BACKGROUND:Platelet-derived growth factor-B (PDGF-B) is an effective pro-angiogenic growth factor, and adeno-associated virus type 9 (rAAV9) has a strong cardiomyocyte targeting affinity, which is an ideal vehicle for ischemic heart disease gene therapy.
OBJECTIVE:To explore the PDGF-B gene transfection of in vitro neonatal rat myocardial cells mediated by rAAV9 against ischemia and hypoxia-induced cardiomyocytes apoptosis.
METHODRat neonatal myocardial cells were isolated and cultured, and then transfected by rAAV9-PDGF-B and empty virus, rAAV9 with enhance green fluorescent protein (eGFP), under multiplicity of infection (MOI) of 105, 106 and 107, respectively. We observed the expression of eGFP under fluorescence microscopy every day, and used flow cytometry to measure transfection efficiency of vector rAAV9. Western blot and immunofluorescence were used to evaluate protein expression of PDGF-B. Myocardial ischemia and hypoxia injury model was established in vitro on the 5th day of transfection of rAAV9-eGFP and rAAV9-PDGF-B with 107 MOI. The number of myocardial apoptosis was measured by TUNEL assay. Western blot was employed to detect the protein expression of Bax and Caspase-3 which were related apoptosis, and the effect and its possible mechanism of PDGF-B gene overexpression against myocardial apoptosis were explored.
RESULTS AND CONCLUSION:rAAV9 vector can efficiently transfect neonatal rat myocardial cells. eGFP and PDGF-B protein expressed in myocardial cells correctly and efficiently, and the expression intensity increased gradual y with the increasing of time course and MOI. The expression became stable on the 5th day, and the transfection efficiency showed significant difference among these groups (P<0.01). Myocardial apoptosis rate was significantly reduced in the rAAV9-PDGF-B group than the rAAV9-eGFP group (P<0.05), and protein levels of Bax and Caspase-3 in the rAAV9-PDGF-B group were significantly lower than those of the rAAV9-eGFP group (P<0.05). These data indicate that overexpression of PDGF-B gene can effectively reduce ischemia and hypoxia-induced myocardial apoptosis, and the possible mechanism might be by inhibiting Bax and Caspase-3 protein expression, which can provide evidence of rAAV9-PDGF-B vector in the gene therapy of ischemic heart diseases.

BACKGROUND:A lot of work has been carried out on the development of the primary cultured rat myocardial cel s at home and abroad. The primary culture technology of rat myocardial cel s becomes more mature, but myocardial cel s from neonatal mice are not easy to be obtained under the same experimental conditions. The mouse genome has more similarities with the human genome, which has a higher research value. OBJECTIVE:To improve the primary culture method of neonatal mouse myocardial cel s, and to obtain myocardial cel s with high purity, vitality and original structure and function. METHODS:The mouse cardiac tissues were treated using an enzyme digestion method to isolate isolated single myocardial cel s:first, the cardiac tissues were digested using trypsin, and then col agenous fibers were treated with col agenase to isolate single myocardial cel s. The concentration and action time of trypsin and type II col agenase were adjusted, and the pH values of reagents and temperature of each step were strictly control ed. RESULTS AND CONCLUSION:At 24 hours after inoculation, the myocardial cel s began to be adherent;at 48 hours, independent pulsation of myocardial cel s could be observed;at 72 hours, myocardial cel s were cross-linked;and at 96 hours, myocardial cel s formed cel clusters and presented with consistent beating. The survival rate and purity of myocardial cel s were both over 95%. This modified method could successful y culture myocardial cel s with high purity and viablility from neonatal mice, and the structure and function of myocardial cel s could be retained. Therefore, it is a feasible culture method.

