Object To explore the protoplast fusion of Dendrobium candidum Wall ex. Lindl. and Gynostemma pentaphyllum. (Thunb) Makino. Methods The two kinds of protoplasts were fused by PEG method, and then cultured in the modified liquid medium containing 2 mg/L BA and 1 mg/L NAA. Results High yield, viability, and pure mesophyll protoplasts were isolated from D. candidum and G. pentaphyllum. Conclusion The first cell division occurres within three days after fusion. And some of the cells are divided three times.

AIM The antisense drug design will be optimized based on bcl 2 mRNA secondary structure simulated with computer. METHODS bcl 2 mRNA second structures were simulated with computer and Mfold software, and the unstable zones on the second structure, as designing antisense zones, were selected. RESULTS Five antisense deoxynucletides were studied and evaluated with experiments of HL 60 and K562 leukemic cells. Two of them exist significant effect of inhibiting grow of HL 60 and K562 leukemic cells with dose of 10 ?mol?L -1 or more. CONCLUSION The designs with computer and corresponding software will be usefully efficient way to look for antisense drugs.

OBJECTIVE:To approach the information of stem cell transplantation in treating myocardial infarction.DATA SOURCES: An online search of Medline database was undertaken to identify articles about stem cell transplantation in treating myocardial infarction published in English from January 1980 to December 2005 using the keywords of "stem cells, myocerdial infarction, cell transplantation".STUDY SELECTION: The literatures were checked primarily, and articles about the stem cell transplantation in treating myocardial infarction were selected, and the repetitive studies and reviews were excluded.DATA EXTRACTION: Totally 106 relevant articles were collected, finally 30 of them were involved and the other 76 were excluded.DATA SYNTHESIS: Myocardial infarction occurs when the arteries that supply blood to the heart muscle (the coronary arteries) become hardened and narrowed. The transplantation of exogenous cells into damaged myocardium, a procedure known as cellular cardiomyoplasty (CCM), can be used to ameliorate the remodeling process by the regeneration of cardiomyocytes and replenishment of the vascular supply. Advances in stem cell therapy now provide wide cell sources for the treatment of cardiovascular disease by CCM such as skeletal myoblasts (satellite cells),embryonic stem cells (ESCs) and bone marrow-derived mesenchymal stem cells (MSCs). Although there are many problems to be resolved, recent in vitro and in vivo animal studies as well as clinical trials have suggested the promising potential of the transplantation of stem cells for the treatment of myocardial infarction.CONCLUSION:Stem cell transplantation acts as a more potential treatment measure for myocardial infarction. There are still some fundamental issues to be addressed for the clinical application in future.

OBJECTIVE: To investigate theresearch condition of mechanism of cardiomyocytes differentiation from embryonic stem (ES) cells and the application potency of ES cells at home and abroad.DATA SOURCES: We searched in Medline database from 1997 to 2005 with the key words of "embryonic stem cell,cardiomyocyte/cardiac myocyte,differentiation" in English.STUDY SELECTION: The retrieved data were selected primarily. Inclusive criterion: articles of differentiation into cardiomyocytes from ES cells.Exclusive criterion: the articles of repetitive research were excluded.DATA EXTRACTION: Totally more than 200 articles were researched,and 26 were included.DATA SYNTHESIS: Some articles of differentiation of ES cells into cardiomyocytes from the construction of primarily experimental methods of cardiomyocytes differentiation from ES cells to electrophysiologic study of ES cells-derived cardiomyocytes in monoplast level were collected. Furthermore, many investigators studied the expression of transcription factors that controlled cardio development in the differentiation of ES cells to cardiomyocytes from the angle of transcription factors. Cardiac transcription factors GATA4, MEF2C and NKx2.5 had been reported to control a cardiac gene program and thus to play a crucial role in transcriptional regulation during cardiogenesis. Meanwhile, calcineurin, MAPKs and other signaling pathways were also involved in the adjustment of cardiomyocyte differentiation. In addition, the purity of differentiated ES-derived cardiomyocyte cultures for myocardial infarction was a key issue.CONCLUSION: When ES cells are cultured within Ebs and in the absence of leukemia inhibitory factor (LIF), some of them can differentiate into cardiomyocytes. ES-derived cardiomyocytes express characteristic contractile proteins, such as MHC and myosin light chain (MLC)-2V. Transcription factors, including GATA4, MEF2C and NKx2.5, play a crucial role in the cardioblast differentiation and trigger the expression of specific genes such as α-actin, MHC and MLC2V. However, t he molecular mechanisms of cardiomyocyte differentiation from ES cells remain largely unexplored. The identification of factors that can promote cardiac differentiation is fundament in order to study cardiac differentiation.

