Objective To construct HaCaT cell lines stably expressing the wild type human GJB6 gene or its mutant by using a Tet-On lentiviral vector, and to lay an experimental foundation for studies on pathogenesis of hidrotic ectodermal dysplasia. Methods The wild-type human GJB6 gene and its mutant (A88V)were amplified by PCR, and then inserted into the Tet-on lentivirus plasmid to construct recombinant lentivirus vectors. The recombinants were identified by gene sequencing and enzymatic digestion. Cultured HaCaT cells were classified into three groups to be transfected with a negative control lentiviral vector (NC group), the lentivirus vector expressing the wild-type human GJB6 gene (WT group), or the lentivirus vector expressing the mutant human GJB6 gene (MU group). Puromycin was used to select HaCaT cell clones stably expressing the GJB6 gene which encodes the connexin 30 (Cx30)protein. The selected HaCaT cell clones were cultured with or without tetracycline for 48 hours, thereafter, real-time PCR(RT-PCR) was performed to detect GJB6 gene mRNA expression, Western-blot analysis to measure expressions of Cx30 and FLAG-tag proteins, and cell counting kit 8 (CCK8)assay to evaluate cellular proliferative activity. Results Enzymatic digestion and gene sequencing showed that recombinant lentivirus plasmids were successfully constructed. RT-PCR showed evidently increased mRNA expression of the GJB6 gene in stably transfected HaCaT cells. Moreover, the expression abundance of the GJB6 gene was 112.369 times higher in the WT group induced by tetracycline than in that without tetracycline treatment (P < 0.05), and 2.249 times higher in the MU group induced by tetracycline than in that without tetracycline treatment (P < 0.05). Western-blot analysis showed that Cx30 and FLAG-tag proteins were stably expressed in the WT group and MU group after induction with tetracycline, while neither of them was observed in the WT group or MU group without tetracycline treatment, or in the NC group. Significant differences were noted in cellular proliferative activity (expressed as the absorbance value at 450 nm)between the MU group with and without tetracycline treatment and between the WT group with and without tetracycline treatment at 4, 8, 12, 24, 36 and 48 hours (all P <0.05), but not between the NC group with and without tetracycline treatment at any of the above time points (all P >0.05). Conclusion HaCaT cell lines which stably express the wild-type GJB6 gene or its mutant(A88V)are successfully constructed.

Objective: To explore the relationship among dark triad, peer relationship, and cyber bullying of middle school students, and to provide suggestions for intervention of cyberbullying behavior of middle school students. Methods: Peer Relationship Scale (PRS), Dirty Dozen (DD) and Cyberbullying Questionnaire (CBQ) were administrated to 1 934 middle school students in Beijing and Yunnan by cluster sampling. Results: Middle school boys scored significantly higher on the dark triad and cyber-bullying than girls ( F =13.45, 50.20, P <0.01). The dark triad of middle school students was positively correlated with cyber-bullying ( r =0.38, 0.40, 0.21, P <0.01), while negative dimensions of peer relationship were positively correlated with cyber-bullying ( r = -0.10 , -0.22, -0.16, P <0.01). Peer relationship had a moderating effect on the relationship between dark triad and cyber-bullying ( β =-0.07， t =-3.24，Δ R 2=0.00， P <0.01). Conclusion Positive peer relationship can reduce the cyber-bullying behavior of middle school students with high dark triad which should be emphasized among middle school students.

Objective To prepare SDF-1 α-loaded nanoliposome ( SNP )-SonoVue complex and investigate its tracing abilities, sustained-release property and effect on migration of bone marrow mesenchymal stem cells (BMSCs). Methods The SNP was prepared to detect its physical characteristics including particle size,zeta potential, morphology, encapsulation efficiency and drug loading.SNP-SonoVue was constructed to detect the sustained release situation of SNP and SNP-SonoVue after low frequency ultrasound ( LIFU ) irradiation, and the connection of SNP-SonoVue was observed by fluorescence microscope. Effects of SNP-SonoVue on migration of BMSCs were detected to evaluate its bioactivity. BMSCs were divided into 6 groups,including Group A: SDF-1α+ 1% serum medium;Group B: SNP- SonoVue+ 1% serum culture medium;Group C:SNP-SonoVue+ 1% serum culture medium + LIFU ( 1 MHz,0.5 W/cm2, expose 30 s stop 30 s, 4 min);Group D: BNP-SonoVue+1% serum medium;Group E:BNP-SonoVue+1% serum medium+LIFU ( 1 MHz, 0.5 W/cm2, expose 30 s stop 30 s, 4 min),Group F:PBS+1% serum culture medium (control group). Its tracing abilitie were investigated in vitro. Results The average particle size of SNP was(220.4±9.9)nm,and the particle dispersion index(PDI) was(0.172± 0.015), the average zeta potential was ( 35.6 ± 1.7) mv. It was showed spherical dispersion by transmission electron microscopy. The encapsulation efficiency was up to 96.7% and the drug entrapment content was 481.76 ng/mg. Flow cytometric showed the suitable conditions for SNP-SonoVue preparation was that the ratio of SNP quality(mg) to Sono Vue microbubbles number(a) was20:(2.8×109)to40:(2.8× 109). Fluorescence microscopy showed that shells of SonoVue microbubbles connected with large numbers of SNP labeled with red fluorescent DiI. Drug release experiment showed that the cumulative SDF-1α release amount of SNP and SNP-SonoVue exposed to LIFU respectively were ( 68.61 ± 3.97 )% and ( 63.21 ± 5.68)% in vitro within 7 days, and the difference was not statistically significant ( P ＞ 0.05 ). Cell migration experiments confirmed that the transfer function of BMSCs in Group A, Ggroup B and group C was significantly higher than that in control group ( P ＜ 0.05 ), but there was no significant difference among the Group A, Ggroup B and group C ( P ＞0.05). In vitro development experiment showed that the SNP-SonoVue complex had obviously enhanced development effect. Conclusions SNP-SonoVue complex is successfully prepared. It has obviously enhanced development effect and can lead to migration of BMSCs.