Many Novel approaches of epigenetic reprogramming of somatic cells have been reported. However,ethical issues caused by somatic nuclear transfer have triggered the development of alternative strategies for reprogramming somatic cells. Recently,many new advances have been acquired for reprogramming somatic cells,which could reverse differentiated somatic cells to a totipotent embryonic state,such as fusion of potential stem cells with somatic cells,incubation of cells in potential cell-free extraction and introduction of defined pluripotency factors into somatic cells. The epigenetic modification in these reprogramming processes,including germ cells and early embryoes,somatic nuclear transfer and other approaches for reprogramming of somatic cells were reviewed. Studies of epigenetics will be benefit for understanding the precise mechanism and improving the efficiency of somatic nuclear reprogramming,which will be eventually applied in the basic study and practice.

Objective To evaluate the efficacy and safety of a collagen dressing for healing of wounds induced by fractional CO2 laser in patients with atrophic facial acne scars.Methods Seventy patients with atrophic facial acne scars were recruited to this study,and randomly divided into a treatment group and a control group.Both the two groups were treated by two sessions of fractional CO2 laser with an interval of one month.After each session of laser therapy,the treatment group were topically treated with a collagen dressing for 20 minutes once a day for 10 consecutive days,while the control group did not apply any collagen dressing.All the patients were followed for 6 months.Efficacy was evaluated by the degree of acute inflammatory reaction,time needed for crust shedding and patient comfort level.The length of downtime as well as incidence of post-inflammatory hyperpigmentation and other adverse reactions were also assessed.Results Compared with the control group,the treatment group showed a decrease in the score for acute inflammatory response (W =312,P ＜ 0.01),time needed for crust shedding (t =2.08,P ＜ 0.05),incidence rate of post-inflammatory hyperpigmentation (x2 =6.06,P ＜ 0.05),length of downtime (t =3.14,P ＜ 0.05),but an increase in self-reported comfort level (W =172,P ＜ 0.01).No new scar formed in any of these patients.Conclusion The collagen dressing is effective in reducing incidence of adverse reactions and improving satisfaction degree of patients with atrophic facial acne scars after fractional CO2 laser therapy.

33 cases were treated by spotted—moxibustionwith Zhuang medicated threads,the effective rate be-ing 84.6～85.0%.The longterm effect was also satis-factory.This therapy can also markedly raised the in-filtration pressure of nocturnal urination.There wasno obvious relation between the therapeutic effect andsyndrome typing.

Objective To observe the epithelial mesenchymal transition (EMT) of podocyte induced by high glucose,and to explore the potential protective mechanism of ursolic acid (UA).Methods The podocytes cultured in vitro were divided into four groups:normal group (glucose 5.5 mmol/L),mannitol group (glucose 5.5 mmol/L+mannitol 19.5 mmol/L),high glucose group (glucose 25 mmol/L) and UA group (glucose 25 mmol/L + UA 5 μmol/L).Podocyte morphology changes were observed by inverted phase contract microscope.The expression of zonula occludens-1 (ZO-1) and α-smooth muscle actin (α-SMA) were detected by immunofluorescence.The expressions of β-catenin and glycogen synthesis kinase-3β (GSK3β) were detected by Western blotting.The expressions of Wnt1,Wnt3a,Wnt5a,Wnt5b and GSK3β were detected by real-time PCR.Results Podocytes showed irregular arborization shape in normal glucose and transited to longer cobblestone-like shape as mesenchyme cell by high glucose culture.Compared with normal group,the expression of ZO-1 protein was down-regulated and the expression of α-SMA was up-regulated by high glucose culture (P ＜ 0.05).The expression of Wnt5a mRNA was down-regulated;β-catenin mRNA and protein were up-regulated (P ＜ 0.05);and GSK3β protein was down-regulated by high glucose culture (P ＜ 0.05).Compared with high glucose group,ursolic acid inhibited podocyte EMT,up-regulated the expression of ZO-1 protein,Wnt5a mRNA,GSK3β (P ＜ 0.05),and down-regulated the expressions of α-SMA protein,β-catenin mRNA and protein (P ＜ 0.05).Conclusion Ursolic acid attenuates high glucose induced epithelial mesenchymal transition of podocyte by inhibiting Wnt/β-catenin signaling pathway.