AimTo explore the effects of bcl-2 antisense oligonucleotide targeting the coding region of the bcl-2 mRNA on apoptosis induced by Vp16 in HL60 and K562 cells. Methods Drug sensitivity was compared by MTT cytotoxicity assay, and expression of bcl-2 protein and apototic cells were assayed by flow cytometry. Results The bcl-2 antisense oligonucleotide of 10 μmol · L- 1 combined with etoposide inhibited expression of bcl-2 protein,increased apoptosis in HL60 and K562 cells and decreased IC50 of etoposide; The antisense oligonucleotide targeting the coding region of the bcl-2 mRNA had stronger effection than the antisense oligonucleotide targeting the translation initiation. ConclusionThe bcl-2 antisense oligonucleotide targeting the coding region of the bcl-2 mRNA can enhances etoposide induced apoptosis of HL60 and K562 cell.

Objective To explore the efficacy of radiofrequency ablation (RFA) combined with splenectomy in patients with small hepatocellular carcinoma (sHCC) associated with hypersplenism.Metheds The data of 100 patients with sHCC associated with hypersplenism who received RFA or hepatectomy combined with splenectomy were analyzed retrospectively.The patients were divided into the observation group and the control group based on the intraoperative approach.Fifty-three patients who received RFA and splenectomy were in the observation group,and the remaining 47 patients who received hepatectomy and splenectomy were in the control group.Multiple intraoperative and postoperative factors were compared between the two groups.Results There were significant differences between the two groups in warm ischemia time,operation time,intraoperative blood loss,hospital stay,and amount of blood transfusion (P ＜ 0.05).The postoperative complication rate of the observation group (7.6％,4/53) was significantly lower than the control group (44.7 ％,21/47) (P ＜ 0.05).There were no significant differences in 1-,3-,and 5-year survival (respectively,100.0％,75.5％,and 67.9％ vs 97.9％,76.6％,and 68.1％) and in disease free survival (96.2％,57.5％,and 41.7％ vs 93.5％,58.3％,and 43.8％ respectively) between the two groups (P ＞ 0.05).Conclusion RFA combined with splenectomy can be considered as an alternative treatment for patients with sHCC associated with hypersplenism.

Objective To detect the micro satellite instability (MSI) from the precancerous and cancerous lesions (GC) and its effect on carcinogenesis.Methods Silver staining single strand conformation polymorphism polymerize chain reaction (PCR SSCP) was used to screen MSI markers at 5 loci in formalin fixed,paraffin embedded tissues of GC (n=30), dysplasia (n=30), intestinal metaplasia (IM) ( n =40) and corresponding normal gastric tissues.Results The abnormal shifting of the single strand DNA was identified in 7(23 3%)out of GC, in 9 (30%) out of dysplasia and in 8(20%) out of IM samples respectively. GC with MSI was associated with distal location of the tumors ( P =0 044). MSI was not detected in low grade IM tissues.Conclusion MSI may be of an early event during gastric carcinogenesis. It contributes probably to the acquisition of a transformed cell phenotype and to the development of gastric cancer.

Objective To investigate the effects of gene transfection with human growth/differentiation factor 5(hGDF5)on the differentiation and proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs).Methods BMSCs were obtained from adult New zealand rabbits and purified by gradient centrifuge.Exogenous recombinant human GDF5 was transfected into BMSCs with liposome method.Then hGDF5 expression at mRNA and protein level was measured separately by reverse transcription-polymerase reaction and indirect immunofluorescence method.Activity of alkaline phosphates(ALP),expression of TypeⅡcollagen(ColⅡ),proteoglycan and growth of the cells were all measured by biological methods to evaluate the effects of hGDF5 gene transfer on the differentiation and proliferation of rabbit BMSCs.Results After hGDF5 gene transfection,BMSCs expressed hGDF5 mRNA and protein,and compared with the control groups,expression of proteoglycan and ColⅡ increased significantly,but no significant difference appeared in ALP activity and cell proliferation.Conclusion Gene transfer with hGDF5 is an effective way to enhance the expression of GDF5 at mRNA and protein.The expression of heterogenetic hGDF5 gene can induce BMSCs' differentiation to chondrogenic cells.But the gene transfection has no obvious effects on the proliferation and ALP activity of BMSCs.

