Animals ; Cardiovascular Diseases ; therapy ; China ; Combined Modality Therapy ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Medicine, Chinese Traditional ; Phytotherapy

Objective Establish rabbit VX2 soft tissue tumor model,and treat it with percutaneous ethanol injection(PEI)under the guidance of magnetic resonance imaging.Make ready for the therapeutic evaluation with functional magnetic resonance imaging. Methods Fifteen healthy New Zealand white rabbits were included in this study.0.2 mL tumor tissue suspensions were injected into the rabbits’posterior limb.14 days later,all rabbits were underwent conventional MRI examination.PET were performed to all the tumors under the guidance of MRI in the next day of the examination.T2 WI was used as guidance and monitoring means.MR com-patible puncture needle with lateral hole was stabed into the lesion center,and inj ected anhydrous ethanol according to the volume of tumors’diameter (1 mL/cm )slowly.the tumors signal characteristics,morphological feature and pathological feature were ob-served pre and post-operation.Results All of the 1 5 rabbits were established soft tissue tumor model successfully;the success rate is 100%.The tumors were oval or round,3-4 cm in diam.MRI scanning showed low signal on T1 WI and high signal on T2 WI be-fore PEI.PEI was performed to all the tumors under the guidance of MR successfully with 3.5 mL ethanol injected into the tumors in average.T2 WI could monitor the ethanol in dispersion and distribution within the tumors clearly.Histologically,tumors were composed of large,uniform,oval/round cells arranged in solid nests which was intensive in the periphery of tumors.Necrosis tissue was apparent in the center of the tumors.10 days after operation,most tissue in the periphery of tumors was coagulative necrosis , only a few tumor cells left.Ranges of necrosis in the tumors center were obviously increased compared with pre-operation.Conclusion Rabbit VX2 tumor of soft tissue model is suitable for the therapeutic evaluation of tumor .It is an animal model which has the characteristic of simple to operate and high rate of suc-cessful.MR T2 WI can monitor the ethanol in dispersion and distribution within the tumors clearly.It is a good guidance and monitoring imaging method of tumor ablation.

AIM: To investigate the apoptosis and molecular mechanism of human hepatocellular carcinoma HepG2 cells induced by ginsenoside Rh4.METHODS: Human hepatocellular carcinoma HepG2 cells were treated with ginsenoside Rh4 at doses of 10, 20 and 40 μmol/L, and the inhibitory effect of ginsenoside Rh4 on HepG2 cell viability was measured by MTT assay.The apoptotic rate of HepG2 cells was analyzed by flow cytometry.The morphological changes of the HepG2 cells were observed by Hoechst 33258 and TUNEL staining.The expression of apoptosis-related proteins Bax, Bcl-2, caspase-3 and caspase-9 was determined by Western blot.RESULTS: Ginsenoside Rh4 promoted apoptosis of HepG2 cells in a dose-dependent manner.TUNEL and Hoechst 33258 staining showed that the cells appeared obvious shrinking, swelling and rupture after treated with ginsenoside Rh4 for 24 h.The results of Western blot showed that with the increasing concentrations of ginsenoside Rh4, the expression of pro-apoptotic proteins Bax, cleaved caspase-3 and caspase-9 increased, while anti-apoptotic protein Bcl-2 decreased gradually.CONCLUSION: Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells, and the main mechanism may be related to down-regulation of Bcl-2 and up-regulation of Bax, cleaved caspase-3, and caspase-9.

OBJECTIVETo investigate the mechanism of senescence delay of human diploid fibroblast (2BS) and protecting telomere length by epimedium flavonoids (EF).

METHODSThe drug sera of EF were used to treat the 2BS. The population doublings of 2BS cells were observed, the mRNA expression of p16 gene were determined by fluorescence real-time quantitative RT-PCR, the telomerase activation of 2BS cells were determined by TRAP-Hyb, the total retinoblastoma (Rb) and phosphorated Rb protein content were detected by ELISA, the telomere length of 2BS cells were determined by telomere restriction fragment (TRF) Southern blot assay.

RESULTSEF could significantly extend the population doublings of 2BS cells, the expression of p16 mRNA was decreased and the content of phosphorated Rb protein were increased by EF. The telomere lengthening of 2BS cells were improved by EF, but the telomerase was not activated.

CONCLUSIONIn senescence human fibroblasts 2BS cells, p16 gene mRNA expression increased, content of phosphorated Rb protein decreased and the telomere length of 2BS shortened, EF might delay the aging of cells through inhibiting the p16 gene expression, promoting the production of phosphorated Rb protein and to protect the length of telomere, but not activating the telomerase.

