Objective To study the apoptosis of multiple myeloma cell line KM-3 induced by NK cells. Methods WST-1 assay was used to detect the killing effect of KM-3 cells treated with NK cells at different effector(E):target(T) ratio. Flow cytometry was applied to analyze Annexin-V+/PI- apoptotic cells and the mitochondrial transmembrane potential. Results NK cells could significantly kill KM-3 cells in a dosand time-dependent manner (P <0.05). After KM-3 cells- were treated with NK cells for 48 hours, the Annexin-V+/PI- cells were increased obviously in dose-dependence (P <0.05). The Annexin-V+PI- cells were increased in time-dependence when treated with NK cells(E:T ratio at 10:1) (P<0.05). The mitochondrial transmembrane potential of KM-3 cells treated with NK cells were significantly decreased in dose-and time-dependence (P < 0.05). Conclusion NK cell can kill KM-3 cells and induce apoptosis in a dose-and time-dependence manner.

In order to optimize the ~(18)O labeling method, two key aspects, peptide dispersion and trypsin deac tivation were discussed o The addition of Rapigest SF in H_2~('8)O and microwave heating enhanced labeling efficiency of α-casein digested peptides(~(18)O/~(16)O) ratio >99%).Chemical modification with tris(2-carboxyeth yl) phosphine (TCEP) and iodoacetamide (IAA) resulted in trypsin deactivated completely.No significant back-exchange from ~(18)O to ~(16)O was observed after labeling in 6 days.The experiment result with peptide mixture from showed that the improved method could be effectively used to label protein and peptide.

Objective To investigate the clinical value of contrast-enhanced ultrasonography(CEUS) in evaluating treatment response of hepatocellular carcinoma after interventional therapy. Methods One hundred and three patients with 136 lesions of hepatocellular carcinoma confirmed by pathology were examined by common color ultrasound (US), contrast-enhanced CT, CEUS and DSA pre- and post-interventional treatment respectively. Results The sensitivity,specificity and accuracy of CEUS for focus judgment after interventional therapy were 95.8%, 95.6% and 98. 5% respectively. The sensitivity, specificity and accuracy of US in detecting tumor deactivation and residue were 92.3% ,77.4% and 83.1% respectively. CEUS were highly consistent with the results of enhanced CT/DSA (Kappa = 0.93) and significantly higher than those of US (Kappa = 0.66). Conclusions CEUS is useful to monitor the efficacy and guide treatment after interventional therapy.

AIM:To establish the murine model of oxygen induced retinopathy ( OIR) and to evaluate the inhibition of retinal neovascularization by erythropoietin ( EPO) blockade.
METHODS: Neonates of C57BL/6 mouse ( P7 ) were exposed to 75%±2% oxygen for 5d and return to normal air environment when 12d ( P12 ) to establish oxygen-induced retinal neovascularization model. The neonates were divided into groups, injected with 0. 5μL solution containing 25ng ( group A), 50ng ( group B), 250ng ( group C) of soluble erythropoietin receptor ( EPO-R) or PBS (group D) at P12, P14 and P16 in the right eye. On P17, the litters were sacrificed and their right eyes were enucleated and fixed with 4% paraformaldehyde, made pathological section. The number of breakthrough internal limiting membrane neovascular nuclei was counted with pathological retinal morphology, understanding theproliferative degree of retinal neovascularization.
RESULTS: The pathological sections showed the neovascular cell nuclei which penetrating the inner limiting membrane in intravitreal EPO receptor injection group was reduced statistically than that in PBS injection group, the difference was statistically significant ( P<0.01). And, neovascular nuclei count differences in the
various concentrations of EPO receptor group was statistically significant (P<0. 01). With the EPO receptor concentration increase, neovascular endothelial cells broken through the internal limiting membrane was reduced.
CONCLUSION: Intravitreal injection of soluble EPO receptor can block EPO and improve neovascularization. The new method is expected to become new treatment of ocular neovascular diseases.

AIM:To investigate the role of bcl-2 and bax expression in apoptosis of pulmonary artery smooth muscle cells(PASMC) of rats induced by Na +/H + exchanger isoform-1(NHE-1) inhibition in vitro. METHODS:Intracellular Ca 2+ concentration ([Ca 2+ ]i) was measured using Fura-2/AM. The expression of bcl-2 and bax mRNA in cells were detected by sqRT-PCR, and the expression of Bcl-2 and Bax protein were examined immunohistochemically. RESULTS:The expression of bax mRNA and Bax protein and [Ca 2+ ]i in cells transfected with NHE-1 ribozyme gene increased significantly compared with those of cells transfected with pLXSN and nontransfected control. Meanwhile, the expression of bcl-2 mRNA and Bcl-2 protein in cells transfected with NHE-1 ribozyme gene decreased significantly. CONCLUSION:Apoptosis of pulmonary artery smooth muscle cells induced by inhibiting NHE-1 may have relevance to the increase in [Ca 2+ ]i and bax expression and the decrease in bcl-2 expression.

