Objective To study the regulatory effects of microRNA-200b (miR-200b) on hypoxia-inducible factors-1α (HIF-1α） in neonatal immature rats with hypoxic-ischemic brain damage (HIBD).Method A total of 240 three-day-old neonatal Sprague-Dawley (SD) rats were randomly assigned into six groups with 40 rats in each group:the hypoxic-ischemic group (HI group),intraventricular injection of miR-200b agomir,intraventricular injection of miR-200b antagomir,intraventricular injection of agomir negative control group,intraventricular injection of antagomir negative control group and the normal control group.The HIBD models of immature neonatal rats were established except for the normal control group.The relative expressions of HIF-1 α in brain tissues of each group were detected using quantitative real-time-PCR at 12 h,1 d,3 d and 7 d after ventricular injection,and the changes of HIF-1α expression in each group were compared.Result (1) Compared with the control group,the expression of HIF-1oα of the HI group began to increase 12 h after the injection of normal saline into the lateral ventricle (P＜0.05),and reached the peak at 1d,with statistically significant difference (P＜0.05),and then gradually decreased to the normal control group level at 7 d.(2) No significant differences of HIF-1α existed among the HI group and the HI+ agomir negative control group and the HI + antagomir negative control group (P＞0.05),and the miR-200b carrier had no significant effects on the expression of HIF-1α.(3)HIF-1α continued to be highly expressed after the injection of antagomir into the lateral ventricle of HI,and was significantly higher than the HI group at 12 h (P＜0.05).No significant differences existed between the HI+antagomir group and the H1 group at 1 d,3 d and 7 d after antagomir injection (P＞0.05).The expression of HIF-1α was constantly lower than the HI group after the injection of agomir,and significantly lower than the HI group at 1d after injection (P＜0.05).Conclusion MiR-200b overexpression inhibits the expression of HIF-1α,and the low expression of miR-200b can increase the level of HIF-1oα in a limited time window.Therefore,miR-200b may participate in the regulation of brain injury in neonatal rats after HIBD by regulating the expression of HIF-1α.

OBJECTIVEThe aim of this study is to optimize conditions for labeling human periodontal ligament stem cells (PDLSCs) using enhanced green fluorescent protein (eGFP) infected by lentivirus vector and to obtain PDLSCs with high stable expressed eGFP.

METHODSPDLSCs were transfected with eGFP by lentivirus vector for 48 h via different multiplicity of infection (MOI) (25, 50, 100, 200 and 400) and the infection efficiency were analyzed by both fluorescent microscope and flow cytometry. The proliferation rate of infected PDLSCs was evaluated by MTT. The infected PDLSCs were further for detection of pluripotent, differentiation ability and alkaline phosphatase (ALP) expression ability.

RESULTSThe infection efficiency for each group were 44.7%, 60.9%, 71.7%, 85.8% and 86.9% respectively. Proliferation of PDLSCs was not affected when MOI was below 200; however, at MOI 400, the proliferation ability was affected compared with control group. The pluripotent and ALP abilities of PDLSCs were not changed by the infection.

CONCLUSIONInfection for 48 h at MOI 200 is optimal for labeling PDLSCs with eGFP using lentivirus vector, and the proliferation and differentiation abilities of PDLSCs are not affected obviously.

Alkaline Phosphatase ; Cell Differentiation ; Genetic Vectors ; Green Fluorescent Proteins ; Humans ; Lentivirus ; Periodontal Ligament ; Stem Cells ; Transfection

Platycodon grandiflorum (Jacq.) A. DC. is a famous medicinal plant commonly used in East Asia. Triterpene saponins isolated from P. grandiflorum are the main biologically active compounds, among which polygalacin D (PGD) has been reported to be an anti-tumor agent. However, its anti-tumor mechanism against hepatocellular carcinoma is unknown. This study aimed to explore the inhibitory effect of PGD in hepatocellular carcinoma cells and related mechanisms of action. We found that PGD exerted significant inhibitory effect on hepatocellular carcinoma cells through apoptosis and autophagy. Analysis of the expression of apoptosis-related proteins and autophagy-related proteins revealed that this phenomenon was attributed to the mitochondrial apoptosis and mitophagy pathways. Subsequently, using specific inhibitors, we found that apoptosis and autophagy had mutually reinforcing effects. In addition, further analysis of autophagy showed that PGD induced mitophagy by increasing BCL2 interacting protein 3 like (BNIP3L) levels.In vivo experiments demonstrated that PGD significantly inhibited tumor growth and increased the levels of apoptosis and autophagy in tumors. Overall, our findings showed that PGD induced cell death of hepatocellular carcinoma cells primarily through mitochondrial apoptosis and mitophagy pathways. Therefore, PGD can be used as an apoptosis and autophagy agonist in the research and development of antitumor agents.
Humans ; Mitophagy ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Cell Line ; Autophagy ; Apoptosis ; Membrane Proteins ; Proto-Oncogene Proteins/genetics* ; Tumor Suppressor Proteins/pharmacology*