ABSTRACT
Objective To construct the recombinant plasmids of knocking down Rho guanine dissociation inhibitor α (GDIα ) gene by using clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) technique, and investigate the effect of Rho GDIα interference on the migration of Hepa 1-6 cells of mouse in order to provide the method of prevention and treatment of liver cancer. Methods To construct and identify the PX458-Rho GDIα-single guide (sg) RNAs by using CRISPR/Cas9 technique. And the Hepa 1-6 cells were transfected by liposomes with PX458-Rho GDIα-sgRNAs for 48 hours respectively, and cells treated with PX458 plasmids were used as control. The migration ability of Hepa 1-6 was checked by wound healing assay and Transwell assay, respectively. Results The expression of Rho GDIα was depressed in group of PX458-Rho GDIα-sgRNAl transfection which was detected by using RT-PCR. The migration distance of Hepa 1-6 in PX458-Rho GDIα-sgRNAl transfection group was significantly promoted comparing with the control group which was transfected with PX458 only, and the cell number of PX458-Rho GDIα-sgRNAl group was more than that in control group by using transwell assay, indicating concluded that knocking down of Rho GDIα promoted the migration ability of Hepal-6 cells. Conclusion The result is explicit that in vivo, Rho GDIα may inhibit the migration of Hepal-6 partially. Overexpression of Rho GDIα might be used as an important method to prevent the metastasize of carcinoma.
ABSTRACT
Objective To construct the clustered regularly interspaced short palindromic repeats / associated protein 9 (CRISPR/ Cas9) plasmid targeting forkhead box J2 (FOXJ2) gene and investigate the effects of FOXJ2 interference on the expression of transforming growth factor-β(TGF-β) / Smads and proliferation in hepatocellular carcinoma cells of mouse. Methods Small guide RNA(sgRNA) sequence of FOXJ2 was designed, linked with PX458 vector and transfected into competent E. coli for proliferation. The recombinant plasmids were sent for sequencing to confirm the accuracy of the sgRNA sequence. The PX458-FOXJ2-sgRNAs plasmids were transfected into Hepa1-6 cells by liposome transfection, respectively. The empty vectors of PX458 were transfected as control group. After 48 hours, the expression of FOXJ2, TGF-β and Smads were obtained by RT-PCR and agarose gel electrophoresis, respectively. The cell proliferation was detected by methylthio tetrazole (MTT) method . Results The CRISPR/ Cas9 plasmids of PX458-FOXJ2-sgRNAs were successfully constructed. The recombinant plasmid of PX458-FOXJ2-sgRNA2 could effectively inhibit FOXJ2 gene expression which induced increasing expression of TGF-β, Smad2 and Smad4 in Hepa1-6 cells comparing to the control group transfected with PX458 only. And the proliferation of Hepa1-6 was promoted in PX458-FOXJ2-sgRNA2 interference group. Conclusion In hepatocellular carcinoma cells of mouse, FOXJ2 gene inhibits the expression of TGF-β, Smad2, Smad4 and cell proliferation partially, which indicates the relationship between FOXJ2 and TGF-β signal pathway. The result provides the target molecule of FOXJ2 for the prevention and treatment of hepatocellular carcinoma.
ABSTRACT
We investigated epidemiological characteristic of scrub typhus in Yongshan County,Yunnan Province,China. The serum samples were collected from the patients with fever for detecting the antibody against Orientia tsutsugamsushi (Ot) by colloidal gold immunoassay assay.Rat traps were used to capture rodents.The spleen tissues of the captured rodents were detected by nested-polymerase chain reaction for rickettsia groEL segment.The groEL segments were sequenced and analyzed the homology with the other known sequences.Thirty-four scrub typhus cases were found in Yongshan County,Yunnan Prov-ince from May 2015 to October 2015.Among them,21 cases were confirmed by laboratory tests and 13 cases were clinical di-agnosis diseases.Of these patients,32.35% of the cases occurred in June.The 32.35% were in the group of the 40-49 year-old,and 79.41% were farmers,94.12% exhibited eschar or skin ulcer(31.25% were observed in groin of these cases),and rash developed in 50%.In 39 spleen tissue samples of Rattus flavipectus,9 samples showed positive for groEL gene Ot,but gro-EL gene of Typhus group rickettsia and spotted fever group rickettsia were negative.Sequence analysis showed that YSP30 was closely related to some Saitama related strains of Ot,such as HSB1,FAR1 and UAP4,while the other 8 strains were closely related to some Karp related strains of Ot,such as UT213,UT221 and SH205.It was confirmed that the Yongshan County was the natural foci of scrub typhus by the serological and molecular biological detections.There are Karp and Saitama genotype related Ots in the natural foci.
ABSTRACT
Objective To investigate the pathophysiological role of serum hydrogen sulfide (H2 S) and its correlation with serum neuron-specific enolase (NSE) level in the process of febrile seizures (FS).Methods Sixty-five children with FS and acute upper respiratory infection were selected as FS group,51 children with acute upper respiratory infection associated with fever were taken as upper respiratory infection group and 43 healthy children in Child Health Section for physical examination were respectively chosen as the healthy control group.All children came from Department of Pediatrics of Zhujiang Hospital,Southern Medical University from Mar.15 to Nov.10,2012.The serum H2S and NSE levels of all groups were measured by Microplate Reader.Results The serum H2S in FS group was significantly lower than that in upper respiratory infection group and healthy control group (all P ＜ 0.01) ; the NSE level in FS group was significantly higher than that in upper respiratory infection group and healthy control group (all P ＜0.01) ;the levels of H2S and NSE had no significant differences between upper respiratory infection group and healthy control group (all P ＞ 0.05).The serum H2S level and NSE level in FS group were negatively correlated (r =-0.279,P =0.024) ; There were significant differences of serum NSE level between seizure frequency less than twice group and seizure frequency greater than or equal to twice group(t =-2.955,P =0.004).The seizure frequency was negatively correlated with serum H2S level (r =-0.269,P =0.03),and positively correlated with serum NSE level (r =0.322,P =0.009).The seizure durations (≥ 5 min) was negatively correlated with serum H2S level (r =-0.532,P =0.019).Conclusion The serum H2S level is expected to be an objective index for eva-luating the seizure brain injury in the early period of FS,and also potentially the important occurrence factors of brain injury.