OBJECTIVETo investigate the effect of homoharringtonine (HHT) on proliferation and apoptosis of CML cell line K562 cells and to explore its possible mechanism through mTOR pathway.

METHODSK562 cells were cultured with different concentrations of HHT or in its combination with mTOR inhibitor rapamycin (RAPA) for 24 hours. The cell viability was analyzed by CCK-8 assay, the cell apoptosis was detected by flow cytometry, the expressions of BCL-6, Caspase-3 and mTOR signal pathway related proteins was assayed by Western blot, the expression of BCL-6 mRNA was determined by RT-PCR.

RESULTSThe HHT inhibited proliferation and induced apoptosis of K562 cells in a concentration-dependent manner(r=0.970). With the increasing of HHT concentration, the expression level mTOR signal pathway related proteins increased(r=0.908), while the mRNA and protein expression levels of BCL-6 decreased(r=-0.961, r=-0.981), as compared with the HHT alone, the combination of HHT with RAPA could down-regulate the expression of mTOR signal pathway related protein and caspase-3, and up-regulated expression of BCL-6.

CONCLUSIONHHT induces apoptosis of K562 cells by inhibiting BCL-6 expression through mTOR signal pathway.

OBJECTIVETo investigate the expression of tripartite-motif protein 25 (TRIM25) and pyruvate kinase M2 (PKM2) protein in non-small cell lung cancer (NSCLC) and explore their role in the occurrence and progression of NSCLC.

METHODSThe expressions of TRIM25 and PKM2 protein were detected in 60 NSCLC specimens and 20 adjacent normal lung tissue (>5 cm from the lesions) with immunofluorescence histochemical method and in 10 fresh specimens of NSCLC with Western blotting. The results were analyzed in relation with the clinicopathological features of the patients.

RESULTSThe positivity rates of TRIM25 expression was 45% in the 60 lung carcinoma specimens, significantly higher than that in the 20 normal lung tissues (10%, P=0.005). TRIM25 protein was expressed in 28.6% of lung adenocarcinoma tissues and in 59.4% of squamous carcinoma tissues (P=0.017). TRIM25 protein expression was positively correlated with the TNM stages and lymph node metastasis of NSCLC (P<0.05). The expressions of PKM2 protein in 60 cases of lung carcinoma was 73.3%,while in 20 cases of normal lung tissues the expressions was 30%(P=0.001). The positivity rates of PKM2 expression differed significantly between lung adenocarcinoma and squamous carcinoma (57.1% vs 87.5%, P=0.008). An inverse correlation was noted between TRIM25 and PKM2 expressions (P=0.026).

CONCLUSIONTRIM25 and PKM2 protein may participate in the occurrence and progression of NSCLC, and their expressions are inversely correlated.

Adenocarcinoma ; enzymology ; Carcinoma, Non-Small-Cell Lung ; enzymology ; Carcinoma, Squamous Cell ; enzymology ; Carrier Proteins ; metabolism ; Humans ; Lung ; pathology ; Lung Neoplasms ; enzymology ; Lymphatic Metastasis ; Membrane Proteins ; metabolism ; Thyroid Hormones ; metabolism ; Transcription Factors ; metabolism ; Tripartite Motif Proteins ; Ubiquitin-Protein Ligases ; metabolism

AIMTo investigate the mechanisms of 17beta-estradiol on the production of endothelin-1 in vascular smooth muscle cells.

METHODSAfter incubation VSMC with various concentrations of 17beta-estradiol (10(-9) - 10(-7) mol/L) or plus L-NAME(10(- 6) mol/L) for different times, the concentration of endothelin-1 was measured. At the same time, the activity of endothelin converting enzyme-1 was analyzed, and the expression of preproET-1mRNA was measured by RT-PCR.

RESULTSIn basal conditions, 17beta-estradiol could inhibit the production of endothelin-1 in VSMC, and the action of 17beta-estradiol had nothing to do with the activity of endothelin converting enzyme-1. L-NAME inhibited the effect of 17-estradiol on the production of endothelin-1 in VSMC. RT-PCR results showed that 17-estradiol inhibited the preproET-1 mRNA expression, and whereas L-NAME reversed this action of 17beta-estradiol.

CONCLUSIONIn basal conditions, 17beta-estradiol decreases the preproET-1 mRNA expression through NO-pathway to inhibit the production of endothelin-1 in cultured VSMC.

Animals ; Cells, Cultured ; Endothelin-1 ; biosynthesis ; Estradiol ; pharmacology ; Muscle, Smooth, Vascular ; cytology ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects

OBJECTIVETo investigate the effect of telomerase reverse transcriptase (TERT) to the proliferation of 5-HT induced pulmonary artery smooth muscle cells (PASMCs).

METHODSThe PASMCs proliferation experiment was performed to detect the effort on PASMCs of 5-HT or ASODN TERT (antisense oligoribonucleotides TERT designed according to the rat TERT mRNA sequence of gene bank). The immunohistochemistry staining experiment and the in situ hybridization experiment were to detect the TERT protein and mRNA expression with 5-HT or ASODN TERT. FITC marked ASODN TERT experiment was done to research the distribution of ASODN TERT in PASMCs.

