OBJECTIVETo develop an effective method for screening recombinant hairpin RNA expression plasmids using single restriction endonuclease analysis.

METHODSThe double-strand DNA fragment containing a ClaI site (the flanking sequences of which were not complementary) was annealed and ligated into small hairpin RNA (shRNA) expression vector pSilencer-4.1 that did not contain ClaI site to construct the circular pSilencer-4.1-ClaI vector. With BamHI and HindIII, the pSilencer-4.1-ClaIwas digested and ligated with the DNA template of green fluorescence protein (GFP) shRNA that did not include a ClaI site. The plasmid DNA of the positive clones was extracted and digested with ClaI, and the inserted DNA sequence of the non-linearized plasmid was identified by sequence analysis.

RESULT AND CONCLUSIONDNA sequencing showed that pSilencer-4.1-ClaI was correctly constructed and the plasmids resistant to ClaI digestion were all recombinant vectors encoding GFP shRNA. The constructed pSilencer-4.1-ClaI can be used as a universal vector to construct the shRNA expression plasmid, and the incorporated ClaI sites may allow efficient screening of recombinant shRNA expression vectors.

Base Sequence ; Gene Expression ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Inverted Repeat Sequences ; Molecular Sequence Data ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; Restriction Mapping ; methods ; Sequence Analysis, DNA ; Time Factors

Objective To explore the perinatal outcomes of women with pulmonary hypertension complicating congenital heart disease(CHD).Methods Clinical data of 45 cases of pregnant women with pulmonary hypertension complicating CHD from Apr 1995 to May 2007 were analyzed and they were divided into three groups:29 cases of slight group[pulmonary hypertension of 30 mm Hg(1 mm Hg=0.133 kPa) to 49 mm Hg],8 cases of moderate group(pulmonary hypertension of 50 mm Hg to 79 mm Hg)and 8 cases of severe group(pulmonary hypertension equal to or higher than 80 mm Hg).The types of CHD,cardiac functional status(New York heart association,NYHA),gestational weeks of pregnancy termination,mode of delivery,pregnancy after CHD operation and outcomes of infants were compared between the groups. Results(1)The highest incidence of CHD were atrial septal defect and ventricular septal defect(58%, 26/45).The rate of pregnant women after CHD operation was 29%(13/45),they were mainly in slight group and their NYHA class were in Ⅰ-Ⅱ.(2)The occurrence rate of NYHA class Ⅲ-Ⅳ was 7/8 in severe group.The rate of NYHA class Ⅰ-Ⅱ as 6/8 in moderate group.The rate of NYHA class Ⅰ- was 97%(28 /29)in slight group.(3)The rate of term delivery was 93%(27/29),preterm labor 3% (1/29),abortion 3%(1/29),and the birth weight was(3153?399)g on average in slight group.The rate of term delivery was 5/8,preterm labor occurred in 3 cases in moderate group.The rate of term delivery was 5/8,preterm labor occurred in 2 cases,and iatrogenic abortion in 1 case in severe group.The average birth weight between slight group and moderate or severe group had a significant difference.(4)Caesarean section rate was 78 %(35/45)among all patients.The rate of cesarean section delivery was 76%(22/29)in slight group,6/8 in moderate group,and 7/8 in severe group.(5)The rate of pregnant women who had portent heart failure or heart failure was 24%(11/45),overall maternal mortality was 4%(2/45).Conclusions The higher the pulmonary hypertension,the worse the outcome of the mother and fetus;The pregnant women with good heart function after cardiac operation would have a good perinatal outcome.Cesarean section is more suitable for those women.

OBJECTIVETo establish an efficient method for screening effective small interference RNA (siRNA) using dual-luciferase reporter assay system.

