Objective To devolope a method for extracting DNA from dried blood spots (DBS)and optimizing the operating procedure,which could be applied to clinical gene diagnosis of thalassemia.And the cross contamination of DBS punching and the storage stability of DBS were studied.Methods A total of 1 50 blood specimens were collected,and DBS were prepared.Circles (3 mm in diameter)were punched in the DBS,and eluted with lysis buffer.The eluting method and operating procedure were opti-mized.Genomic DNA extracted from the elution solution by magnetic beads,and were performed thalassemia gene test.Finally jud-ging whether the results of DBS and whole blood were consistent.Two methods of thalassemia gene test were used in DBS and the compatibility of DBS processing method was verified.Judging whether there was cross contamination of DBS punching by the thalassemia gene test results of blank hole which were punched in the blank filter paper between thalassemia positive DBS.The DBS storage stability in thalassemia gene test was verified by detecting the DBS which were dry stored at room temperature for 6 and 9 months.Results 5 circles (3 mm in diameter)DBS were vibrating eluted at 55 ℃ for 1 hour,the DNA concentration extracted from the elution solution was 10-20 ng/μL,which was dissolved in 50 μL solution,and the DNA quality was good.The thalassemia gene test results of DBS and whole blood were the same,and the DBS results of two thalassemia gene test methods were the same too. The cross contamination of DBS punching was not detected in thalassemia gene test.The DBS which were dry stored at room tem-perature for 6 and 9 months could be stably performed thalassemia gene test.Conclusion DBS could be used to perform thalassemia gene test,which is accurate,convenient and stable.It is an ideal way for specimen referral of thalassemia gene test.

Objective: To explore the associations between parental marital status with bullying and self injurious behavior among primary and secondary school students, and to provide intervention support for the prevention of self injurious behavior of primary and secondary school students. Methods: A total of 11 107 primary and secondary school students in Nanyang, Anyang and Xinxiang city from central China were selected using multistage clustering sampling method. A questionnaire survey regarding bullying was administered. Results: Report rate of bullying in boys (18.1%) was higher than that in girls (9.8%), while report rate of self injurious behavior in girls (3.9%) was higher than that in boys(3.2%)( χ 2=155.56, 4.64, P <0.05). The severity of bullying ( r =0.44) and types of bullying ( r =0.42) were positively correlated with self injurious behaviors( P <0.01), while parental marital status was negatively correlated with self injurious behavior( r=-0.11, P <0.01). Parental marital status negatively moderated the relationship between severity of bullying( β =-0.01), types ( β =-0.05) with self injurious behavior( P <0.01). Conclusion Parental marital status plays a moderating role in the association between bullying and self injurious behavior among primary and middle school students.The lower parental marital status, the higher rate of self injurious behavior among bullied children. Comparison of bullied rates among primary and secondary school students with different characteristics.

Objective: To analyze epidemiological characteristics of campus bullying among primary and middle school students in central China to explore its relation with mental health problems, and to provide a reference for the campus bullying prevention. Methods: Stratified cluster sampling method was used to select primary and middle school 10 581 students from Anyang, Nanyang and Xinxiang cities of Henan Province, Middle School Students Mental Health Scale and the Self designed Scale of Adolescent Bullying Behavior were used to analyze the relationship between mental health problems with campus bullying behavior. Results: The total report rate of bullying penetrator was 12.5% among students in the three cities. Among primary and middle school students with mental health problems such as hostility, interpersonal stress, academic pressure and emotional imbalance, the detection rate of bullying behavior was 24.2%, 20.3%, 19.4% and 20.1%, respectively. The results of multivariate analysis showed that hostility symptoms ( OR =3.78, 95% CI =1.71-8.32), interpersonal stress ( OR =3.50, 95% CI = 1.62 -7.57), academic pressure ( OR = 1.62 , 95% CI =1.21-2.16) and emotional imbalance ( OR =2.80, 95% CI =1.41-5.56) showed a significant impact on campus bullying ( P <0.05). Conclusion Mental health problems of primary and middle school students are closely related to the occurrence of bullying behavior. It is necessary to pay attention to the mental health education of bullies and intervene bullying behaviors from the source.

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.
Swine ; Animals ; Induced Pluripotent Stem Cells/metabolism* ; Receptors, Cell Surface/genetics* ; Antigens, CD/metabolism* ; Porcine respiratory and reproductive syndrome virus/genetics*

Female ; Humans ; Mutation ; Pregnancy ; Prenatal Diagnosis ; alpha-Globins ; genetics