RESULTS5-HT promoted PASMCs proliferation in a dose-dependent manner (10(-9) - 10(-5) mol/L). 5-HT also significantly increased TERT expression at protein and mRNA levels as shown by immunohistochemistry staining and the in situ hybridization studies. This effect could be blocked by ASODN TERT in a time and dose-dependent manner.

CONCLUSIONSOur experiments show TERT is one of the key factors in the procession of 5-HT induced PASMCs proliferation. ASODN TERT might be a potential therapy agent for pulmonary hypertension.

Animals ; Cell Proliferation ; Cells, Cultured ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; Pulmonary Artery ; cytology ; RNA Replicase ; RNA, Messenger ; genetics ; Rats ; Serotonin ; pharmacology ; Telomerase ; pharmacology

Clinical epidemiologic data and animal experimental studies regard estrogen as being protective against the development of cardiovascular diseases. The mechanisms by which estrogen affects the development of vascular diseases are not clear. Recent studies demonstrated that the cardiovascular protective effects of estrogen are closely related to nitric oxide (NO) pathway. Our previous study proved that estrogen inhibited the proliferation and oncogene expression of vascular smooth muscle cells (VSMCs) induced by endothlin 1 (ET-1) and serum,this effect was mediated by NO release. In the present study, we investigated the role of inducible nitric oxide synthase (iNOS) in the VSMCs cycle arrest induced by 17 beta-estradiol (E(2)). The effects of E(2) on iNOS activity and protein expression in cultured rat VSMCs and the influence of NOS inhibitor N(G)-nitro-L-arginine methylester (L-NAME) on the inhibitory effect of E(2) on cell cycle were investigated. NOS assay kit was used to measure the activity of iNOS and protein expression of iNOS was determined by Western-blot. Cell cycle analysis was accessed by flow cytometry. The results obtained showed that E(2) increased iNOS activity of VSMCs but not in a dose-dependent manner. E(2) 10 nmol/L increased the iNOS activity of VSMCs distinctly at two time points: 30 min and 12 h. These effects were significantly inhibited by estrogen receptor (ER) antagonist Tamoxifen (0.1 micromol/L) and NOS inhibitor L-NAME (1 micromol/L). E(2) increased iNOS protein expression of VSMCs in a dose-dependent manner. The effect of E(2) on iNOS protein expression of VSMCs started at 3 h, distinctly increased at 12 h and then decreased. Tamoxifen significantly inhibited the E(2)-induced iNOS protein expression of VSMCs. ET-1 increased cell percentage of S phase and G(2)+S/G(1). This effect was inhibited by E(2). L-NAME significantly attenuated the inhibitory effect of E(2) on cell cycle of VSMCs. The results suggest that E(2) induced G(1) arrest of VSMCs, which was associated with an increase in iNOS activity and protein expression of VSMCs. These effects were at least mediated by estrogen receptor partly.
Animals ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Cells, Cultured ; Endothelin-1 ; metabolism ; Estradiol ; pharmacology ; Estrogen Antagonists ; pharmacology ; Female ; Muscle, Smooth, Vascular ; cytology ; Nitric Oxide Synthase ; metabolism ; physiology ; Nitric Oxide Synthase Type II ; Rats ; Tamoxifen ; pharmacology

Rat vascular smooth muscle cells (VSMC) were used to study the effect of 17beta -estradiol (E(2)) on cellular proliferation (cell counting), DNA synthesis ((3)H thymidine incorporation), MTT, c -fos mRNA expression and nitric oxide (NO) release. The results obtained showed that E(2) (10(-12) 10(-8) mol/L) induced NO release from VSMC in a concentration-dependent manner; 10(-8) mol/L E (2)significantly inhibited VSMC cellular proliferation and DNA synthesis induced by 10% FCS and 10(-7) mol/L ET-1, which was obviously reversed by 10(-7)mol/L tamoxifen and 10(-6) mol/L L-NAME; after a pretreatment for 24 hours, 10(-8)mol/L E(2) significantly inhibited VSMC c -fos mRNA expression induced by 10(-7)mol/L ET-1, which was also obviously reversed by 10(-6) mol/L L-NAME. These results suggest that the inhibitory effects of E(2) on VSMC cellular proliferation and c -fos mRNA expression are closely related with NO release in VSMC, which is, at least, partly medicated by ER.
Animals ; Cell Division ; drug effects ; Cells, Cultured ; Estradiol ; pharmacology ; Female ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Nitric Oxide ; metabolism ; physiology ; Proto-Oncogene Proteins c-fos ; biosynthesis ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Sprague-Dawley