Objective To investigate the effects of leptin on Th17 cells and the possible mechanism. Methods The leptin-deficient ( ob/ob) mice and their homologous wild-type mice were used in the study.The percentages of Th17 cells in peripheral blood samples, spleen tissues and lymph nodes were measured by flow cytometry ( FCM) analysis.The splenic CD4+T cells, separated from the ob/ob mice and the wild-type mice by using magnetic beads,were respectively cultured with leptin at various concentrations and with anti-leptin neu-tralization antibody to evaluate the effects of leptin on Th17 cells.The quantitative real-time PCR was performed to analyze Th17 cell-related cytokines at transcriptional levels.The levels of IL-6 and IL-17A in the supernatants of CD4+T cell culture were measured with Luminex technology.Results Compared with the wild-type mice, the ob/ob mice showed lower percentages of Th17 cells in both peripheral blood samples and spleen tissues (0.49%±0.03%vs 1.29%±0.1%, 1.56%±0.22%vs 2.47%±0.11%).There was a decrease in the percentages of Th17 cells upon the in vitro treatment of CD4+T cells from wild-type mice with anti-leptin antibody.The per-centages of Th17 cells were increased in a dose-dependent manner upon the in vitro treatment of CD4+T cells from ob/ob mice with leptin.Moreover, the levels of IL-17A and IL-6 and the transcriptional levels of RORγt, IL-17A and IL-6 in leptin deficiency group were lower than those of wild-type group, but were increased upon the treatment with leptin.No significant difference with the transcriptional levels of TGF-βand IL-23 was ob-served between the two groups with and without intervention.Conclusion Leptin deficiency seriously hampered the generation of Th17 cells in mice and resulted in a decreased expression of RORγt, IL-17A and IL-6 at mRNA level.The treatment of CD4 T cells with leptin might promote the generation of Th17 cells through up-regulating the transcription of RORγt and IL-6.

Objective To explore the effects of hammerhead ribozyme on the expression and activity of NHE 1 and pHi in pulmonary artery smooth muscle cells (PASMCs) of rats and its role in PASMCs proliferation in vitro . Methods According to the secondary structure of NHE 1 mRNA in rats, NHE 1 specific hammerhead ribozyme was designed with the assistance of computer. The recombinant vector of retroviral plasmid pLXSN and hammerhead ribozyme, PRZ, was transfected into the cultured PASMCs. G418 resistant cell clones were screened with 100 ?g/ml G418. Then, the expression of NHE 1 mRNA was detected by RT PCR, intracellular pH(pHi) value and recovery rate of pHi after intracellular acid loading were measured by fluorescent probe BCECF. 22 Na uptake and 3H TdR incorporation were determined, respectively. Results Compared with the cells transfected with pLXSN and non transfected cells, NHE 1 mRNA, pHi value, pHi recovery rate, 22 Na uptake and 3H TdR incorporation decreased significantly in cells transfected with recombinant vector PRZ. No significance was found between the pLXSN transfected group and non transfected group. Conclusion Hammerhead ribozyme can cleave the target NHE 1 mRNA specifically, reduce the expression of NHE 1 mRNA, induce intracellular acidosis and consequently suppress the proliferation of PASMCs.

To study the regulation of androgen receptor (AR) expression in human prostate cancer LNCaP cells by triptolide (TP) and the possible mechanism, by using qRT-PCR and Western blot, the AR mRNA and protein levels in TP treated LNCaP cells were detected, and the AR protein level in TP and NF-κB inhibitor treated LNCaP cells was also detected; a series of pGL3-AR promoter reporter gene vectors were built using restriction-free cloning method, and the vectors were employed to investigate the effects of TP on the transcriptional activity of AR promoter in LNCaP cells; the upstream proteins which may play regulatory roles were detected using western blot assay. After treated LNCaP cells with TP for 48 h, AR mRNA and protein expressions decreased with increasing TP concentration. The expression of AR target gene PART1 and prostate specific antigen (PSA) was also downregulated by TP treatment; a series of pGL3-AR promoter reporter vectors were constructed and validated by sequencing and luciferase activity; the results of dual luciferase reporter assay showed that TP downregulated AR at the transcriptional level; PI3K/AKT/NF-κB pathway which is associated with AR promoter activity was drowregulated by TP. In conclusion, our results demonstrated that the transcriptional activity of AR in LNCAP cells was downregulated by TP, and PI3K/AKT/NF-κB pathway may be involved in the regulation mechanism.
Cell Line, Tumor ; Diterpenes ; pharmacology ; Down-Regulation ; Epoxy Compounds ; pharmacology ; Genetic Vectors ; Humans ; Male ; NF-kappa B ; antagonists & inhibitors ; Phenanthrenes ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Promoter Regions, Genetic ; Prostate-Specific Antigen ; metabolism ; Prostatic Neoplasms ; metabolism ; RNA, Messenger ; Receptors, Androgen ; metabolism ; Signal Transduction ; Transcriptional Activation