ABSTRACT
The use of two inhibitors of Mek1/2 and Gsk3β (2i) promotes the generation of mouse diploid and haploid embryonic stem cells (ESCs) from the inner cell mass of biparental and uniparental blastocysts, respectively. However, a system enabling long-term maintenance of imprints in ESCs has proven challenging. Here, we report that the use of a two-step a2i (alternative two inhibitors of Src and Gsk3β, TSa2i) derivation/culture protocol results in the establishment of androgenetic haploid ESCs (AG-haESCs) with stable DNA methylation at paternal DMRs (differentially DNA methylated regions) up to passage 60 that can efficiently support generating mice upon oocyte injection. We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations. Furthermore, we demonstrate that TSa2i-treated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation. Strikingly, AG-haESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells, in part through the enhanced proliferation of H19-DMR hypermethylated cells. Together, we establish AG-haESCs that can long-term maintain paternal imprints.
ABSTRACT
Objective To evaluate the effect of small interfering RNA (siRNA)targeting survivin gene on the expression of survivin and proliferation,apoptosis,migration and invasion of a human cutaneous squamous cell carcinoma cell line A431 in vitro.Methods A survivin-specific siRNA was designed and synthesized.Cultured A431 cells were divided into 3 groups to be transfected with 50.0 nmol/L liposome complexes containing survivin-specific siRNA (survivin siRNA group),50.0 nmol/L liposome complexes containing unrelated siRNA (negative control group) and 50.0 nmol/L prepared vesicles (blank control group).Real-time quantitative reverse transcription-PCR (RT-PCR) and Western blot analysis were performed to determine the mRNA and protein expression of survivin in A431 cells,respectively.Methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate cellular proliferative activity,flow cytometry using annexin-V/propidium iodide (PI) staining to detect cell apoptosis,Transwell assay to estimate migratory and invasive activities of A431 cells,and flow cytometry to detect cell cycle changes.Results At 48 hours after transfection,the mRNA and protein expression of survivin both significantly differed among the survivin siRNA group,negative control group and blank control group (mRNA:0.56 ± 0.15,0.88 ± 0.37,0.90 ± 0.43,F =276.67,P ＜ 0.001;protein:0.59 ± 0.04,0.86 ± 0.05,0.91 ± 0.07,F =243.61,P ＜ 0.001),the survivin siRNA group showed significantly lower mRNA and protein expression of survivin compared with the negative control group and blank control group (all P ＜ 0.05),and there were no significant differences between the negative control group and blank control group (both P ＞ 0.05).Repeated measures analysis of variance showed that the transfection with survivin siRNA could significantly inhibit the proliferation of A431 cells (F =13.19,P =0.004),the proliferation inhibition rate was significantly higher in the survivin siRNA group than in the negative control group and blank control group (both P ＜ 0.05),and no significant difference was observed between the negative control group and blank control group (P ＞ 0.05).At 24 hours after transfection,the apoptosis rate significantly differed among the 3 groups (F =83.97,P =0.002).The survivin siRNA group showed a significantly higher apoptosis rate compared with the negative control group and blank control group (both P ＜ 0.05),and there was no significant difference between the negative control group and blank control group (P ＞ 0.05).At 48 hours after transfection,the survivin siRNA group showed a significantly higher proportion of cells at G2/M phase,but lower number of migratory cells and invasive cells compared with the negative control group and blank control group (all P ＜ 0.05).Conclusion Survivin-specific siRNA can inhibit the expression of survivin gene and the proliferation of A431 cells,promote cell apoptosis,and suppress cell migration and invasion,indicating that survivin may serve as a genetic target for the treatment of cutaneous squamous cell carcinoma.
ABSTRACT
Objective To evaluate effects of antisense oligonucleotides against microRNA-155 (miRNA-155) on the proliferation,apoptosis,migration and invasion of a human cutaneous squamous cell carcinoma cell line A431.Methods A431 cells were divided into 3 groups:nonsense oligonucleotide group transfected with nonsense control oligonucleotides using liposomes,antisense oligonucleotide group transfected with antisense oligonucleotides against microRNA-155 using liposomes,and blank control group treated with Dulbecco's minimum essential medium (DMEM) containing Lipofectamine 2000.Real-time quantitative polymerase chain reaction (qRT-PCR)was performed to determine the expression of miRNA-155 in A431 cells:Methyl thiazolyl tetrazolium (MTT) assay was conducted to estimate cellular proliferative activity at 24,36,72,96 and 120 hours after transfection,flow cytometry to detect apoptosis and cell cycle changes,and Transwell assay to evaluate the migration and invasion of A431 cells.Statistical analysis was carried out by one-way analysis of variance (ANOVA) for intergroup comparisons and by least significant difference (LSD)-t test for multiple comparisons.Results After transfection,there were significant differences in the expression of miRNA-155 among the nonsense oligonucleotide group,antisense oligonucleotide group and blank control group (0.98 ± 0.02,0.28 ± 0.18,1.00 ± 0.01 respectively,F =634.57,P ＜ 0.001),and the expression of miRNA-155 was significantly lower in the antisense oligonucleotide group than in the blank control group and nonsense oligonucleotide group (both P ＜ 0.05).At 72,96 and 120 hours,there were significant differences in the survival rate of A431 cells among the 3 groups (all P ＜ 0.05),and the antisense oligonucleotide group showed a significantly lower survival rate of A431 cells compared with the blank control group and nonsense oligonucleotide group (all P ＜ 0.05).Additionally,the proportions of cells at G0/G1 phase and at S phase,and the cellular proliferative index all significantly differed among the 3 groups (F =23.46,36.81,19.35,respectively,P ＜ 0.01).The antisense oligonucleotide group showed significantly higher proportion of cells at G0/G1 phase (74.63％ ± 2.13％),but lower proportion of cells at S phase (9.88％ ± 1.83％) and cellular proliferative index (25.36 ± 2.13) compared with the blank control group(62.92％ ± 2.56％,18.86％ ± 2.78％,37.08 ± 2.56,respectively,all P ＜ 0.05) and nonsense oligonucleotide group (63.75％ ± 3.06％,18.33％ ± 3.72％,36.25 ± 3.06,respectively,all P ＜ 0.05).Additionally,the antisense oligonucleotide group showed significantly lower numbers of migratory cells and invasive cells compared with the blank control group and nonsense oligonucleotide group (all P ＜ 0.05).Conclusion Transfection of A431 squamous cell carcinoma cells with antisense miRNA-155 oligonucleotides can decrease the expression of miRNA-155,effectively inhibit the proliferation,migration and invasion of A431 cells,and promote cell apoptosis.
