Objective To investigate the feasibility of DNA sequencing analysis in molecular diagnosis for spinal muscular atrophy (SMA). Methods Two pairs of primers were utilized to amplify the region including 5 different bases in SMA-causative gene SMN1 and its homologue copy SMN2 by polymerase chain reaction (PCR). The first primer amplified a fragment 501 bp long spanning from SMN intron 6 to intron 7 targeting four different bases (g.31957, 32006, 32154 and 32269). The second primer reversely amplified a 189 bp long fragment within SMN exon 8 including one base-pair differ-ence (g.32734). PCR procedure was followed by Sanger sequencing technique to identify the 5 different bases. SMA patients caused by SMN1 homozygous deletion were distinguished from carriers or normal controls by absence of SMN1 specific bas-es in sequence chromatograms. This assay was performed in 7 SMA suspected patients and their parents. The specimens were also detected by PCR- restriction fragment length polymorphism (RFLP) method. Results It was found that 6 of 7 SMA suspected patients showed only SMN2 specific bases at the 5 different base positions among the region from intron 6 to exon 8, which meant the patient displaying only SMN2-specific nucleotide a, T, g, g and A at g.31957, 32006, 32154, 32269 and 32734, while their parents (carriers) showed a/g, T/C, g/a, g/a and A/G at the same sites. SMN1 gene was deleted in the patient, and the deletion region was inferred from intron 6 to exon 8. Because carriers had both SMN1 and SMN2 genes, they can be discriminated from the SMN1 deleted patient. One of 7 patients yield an unique sequence chromatogram of a, T, g, g and A/G, indicating that exon 8 of SMN1 was not deleted in this patient. Conclusion DNA sequencing analysis is an alter-native simple method for detecting SMA caused by homozygous deletion of SMN1. We recommend to replace the widely used PCR-RFLP method with DNA sequencing assay.

The effects of barium chloride(BaCl2)on several items of the immune functions of mice were observed.After the mice had drunk water containing 10 mg/L and 100 mg/L of BaCl2 for one month,evident histological changes of their liver were observed but no remarkable changes were found in the spleen,the lymph nodes,the thymus,the bone marrow and the peripheral leucocytes.In the 100 mg/L group,the transformation rate of the ConA-stimulated lymphocytes of the spleen was significantly lower than that of the control(P

Objective To explore the changes in inositol requiring enzyme 1 (IRE1),apoptosis signal regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) mRNA levels in peripheral blood CD4+ T cells of children with acute paraquat (PQ) poisoning.Methods Blood samples of 30 cases of acute PQ poisoning (PQ group),who visited Tianjin Children's Hospital from June 2014 to June 2016,with 18 male and 12 female,aged from 2 to 14 years old,were collected,and the clinical and laboratory data were documented.Peripheral venous blood samples were collected after paraquat was taken.Thirty healthy children at the same age and of the same sex were selected as a healthy control group,18 male and 12 female,aged from 2 to 14 years old.CD4+ T cells in the peripheral blood were separated,and IRE1,ASK1 and JNK mRNA levels in peripheral blood CD4+ T cells were measured by real time polymerase chain reaction (Real-time PCR) method.Specificity of PCR products was validated through agarose gel electrophoresis.The data were statistically analyzed by SPSS 13.0 software.Results All of the 30 children had mucosal lesions,nausea,vomiting and abdomen pain,19 cases with oliguria and anuria,16 cases with alimentary tract bleeding,12 cases with headache and dizziness,11 cases with short of breath,dyspnea and difficult breathing,8 cases with convulsion,5 cases with jaundice.The IRE1,ASK1 and JNK mRNA levels in PQ group were significantly higher than those in healthy control group (1.70 ± 0.16 vs.1.02 ± 0.18,3.56 ± 0.85 vs.1.05 ± 0.31,5.22 ± 0.87 vs.1.01 ± 0.33,t =15.26,15.21,24.78,all P ＜ 0.01).Conclusions PQ increased the expressions of IRE1,ASK1 and JNK in peripheral blood CD4+ T cells,which may be related to PQ-induced oxidative stress and immune activation and lead to a complex cytokine network via endoplasmic reticulum stress and CD4+ T cell apoptosis and then results in the occurrence and development of multiple organ failure.