Objective To evaluate the effects of dexmedetomidine on hypoxic pulmonary vasoconstriction and oxygenation during one- lung ventilation (OLV) undergoing esophagectomy. Methods Fifty-six adult patients undergoing esophagectomy and requiring OLV were selected.During inhalational anesthesia with sevoflurane, patients were randomized to receive either dexmedetomidine (dexmedetomidine group,28 patients)or saline placebo(control group,28 patients). The bolus dose of 0.3 μg/kg over 10 min followed by a maintenance dose of 0.6 μg/(kg·h)was used in dexmedetomidine group. The arterial blood gas samples were obtained to evaluate the effects of dexmedetomidine on oxygenation in three times:T1:double-lung ventilation 10 min after anesthetic intubation;T2:OLV 10 min;T3:60 min after continuous infusion of dexmedetomidine. Outcomes included differences in hemodynamic parameters(heart rate and mean arterial pressure), end-tidal sevoflurane concentration, ephedrine dose and atropine dose.Results The levels of pH, arterial partial pressure of carbon dioxide(PaCO2)in two groups had no significant differences(P>0.05).The level of oxygenation index in two groups at T3had significant difference: (153.29 ± 19.00) mmHg(1 mmHg=0.133 kPa)vs. (117.79 ± 12.00) mmHg, 1 mmHg = 0.133 kPa, P < 0.01. At T3, the level of heart rate in dexmedetomidine group was significantly lower than that in control group:(68 ± 11)times/min vs.(89±13)tims/min;meanwhile, the level of end-tidal sevoflurane concentration in dexmedetomidine group was significantly lower than that in control group: (2.9 ± 0.8)% vs. (4.2 ± 0.1)%; there were significant differences (P < 0.01). The ephedrine dose in two groups had no significant difference(P>0.05).Conclusions Dexmedetomidine may provide clinically relevant benefits by improving oxygenation and decreasing the requirement of inhalational anaesthetic agents, thereby limiting its effect on hypoxic pulmonary vasoconstriction during OLV in adults undergoing esophagectomy surgical procedures.

Objective To explore the alteration of the peripheral thyroid hormone level in patients with chronic alcohol dependence and its relationship with depressive emotion.Methods Data were collected from 21 male patients with chronic alcohol dependence (case group) and 21 male health control (control group).Hamilton Depression Scale-24 (HAMD-24) and Alcohol Dependence Screening Scale (MAST) were used.The level of thyroid stimulating hormone (TSH),free triiodothyronine (FT3),free thyroxine (FT4),triiodothyronine (T3) and four iodine thyroxine (T4) of case group and control group were separately determined.The level of above-mentioned hormones and scale score of case group were evaluated in the first and sixth week of the period of temperance.Data of control group were collected once at baseline.Results The level of FT3 in case group was decreased significantly in the first week of the period of temperance compared with the control group ((4.34± 1.01) pmol/L,(5.85 ± 0.78) pmol/L,t =5.56,P＜ 0.01),while the level of TSH in case group was increased((2.62±0.69) uIU/ml,(1.70±0.81)uIU/ml,t=-3.96,P＜0.01).The level of FT3 and T3 in case group decreased significantly in the sixth week of the period of temperance comparedwith the control group(FT3:(5.22±0.92) pmol/L,(5.85±0.78) pmol/L,t=2.45,P＜ 0.05;T3:(1.54±0.54)pmol/L,(1.84±0.34)pmol/L,t=2.92,P＜0.01).The level of FT3 of the case group in the first week decreased significantly compared with the sixth week in the period of temperance (t=-4.12,P＜0.01),while the level of FT4 were increased significantly (t=-3.93,P＜0.01).In case group,the scores of HAMD-24 and the level of T3 (r=-0.465,P＜0.05),TSH (r=-0.489,P＜0.05) were negative correlation in the first week of the period of temperance.No correlation were observed between the score of HAMD-24 and the level of FT3,FT4,TSH,T3,T4 (r=-0.303-0.047,all P＞ 0.05)in the sixth week of the period of temperance.Conclusion The level of serum thyroid is disordered in patients with chronic alcohol dependence.The disorder will recover in the period of temperance and is associated with depressive emotion.