In the present study, confluent bovine aortic endothelial cells (BAECs) were used to study the rapid nongenomic effects of 17beta-estradiol and the membrane impermeable conjugated 17beta-estradiol (E(2)BSA) on the activation of endothelial nitric oxide synthase (eNOS) and mitogen activated protein kinase (MAPK). eNOS activation was assessed in whole cells by measuring [(3)H]L-arginine conversion to [(3)H]L-citrulline. MAPK activity was determined by Western blotting. The results obtained show that the addition of various concentrations of E(2) (0.001-1 micromol/L) resulted in 122+/-29, 186+/-17, 83+/-20 and 157+/-29% increases in eNOS activity, respectively, in BAECs within 15 min of exposure to the hormone. E(2) (0.01 mol/L)-stimulated eNOS activity was detectable during 5-, 15- and 30- min incubation which yielded increases of 37+/-6, 56+/-9 and 38+/-8%, respectively. The increase reached a plateau from 15 through 30 min and rapidly declined thereafter. E(2)BSA 17.5 ng/ml also enhanced eNOS activity by an increase of 35+/-9% above the basal activity. The effect of E(2) and E(2)BSA on eNOS activation was unaffected by actinomycin D 25 microg/ml but was obviously inhibited by tamoxifen (0.1 micromol/L) and PD98059 (50 micromol/L). Compared with control E(2) and E(2)BSA stimulation of BAECs for 15 min caused an increase in MAPK activity by 428+/-17 and 360+/-14% respectively. This effect was blocked by tamoxifen. These results suggest that there might be the membrane estrogen receptor localized on BAECs, which mediates the rapid nongenomic effect of estrogen on eNOS activation through MAPK pathways.
Animals ; Aorta ; cytology ; Cattle ; Cell Membrane ; metabolism ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Estradiol ; pharmacology ; Mitogen-Activated Protein Kinases ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Receptors, Estrogen ; physiology

OBJECTIVETo evaluate therapeutic effects of Wallis interspinous dynamic stabilization in treating ASD after lumbar spinal fusion.

METHODSTotally 40 patients (included 16 males and 24 females, aged 25 to 60 years old) with degenerative disc disease were treated with posterior interbody fusion. Among them, 20 cases (treatment group) were treated with posterior interbody fusion combined with Wallis interspinous dynamic stabilization, while other 20 cases (control group) only treated with posterior interbody fusion. JOA score and VAS score were compared after inserted Wallis interspinous dynamic stabilization at 1 month and 3 years, and changes of intervertebral disc height of adjacent segment and cross-sectional area of the canal were tested and compared.

RESULTSAll patients were followed up from 3 to 5 years with an average of 3.6 years. All injuries were healed at stage I and the pain were released after treatment. There were no significant meaning in JOA score and VAS score at 1 month after treatment between two groups (P>0.05), while had meaning at 3 years (P<0.05). There were no statistical significane in intervertebral disc height of adjacent segment and cross-sectional area of the canal at 1 month after treatment (P>0.05), while had statistical meaning at 3 years (P<0.05).

CONCLUSIONThere is no difference in immediate effects between two groups. Both of them can obtain good results for effective decompression. Medial-term effectiveness of treatment group is obviously better than control group, which depends on Wallis interspinous dynamic stabilization to plays good biology effects and effective accelerate adjacent degeneration caused by lumbar fusion.

Adult ; Decompression, Surgical ; Female ; Humans ; Intervertebral Disc Degeneration ; surgery ; Lumbar Vertebrae ; surgery ; Male ; Middle Aged ; Spinal Fusion ; Treatment Outcome

Using cell culture, radioimmunoassay for endothelin and RT-PCR, the effect of aldosterone on the endothelin secretion of ventricular fibroblasts was studied. The results showed that aldosterone (1×10－7 mol/L) promoted the expression of ppET-1 mRNA, which began to increase in 2 hours and attained the highest level in 4 hours, thereafter decreased; aldosterone increased the endothelin level in ventricular fibroblasts and fibroblast conditioned growth medium (FCGM) as well, which was blocked by spironolactone (1×10－6 mol/L), an aldosterone receptor antagonist. The results suggest that aldosterone can increase endothelin secretion by ventricular fibroblasts, which can be inhibited by its receptor antagonist spironolactone.

Objective To observe the immunohigical effects of three doses of H2 strain live attenuated hepatitis A vaccine 8 years after the administration and to compare with that of one dose of the vaccine. Methods In a country area, 110 children of 1 to 7 years old susceptible to HAV were screened and administered with one dose of the vaccine, as group B ; Group A were 42 children from one of the villages and administered with 3 doses of the vaccine according to 0, 2, 6 month schedule. Blood samples were taken for the children 1, 2, 6, 7, 8,12, 24, 36 and 96 months after the administrations respectively and detected for anti-HAV antibody. Results For group B, the sero conversion rate of anti-HAV and GMC reached peak at 92.2% and 126.2 mIU/ml respectively,and then, began to drop with time; For group A, after 2 dose of the vaccine, the sero-conversion rate reached 100%, and the GMC reached peak of 2 739 mIU/ml one month after the third dose at 7 months. So that, group A has a better short-term immunological effects than that of group B. During 36 through 96 months, the anti-HAV positive rate in group B was 75%-71% and 80-89 mIU/ml respectively, and comparatively in group A were 100% and 918.2-480.6 mIU/ml respectively. The differences between group A and B were significantly important. Conclusion A 3-dose schedule administration of H2 strain live attenuated hepatitis .A vaccine has better immunological effects than 1-dose schedule in 8years and further observations are needed.