Animals ; Cells, Cultured ; Cellular Senescence ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; Epimedium ; chemistry ; Fibroblasts ; cytology ; Flavonoids ; pharmacology ; Male ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Retinoblastoma Protein ; metabolism ; Telomerase ; biosynthesis ; Telomere ; genetics ; metabolism ; Transduction, Genetic

To construct a rabies virus mutant, the psi region was replaced by the coding region of human cytochrome c gene, and the coding region for cytoplasmic domain of glycoprotein G was deleted in the full-length of genomic cDNA of rabies virus strain SRV9. The mutant plasmid and the plasmids with N, P, L and G structural proteins of wild type SRV9 were co-transfected into BHK-21 cells. It was shown by IFA that there were many specific fluorescence in the BHK-21 cells, and typical rabies virus virions were observed by electronic microscope. These results demonstrated that the mutant rabies virus was successfully rescued. The genetically modified SRV9 stain has promise to provide invaluable experimental tool to develop attenuated live rabies vaccine.
Animals ; Cell Line ; Cricetinae ; DNA, Complementary ; genetics ; DNA, Viral ; genetics ; Genome, Viral ; genetics ; Humans ; Microscopy, Immunoelectron ; Mutation ; Rabies virus ; genetics ; ultrastructure

To explore the feasibility of real-time quantitative PCR (QRT-PCR) for selecting cell strains which overexpress a certain transgene, expression level of RbAp46 was detected in transfected cell strains by using optimal real-time PCR with SYBR Green I. Meanwhile, semi-quantitative RT-PCR and Western blot were performed to compare with the QRT-PCR. The results showed that values of RbAp46(N) were 2064.42 +/- 253.47, 860.94 +/- 291.07, 234.456 +/- 31.08, 18.17 +/- 5.14 and 1.46 +/- 0.54 in K562/RbAp46, K562/CMV, SHG44/RbAp46 monoclone, SHG44/RbAp46 multiclone and SHG44/CMV, respectively. The results were consistent with that determined by semi-quantitative RT-PCR and Western blot. It is concluded that QRT-PCR provides a highly efficient and reproducible method for selection of transfected cell subclones at different level of transgene expression.
Blotting, Western ; Carrier Proteins ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; K562 Cells ; Nuclear Proteins ; genetics ; metabolism ; Organic Chemicals ; chemistry ; RNA, Neoplasm ; metabolism ; Retinoblastoma-Binding Protein 7 ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Transfection ; Transgenes ; genetics

In order to study the potential effects of exogenous WT1 gene isoform on apoptosis in leukemia cell line NB4 and its possible molecular mechanisms, the eukaryotic expression recombinant vector (pCB6(+)/WTA) containing full-length human WT1 isoform (WTA: -17aa/-KTS) cDNA and the vacant vector-alone were introduced into the leukemia cell line NB4 respectively by electroporation. The WTA mRNA and protein in cells were detected by RT-PCR and Western blot. Binding of Annexin V were tested by flow cytometry and agarose gel electrophoresis to verify whether exogenous WTA could induce apoptosis of NB4 cells. Expressions of p21, p53, bcl-2, bcl-XL and c-myc genes were determined by semi-quantitative RT-PCR after introducing recombinant vectors into the NB4 cells. The results showed that in exposure to As(2)O(3) at 0.8 micromol/L for 48 hours, the NB4/WTA cells exhibited the morphological hallmarks of apoptosis, the marked DNA ladder shown by gel electrophoresis, and the enhanced apoptosis rate marked by Annexin V. RT-PCR showed an increase in p21 and c-myc genes expression, a decrease in bcl-2 and a relative constant expression of p53, bcl-XL in NB4/WTA cells. It is concluded that the introduction and expression of exogenous WTA gene can lead to apoptosis of NB4/WTA cells by down-regulating the Bcl-2 gene expression and up-regulating the p21 and c-myc genes expression.
Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Leukemia ; genetics ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Proto-Oncogene Proteins c-myc ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Tumor Suppressor Protein p53 ; genetics ; WT1 Proteins ; genetics ; metabolism ; physiology ; bcl-X Protein ; genetics

