56) pg/ml in CD8(+) T cells+ gastric cancer cells group (P<0.01). However, the percentage of target cells was (14.47±3.14)% and γ-IFN secretion level was (70.45±19.43) pg/ml in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells+ anti-IL-9 neutralizing antibody group, which were lower than those in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells group (P<0.01). Conclusion: Tumor-infiltrating Th9 cells and the secreting IL-9 promote the activity of CD8(+) T cells in gastric cancer patients, and enhance anti-tumor immune response.

[Study of the effects of long-term outcomes of autologous peripheral blood stem cell reinfusion in patients with decompensated cirrhosis].

Objective: Autologous peripheral blood stem cells (PBSC) derived from bone marrow can promote liver regeneration and improve the liver function of patients, but there are few studies on its effect on the long-term outcomes in patients with decompensated cirrhosis. Based on previous work, this study observed the clinical outcomes of PBSC treatment in patients with decompensated cirrhosis for 10 years, in order to provide more data support for the safety and efficacy of stem cells in clinical applications. Methods: Data of patients with decompensated liver cirrhosis who completed PBSC treatment in the Department of Gastroenterology of the First Affiliated Hospital of Air Force Military Medical University from August 2005 to February 2012 were included. The follow-up endpoint was death or liver transplantation, and patients who did not reach the follow-up endpoint were followed-up for at least 10 years. The patients with decompensated liver cirrhosis who met the conditions for PBSC treatment but did not receive PBSC treatment in our hospital during the same period were used as controls. Results: A total of 287 cases with decompensated liver cirrhosis had completed PBSC treatment, and 90 cases were lost to follow-up within 10 years after surgery. A total of 151 cases with complete survival follow-up data were included in the control group. There were no statistically significant differences in baseline information such as gender, age, etiological composition and liver function score between the two groups. The 10-year survival rate was higher in PBSC than control group (37.56% vs. 26.49%, P<0.05). Cholinesterase, albumin, international normalized ratio, Child-Turcotte-Pugh score, model for end-stage liver disease score, and other indicators were gradually recovered within 3 months to 1 year after PBSC treatment, and stabilized at a more desirable level in the long-term after follow-up for up to 10 years. There was no statistically significant difference in the incidence of liver cancer between the two groups (25.22% vs.31.85%, P=0.267). The age of onset of hepatocellular carcinoma was later in PBSC than control group [(56.66±7.21) years vs. (52.69±8.42) years, P<0.05]. Conclusions: This long-term observational follow-up study of more than ten years confirms that PBSC treatment can bring long-term benefits to patients with decompensated cirrhosis, with good long-term safety, thus providing more data support on the safety and efficacy of stem cells for clinical applications.

[Family genetic analysis of paternal Dystrophin gene mutations in a case of female Duchenne muscular dystrophy].

A pedigree genetic analysis of a female Duchenne muscular dystrophy (DMD) inherited from paternal chimerism was conducted to explore the genetic diagnosis strategy. No large deletions/duplications was found in the DMD gene of the proband. Next-generation sequencing (NGS) results showed that the proband had a heterozygous mutation in the DMD gene c.4707C>A (p.C1569X). This locus has not been reported in the literature and is considered as a pathogenic mutation. Sanger sequencing revealed that the father of the proband carried the same mutation, and the mosaic ratio was about 17.7%. The specific enzyme digestion test showed that the proband had maternal skewed X-inactivation. DMD a recessive inherited disease of the X chromosome, exists in female patients, and very few of them are inherited from paternal origin. Female patients need to pay close attention to skewed X-inactivation and suspected new mutations. Mosaic is not excluded, especially the inheritance of paternal mosaicism with normal phenotype. Prenatal gene screening is necessary for reproduction.

[Impacts of high-voltage electrical burn on serum platelet-related factors and platelet aggregation number in rats and the interventive effect of Xuebijing].