Objective To investigate the effect of oleanolic acid pretreatment on hepatic ischemiareperfusion (I/R) injury in rats. Methods One hundred and twenty-eight male SD rats weighing 230-250 g were randomly divided into 4 groups (n = 32 each): sham operation group (group S), I/R group, 0.5% sodium carboxymethyl cellulose group (group CMC) and oleanolic acid preconditioning (group OA). Partial liver ischemia was produced by clamping hepatic portal vein and hepatic arteries for 60 min with atraumatic mini-clamp, followed by 12 h of reperfusion in group I/R, CMC and OA. Oleanolic acid suspension 100 mg/kg was infused intragastrically in group OA, while the equal volume of 0.5% CMC-Na (in group CMC) and drinking water (in group S and I/R) was infused intragastrically instead once a day for 7 days, and then hepatic I/R was performed at day 8. The left liver was removed and blood sample was taken from inferior vena cava at 0, 3, 6 and 12 h ofreperfusion for determination of serum alanine amino transferase (ALT) activity, superoxide dismutase (SOD)activity, malondialdehyde (MDA) and glutathione (GSH) content, and expression of phospho-phosphatidylinositol3-kinase (p-PI3K), Akt, p-Akt, Bcl-2, Bax, p-Bad and Bad in the liver, and microscopic examination. Results Serum ALT activity and MDA content in the liver were significantly increased, SOD activity and GSH content in the liver were significantly decreased, expression of p-PI3K, p-Akt, Bax, Bad and p-Bad was up-regulated, and Bcl-2 expression was down-regulated during reperfusion in group I/R, CMC and OA as compared with group S (P ＜0.05). Compared with group I/R, serum ALT activity and MDA content in the liver were significantly decreased, SOD activity and GSH content in the liver were significantly increased, expression of p-PI3K, p-Akt,Bcl-2 and p-Bad was up-regulated, and expression of Bad and Bax was down-regulated during reperfusion in group OA (P ＜ 0.05), but no significant change was found in the indexes mentioned above in group CMC (P ＞ 0.05).Serum ALT activity and MDA content in the liver were significantly lower, SOD activity and GSH content in the liver were significantly higher, expression of p-PI3K, p-Akt, Bcl-2 and p-Bad was significantly higher, and expression of Bad and Bax was significantly lower during reperfusion in group OA than in group CMC (P ＜ 0.05).The pathological changes in the liver were milder in group OA than in group I/R. Conclusion Oleanolic acid pretreatment can alleviate hepatic I/R injury by activating PI3K/Akt signaling pathway and inhibiting apoptosis.

Objective To investigate the expression of microRNA let-7a in formalin-fixed paraffinembedded (FFPE) melanoma tissue and its effect on the proliferation of A375 cell lines. Methods Tissue samples were obtained from 13 patients with malignant melanoma and 10 with congenital pigmented nevus,then fixed with formalin and embeded with paraffin. microRNAs were isolated from these samples and reversely transcripted to cDNA with stem-loop primer. Then, real time quantitative PGR was performed with Taqman MGB probe and matched primers to measure the expression of microRNA let-7a. cy3-Labeled negative control siRNA was transfected into A375 cells followed by the examination of transfection efficiency under fluorescense microscopy. Subsequently, hsa-let-7a mimics were transfected into A375 cells to observe their effect on the proliferation of these cells. Results microRNAs were successfully isolated from FFPE tissues, and quantitative PGR showed a significant reduction in the expression of microRNA let-7a in melanoma tissue compared with nevus tissue (t = 2.364, P ＜ 0.05). The transfection efficiency was found to be about 80% ～90%. After transfection with hsa-let-7a mimics, the proliferation of A375 cells was inhibited and the inhibition rate amounted to 32.7% at 24 hours. Conclusions The expression of microRNA-let-7a is attenuated in melanoma, and overexpression of let-7a can inhibit the proliferation of A375 cells, which implies that let-7a functions as a tumor suppressor gene in melanoma.