In order to investigate the application of recombinant adeno-associated virus (rAAV) vector containing Tet regulation system and HSVtk gene in cancer gene therapy, pAAV/TRE/HSVtk/Tet-On was constructed and identified with PCR and restriction enzyme digestion. Packaging cells HEK293 were cotransfected with plasmids pAAV/TRE/HSVtk/Tet-On, pAAV-RC and pAAV-helper to produce infectious rAAV, and CsCl2 densitygradient centrifugation method was performed for purification and concentration of rAAV. The viruses were then transduced into MCF-7 cells. The results of dot blot hybridization indicate that the rAAV can transfer the target gene into MCF-7 cells. MTT assay showed that GCV could kill AAV-infected MCF-7 cells under the induction of Dox. The data demonstrated that rAAV containing Tet regulation system and HSVtk gene was successfully obtained, and could be used for further investigation of in vivo and in vitro experiments.
Cell Line, Tumor ; Dependovirus ; genetics ; metabolism ; Doxycycline ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; genetics ; Transfection

OBJECTIVETo evaluate target-controlled infusion (TCI) of remifentail-propofol and the balanced anesthesia of fentanyl-isoflurane during the operation with suspension laryngscope.

METHODSSixty ASA I-II patients scheduled for the surgery through suspension laryngoscopy were randomly divided into two groups: TCI group and control group. In TCI group, anesthesia was maintained with TCI remifentanil-propofol which was stopped at the end of operation. The target plasma concentration of remifentanil was set at 6 microg/L and propofol at 3 mg/L. In control group, anesthesia was induced with intravenous fentanl 2.5 microg/kg and propofol 1-2 mg/kg, maintained with fentanl 0.03 microg.kg(-1). min(-1) and 1% isoflurane which was stopped at the end of surgery. Intubation was facilitated with succinylcholine 1-1.5 mg/kg.MAP, HR, ECG, S(p)O(2) and P(ET)CO(2) were monitored during anesthesia. The following parameters were recorded and compared between two groups: (1) the changes in blood pressure (BP), heart rate(HR) and S(p)O(2) at different time point; (2) recovery profile including the time of response to verbal commands, autonomous breathing, tracheal extubation, orientation recovery, discharging from PACU after operation; (3) OAAS scores after operation; (4) postoperative complications; (5) unexpected events and awareness during operation.

RESULT(1) The hemodynamics were stable while the target plasma concentration of remifentanil was set at 6 microg/L and propofol at 3 mg/L. (2) During tracheal intubation, suspension laryngoscope was inserted, and extubation MAP was significantly lower in TCI group than that in control group; (3) There were no significant differences in hemodynamic values and S(p)O(2) of different time points between two groups. Study group was faster than control group on recovery profile including the time of response to verbal commands, autonomous breathing, tracheal extubation, orientation recovery and discharging from PACU. There was respectively one unexpected event in both groups.

CONCLUSIONRemifentanil supplemented with isoflurane anesthesia can achieve the optimal hemodynamic stability during the operation with suspension laryngoscopy and better recovery profile from anesthesia than fentanyl.

Adolescent ; Adult ; Anesthetics, Combined ; administration & dosage ; Anesthetics, Intravenous ; administration & dosage ; Blood Pressure ; Female ; Heart Rate ; Humans ; Laryngoscopy ; methods ; Male ; Middle Aged ; Piperidines ; administration & dosage ; Propofol ; administration & dosage

OBJECTIVETo study the potential effects of exogenous WT1 gene isoform on the differentiation of leukemia cell line NB4 and its possible molecular mechanisms.

METHODSThe recombinant eukaryotic expression vector (pCB6 + /WTA) containing full-length human WT1 isoform (WTA: -17AA/ -KTS) cDNA and the blank pCB6 + vector were transfected into leukemia cell line NB4 by electroporation. The WTA mRNA and protein in cells were detected by RT-PCR and Western blot. Cell morphology, NBT reduction and CD11b antigen expression in NB4 cells were assayed to evaluate cell differentiation. Expression of PML/RARalpha, p21 and c-myc genes was determined by semi-quantitative RT-PCR after transfection.

RESULTSCompared with NB4/WTA cells, NB4 and NB4/CMV (NB4 cells transfected with pCB6 + vector) cells had higher morphological differentiation rates and higher CD11b expression levels after exposure to ATRA for 48 hours. The percentage of NBT reduction in NB4/WTA cells was lower than that in control groups. The difference in NBT reduction rate between NB4/WTA and control cells was gradually increased after treated with ATRA for three days. The expression levels of PML/RARalpha, p21 and c-myc genes in NB4/WTA cells were notably increased.

CONCLUSIONOverexpression of exogenous WTA gene could partially inhibit the differentiation of NB4 cells by up-regulating the expression of PML/RARalpha, p21 and c-myc genes.

Cell Cycle ; Cell Differentiation ; genetics ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; metabolism ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; RNA, Messenger ; genetics ; Transfection ; WT1 Proteins ; genetics ; metabolism