Objective: To explore the effect of high-voltage electrical burn on platelet function and rheological behavior in rats and the interventive effect of Xuebijing. Methods: A total of 280 Sprague Dawley rats of clean grade (aged 8-10 weeks, male and female unlimited) were divided into sham injury group, simple electrical burn group, electrical burn+ saline group, and electrical burn+ Xuebijing group according to the random number table, with 70 rats in each group. Rats in sham injury group were not conducted with electrical current to cause sham injury. Rats in the other three groups were given electrical current with output voltage of 2 kV and current intensity of (1.92 ± 0.24) A for 3 s, which caused high-voltage electrical burn wounds, each with an area of 1 cm×1 cm distributed in the left forelimb at the current inlet and the right hindlimb at the current outlet respectively. Rats in sham injury group and simple electrical burn group were not treated after injury. At post injury minute 2 and on post injury day (PID) 1, 2, 3, 4, 5, and 6, rats in electrical burn+ saline group and electrical burn+ Xuebijing group were intraperitoneally injected with 6 mL/kg saline and 6 mL/kg Xuebijing, respectively. Survival conditions of rats were recorded during the experiment. At 15 min before injury and at post injury hour (PIH) 1, 8, 24, 48, 72, and on PID 7, 10 rats in each group were respectively selected according to the random number table to sacrifice after collection of 5 mL blood under the direct vision of heart. Blood in the volume of 0.05 mL from each rat was taken to make blood smear, and platelet aggregation number was counted under 400 fold field of view using multiple projection microscope. The remaining blood samples were centrifuged to collect supernatant, and the content of platelet-derived growth factor (PDGF), thrombopoietin (TPO), and platelet activating factor (PAF) was detected by enzyme-linked immunosorbent assay. Data were statistically analyzed with analysis of variance for factorial design and Student-Newman-Keuls method. Results: All rats in sham injury group and simple electrical burn group survived during the experiment. One rat in electrical burn+ saline group died on PID 6, and one rat on PID 5 and one rat on PID 6 died in electrical burn+ Xuebijing group. The levels of all indexes among the 4 groups were close at 15 min before injury. The serum content of PDGF, TPO, and PAF and platelet aggregation number of rats in the three electrical burn groups at all time points after injury were higher or more than those in sham injury group, and the first three indexes reached the peak at PIH 8. The serum platelet aggregation number of rats in simple electrical burn group reached the peak at PIH 48, and that in electrical burn+ saline group and electrical burn+ Xuebijing group reached the peak at PIH 72. Among them, the serum content of PDGF of rats in electrical burn+ Xuebijing group at PIH 48, 72 and on PID 7 ((12.8±4.0), (11.6±4.4), (11.0±3.6) ng/mL, respectively) was close to that in sham injury group ((10.4±2.0), (10.4±2.5), (9.8±3.3) ng/mL, respectively, P>0.05). The serum content of TPO of rats in electrical burn+ Xuebijing group at PIH 24, 72 and on PID 7 ((200±52), (192±36), (193±32) ng/mL, respectively) was close to that in sham injury group ((182±30) , (184±41), (183±33) ng/mL, respectively, P>0.05). The serum content of PDGF, TPO, and PAF and platelet aggregation number of rats in electrical burn+ Xuebijing group at every time point after injury was generally lower or less than that in electrical burn+ saline group and simple electrical burn group. Conclusions: Application of Xuebijing treatment after high-voltage electrical burn can decrease the content of PDGF, TPO, and PAF in the serum and reduce the number of platelet aggregation, thereby inhibit platelet activation and improve platelet rheology.

[Study of de novo point mutations in known genes among patients with unexplained intellectual disability or developmental delay].

Objective: To analyze the de novo point mutations in known genes among patients with unexplained intellectual disability (ID) or developmental retardation (DD). Methods: A total of 120 outpatients with ID or DD were recruited in the Department of Neurology, Affiliated Children's Hospital of Capital Institute of Pediatrics between September 2015 and April 2017. Target gene sequencing was used to screen the candidate gene. The sequencing data were analyzed by a variety of bioinformatics software. Combining with the phenotypes of the patients, the candidate genetic/genomic variants were identified from next-generation sequencing data. The final pathogenicity of the genetic/genomic variants were interpreted according to the guideline of the American College of Medical Genetics and Genomics (ACMG) for variants after segregation analysis in the parents and necessary family members by Sanger sequencing. The comprehensive physiological function and signaling pathways of 20 disease genes with de novo point mutation discovery was also studied. Results: Among the 120 patients, 23 patients were found to carry clear pathological changes, and the incidence of de novo point variation was 19.2%. The patients included 12 males and 11 females, with an age of 2 months to 6-year-6-month. Five patients were diagnosed with early onset of epileptic encephalopathy. Seven had mental retardation type 5, 6, 8, 19, 20, 22, 39, respectively. Weill-Marchesani syndrome type 2 was found in one case, Wiedemann-Steiner syndrome in one case, Coffin-Siris syndrome in two cases, Rubinstein-Taybi syndrome in one case, GLUT1 deficiency syndrome in one case, Rett syndrome in one case, cardio-facio-cutaneous syndrome 3 in one case, neurodegeneration with brain iron accumulation in one case, corpus callosum local dysplasia in one case, and congenital fibrosis of the extra-ocular muscles in one case. A total of 20 novel mutations were reported in this study. No somatic mutation was found in the samples of 6 patients with mutation and their parents' peripheral blood DNA samples by amplicon-based deep sequencing. This study found that the main disease genes were involved in chromatin remodeling, transcriptional regulation, autophagy body assembly, MAPK signal pathway, DNA methylation, potassium, sodium ion transport, cell skeleton assembly and skeletal muscle development. These genes were significantly enriched in the following biological processes: Ras signaling pathways, transcription factor binding and cancer related signaling pathway. Conclusions: The etiology of children affected with intellectual disability or developmental delay is complex. Harmful de novo point mutation plays an important role in these diseases. Targeted exome/genome sequencing based on the core family is helpful for the molecular diagnosis of patients and the discovery of more genes.