Objective To explore the feasibility of using composite scaffolds of rabbit oral epithelial cells and polycaprolactone (PCL) electrospun fibers for urethral repair. Methods The 25%PCL was prepared using a 5:1 by volume mixture of trichloromethane and anhydrous methanol, and PCL fiber tubular scaffolds were obtained by electrospinning. Rabbit oral mucosa epithelial cells (1.5 × 105) were implanted on the PCL scaffold. Subsequently, they were embedded in nude mice subcutaneous, explanted in 2 weeks. PCL fiber tubular scaffolds without rabbit oral mucosa epithelial cells were used as control. The complex urethral scaffolds were evaluated by immunofluorescence staining with cytokeratin antibody and HE staining. Results Compared with blank PCL group, the rabbit oral mucosa epithelial cell group showed a good cellularization. Rabbit oral mucosa epithelial cells formed a dense cell layer on the surface of PCL lumen, which suggested that rabbit oral mucosa epithelial cells can proliferate on the surface of PCL lumen. Conclusion Rabbit oral epithelial cells can be used as one of the seed cells for tissue engineered urethral scaffolds, and it is possible to construct tissue engineering substitute materials for urethral repair by rabbit oral epithelial cells combined with PCL.

Objective:To explore the cytogenetic characteristics of hypospadias in children by karyotype analysis.Methods:From June 2008 to May 2018, 45 children with hypospadias in Tianjin Children's Hospital had cytogenetic abnormalities. Their median age was 10 months(range 3 hours to 5 years old). Of the 45 cases, 20 were proximal hypospadias, 1 was middle hypospadias. All 24 cases had varying degrees of genitourinary malformations. Among them, 15 cases had unilateral or bilateral cryptorchidism, 5 cases had scrotal division, 3 cases had penile scrotal transposition, 3 cases had small penis, 3 cases had indirect inguinal hernia, 1 case had repeated urethra, 1 case had hydrocele and 1 case had concealed penis. To the other systemic malformations, there was 1 with cleft lip and palate and 1 with congenital heart disease. G-banding karyotype analysis of peripheral blood lymphocytes was performed in all 45 cases.Results:Among the 45 cases of hypospadias with abnormal karyotypes, with an abnormal rate of 14.0%, 28 cases (62.22%) had sex chromosome abnormalities, including (47, XXY), (46, XX/47, XXY), (45, X0/47, XYY), etc. Sexual inversion occurred in 8 cases (17.78%), all of which were 46, XX. There were 4 autosomal abnormalities (8.89%), including (46, XY, 9p+ ), (46, XY, 10p+ ) and (46, XY, 1q+ ). Chromosome polymorphism was found in 4 cases (8.89%), including [46, XY, inv(9)] and [46, XY, 16qh+ ], and the equilibrium translocation of 1 case (2.22%) was [45, XY, -21, -22, + t(21; 22)]. Among the 45 cases, 8 sex reversal children with (46, XX) chromosome karyotype were all proximal hypospadias.Conclusions:Children with hypospadias may be associated with chromosomal karyotype abnormalities, including sex chromosomal abnormalities, autosomal abnormalities, chromosome polymorphism and balanced translocation. Among them, sex chromosome abnormality was the most common and balanced translocation was the least.