Objective To explore the integration of the digital imaging medical records system and the hospital information system ( HIS), for a hospital-wide information platform for digital medical records management. Methods All the medical records were classified as " to copy and not to copy" by sampling, and an item corresponding table was developed for both HIS medical record files and imaging records copying types. The imaging copying system for paper-based medical records only covers those must-copy ones, while those medical record files without need to be copied were directly transcoded via the interface to the imaging medical records system from the HIS system. This makes digital imaging medical records complete. Results The digital imaging medical record system is thus integrated, and the cost of making imaging medical records was sizably reduced without compromising the quality and integrity of medical records. Conclusions Imaging medical records produced by copying paper-based ones are integrated with those directly sent via the interface to the imaging medical records system, forming complete digital imaging records, at a much lower cost.

Objective To investigate the relationship of programmed death ligand-1 (PD-L1) with clinicopathological characteristics,radiosensitivity and prognosis of the patients with esophageal squamous cell carcinoma(ESCC). Methods Ninety ESCC patients who received radical radiotherapy diagnosed by ESCC in Affiliated Hospital of North Sichuan Medical College were enrolled. Twenty cases of normal esophageal mucosa were used as the controls. The expression of PD-L1 was detected by using immunohistochemical SP method. Results The expression of PD-L1 protein was not correlated with age, gender, the maximum diameter of tumor, the length of lesion, the local aggressive of tumor, clinical stage and primary tumor volume (all P> 0.05). However, it was statistically correlated with the lymphatic metastasis (χ2= 4.404, P= 0.036). Meanwhile, PD-L1 positive expression was sensitive to radiation(χ2=4.888, P< 0.05). Single factor analysis showed that the maximum diameter of tumor and radiosensitivity were correlated with progression-free survival (PFS) (χ2=6.239,P =0.013;χ2=6.852,P =0.008; χ2= 6.312, P= 0.012) and overall survival (OS) (χ2=8.170, P = 0.004; χ 2= 4.261, P = 0.039; χ2= 5.003, P= 0.025) of ESCC patients. Multifactor analysis showed that the radiosensitivity and the maximum diameter of tumor affected PFS (OR= 0.512, 95 % CI 0.275-0.954, P= 0.035) and OS (OR= 0.507, 95 % CI 0.266-0.968, P= 0.039) in ESCC patients, respectively.Conclusions The level of PD-L1 expression is increased significantly in ESCC tissues compared with the normal esophageal mucosa tissues. PD-L1 may be a novel biomarker for predicting metastatic potential and radiosensitivity in ESCC patients, rather than the prognosis predictors of ESCC patients treated by radiotherapy.

Objective: To explore the relationship among osteopontin(OPN), Interleukin-17A(IL-17A), anti-MBP auto-antibody and autism spectrum disorder(ASD), and to provide the theoretical basis for the etiology and pathogenesis of ASD. Methods: Forty autistic children and forty matched healthy children were enrolled in this case-control study. The levels of OPN, IL-17A, anti-MBP autoantibody in serum were measured by enzyme-linked immunosorbent assay (ELISA). The associations between those metabolic levels and the severity and intelligence of ASD children were performed by Pearson or Spearman correlation. Results: Children with ASD had higher serum levels of OPN, IL-17A [(296.89±162.95),0.93] pg/mL compared to healthy control[(217.98±113.39), 0.62] pg/mL(P<0.05). Serum OPN, IL-17A, and anti-MBP auto-antibody levels in ASD group were not correlated with the scores of ABC, CARS, and PPVT(P>0.05). However, anti-MBP auto-antibodies level in children with ASD were positively correlated with OPN and IL-17A levels, respectively(r=0.35, 0.34, P<0.05). Conclusion It was obvious that the ASD children were found with neuroimmunologic abnormality, and the underlying mechanism needs to be further explored.