Objective To investigate the effect of BRAFV600E mutation on the invasion capacity of a human melanoma cell line, A375. Methods Plasmids containing short hairpin RNAs (shRNA) specific for BRAF gene were prepared in previous study, and used to transfect A375 cells. Those cells transfected with negative plasmid and untransfected cells served as the controls. Transwell chambers were used to examine the invasion ability of melanoma cells in vitro. RT-PCR and Western blotting were performed to detect the mRNA and protein expressions of matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF), respectively, before and after the transfection. The activity of MMP-2 was also studied with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results Compared with the negative control, the specific shRNA decreased the mRNA and protein expressions of MMP-2 by 35% and 85%, respectively, and those of VEGF by 45% and 14%, respectively. Additionally, the number of cells invading through Matrigel chambers reduced by 69% in those cells transfected with the positive plasmid. Conclusions The mutant BRAFV600E has the potential to enhance the invasion capacity of melanoma cells, whereas specific shRNA could suppress the increase in metastasis capacity likely by inhibiting the production of VEGF and MMP.

Objective To use tetraploid embryo complementation combined with gene transfer to produce genetically modified embryonic stem cells (EsCs) clones. Methods In this study, EGFP was introduced into ESCs by electroporation, and transfected positive cells were selected by G418 resistance. The tetraploid embryos were obtained from diploid blastomere electrofusion which preformed at 2-cell stage. Afterwards, 19-21 EGFP-ESCs were inserted into each tetraploid blastocyst cavity by piezo drilled microinjection,then the injected blastocysts were transferred into the uterus of pseudo-pregnancy at 2.5-day or the oviduct of 0.5-day female mice. Results The transfected ESCs maintained normal karyotype even after long-term passage (2n=40). The rate of fusion was 95.07%, and the developmental rate of tetraploid blastocyst was 95%.Totally 410 injected blastocysts were obtained. Unfortunately, we have not got any vital offsprings, except 151 implantation sites (pseudo-pregnancy 2.5 days:29.41%;the oviduct of half one day:64.37%). Furthermore, scattered EGFP expressions in transgenic fetus were observed under invert fluorescent microscope. Conclusion The transfected ESCs were observed in transgenic fetus, and the implantation rate in oviduct was higher than that in uterine.

