Objective To investigate the effects of recombinant trichosanthin(rTCS) on methylation status and expression level of p27 gene in HeLa cells.Methods HeLa cells was treated by different concentration(20 ?g/mL,40 ?g/mL,and 80 ?g/mL) of rTCS for 48 h and then methylation-specific polymerase chain reaction(MSP) was used to detect the promoter methylation status of the p27 gene,real-time PCR was used to detect levels of p27 and DNMT1 mRNA,and Western blotting assay was used to detect expression level of p27 protein before and after treatment with rTCS.Results Low expression level and promoter methylation status of the p27 gene were detected in HeLa cells.Treatment with 40 ?g/mL rTCS totally demethylated p27 promoter.Treatment with 20 ?g/mL,40 ?g/mL or 80 ?g/mL rTCS resulted in a 2.22-,4.00-or 6.03-folds increase in p27 mRNA level,respectively,and also a great increase in p27 protein level.A high DNMT1 expression level was observed in HeLa cells and treatment with 40 ?g/mL rTCS resulted in a 78% decrease at the DNMT1 mRNA expression.Conclusion rTCS could reverse promoter hypermethylation and re-activate the expression of p27 gene by inhibiting DNMT1 expression in HeLa cells,which indicates its potential use in cancer therapy.

Objective To prepare a kind of superparamagnetic iron oxide (SPIO) and doxorubicin loaded multifunctional polymer microbubbles (MPMBs),and to explore its potential application as an ultrasound(US)/magnetic resonance (MR) imaging contrast agent in vitro.Methods The MPMBs and normal polymer microbubbles (PMBs) were made by double emulsion and freeze-drying methods.The physical property,drug encapsulation efficiency and the drug-loading efficiency of MPMBs were determined,and the release property and US/MR imaging enhancement of MPMBs were observed.Results The MPMBs had a regular shape and narrow size distribution.The drug encapsulation efficiency was (60.20±2.69) ％,and the drug-loading efficiency was (6.02 ± 0.27) ％.The in vitro release experiment showed that ultrasound can promote the release of doxorubicin in MPMBs.US imaging in vitro showed that the enhancement of MPMBs was better than PMBs,and MR imaging in vitro conformed that MPMBs could well enhance MR imaging.Conclusions The MPMBs is a multifunctional contrast agent with the treatment function as well as US/MR dual-mode imaging enhancement effect.

Objective To prepare polyclactic-co-glycolic acid(PLGA) polymer ultrasound contrast agent loading sudan black dye (SB-PLGA) and study the enhancement effect of it on rabbit lymph nodes imaging and as a sentinel lymph node (SLN) biopsy in the feasibility and value of the indicator.Methods SB-PLGA ultrasound contrast agent was made by double emulsion and freeze-drying methods.Thigh subcutaneous rabbit VX2 tumor model of eight lymph nodes were set up with the foot pad subcutaneous injection of contrast agents to get contrast-enhanced ultrasound imaging of the popliteal lymph node and the second inguinal lymph node stations.Then the blue stained lymph nodes were resected by popliteal lymph node dissection and the second station lymph nodes were also observed after 30 mins respectively,lymph nodes were examined by frozen sections HE staining in parallel.Results SB-PLGA microbubbles had a tight size distribution.Ultrasound imaging can be significantly enhanced lymph node imaging,the second station lymph node imaging was not obvious.Popliteal lymph node dissection for lymph node staining,30minutes later,frozen sections HE staining were seen within the lymph node contrast agent present in the lymphatic sinus and a large entry of macrophages.Inguinal lymph node dissection showed the second station of lymph nodes no blue staining.Conclusions SB-PLGA ultrasound contrast agent is good for sentinel lymph node imaging and biopsy indicator.Contrast agent retention and accumulation in the lymph nodes by the uptake of macrophages may be associated with the mechanisms of lymph node enhanced imaging.