BACKGROUND: Reactive oxygen species (ROS), including superoxide anion (O2) and hydrogen peroxide (H2O2), have been recognized as specific second messengers in signaling cascades involved in the growth and differentiation of cells.The generation of excessive ROS initiates cardiomyocyte apoptosis. This paper is aimed to corroborate the hypothesis that excessive amounts of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 4-dependent ROS can suppress cardiomyocyte differentiation from embryonic stem (ES) cells by induction of apoptosis.OBJECTIVE: To investigate the role of NOX4 ROS on cardiomyocyte differentiation.DESIGN: Randomized and controlled trial observation.SETTING: Beijing Institute of Geriatrics, Beijing Hospital, Key Laboratory of Geriatrics, Ministry of Health.MATERIALS: ES cells were preserved by the laboratory of Beijing Institute of Geriatrics, Beijing Hospital, Key Laboratory of Geriatrics, Ministry of Health, while other reagents were purchased from Sigma Company without specific notification.METHODS: The experiment was carried out in the Beijing Institute of Geriatrics, Beijing Hospital, Key Laboratory of Geriatrics, Ministry of Health from October 2003 to August 2005. ①Mouse ES cells were differentiated into embryoid bodies (Ebs). At day 4, Ebs were managed for one hour with different concentrations (1, 10, 100, 1 000 nmol/L) of H2O2, and generation of cardiomyocytes was observed at day 8 to analyze the effect of ROS on cardiomyocyte differentiation.② CGB8 cells transfected with pcDNA3.1 and pcDNA3.1-NOX4 respectively were adopted, while those untransfected CGB8 cells were taken as controls. A quantitative NBT (nitro blue tetrazolium) test was used to measure NOX4 ROS generation. The levels of NOX4 mRNA and MLC2v protein were assayed by RT-PCR and western blotting, respectively.③ROS scavenger N-acetylcysteine (5 mmol/L) and catalase (200 U/mL) managed for 2 hours before transcription were taken as controls to observe the apoptosis of CGB8 cell after NOX4 overexpressed and the expressions of p21, p53 and Bcl-2 of NOX4-transfected CGB8 cells simultaneously. And the apoptosis of ES cells was detected by Hoechst staining, TUNEL assay and DNA fragmentation.MAIN OUTCOME MEASURES: ①the role of ROS on cardiomyocyte differentiation.②the expression of NOX in ES cells.③ the production of ROS, differentiation of cardiomyocyte and apoptosis of ES cells after NOX4 overexpressed.④the feasibility that p53, p21 and Bcl-2 are involved in the apoptosis induced by the overexpression of NOX4.RESULTS: ①Different concentrations of ROS play different roles on cardiomyocyte differentiation. The exposure of Ebs to 1-100 nmol/L H2O2 for 2 hours at day 4 of culture leaded to an enhanced beating activity (P ＜ 0.01 or 0.001), whereas 1 μmol/L H2O2 depressed cardiomyocyte differentiation as compared with control conditions (P ＜ 0.001). ②NOX4 was highly expressed in ES cells, while NOX1 and NOX2 were absent and only a weak band of NOX3 was detected. The results from RT-PCR revealed that NOX4 overexpressed in CGR8 transfected with pcDNA3.1-NOX4 as compared to the control.③NBT result proved that highly-expressed NOX4 produced the excessive amounts of ROS (P ＜ 0.05). The beating activity was remarkably reduced in NOX4-overexpressing Ebs as compared to wild-type or mock-transfected Ebs (P ＜0.01). Moreover, a severe reduction of MLC2v protein in NOX4-overexpressing Ebs was also observed by western blot analysis.④p21 and p53 may be responsible for the NOX4-induced apoptosis. P53-/- cells R72D27 transfected by NOX4 did not induce apoptosis. NOX4 overexpression induced ES cell apoptosis could be prevented by overexpression of Bcl-2.CONCLUSION: The findings of this paper highlight the role of NADPH oxidase NOX4 in cardiomyocyte differentiation from ES cells. P53, p21 and Bcl-2 could be involved in the apoptotic pathway.

AIM: To investigate whether the bcl-2 antisense oligonucleotide increases the sensitivity of HL60 and K562 cell lines to daunorubicin. METHODS: IC50 for HL60 and K562 was determined with MTT method, the expression levels of Bcl-2 protein were assayed by immunofluorescence using fluoresce isothiocyanate labeling. In addition, apoptosis was detected by morphological observation and flow cytometric analysis of DNA fragmentation. RESULTS: It was found that the two oligonucleotides directed against the coding region and the translation initiation of bcl-2 mRNA, combined respectively with daunorubicin, inhibited expression of bcl-2 protein, increased apoptosis in HL60 and K562 cells, and decreased IC50 of daunorubicin significantly ( P