Objective To observe the lung protection of Astragalus membranaceus against radiotherapy to intermediate-stage and terminal thoracic neoplasm, and its influence on TNF-α and ET expression.Methods The patients with intermediate-stage and terminal thoracic neoplasm under radiotherapy were divided into a treatment group and a control group.Patients in the treatment group took 10 ml of Asragalus membranaceus twice a day.for consecutive 6 months from the beginning of radio therapy.TNF-α and ET in the plasma were measured before and after the radiotherapy.The clinical symptom,iconographic changes and lung diffusion were observed from the 15th day of radiotherapy.Results The TNF-α and ET in plasma afterthe radiotherapy were(2.48±0.75)as/ml and(69.32±23.03)pg/ml for the treatment group,and(5.12±1.01)ns/ml and(97.87±37.83)pg/ml for the control group with the statistial difference(x2=7.49,6.57,P<0.001).The decrease of CO diffusion 5 and 10 months after the radiotherapy in the treatment group was statistically different compared with that in the control group(x2=3.98,3.78,P<0.05).There was a statistical difference of the incidence of acute radiation pneumonitis and pulmonary fibrosis between these two groups(P<0.05).Conclusions Astragalus membranaceus could inhibit the excess expression of TNF-α and ET in plasma and reduce the deterioration of diffusion after radiotherapy,so that it can be used for intervention of lung injuries from radiotherapy.

Objective To analyze the clinical and OTC gene mutation characteristics of 1 case with ornithine transcarbamylase deficiency (OTCD) and to deepen the understanding of OTCD.Methods One case of 14-month female OTCD patient was analyzed.Clinical data of the child patient was collected and venous blood 2 mL from the patient and her parents was extracted respectively.Polymerase chain reaction was used to amplify the fragment where various exon of OTC and its neighboring intron were distributed, followed by direct sequencing to detect mutation.Results It was showed that late-onset OTCD child patient had contracted the disease for 3 months,with intermittent drowsiness, vomiting and psychomotor development regression.Cerebellar ataxia was the main symptom of the child patient when she was taken to Tianjin Children's Hospital.According to brain MRI, the lesion was severe.Blood chemistry showed mild hepatic lesion and increased blood ammonia.According to urine gas chromatography mass spectrometry analysis,there was a rise in uracil and orotic acid.OTC genetic testing showed the child patient and her mother were in the 8th exon,c.852C ＞ G (p.Y284X).Missense mutation occurred in this locus.The mother had normal phenotype.Conclusions Clinically OTCD has the symptoms of hyperammonemia and the resulting in varying degrees of damage to the nervous system and the liver.Without clinical specificity, this disease is easy to be misdiagnosed.Methods like blood ammonia and urine metabolic disease screening, blood amino acid analysis and genetic testing help confirm the disease earlier.As for treatment, early intervention and chronic control of blood ammonia level to guard against hyperammonemia will lead to better curative effect.

Objective To evaluate the efficacy of self?made breathing circuit joint for intermittent positive pressure ventilation ( IPPV) in patients with central airway obstruction undergoing interventional fi?beroptic bronchoscopy ( FOB) . Methods Sixty?two patients of both sexes with central airway obstruction requiring tracheal intubation, aged 60-80 yr, with body mass index of 20-26 kg∕m2 , of American Society of Anesthesiologists physical status Ⅲ or Ⅳ and Medical Research Council dyspnea scale grade Ⅲ or Ⅳ, undergoing interventional FOB under general anesthesia, were divided into 2 groups ( n=31 each) using a random number table:high frequency jet ventilation ( HFJV) group and IPPV group. The patients were tra?cheally intubated after induction of general anesthesia. The self?made breathing circuit joint was connected, then the anesthesia machine was connected to perform IPPV, and the ventilator settings were adjusted to maintain the end?tidal pressure of carbon dioxide 35-45 mmHg in group IPPV, and HFJV was used in group HFJV. Before induction ( baseline) , at 10, 20, 30 and 40 min after start of operation, and at the end of operation, arterial blood samples were collected for blood gas analysis, the pH value, arterial oxy?gen partial pressure, and arterial carbon dioxide partial pressure were recorded. The development of hyper?capnia was recorded. Results Hyoxemia was not found in the two groups. The incidence of hypercapnia was 74%, and in addition the incidence of severe hypercapnia was 10% in group HFJV. The incidence of hypercapnia was 16%, and all the patients presented with permissive hypercapnia in group IPPV. Com?pared with group HFJV, the incidence of hypercapnia was significantly decreased, and the pH value and arterial oxygen partial pressure were increased, and arterial carbon dioxide partial pressure was decreased from 10 min after start of operation to the end of operation in group IPPV (P<0.05). Conclusion The self?made breathing circuit joint provides better efficacy than HFJV when used for IPPV in the patients with central airway obstruction undergoing interventional FOB.