Objective To explore the effect of expression of protein kinase C receptor 1(RACK1) induced by lipopolysaccharide (LPS) on Sonic hedgehog(SHH) signaling pathway in rat puhnonary microvascular endothelial cells (RPMVEC).Methods The healthy male SPF grade SD rat with 100-120 g body weight were gotten from the laboratory animal center of Anhui province.Using immunocytochemistry method,the expression of RACK1 protein in RPMVECs was detected,cultured RPMVECs were randomly divided into different groups as LPS dose-dependent group,SAG(smoothened Agonist,a SHH signaling pathway specific agonist) dose-dependent group,LPS time-dependent group,SAG time-dependent group and LPS+SAG group.In LPS dose-dependent groups,RPMVECs were cultured with 0.1,1,10 mg/L LPS for 8 h.In LPS time-dependent groups,RPMVECs were cultured with 10 mg/L LPS for 0,2,4,8,12,24 h.In SAG dose-dependent groups,RPMVECs were cultured with 0.1,1,10 μ mol/L for 8 h.In SAG time-dependent groups,RPMVECs were cultured with 1 μ mol/L SAG for 0,2,4,8,12,24 h.In LPS+SAG group,RPMVECs were cultured with 1 μ mol/L SAG 8 h after 10 mg/L LPS treatment for 1 h.In addition,blank group,LPS group and SAG group were set for control.Western blot were used to detect the level of RACK1 and RT-PCR were used to detect the expression of GLI-1 mRNA after intervention.Results Immunocytochemistry revealed that RACK1 were present in RPMVEC.1.In LPS dose-dependent groups (0,0.1,1,10 mg/L),the level of RACK1 elevated as LPS dose increased correspondingly with inter-group difference (P＜0.05);the relative expression levels of GLI-1 mRNA were (1.109 + 0.063),(1.039 + 0.135),(0.813 ± 0.066),(0.770 + 0.105),(1 mg/L vs.10 mg/L,P＞0.05;the rest P＜0.05).In LPS time-dependent groups,the relative expression level of RACK1 at 2 h (0.370 + 0.010) was higher than that at 0 h (0.329 ± 0.008),peaked at 12 h (1.296 ± 0.048),and compared with 0 h,there was significant differences (F=1 272.204,P＜0.05).The relative expression level of GLI-1 mRNA was decreased at 2 h (0.929 ±-0.007),and compared with 0 h(1.089 ± 0.042),there was significant differences (F=306.609,P＜0.05).2.In SAG dose-dependent groups,there was no significant difference in level of RACK1 between groups(all P＞0.05).The relative expression levels ofGLI-1 mRNA were (1.109 ± 0.063),(1.169 ± 0.052),(3.468 ± 0.128),(3.434 ± 0.054),(0 μ mol/L vs.0.1 μ mol/L and 1 μ mol/L vs.10 μ mol/L,P＞0.05,the rest P＜0.05).Among SAG time-dependent groups,there was no significant difference in levels of RACK1 protein(P＞0.05).The relative expression level of GLI-1 mRNA increased at 2 h (3.027 ± 0.065),and compared with 0 h (2.651 + 0.123),there was significant differences (F=132.841,P＜0.05).3.In LPS+SAG intervention groups,the expression of RACKI was lower than that in LPS group (0.831 ±0.040 vs.1.189 ± 0.149,P＜0.05),and the expression of GLI-1 mRNA was higher than that in LPS group (2.720 + 0.130 vs.0.796-4-0.082,P＜0.05).Conclusions The LPS up-regulates the expression of RACK1 in RPMVECs,and the activated SHH signaling pathway can down-regulate the expression of RACK1 induced by LPS in RPMVECs.

OBJECTIVETo observe the effects of high expression of recombinant trichosanthin (rTCS) on the cell proliferation and cell cycle of human cervical cancer Caski cells.

METHODEukaryotic expression plasmid pcDNA3.1(-)/6His-TCS was constracted and stably transfected into Caski cells. RT-PCR,Western-blot were used to select the clones with rTCS high-expressing. Using pcDNA3.1(-)-transfected cells as the control, MTT assay and flowcytometry were used to elucidate the effects of rTCS high expression on cell growth and cycle regulation in Caski cells.

RESULTThe Caski cells with stable high expression of rTCS was successfully established, which could inhibit the cell growth (P<0.01) and arrest Caski cells in G1 and G2 phases (P<0.05) obviously.

CONCLUSIONHigh expression of rTCS can inhibit the growth of Caski cervical cancer cells, which might provide a new pathway for the therapy of cervical cancer.

Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Female ; Humans ; Recombinant Proteins ; pharmacology ; Transfection ; methods ; Trichosanthin ; pharmacology ; Uterine Cervical Neoplasms ; pathology

Objective To explore the related genes that play a key regulatory role in cisplatin resistance in lung adenocarcinoma. Methods Bioinformatics methods were used to download the differentially-expressed genes between cisplatin sensitive group and drug resistant group in patients with lung adenocarcinoma in TCGA database and GDSC database. GO function analysis and KEGG pathway enrichment analysis were carried out to analyze the differentially-expressed genes. The protein-protein interaction network was constructed and hierarchical cluster analysis was carried out to screen the key genes. The key genes were verified at the cell level by real-time fluorescence quantitative PCR and ELISA. Then the expression of the selected key gene in A549/DDP cells was silenced by siRNA and its sensitivity to cisplatin was detected. Results We screened out 178 differentially-expressed genes. After cluster analysis, CXCL9, CXCL10, NKX2-1 and SFTPA1 were regarded as the key genes of cisplatin resistance in lung adenocarcinoma. CXCL10 was temporarily selected for subsequent verification and function experiment. The mRNA expression of CXCL10 in A549/DDP cells was significantly higher than that in A549 cells (P < 0.001), and the expression of CXCL10 protein in the supernatant of A549/DDP cells was higher than that in A549 cells, which were consistent with the prediction of bioinformatics. The sensitivity of A549/DDP cells to DDP increased after silencing CXCL10 expression. Conclusions CXCL10 is a key gene to regulate cisplatin resistance in lung adenocarcinoma. Downregulating the expression of CXCL10 can become a potential target for reversing cisplatin resistance in lung adenocarcinoma.