Objective To observe the effect of saxagliptin combined insulin four times to strengthen the vola-tility on blood glucose variability in patients with type 1 diabetes.Methods According to random number table meth-od,60 patients with type 1 diabetes were divided into DPP4 group(28 cases)and the control group(32 cases).The control group was given insulin four times to strengthen the volatility(insulin aspart/insulin lispro +insulin glargine /insulin detemir),the DPP4 group on the basis of insulin four times to strengthen the volatility plus the saxagliptin 5mg/d,all patients into the group after1 -3D and 13 -15D using CGMS(Medtronic)continuously monitor the blood glucose.Results (1)Within the group comparison:the DPP4 group:1 -3d after treatment:MAGE and SDBG,MBG, LAGE,PT10.0,PT3.9 were lower than before treatment,including MAGE [(6.91 ±1.63)mmol/L vs.(6.31 ± 1.42)mmol/L,t =0.993],SDBG[(2.63 ±0.81)mmol/L vs.(2.41 ±0.51)mmol/L,t =0.751],MBG[(11.51 ± 1.24)mmol/L vs.(10.87 ±2.01)mmol/L,t =1.077],LAGE[(9.43 ±1.73)mmol/L vs.(8.56 ±1.97)mmol/L, t =1.125],PT10.0[(12.99 ±5.61)% vs.(9.66 ±5.03)%,t =1.427],PT3.9[(5.51 ±2.43)% vs.(5.07 ± 2.44)%,t =1.141],there were statistically significant differences compared with before treatment(all P <0.05), 1 -3d after treatment,SDBG[(2.77 ±0.73)mmol/L vs.(2.14 ±0.69)mmol/L,t =1.547],MBG[(11.67 ± 1.46)mmol/L vs.(9.76 ±1.58)mmol/L,t =1.1.326]were decreased,but there were no statistically significant differences compared with before treatment(all P >0.05);13 -15d after treatment:MAGE[(6.88 ±1.49)mmol/L vs.(2.97 ±0.86)mmol/L,t =3.021],SDBG[(2.77 ±0.73)mmol/L vs.(1.12 ±0.43)mmol/L,t =1.964],MBG [(11.67 ±1.46)mmol/L vs.(7.44 ±0.93)mmol/L,t =2.760],LAGE[(9.55 ±1.77)mmol/L vs.(6.53 ±1.21)mmol/L, t =2.409],PT10.0[(13.58 ±5.14)% vs.(4.72 ±2.37)%,t =2.657],PT3.9[(5.36 ±2.05)% vs.(3.05 ± 2.60)%,t =1.840]were decreased,there were statistically significant differences compared with before treatment (P <0.05 or P <0.01 );the control group:1 -3d after treatment:MAGE [(6.91 ±1.63)mmol/L vs.(6.31 ± 1.42)mmol/L,t =0.993],SDBG[(2.63 ±0.81)mmol/L vs.(2.41 ±0.51)mmol/L,t =0.751],MBG[(11.51 ± 1.24)mmol/L vs.(10.87 ±2.01)mmol/L,t =1.077],LAGE[(9.43 ±1.73)mmol/L vs.(8.56 ±1.97)mmol/L, t =1.125],PT10.0[(12.99 ±5.61)% vs.(9.66 ±5.03)%,t =1.427],PT3.9[(5.51 ±2.43)% vs.(5.07 ± 2.44)%,t =1.141]were lower than before treatment,but compared with before treatment,there were no statistically significant differences(all P >0.05 );13 -15d after treatment:MAGE [(6.91 ±1.63 )mmol/L vs.(6.07 ± 1.36)mmol/L,t =1.223],SDBG[(2.63 ±0.81)mmol/L vs.(1.91 ±0.93)mmol/L,t =0.984],MBG[(11.51 ± 1.24)mmol/L vs.(8.82 ±1.13)mmol/L,t =1.808],LAGE[(9.43 ±1.73)mmol/L vs.(7.06 ±1.57)mmol/L, t =1.963],PT10.0[(12.99 ±5.61)% vs.(6.74 ±3.35)%,t =2.012],PT3.9[(5.51 ±2.43)% vs.(4.73 ± 2.57)%,t =1.541]were decreased,there were statistically significant differences in MBG,LAGE,PT10.0 compared with before treatment(all P <0.05).Group comparision:1 -3d after treatment:the DPP4 group:MAGE[(4.81 ± 1.15)mmol/L vs.(6.31 ±1.42)mmol/L,t =2.351],SDBG[(2.14 ±0.69)mmol/L vs.(2.41 ±0.51)mmol/L, t =1.332],MBG[(9.76 ±1.58)mmol/L vs.(10.87 ±2.01)mmol/L,t =0.856],LAGE[(7.74 ±1.88)mmol/L vs.(8.56 ±1.97)mmol/L,t =2.102],PT10.0 [(7.47 ±4.96)% vs.(9.66 ±5.03)%,t =2.667],PT3.9 [(4.64 ±2.14)% vs.(5.07 ±2.44)%,t =1.890]were all significantly lower than the control group,there were statistically significant differences in MAGE,LAGE,PT10.0 between the two groups(all P <0.05).13 -15d after treatment:the above indictors,the DPP 4 group was decreased obviously compared with the control group,MAGE [(2.97 ±0.86)mmol/L vs.(6.07 ±1.36)mmol/L,t =2.854],SDBG[(1.12 ±0.43)mmol/L vs.(1.91 ± 0.93)mmol/L,t =2.328],MBG[(7.44 ±0.93)mmol/L vs.(8.82 ±1.13)mmol/L,t =2.125],LAGE[(6.53 ± 1.21)mmol/L vs.(7.06 ±1.57)mmol/L,t =2.111],PT10.0[(4.72 ±2.37)% vs.(6.74 ±3.35)%,t =2.312] and PT3.9 [(3.05 ±2.60)% vs.(4.73 ±2.57)%,t =2.237],there were statistically significant differences between the two groups (P <0.05 or P <0.01).Conclusion The combination of DPP4 inhibitors and insulin four renforcement can improve blood glucose fluctuation in patients with type 1 diabetes,reduce the dosage of insulin and not increase incidence of hypoglycemic events.

Objective To investigate the effect of transcranial direct current stimulation (tDCS) on aphasia recovery after stroke. Meth-ods From April, 2012 to January, 2013, 20 aphasic patients after stroke were enrolled in an A-B experiment design. During phase A, ten times of sham tDCS and language training (five days a week) were implemented, then ten times language training combined with tDCS (five days a week) were implemented in phase B. The treatment lasted for four weeks. Picture naming was measured for all patients before and af-ter treatment both in phase A and phase B. Results The D-value scores of picture naming before and after treatment were significantly more in phase B than in phase A in both treatment items and non-treatment items (t>3.030, P<0.05). Conclusion tDCS could raise the accuracy of picture naming in patients with aphasia after stroke.