Objective:To explore the relationship between the type of mutation and clinical features, prognosis, and clinical characteristics of chronic granulomatous disease (CGD) caused by compound heterozygous mutations in the NCF2 gene in children. Methods:The clinical data of 1 case of neonatal CGD caused by compound heterozygous mutations of NCF2 gene at Tianjin Children′s Hospital in August 2019 was analyzed, and domestic and international literatures were searched to summarize the clinical characteristics, gene mutation type and prognosis of CGD caused by NCF2 mutation. Results:The diagnosis of CGD was confirmed by the presence of compound heterozygous mutations c. 196_197insA (p.Arg66Glnfs23X) and c. 1180T>G (p.Tyr394Asp) in the NCF2 gene, accompanied with the clinical manifestations of fever, cough, multiple clumps and nodules in the chest CT at 25 days after birth, and the neutrophil respiratory burst test stimulation index(SI) 23.This new mutation was not reported in the Human Genetic Mutation Database.The child had a residual portion of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and was followed up until the age of 9 months with an antifungal drug without recurrent infection.A total of 101 cases of CGD patients with NCF2 gene mutation were reported in domestic and international databases.Totally, 33 cases had SI results, with 22 cases below 3, 11 cases above 3, and 8 cases of missense mutations. Conclusions:c. 196_197insA and c. 1180T>G are new mutations in NCF2 gene that can lead to CGD.CGD patients containing missense mutations in the NCF2 gene may have more residual NADPH oxidase activity.

Objective To explore pathogenic composition of hand,foot and mouth disease(HFMD),and the molecular typing of enterovirus in Tianjin Children′s Hospital in 2016.Methods Single center study.A total of 327 samples of HFMD cases which collected from Tianjin Children′s Hospital from March to November in 2016 were tested for nucleotide acid of enterovirus(EV),human enterovirus 71(EV71),Coxsackievirus A 16(CA16)by real-time reverse transcription polymerase chain reaction(RT-PCR),among which 104 sample of other EV positive were selected to amplify and sequence the whole VP1 region by using RT-PCR.Homology was analyzed and phylogenetic tree were constructed by comparison of the sequence with all subgenotype of EV by Chromas1.62 and MEGA6.06.EV positive rate in different age groups were compared by SPSS20.0.Results Of all the 327 HFMD cases tested,there were 272 EV positive cases,the constituent ratio of EV71,CA16 and other EV were 55.1％(150/272),6.6％(18/272)and 38.2％(104/272)respectively.The EV positive rates of different age groups(69.0％-90.9％)were different significantly(x2=15.897,P=0.044),the 3 years-old-group had the highest EV positive rate than that of the other age groups(90.9％,40/44).Of all 104 samples of other EV tested,34 were CA10(12.5％)and 14 were CA6(5.1％).Phylogenetic analysis of 11 EV71 VP1 and 20 CA10 VP1 showed that the EV71 and CA10 strains belonged to genotype C4a and genotype G,respectively.ConclusionsEV71 was still the predominant pathogen in spite of the constituent ratio of other EVs increased markedly in 2016 in Tianjin.It is important to enhance etiological monitoring for control and prevention of HFMD.