METHODSBased on the siRNA expression vector pSilencer-4.1, 3 candidate green fluorescence protein (GFP) gene siRNA expression plasmids, namely pSi-GFPsiRNA1, pSi-GFPsiRNA2, and pSi-GFPsiRNA3, along with the negative control pSi-Negative, were constructed. Using the pGL3-promoter vector, the GFP-luciferase (GFP-LUC) expression plasmid pGL3-GFPf was constructed with the same Kozak consensus translation initiation site and start codon ATG for GFP-LUC coding sequence. The GFP fragment containing the target sequences of 3 GFP siRNAs was introduced into the 3' untranslate region of LUC in the modified pGL3-promoter vector to construct the plasmid pGL3-GFPp. The GFP siRNAs expression plasmids and Renilla luciferase reporter vector pRL-TK were co-transfected with pGL3-GFPf or pGL3-GFPp into the HEK293 cells, respectively. The luciferase activities were determined by dual-luciferase reporter assay, and the GFP mRNA expressions were detected by real-time quantitative PCR.

RESULTSIn the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPf, the luciferase activities were reduced obviously, and the reduction was more significant in cells transfected with GFPsiRNA1 compared with the control cells (P<0.01).GFP mRNA levels were also markedly lowered in cells transfected with GFPsiRNA1 as shown by real-time PCR (P<0.01). In addition, the results of dual-luciferase reporter assay and real-time PCR showed that among the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPp, the GFP expression was inhibited most obviously by GFPsiRNA1 (P<0.01).

CONCLUSIONThe dual-luciferase reporter assay system provides a useful method for screening effective siRNAs targeting specific genes.

Animals ; Cell Line ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; Luciferases ; genetics ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

We report two rare cases of primary choriocarcinoma in the pineal region verified histologically. In both cases, the pre-operative serum level of human chorionic gonadotropin (HCG) was significantly elevated to 128-/+935.7 and 9 -/+088.9 mIU/ml, respectively, and serum alpha-fetoprotein (AFP) was negative. The tumors were microsurgically removed, and postoperative hydrocephalus were treated by endoscopic third ventriculostomy. Both patients underwent chemotherapy and radiotherapy. After adjunctive treatment, the serum HCG decreased within normal range. During the two-year-long follow-up, no radiological (MRI) evidence was found to suggest recurrence in MR imaging, and the serum HCG was normal in one patient, but mildly elevated in the other. HCG measurement can be crucial to the diagnosis and post-treatment monitoring of choriocarcinoma, and radical surgical tumor removal and combined modality therapy including chemotherapy and radiotherapy may ensure good results.
Adolescent ; Child ; Choriocarcinoma ; blood ; diagnosis ; surgery ; therapy ; Combined Modality Therapy ; Follow-Up Studies ; Humans ; Magnetic Resonance Imaging ; Male ; Pinealoma ; blood ; diagnosis ; surgery ; therapy ; Recurrence ; Testicular Neoplasms ; blood ; diagnosis ; surgery ; therapy ; Treatment Outcome

OBJECTIVETo construct an eukaryotic expression vector for miRNA-1-2 that can be expressed in rat H9C2 cardiomyocytes.

METHODSThe precursor miRNA (pre-miRNA) DNA template for miRNA-1-2 was designed and generated by PCR amplification. The DNA template was inserted into the hairpin RNA expression vector pSilence-4.1-neo and identified by DNA sequencing analysis. The recombinant plasmid DNA was then transfected into H9C2 cells via Lipofectamine, and the green fluorescence protein expression vector pEGFP-N3 served as the transfection marker. Twenty-four hours after transfection, the total cellular RNA was extracted using TRIzol reagent, and thermoscript reverse transcriptase (RT)-PCR was performed to determine miRNA-1-2 precursor expression.

RESULTS AND CONCLUSIONDNA sequencing indicated that the miR-1-2 expression plasmid was correctly constructed. The precursor miRNA-1-2 was successfully expressed in the H9C2 cells, and the expression of Hand2 protein could be efficiently inhibited by miRNA-1.

Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Cell Line ; Down-Regulation ; Green Fluorescent Proteins ; genetics ; metabolism ; MicroRNAs ; genetics ; Myocytes, Cardiac ; cytology ; metabolism ; Plasmids ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Rats ; Transfection

The effects of ketamine on transient outward potassium current (I(to)) of isolated human atrial myocytes were investigated to understand the mechanism of part of its effects by whole-cell patch-clamp. Atrial myocytes were enzymatically isolated from specimens of human atrial appendage obtained from patients under going cardiac valve displacing. Ito is recorded in voltage-clamp modes using the patch-clamp technique at room temperature. Currents signals were recorded by an Axopatch 200B amplifier with the Digidata 1322A-pClamp 9.0 data acquisition system. Ketamine decreased I(to) of human atrial myocytes in a dose-dependent manner. The current-voltage curve was significantly lowered, 30, 100, 300, and 1000 micromol x L(-1) ketamine decreased respectively I(to) current density about (13.62 +/- 0.04)%, (38.92 +/- 0.05)%, (72.24 +/- 0.10)% and (83.84 +/- 0.05)% at the potential of 50 mV, with an IC50 of 121 micromol x L(-1). The I(to) activation curve, inactivation curve and the recovery curve were not altered by ketamine. So, ketamine concentration-dependently decreased I(to) of human atrial myocytes.
Adolescent ; Adult ; Aged ; Anesthetics, Dissociative ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Heart Atria ; cytology ; Humans ; Ketamine ; administration & dosage ; pharmacology ; Male ; Middle Aged ; Myocytes, Cardiac ; cytology ; drug effects ; physiology ; Patch-Clamp Techniques ; Potassium Channels ; drug effects ; Young Adult

OBJECTIVETo investigate whether Leptotrombidium scutellare could be naturally infected by both Hantaan virus (HV) and Orientia tsutsugamushi (OT) and transmission status by stinging.

METHODS3459 Leptotrombidium scutellares from mice bodies and 3265 which were free were collected in the epidemic area of hemorrhagic fever with renal syndrome (HFRS) and tsutsugamushi disease.15 days later, the suspensions of lung and spleen of mice with 6 in a group stung by 1, 5 or 10 infected mites were injected intra-cerebrally into other mice for the detection of HV and OT in the next 6 generations of the mice, with immunofluorescent antibody technique (IFAT) and Giemsa staining technique. The passages of Vero-E6 cells inoculated on the aseptic filtrations from different number of infected mites were used to detect HV and OT pathogens. HV-RNA and OT-DNA were detected by PCR.

RESULTSAfter passage, HV positive mouse body mite group out of both 5 and 10 mites in the sixth generation, OT positive mouse body mite group out of the 10 mites in the sixth generation, both HV and OT positive mouse body mite group out of 1 mite in the fifth and sixth generation, both HV and OT positive mouse body mite group out of 5 and 10 mites in the sixth generation, and free mites group out of 1, 5 and 10 mites in the sixth generation, were found one mouse infected by both HV and OT, respectively. Out of the fourth generation of Vero-E6 cells, one sample was found both HV and OT positive out of 5 and 10 HV and OT mouse body mite group, respectively. In the sixth generation, both HV and OT positive cells were detected in one mouse mite group and the 1, 5, 10 free mite groups, respectively. HV-RNA and OT-DNA were all detected by PCR.

CONCLUSIONBoth HV and OT could be coexisted in wild Leptotrombidium scutellare and transmitted by stinging.

Animals ; Hantaan virus ; Hemorrhagic Fever with Renal Syndrome ; transmission ; Insect Bites and Stings ; Mice ; Mice, Inbred Strains ; Mites ; parasitology ; virology ; Murinae ; Orientia tsutsugamushi ; Scrub Typhus ; transmission ; Trombiculidae

OBJECTIVETo evaluate the efficacy and safety of the Qingrelishi-category Chinese medicine (for dispelling heat and resolving dampness) in the treatment of chronic prostatitis.

METHODSRandomized clinical trials or controlled clinical trials comparing Qingrelishi with plant america, other herbal medicine and Western medicine in the treatment of chronic prostatitis were identified by electronic and manual retrieval and analysis. The methodological quality of the included trials was assessed and Meta-analysis was performed with Revman 4. 2 software.

RESULTSForty-four randomized clinical trials or controlled clinical trials (n=5746) were identified. The methodological quality ranked high in three double-blind trials and the others ranked low. Meta-analysis indicated that Qingrelishi was more effective than Nankangpian( RR 1.22, 95% CI 1.10-1.35) and Prostate( RR 1.26, 95% CI 1.13-1.41) in the treatment of chronic prostatitis. Subgroup analysis revealed that Qingrelishi was more effective than Qianliekang (RR 1.32, 95% CI 1.19-1.45) and quinolones antibiotic (RR 1.34, 95% CI 1.15-1.57). There were no significant differences in efficacy either between Qingrelishi and a-receptor blocker and Puleanpian or between Qingrelishi plus quinolone antibiotics and quinolone antibiotics alone. Eighteen articles reported side effects and no serious adverse events were reported.