ABSTRACT
Objective To estimate the clinical application value of B ultrasound-guided visual painless artificial abortion operation. Methods One hundred and twenty women intented to terminate early pregnancy were randomly divided into 2 groups: group A (observation group) with ultrasound-guided visual painless artificial abortion operation and group B (control group) with traditional artificial abortion. Results Compared with the control group,the observation group has the advantages of less bleeding,less operation time, shorter bleeding time and less complications. There were statistical significances between two groups. Conclusion B ultrasound-guided visual painless artificial abortion operation is a safe and effective method, and it is worth promoting.
ABSTRACT
ObjectiveTo summarize the experience of totally thoracoscopic operation for congenital heart diseases.MethodsFrom September 2000 to November 2011,1281 patients with congenital heart disease,including 1016 cases of atrial septal defect,110 cases of ventricular septal defect,61 cases of atrioventricular tube defects,33 cases of tetralogy of Fallot,24 cases of part anomalous pulmonary venous connection,12 cases of pulmonary valve stenosis,9 cases of patent ductus arteriosus,8 case of triatriatum,7 cases of unroofed coronary sinus syndrome,and 1 case of total anomalous pulmonary venous connection were treated totally under thoracoscope.Surgical procedures were performed through 3 troears inserted at the right chest wall,and catheters were placed in the right femoral artery and vein ( or in the right atrium,femoral vein) to set up extracorporeal circulation.The ascending aorta was cross-clamped with long tailor-made forceps and the myocardium was protected by coronary perfusion with cold crystalloid (blood) cardioplegia.ResultsAll the operations were completed successfully.The mean extracorporeal circulation and cross-clamping time were ( 42 ± 16 ) min and ( 21 ± 9 ) min respectively.Postoperative ventilation was withdrawn in(4.1 ± 1.5 ) h,and the patients were discharged from the hospital in(7.1 ± 1.4) d.35 of the patients had postoperative complications,including 16 cases of right pneumothorax (healed by thoracentesis),12 cases of subcutaneous emphysema ( healed by bandaged chest) and 7 cases of fat liquefaction of the incision at the right axillary( 3 cases) and groin(4 cases) ( delayed healing).No severe complications occurred in this series.UCG performed 4 - 8 days after the operation revealed no residual shunt.Follow-up up to 3 months to 9 years were available in 914 cases.During the period,the heart function was confirmed as level Ⅰ - Ⅱ.ConclusionTotally thoracoscopic cardiac surgery is feasible,safe,and minimal invasive for patients,resulting in quick recovery and good cosmetic outcomes.
ABSTRACT
ObjectiveTo Summarize the experience of totally thoracoscopic operation for atrial myxoma.Methods From December 2007 to November 2011,44 patients with atrial myxoma,including 37 cases of Left atrial myxoma,6 cases of Right atrial myxoma,1 case of double atrial myxoma were treated totally under thoracoscope.Surgical procedures were performed through 3 troears inserted at the right chest wall,and catheters were placed in the right femoral artery and vein (or in the right atrium,femoral vein)to set up extracorporeal circulation.The ascending aorta was cross-clamped with long tailor-made forceps and the myocardium was protected by coronary perfusion with cold crystalloid (blood) cardioplegia.ResultsAll the operations were completed successfully.The mean extracorporeal circulation and cross-clamping time were (49 ± 18 )min and (28 ± 10) min respectively.Postoperative ventilation was withdrawn in(3.8 ± 1.4) h,and the patients were discharged from the hospital in (6.8 ± 1.3 ) d.3 of the patients had postoperative complications,including 1 case of fat liquefaction of the incision at the right groin ( delayed healing),2 cases of subcutaneous emphysema ( healed by bandaged chest).No severe complications occurred in this series.UCG performed 3 - 5 days after the operation revealed surgical results were satisfactory.Followup up to 2 months to 4 years were available in all cases.During the period,the heart function was confirmed as level Ⅰ.ConclusionVideo-assisted thoraeoseopie cardiac surgery is feasible,safe,and minimal invasive for patients,resulting in quick recovery and good cosmetic outcomes.