Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paper we examine the effect of CBHA treatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 μmol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHA treatment. Meanwhile, CBHA treatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNT embryos can be modified by CBHA treatment.
Acetylation ; Animals ; Blastocyst ; cytology ; Cell Nucleus ; metabolism ; Cinnamates ; pharmacology ; Embryo, Mammalian ; drug effects ; metabolism ; Embryonic Development ; drug effects ; Epigenesis, Genetic ; Female ; Gene Expression ; Histone Deacetylase Inhibitors ; pharmacology ; Histones ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; In Vitro Techniques ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Nuclear Transfer Techniques ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Swine

Objective To evaluate the clinical efficacy of gemcitabine-oxaliplatin (GEMOX) regimen combined with targeted therapy for advanced gallbladder cancer.Methods The retrospective descriptive study was conducted.The clinical data of 21 patients with advanced gallbladder cancer who were admitted to the Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine between January 2016 and December 2017 were collected,including 8 males and 13 females,aged from 28 to 80 years,with the age of (58± 12)years.Patients received GEMOX regimen combined with targeted therapy.According to the results of gene test,patients selected tageted therapy with Cetuximab,Herceptin or Apatinib.Observation indicators:(1) gene test situations;(2) situations of GEMOX regimen combined with targeted therapy;(3) adverse reactions of GEMOX regimen combined with targeted therapy.Measurement data with normal distribution were represented as Mean±SD,and measurement data with skewed distribution were described as M (range).Count data were represented as absolute number or percentage.The survival curve and rate were respectively drawn and calculated using the Kaplan-Meier method.The survival analysis was done using the Log-rank test.Results (1) Gene test situations:of 21 patients,19 were confirmed as K-ras wild type,including 13 of single K-ras wild type,4 of K-ras wild type combined with human epidermal growth factor receptor 2 (HER2),2 of K-ras wild type combined with vascular endothelial growth factor receptor 2 (VEGFR2);5 were detected positive HER2,including 1 of single positive HER2,4 of positive HER2 combined with K-ras wild type;3 were detected positive VEGFR2,including 1 of single positive VEGFR2,2 of positive VEGFR2 combined with K-ras wild type.Two and 19 patients had 0 and l of Eastern Cooperative Oncology Group score.(2) Situations of GEMOX regimen combined with targeted therapy:all the 21 patients underwent ≥ 2 courses of GEMOX regimen combined with targeted therapy.Among the 21 patients,0,4,9 and 8 were respectively detected in the complete remission (CR),partial remission (PR),stable disease (SD) and disease progression (PD).Fourteen patients (13 of single K-ras wild type and 1 of K-ras wild type combined with positive VEGFR2) received GEMOX regimen combined with Cetuximab therapy,including 0 with CR,4 with PR,5 with SD and 5 with PD;5 patients (1 of single positive HER2 and 4 of positive HER2 combined with K-ras wild type) received GEMOX regimen combined with Herceptin therapy,including 0 with CR,0 with PR,2 with SD and 3 with PD;2 patients (1 of single positive VEGFR2 and 1 of positive VEGFR2 combined with K-ras wild type) received GEMOX regimen combined with Apatinib therapy,including 0 with CR,0 with PR,2 with SD and 0 with PD.The objective response rate was 19.0％ (4/21) and disease control rate was 61.9％(13/21) in the 21 patients.The median onset time was 1.8 months in the 21 patients.The 3-,6-and 9-month progression free survival (PFS) rates and median PFS time were respectively 90.5％,71.4％,58.5％ and 10.7 months.The 6-and 12-month overall survival rates were respectively 90.2％ and 58.6％,and median overall survival (OS) time was 15.5 months in the 21 patients.The PFS and OS time were 8.4 months and 10.4 months in the 7 patients combined with jaundice,10.5 months and 14.8 months in the 14 patients without jaundice,with no statistically significant difference (x2 =0.868,0.774,P＞0.05).(3) Adverse reactions of GEMOX regimen combined with targeted therapy:the most common adverse events were skin rash and digestive tract reactions.No serious adverse event occurred during the therapy.All the adverse events were improved after symptomatic treatments.Conclusion GEMOX regimen combined with targeted therapy for advanced gallbladder cancer has good outcomes,less adverse reactions and higher safety.

OBJECTIVETo prepare cisPLLAtin-loaded polylactic acid/cnts, and to study the anti-tumor effect of 5-FU-PLLA-CNTs on human gastric carcinoma cell lines(MGC803 and MNK45).

METHODS5-FU-PLLA-CNTs were prepared with ultrasound emulsification. The morphology of 5-FU-PLLA-CNTs was determined by scanning electron microscope(SEM), and its drug loading and drug release curve in vitro were detected by UV-Vis-NIR spectrophotometer. Cells were divided into experiment, positive control and negative control groups. CCK8 method was used to test the cytotoxic effect of 5-FU-PLLA-CNTs in different concentrations on MGC803 and MNK45 cell proliferation. Flow cytometry was employed to measure the apoptotic rate of MGC803 and MNK45 cells before and after the intervention of 5-FU-PLLA-CNTs.

