Objective To establish a universal stem loop primer (USLP) based real-time PCR method to scan mature miR profile and quantify it's expression.Methods The common universal stem-loop primer pairs were re-designed; 8 random nucleotides were introduced at 3 ' end for reverse transcription of the mature miR,establishing a miR scanning and quantifying system based on SYBR Green Ⅰ PCR (improved USLP method).10-fold gradient diluted standard miRNA-155 cDNA ( 1 ～ 109 copies/μ1) were utilized to evaluate the sensitivity of this method.The specificity was verified by melting curve assay; the precision was assessed by intra-assay coefficient of variation (ICV) of threshold cycle (Ct value) through 20 repeated detections of the standard miR-155 cDNA (2 × 105,2 × 106,2 × 107 copies/μl) ; cost of the primers and time were evaluated,compared with that of the conventional USLP method.Peripheral blood samples were cultured with phytohaemagglutinin (PHA) for0 h,16 h,24 h,48 h and 72 h,and 87 candidate miR that may be associated with human immunity from PubMed data were scanned and quantified from the cultured T cells.Results The sensitivity of the improved USLP method was 103 copies/μl of standard miR-155 cDNA.Melting curve assay showed a single melting peak at 80 ℃,suggesting the excellent PCR specificity of miR-155.Precision of our method quantifying miR-155 was acceptable (ICV ＜ 2.5％ ).Compared with the traditional stem loop primers,our method saved 75％ cost of primers ( 1 917 bp vs 7 851 bp) and 60％ test time of reverse transcription (85 min vs 205 min).By our method,85 of the 87 miR expression in T cells had no significant difference after the PHA stimulation; the expression of miR-150 (72 h) decreased by 10 times and that of miR-155 (48 h) increased 8 times after culture with PHA (Z =-2.032,P =0.042;Z =- 2.023,P =0.043,respectively ).Conclusions The improved USLP method is fast,precise,sensitive,and cost-effective.It could be used for miR profile scanning and quantifying in T cells.

Objective To investigate the efficiency of Trichostatin A (TSA) in inducing cell apoptosis and altering the Notch pathway genes expression in PANC-1 cells line.Methods The survival rate and apoptosis of PANC-1 cells were measured by MTT assay and Hoechst 33258 staining,respectively.mRNA expression levels of the genes,numb,gcn512,dll3,hes6,eaf2,cytohesins,in PANC-1 cells were assessed by real-time quantitive PCR.Western blot was used to measure the expression of bcl-2,bax,actived caspase-3 and NICD protein which was the biologically active form of Notch-1.Results After culturing with 0.1,0.2,and 0.4 μmol/L TSA for 24 hours,the cellular survival rate of PANC-1 cells significantly decreased to 72％,58％ and 39％,respectively.The survival rate of PANC-1 was negatively correlated to time length of culture with TSA.Increased apoptosis of PANC-1 cells after 12,24 and 36 h culture with TSA was detected by Hoechst 33258 staining.Western blotting showed that the expression of bax,actived caspase-3 and NICD protein increased while the bcl-2 protein decreased after culture with TSA.In real time quantitive PCR assessment,the mRNA expression of numb and hes6 in PANC-1 cells were upregulated by TSA (P ＜ 0.05),while the mRNA expression of gcn512 and dll3 were down-regulated by TSA (P ＜ 0.05).While mRNA expressions of eaf2 and cytohesin1,2,3,4 were not affected by TSA.Conclusions TSA induces apoptosis of pancreatic cancer cell line PANC-1.The Notch signal pathway may be involved in inducing cellular apoptosis of PANC-1 when cultured with TSA.

Objective To prepare the erythrocyte membrane camouflaged gefitinib(GEF)nanomedi-cine,and to observe its in vivo half-life and blood drug concentration by using the lung cancer bearing nude mouse model and explore its tumor suppression effect and tumor suppression mechanism.Methods The GEF standard curve was established by HPLC.A549 tumor bearing nude mice were constructed and divided into the GEF group,GEF nanoparticles group(GEF-NPs group),erythrocyte camouflaged GEF nanoparticles group(RBC@GEF-NPs group).The plasma drug concentration at different time points was detected by HPLC,the cell cycle was detected by flow cytometry and the tumor cellular apoptosis was detected by TUNEL.Results The tumor volume in the RBC@GEF-NPs group was significantly smaller than that in the GEF group and GEF-NPs group(P＜0.05);the in vivo half-life period in the RBC@GEF-NPs group was sig-nificantly longer than the GEF group and GEF-NPS group(18.90 h vs.2.44 h vs.10.50 h,P＜0.05),and the difference was statistically significant(P＜0.05);the GEF drug concentration in the tumor tissues of the RBC@GEF-NPs group was 48.5 ng/mg,which was 5 times and 2 times of that in the GEF group(10.8 ng/mg)and GEF-NPs group(24.7 ng/mg)respectively(P＜0.05).Compared with the GEF group,the proportion of G0/G1 phase cells in the RBC@GEF-NPs group was increased from 55.5％to 71.35％(P＜0.05)and which in the G2/M stage was decreased from 10.05％to 4.22％(P＜0.05),which in the S stage was decreased from 34.62％to 24.43％(P＜0.05);the cellular apoptosis index in the RBC@GEF-NPs group was significantly higher than that in the GEF group and GEF-NPs group[(23.42±2.47)％vs.(8.34±1.32)％vs.(14.32±2.59)％,P＜0.05],and the differences were statistically significant.Conclusion RBC@GEF-NPs could in-crease the half-life and blood concentration of A549 lung cancer bearing nude mice,block and inhibit the lung cancer growth by promoting tumor cell apoptosis and G1 phase arrest.

Objective To investigate whether the population pharmacokinetics (PPK)models can optimize the initial dosage of individualized Valproic acid (VPA)in children with epilepsy. Methods The epileptic children without taking VPA previously were recruited from October 2015 to May 2017 at the Department of Pediatrics,Peking University First Hospital,and they were divided into the PPK model group and the traditional empirical method group by randomized method. The initial VPA dosages for the PPK model group were calculated by PPK model,whereas those of the traditional empirical method group were dosed at 20-25 mg/(kg·d)regularly. The steady-state serum trough concentrations of VPA were extracted,and then the number and percentage of the patients whose serum trough concen-trations of VPA were 50-100 mg/L in the 2 groups were analyzed and compared with prospectively randomized me-thod. Results Totally 65 epileptic children were recruited and they were randomly divided into the traditional empirical method group (32 cases)and the PPK model group (33 cases). Twenty-seven children in the traditional empirical method group were observed,and 12 children had local epilepsy attack and 15 had generalized seizures;whereas among 29 cases in the PPK model group,there were 12 local attack of epilepsy and 17 had generalized seizures. VPA add-on therapy was administrated in 9 cases and 15 cases in the traditional empirical method group and the PPK model group, respectively. There were 5 cases,21 cases and 1 case with VPA serum concentrations of ＜50 mg/L,50-100 mg/L and＞100 mg/L in the traditional empirical group;while there were 9 cases,20 cases and 0 case in the PPK model group. The VPA serum concentrations of 21 cases (77. 8%,21/27 cases)in the traditional empirical method group and 20 ca-ses (69. 0%,20/29 cases)in the PPK model group were 50-100 mg/L,respectively,and the difference was not sta-tistically significant(P＞0. 05). Conclusion Although the study doesn't suggest that the established PPK model of VPA in Chinese epileptic children is superior to the traditional empirical method,the PPK model might be potentially valuable for optimized individualized dosage adjustment for those with serum trough concentrations not in the reasonable range by the traditional empirical method and with clinical seizure or brain firing activities.