Aim To investigate the effects of ampelopsin on induction of apoptosis in human hepatocellular carcinoma Bel-7402 cells.Methods Bel-7402 cells were treated with ampelopsin with different concentrations for 24,48 and 72 h.The cell proliferation was detected by MTT assay.The morphological change of cells was observed through microscope observation by fluorescence staining.DNA fragmentation was visualized by agarose gel electrophoresis.The apoptosis rate was analyzed by flow cytometry.The expressions of caspase-3,Bcl-2 and Bax protein were detected by Western blot.Results Ampelopsin inhibited the proliferation of Bel-7402 cell line in a dose-and time-dependent manner.The IC50 values were 89.6?16.1,36.2?6.5 and 15.3?3.0 mg?L-1 at 24,48 and 72 h,respectively.The fluorescence microscope showed clearly cell apoptosis with apoptotic body.Agarose gel electrophoresis result showed that Bel-7402 treated with ampelopsin produced a DNA ladder band.The sub-G1 peak was detected and resulted in dose-and time-dependent increasing of the population of sub-G1 DNA content by flow cytometry.The expression of Bcl-2 protein was down-regulated,while the expression of Bax protein was up-regulated.The pro-caspase-3 protein was down-expressed and activated.Conclusions Ampelopsin could inhibit Bel-7402 proliferation through inducing cell apoptosis.The mechanism might be that ampelopsin could directly or indirectly enhance the level of anti-apoptosis protein Bcl-2 and decrease the level of apoptosis protein Bax.The pathway of pro-caspase-3 activated was initiated and effector caspase-3 was sequentially activated.

External applies and washing of traditional Chinese medicine has sound curative effect in treating muscular and bone pain.The author reported some his experiences on the treatment of muscular and bone pain with hot applies and tendon-relaxing and collaterals-activating lotion.

Objective To qualitatively analyze the expressions of key marker genes involved in osteogenic differentiation of bone marrow stromal cells (BMSCs) of mice at defined stages in vitro using real-time RT-PCR. Methods The BMSCs of mice were cultured in vitro and induced to osteoblasts for 7, 14, and 21 days. The total RNA was extracted from the cultured cells and the gene expression levels of collagenⅠ, bone alkaline phosphatase (APL), osteopontin (OPN) and bone sialoprotein (BSP) in the osteoblasts at the defined stages were determined by real-time RT-PCR. Results The ColⅠmRNA levels in 14 and 21 days were (0. 75?0. 04) and (0. 34?0. 03) times respectively of that in 7 days. ALP, OPN and BSP mRNA levels in 21 days were (19. 70?2. 36), (150. 12?9. 31) and (7. 73?0. 58) times of those in 7 days respectively. Conclusions ColⅠexpression levels tend to gradually decline while ALP, OPN and BSP levels gradually increase along with the culture time. Real-time RT-PCR is a reliable method for investigation of gene expressions in BMSCs cultured in vitro.

OBJECTIVETo study the anti-halitosis effect of sugar-free chewing gum through their influence on odor induced by cysteine.

METHODSTen volunteers were randomly divided into the treatment group and the untreated group; each group consisted of five volunteers. All volunteers consented to participate in a test in which breath odor was induced by cysteine. After the test, the treatment group chewed sugar-free chewing gum for 1 min, whereas the untreated group did not undergo any treatment. The effectiveness was determined by the percent reduction of H2S, CH3SH, and (CH3)2S response after the volunteers chewed gum for 1, 10, and 20 min.

RESULTSAt 1, 10, and 20 min, H2S of the treatment group was reduced by 82.68%, 92.27%, 97.47%, respectively, CH3SH was reduced by 65.49%, 73.79%, and 82.89%, respectively, and (CH3)2S was reduced by 60.45%, 73.82%, and 59.72%, respectively. The differences between the two groups at different times were significant (P < 0.05).

CONCLUSIONChewing gum can effectively inhibit cysteine-induced odor.

Objective To analyze the expression and regulation of HES1 in sperms with low motility.Methods Thirty semen samples from asthenospermia patients and 20 semen samples from healthy and fertile adults were collected,total RNAs were extracted to produce cDNAs probes.Hybridization with Phalanx OneArray~(TM) containing 30 968 probes was carried out after the labeled cDNAs were purified by PCR product purification kit.Realtime RT-PCR was used to analyze the expression of hsa-miR-487a and hsa-miR-193b;the expression of the target genes of hsa-miR-487a and hsa-miR-193b were searched from gene-expression profiles in asthenospermia patients' sperms.Results The expression level of HES1 in low motility sperms was up-regulated.The expression level of hsamiR-193b in low motility sperms was 2.19 times higher than that in high motility sperms,hsa-miR-487a was 0.43% of that in high motility sperms.Conclusion The expression level of HES1 in low motility sperms was up-regulated.Hsa-miR-487a and hsa-miR-193b may affect the expression of HES1 and so regulate sperm motility.

[Objective]To assess the clinical effect and radiological results of three methods of anterior cervical decompression and reconstruction for the treatment of multilevel cervical myelopathy.[Method]Fifty-two patients with continuous multilevel cervical myelopathy treated by the same team were divided into three groups.Group Ⅰ was performed the twosegment-corpectomy and reconstructed with a long titanium mesh and plate.Group Ⅱ was performed the three-level-discectomy and fused with three cages.Group Ⅲ was performed the combination with corpectomy and discectomy.The parameters included intervertebral height,JOA score.The data was compared statistically with Student s T-test or ANOVA.[Result]JOA score in all groups was improved post operation,but there was no significant difference among the three groups.The post-operation intervertebral height lose of group Ⅰ was more significant than other two groups,three cases of titanium meshs subsidence happened in group Ⅰ and one same case happened in group Ⅲ.[Conclusion]All the three surgical methods of anterior decompression and fusion can procure wonderful clinical effect,but group Ⅱ and Ⅲ were better than group Ⅰ in the stability and the maintenance of intervertebral height post operation.