RESULTSDeep layer film of 5-FU-PLLA-CNTs was successfully established, whose drug-load rate was(4.54±0.43)%, entrapment rate was(21.56±2.36)%. In vitro release test showed release rate within 24 h of 5-FU-PLLA-CNTs was 23.9% in a as lowly increasing manner, and accumulating release rate was 85.3% at day 31. CCk8 experiment revealed, as compared to control group, 5-FU-PLLA-CNTs significantly inhibited the proliferation of two cell lines in dose-dependent and time-dependent manner. The best 5-FU-PLLA-CNTs concentration of inhibition for human gastric cancer cell lines was 1 mg/well. Flow cytometry indicated the apoptotic rate of MGC803 and MNK45 cells in experiment group treated by 1 mg/well 5-FU-PLLA-CNTs significantly increased as compared to negative control group (P<0.05), while the difference was not significant as compared to positive control group (P>0.05).

CONCLUSIONThe 5-FU-PLLA-CNTs has good drug sustained-release capacity, and can significantly kill and inhibit the proliferation of MGC803 and MNK45 cell lines.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Delayed-Action Preparations ; Fluorouracil ; pharmacology ; Humans ; Lactic Acid ; pharmacology ; Nanotubes, Carbon ; Polyesters ; Polymers ; pharmacology ; Stomach Neoplasms ; pathology

ObjectiveTo explore the intervention mechanism of Xiangsha Liujunzi Tang in rats with functional dyspepsia （FD） based on the Ras homolog gene family member A （RhoA）/Rho-associated coiled-coil containing protein kinase 2 （ROCK2）/Myosin phosphatase target Subunit 1 （MYPT1） pathway. MethodSixty male SD suckling rats in SPF grades were randomly divided into blank group （n=10） and model group （n=50）. The comprehensive modeling method （gavage administration of iodoacetamide+exhaustion of swimming+disturbance of hunger and satiety） was used to replicate the rat model of FD. After successful replication of the model， the rats in the model group were randomly divided into model group， mosapride group， and high， middle， and low-dose Xiangsha Liujunzi Tang groups， with 10 rats in each group. Rats in the blank group and model group were given 10 mL kg^-1·d^-1 normal saline， those in the mosapride group were given 1.35 mg·kg^-1·d^-1 mosapride， and those in the high， middle， and low-dose Xiangsha Liujunzi Tang groups were given 12， 6， and 3 g·kg^-1·d^-1 Xiangsha Liujunzi Tang， respectively. The intervention lasted 14 days. The general living conditions of rats were observed before and after modeling and administration， and the 3-hour food intake and body mass of rats were measured. After intervention， the intestinal propulsion rate of rats was measured， and the pathological changes in the gastric tissue were observed by hematoxylin-eosin （HE） staining. The content of choline acetyl transferase （ChAT） and vasoactive intestinal peptide （VIP） in the medulla oblongata and gastric tissue homogenate was determined by enzyme-linked immunosorbent assay （ELISA）， the distribution of adenosine triphosphate （ATP） enzyme in gastric antrum smooth muscle was observed by frozen section staining， and the protein expression levels of RhoA， ROCK2， and phosphorylated-myosin phosphatase target subunit 1 （p-MYPT1） in the gastric tissue were detected by Western blot. ResultCompared with the blank group， the model group had withered hair， lazy movement， slow action， poor general living condition， lower 3-hour food intake， body mass， and lower intestinal propulsion rate （P<0.05）， whereas no obvious abnormality in gastric histopathology. In the model group， the content of ChAT in the medulla oblongata and gastric tissue decreased， the content of VIP in gastric tissue increased， the distribution of ATP enzyme in gastric antrum smooth muscle decreased significantly， and the protein expression levels of RhoA， ROCK2， and p-MYPT1 in the gastric tissue decreased significantly （P<0.05）. As compared with the model group， the general living condition of rats in each intervention group was significantly improved， and the 3-hour food intake， body mass， and intestinal propulsion rate were significantly increased （P<0.05）. There was no significant difference in gastric pathology in the intervention groups. The content of ChAT in the medulla oblongata and gastric tissue increased significantly， the content of VIP in the gastric tissue decreased， the distribution of ATP enzyme in gastric antrum smooth muscle increased significantly， and the protein expression levels of RhoA， ROCK2， and p-MYPT1 in the gastric tissue increased significantly （P<0.05）. The intervention effect of Xiangsha Liujunzi Tang group on the above indexes was dose-dependent. ConclusionXiangsha Liujunzi Tang can effectively improve the general living condition and gastric motility of rats with FD， and its specific mechanism may be related to the activation of the RhoA/ROCK2/MYPT1 pathway in the gastric tissue to regulate smooth muscle relaxation and contraction and promote gastric motility.