CONCLUSIONQingrelishi may be effective in the treatment of chronic prostatitis. However, the evidence is not strong due to the generally low methodological quality and the variations of the herbs. More randomized clinical trials are required.

Chronic Disease ; Databases, Bibliographic ; statistics & numerical data ; Drugs, Chinese Herbal ; adverse effects ; therapeutic use ; Humans ; Male ; Meta-Analysis as Topic ; Phytotherapy ; Prostatitis ; drug therapy ; Treatment Outcome

AIM:To investigate the role of microRNA (miR)-199a-5p in myocardial fibrosis and the potential target of miR-199a-5p.METHODS:C57BL/6 mouse cardiac fibroblasts were isolated and cultured for cellular experimen-tal study.Dual-luciferase reporter assay was performed to confirm the interaction between miR-199a-5p and the 3'-untrans-lated region (3'-UTR) of silent information regulator 1 (SIRT1).The expression of SIRT1 and fibrosis markers collagen (Col) 1a1, Col3a1 and α-smooth muscle actin (α-SMA) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively.RESULTS:The expression levels of miR-199a-5p, Col1a1, Col3a1 andα-SMA were marked-ly increased in cardiac fibroblasts after treatment with angiotensin Ⅱ(AngⅡ).The over-expression of miR-199a-5p signif-icantly increased the expression of Col1a1, Col3a1 andα-SMA in cardiac fibroblasts.Moreover, the results of dual-lucifer-ase reporter assay revealed that miR-199a-5p interacted with the 3'-UTR of SIRT1.miR-199a-5p inhibited SIRT1 expres-sion at post-transcriptional level.Meanwhile, miR-199a-5p mimic, in parallel to SIRT1 siRNA, inhibited SIRT1 expres-sion, increased the expression of Col1a1, Col3a1 and α-SMA in cardiac fibroblasts.Inactivation of NF-κB signaling con-tributed to the decrease in miR-199a-5p in Ang II-treated cardiac fibroblasts .CONCLUSION:SIRT1 is a target gene of miR-199a-5p, which mediates the pro-fibrotic effect of miR-199a-5p on cardiac fibroblasts .

Aim To study whether there was arterial heterogeneity and association with L-type calcium channel (LCC) in different parts of arteries in re-sponse to certain vasoconstrictor. Methods The aor-ta, renal arteries and coronary arteries were dissected from rats. Arterial ring contractions induced by pheny-lephrine (Phe), 5-hydroxyl tryptamine (5-HT) or U46619 in concentration-dependent manner were meas-ured using the Multi Myograph system and the response to nifedipne was observed. Results (1) Phe had no obvious effect on the tension of coronary artery,but in-duced concentration-dependent vasoconstriction in aor-ta and renal artery,and pEC50of aorta was significantly higher than that of renal artery (P<0.05). The inhi-bition rate of nifedipine on the aortic contractile re-sponses was significantly higher than that of renal arter-y (P<0.05). (2) The contraction induced by 5-HT on aorta was not obvious, but was significant on renal artery and coronary artery. The inhibitory rate of nife-dipine on coronary artery vasoconstriction was signifi-cantly higher than that of renal artery (P <0.05). (3) U46619 could induce aorta,renal artery and coro-nary artery concentration- dependent contraction, but the Emaxof them were both higher than that of renal ar-tery (P<0.05). And the pEC50of aorta was the lar-gest (P<0.05). Nifedipine significantly inhibited the contraction of aorta, renal artery and coronary artery induced by U46619 with the greatest inhibitory rate on the coronary artery vasoconstriction and minimal inhibi-tion on aortic vasoconstriction. Conclusions The re-sponse to certain vasoconstrictor is different among aor-ta, renal artery and coronary artery in rats, and the contraction mediated by L-type calcium channel is also different.