Objective: To understand the status of doctor visit, rehabilitation and foster care of children with autism spectrum disorders in Heilongjiang Province, and to provide a scientific reference for improving ASD rehabitation education system and making the related policies. Methods: Eight autism rehabilitation institutions were selected in Heilongjiang Province by stratified cluster sampling, 357 primary caregivers of ASD children participated in the survey by using the questionnaire "ASD Children’s Rehabilitation Education Status and Needs". Results: The average age of abnormal behavior found of ASD children was (31.08±12.96)months, and the average age of first doctor visit was (35.88±13.20) months, the average age of diagnose was (38.64±13.20) months, and initial rehabilitation was (43.56±16.08)months. The proportion of children who had been diagnosed and have trained in the rehabilitation institutions before the age of 3 years was only 39.0% and 32.0%, respectively. The proportion of rehabilitation out of home town was 47.3%, and rehabilitation >20 hours per week was 73.4%. The proportion of fathers’ and mothers’ work lives affected was 34.5% and 67.8%, respectively, the differences were of statistical signficance(χ2=226.32, P<0.01). About 41.2% of ASD families received government financial support. Conclusion The average age of diagnose is late prolonged, and the proportion of children diagnosed and training before the age of 3 years was relatively low. There were obvious regional differences of rehabilitation education resources distributions of ASD in Heilongjiang Province. The results also demonstrate the need to sustain and enhance the coverage rate of the government financial support.

OBJECTIVEThe present study describes an improved in vitro culture method for obtaining high purity, vital and fully functional cardiomyocytes from neonatal rat.

METHODSAfter cutting ventricular tissue with improved method, ventricular tissues were digested with low concentrations of trypsin overnight at 4 °C, and then underwent collagenase II digestion. Thereafter, cardiomyocytes were purified by combined differential adhesion and chemical inhibition methods.

RESULTSAdherent cardiomyocytes were seen at 12 h after culture, spontaneously beating cardiomyocytes were observed at 24 h after culture, crosslinked cardiomyocytes were found at 48 h after culture, adhesion clustered cardiomyocytes were seen at 72 h after culture, dense network formed from inter-connected was evidenced together with radial arranged cell clusters and cell pseudopodia 96 h the mutual contact woven into and formed radically ordering cell clusters and island-like beating cardiomyocytes at 96 h after culture. The cell survival rate and purity were more than 98%.

CONCLUSIONFully functional spontaneous beating cardiomyocytes can be obtained by the use of this improved primary neonatal rat cardiomyocytes culture method.

OBJECTIVE: To analyze a child with 11β hydroxylase deficiency (11β-OHD) due to CYP11B2/CYP11B1 chimeric gene. METHODS: Clinical data of the child who was admitted to Henan Children's Hospital on August 24, 2020 were retrospectively analyzed. Peripheral blood samples of the child and his parents were collected and subjected to whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing. RT-PCR and Long-PCR were carried out to verify the presence of chimeric gene. RESULTS: The patient, a 5-year-old male, had featured premature development of secondary sex characteristics and accelerated growth, and was diagnosed with 21 hydroxylase deficiency (21-OHD). WES revealed that he has harbored a heterozygous c.1385T>C (p.L462P) variant of the CYP11B1 gene, in addition to a 37.02 kb deletion on 8q24.3. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.1385T>C (p.L462P) was rated as a likely pathogenic variant (PM2_Supporting+PP3_Moderate+PM3+PP4). The results of RT-PCR and Long-PCR suggested that CYP11B1 and CYP11B2 genes have recombined to form a CYP11B2 exon 1~7/CYP11B1 exon 7~9 chimeric gene. The patient was diagnosed as 11β-OHD and effectively treated with hydrocortisone and triptorelin. A healthy fetus was delivered following genetic counseling and prenatal diagnosis. CONCLUSION 11β-OHD may be misdiagnosed as 21-OHD due to the potential CYP11B2/CYP11B1 chimeric gene, which will require multiple methods for the detection.
Child, Preschool ; Humans ; Male ; Adrenal Hyperplasia, Congenital/genetics* ; Cytochrome P-450 CYP11B2/genetics* ; Exons ; Retrospective Studies ; Steroid 11-beta-Hydroxylase/genetics*