[Clinical manifestation and gene analyses of 15 patients with intellectual disability or developmental delay complicated with congenital nystagmus].

Objective: To analyze the clinical and genetic features of 15 cases with intellectual disability or developmental delay (ID/DD) complicated with congenital nystagmus. Method: The clinical characteristics and the results of laboratory tests, images and genetics of 15 patients with ID/DD complicated with congenital nystagmus, confirmed by gene diagnosis in the Department of Neurology, Children's Hospital Affiliated to Capital Institute of Pediatrics from March 2015 to October 2016, were retrospectively analyzed. The physiological function of 13 disease genes and the molecular signaling pathways were also comparatively studied. Result: The patients included 11 males and four females, with an age of 2 months-15 years (median age 27 months). The result of multiplex ligation-dependent probe amplification was positive in two patients only with hypomyelination on head MRI. Positive results were found in 13 patients with or without abnormal head MRI or other deformities using targeted capture technology and next generation sequencing. Two patients were diagnosed with Pelizaeus-Merzbacher disease, two had hypomyelination with an atrophy of the basal ganglia and cerebellum and two had oculocutaneous albinism. Pelizaeus-Merzbacher-like disease was found in one case, cerebro-oculo-facio-skeletal syndrome in one case, Rubinstein-Taybi syndrome in one case, mental retardation type 5 in one case, methylmalonic aciduria combined with hyperhomocysteinemia in 1 case, ataxia telangiectasia in one case, hypomyelinating leukodystrophy type 8 in one case, Marinesco-Sjögren syndrome in one case and CHARGE syndrome in one case. A total of 12 novo mutations were reported in this study. Conclusion: The causes of children with ID/DD complicated with congenital nystagmusis are complex. Comprehensive clinical and auxiliary examinations should be performed to improve the accuracy of the diagnosis. Reasonable application of different genetic testing methods can significantly improve the diagnostic accuracy of molecular genetic etiology in children with ID/DD.

[Application of single nucleotide polymorphism-microarray and target gene sequencing in the study of genetic etiology of children with unexplained intellectual disability or developmental delay].

Objective: To evaluate the application of single nucleotide polymorphism (SNP)-microarray and target gene sequencing technology in the clinical molecular genetic diagnosis of unexplained intellectual disability(ID) or developmental delay (DD). Method: Patients with ID or DD were recruited in the Department of Neurology, Affiliated Children's Hospital of Capital Institute of Pediatrics between September 2015 and February 2016. The intellectual assessment of the patients was performed using 0-6-year-old pediatric examination table of neuropsychological development or Wechsler intelligence scale (>6 years). Patients with a DQ less than 49 or IQ less than 51 were included in this study. The patients were scanned by SNP-array for detection of genomic copy number variations (CNV), and the revealed genomic imbalance was confirmed by quantitative real time-PCR. Candidate gene mutation screening was carried out by target gene sequencing technology.Causal mutations or likely pathogenic variants were verified by polymerase chain reaction and direct sequencing. Result: There were 15 children with ID or DD enrolled, 9 males and 6 females. The age of these patients was 7 months-16 years and 9 months. SNP-array revealed that two of the 15 patients had genomic CNV. Both CNV were de novo micro deletions, one involved 11q24.1q25 and the other micro deletion located on 21q22.2q22.3. Both micro deletions were proved to have a clinical significance due to their association with ID, brain DD, unusual faces etc. by querying Decipher database. Thirteen patients with negative findings in SNP-array were consequently examined with target gene sequencing technology, genotype-phenotype correlation analysis and genetic analysis. Five patients were diagnosed with monogenic disorder, two were diagnosed with suspected genetic disorder and six were still negative. Conclusion: Sequential use of SNP-array and target gene sequencing technology can significantly increase the molecular genetic etiologic diagnosis rate of the patients with unexplained ID or DD. Combined use of these technologies can serve as a useful examinational method in assisting differential diagnosis of children with unexplained ID or DD.

[Diagnostic value of serum copper/zinc ratio in gynecologic tumors].

Serum copper/zinc levels were determined in 39 patients with gynecologic tumors and 20 normal individuals. Of these 39 patients, twenty-two had primary malignant tumors and seventeen had benign growths. All the patients were divided into two groups: treated and untreated. The serum Cu/Zn ratio in the untreated benign tumors was significantly higher than of the normal controls (P less than 0.05). The ratio was much higher in the untreated Stage II-IV malignant tumors than that of the benign group (P less than 0.01). The measurement of serum Cu/Zn ratio is also helpful in observing the clinical course of the disease.