The forced oscillation technique (FOT) is a noninvasive method for respiratory mechanics measurement. For the FOT, external signals (e.g. forced oscillations around 4-40 Hz) are used to drive the respiratory system, and the mechanical characteristic of the respiratory system can be determined with the linear system identification theory. Thus, respiratory mechanical properties and components at different frequency and location of the airway can be explored by specifically developed forcing waveforms. In this paper, the theory, methodology and clinical application of the FOT is reviewed, including measure ment theory, driving signals, models of respiratory system, algorithm for impedance identification, and requirement on apparatus. Finally, the future development of this technique is also discussed.
Algorithms ; Electric Impedance ; Oscillometry ; Physical Therapy Modalities ; Respiratory Mechanics

BACKGROUND: How to deal with bone defect is a big problem to surgeons. In recent years, the development in the technology of molecular biology and tissue engineering provides broad prospect for the clinical treatment of bone defect, which is one of the important study directions in department of orthopedics. The transforming growth factor-beta 1(TGF-β1),one of the important factors in bone formation, plays an important role in bone metabolism and recovery.OBJECTIVE: To observe the therapeutic effects of bone defects with osteoblasts transfected . By TGF-β1 combining with biomimetic biodegradable polymer scaffolds.DESIGN: Randomized controlled trial.SETTING: Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Siderophilin, trypsin, 3H-proline and sirius red, etc.MATERIALS: The experiment was completed in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from March to August in 2003. Twenty healthy adult Sprague-Dawley(SD) rats of SPF grade, weighing 200-250 g, were provided by the Experimental Animal Center of Tongji Medical College.METHODS: The osteoblasts transfected by TGF-β1 gene, combining with poly-DL-lactic acid scaffolds modified with poly-L-lysine, were transplanted into rat tibia defect. Radiographs and histological analysis were performed to evaluate the repair effects.MAIN OUTCOME MEASURES: The X-ray evaluation and histology observation were performed at the 4th and 8th weeks after the operation.RESULTS: Totally 20 SD rats were included in result analysis without one rat missing. ①In the experiment group, X-ray image indicated callus formation, while histology observation showed osteoid tissue and new bone formation, and osteoblasts attached to the surface of the materials after 4 weeks. Eight weeks later, the defect was essentially repaired, and the bone density of new bone was similar to that of the autogenous bone. ②In the control group, there was no formation of callus and osteoid tissue, and few osteoblasts attached to the surface of the materials, and a lot of lymphocytes infiltrated and blood capillary grew in the lacune of materials after 4 weeks. Eight weeks later, the imbedded materials were substituted mostly by fibrous tissue.CONCLUSION: The ideal repair effect of bone defect can be obtained through the combination of molecular biology with tissue engineering.

Objective To construct and identify the recombinant vector pcDNA3. 1 (-) B/myc-BRMS 1 carrying breast-cancer metastasis suppressor 1 (BRMS 1) which can express in eukaryote cells and which will provide the basis for further researching the mechanisms of metastasis suppression and working on cancer metastasis gene ther-apy. Methods To isolate total RNA from MCF - 7 cells and design a pair of primers, and coding sequence of aRMS 1 cDNA were amplified from human breast cancer cells MCF -7 by reverse transcription-polymerase chain reaction (RT-PCR). Then the product was inserted to the PcDNA3. 1/myc-His (-) B plasmid. The recombined pcDNA3. 1 (-)B/myc-BRMS1 was identified by gene sequence analysis,then recombinants was transfected into HEK-293 cells and was identified by Western blot. Results The recombinant of pcDNA3.1 (-) B/myc-BRMS1 was structurally confirmed by analysis of sequencing. The inserted fragment in the vector was in the right direction and its sequence was structurally confirmed to be consistent with CDS sequence of human BRMSI cDNA that of the published data. GenBank, [AF159141]. The recombinants was transfected into HEK-293 cells ,then the cells expressed protein tagged c-myc identified by Western blot indicated it can express in eukaryote cells. Conclusion cDNA of human BRMS1 can be successfully cloned and inserted into Eukaryote-expression vector. The newly constructed vector may serve as the potential tool to conduct further comprehensive experiments in future on BRMS1 function and on gene therapy.

BACKGROUND:Human leukocyte antigen (HLA), the major histocompatibility complex of human, plays an important function in the transplant rejection. Decreasing the expression of HLA wil prolong the survival time of transplants.
OBJECTIVE:To design smal interfering RNA (siRNA) of HLA-A2 and to detect the effect of siRNA-HLA-A2 on the expression of HLA-A2.
METHODS:Four kinds of siRNA-HLA-A2 domains were designed, and recombinant lentivirus expression vector were formed. The 293T cells, highly expressing HLA-A2, were infected in vitro. Then the knockout efficacy of four domains was detected to select the highly efficient siRNA-HLA-A2 target sequences. The human embryo lung fibroblasts were cultured in vitro and infected with the lentivirus carrying the target sequence. The infecting efficiency of LV-siRNA-HLA-A2 was observed under the fluorescence microscope and the silence function of this siRNA in human embryo lung fibroblasts was detected by western blot analysis.
RESULTS AND CONCLUSION:According to the mRNA sequence of HLA-A2 in Genbank, three siRNAs were designed and synthesized. In vitro, the over expression of HLA-A2 in 293K cells was successful y silenced. The HLA-A2 expression in human embryo lung fibroblasts was also efficiently silenced after the human embryo lung fibroblasts were infected by the highly efficient siRNA of HLA-A2. The efficacy was up to 80%.