By taking the course of laboratory hematology in Curtin University in Australia as an ex-ample, this paper introduced the characteristics of its teaching mode, teaching contents, teaching methods, assessment methods and laboratory practice. The advantages and disadvantages of education characteristics between Australia and China were compared and analyzed on the training methods and the training objec-tives, respectively. The education characteristics of laboratory hematology in Curtin University provided an important inspiration for our education reform in the field of medical laboratory in the future.

Background and purpose:The human oncogene B-cell-speciifc moloney murine leukemia virus integration site 1 (Bmi-1) is an important member of the polycomb group family, and it regulates cell proliferation and senescence via INK4a/ARF locus. This study investigated the effects of Bmi-1-siRNA on the proliferation of lung adenocarcinoma cell line SPC-A1 cells with INK4a/ARF locus and clarify the mechanism of Bmi-1-mediated effect on proliferation of lung adenocarcinoma cells. Methods:In this study, we chose the most efifcient siRNA chain the pGeneshl-2-Bmi-1 sense-1 and inserted into a pSUPER-retro-neo retroviral vector. The packaged si-Bmi-1 pSUPERret-ro-neo retroviral vector was stably transfected into lung adenocarcinoma SPC-A1 cell line. The stably transfected cells were cultured and passed. After transfection, the levels of Bmi-1 mRNA and protein expression of SPC-A1 cells were analyzed by RT-PCR and Western blot respectively. Trypan blue, MTT and plate colony forming assay were performed to observe the proliferation capibility of SPC-A1 cells and evaluate the cloning forming ability in vitro. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of SPC-A1 cells. Cell cycle distribu-tion was analyzed by lfow cytometry (FCM) in SPC-A1 cells. The expression levels of proliferation proteins including p16INK4a, p53, Cyclin D1, PTEN, Akt and Ser473p-Akt were analyzed by Western blot. Results:The mRNA and protein expression levels of Bmi-1 were signiifcantly reduced in SPC-A1-Bmi-1-siRNA cells transfected with pSUPER-retro-neo retroviral vector. Knockdown of Bmi-1 could inhibit the growth, colony formation in vitro and tumorigenesis in vivo of SPC-A1 cells (P<0.01). The transfected SPC-A1 cells were arrested in G1 phase [(64.6±1.2)%, P<0.05]. Compared with two control groups, p16INK4a, p53 and Akt were not affected (P>0.05), while Cyclin D1 and Ser473p-Akt were downregulated (P<0.01) and PTEN was up-regulated (P<0.01) in the SPC-A1-Bmi-1-siRNA cells. SPC-A1-Bmi-1-siRNA cells were treated with various concentrations of PTEN inhibitor to determine expression levels of PTEN, Bmi-1 and Ser473p-Akt protein. Ablation of PTEN rescued Bmi-1 and Ser473p-Akt expression in SPC-A1-Bmi-1-siRNA cells. Conclusion:Knockdown of Bmi-1 gene can arrest the proliferation of SPC-A1 cells through G0/G1 phase arrest by inhibiting Cyclin D1 expression indirectly, which may be not associated with p16INK4a signaling pathway.

BACKGROUND:There are several reports about the application of fresh bone marrow aspirate being injected directly to repair partial ligament injury, but the application about fresh bone marrow aspirate directly being planted on scaffolds to build tissue-engineered ligament is rarely mentioned.
OBJECTIVE:To evaluate the feasibility of applying fresh bone marrow aspirate planted directly on scaffolds to construct tissue-engineered ligament
METHODS:We constructed fibroin fiber/smal intestinal submucosa composite scaffold, then planting fresh bone marrow directly to built bone marrow seeding group and planting seed cel s (bone marrow mesenchymal stem cel s) on the scaffold to built cel seeding group. The control group had no treatment. After that, we detected the density of cel adhesion, cel proliferation ability and extracel ular matrix secretion. Then, the composite in the bone marrow seeding group was implanted into the broken anterior cruciate ligament in rabbits, and material biocompatibility in vivo was evaluated after 12 weeks.
RESULTS AND CONCLUSION:After 4 hours of incubation, bone marrow seeding group was significantly higher than the cel seeding group in cel adhesion density and proliferation rate (P<0.05). Bone marrow seeding group and cel seeding group showed higher type I, III col agen secretion compared with the control group (P<0.05), but the col agen secretion of bone marrow seeding group and cel seeding group showed no significant difference. Composite cel scaffold implantation in vivo did not cause fatal immune rejection and severe inflammatory reaction, and no significant ligament regeneration and vascularization occurred. These findings indicate that fresh bone marrow aspirate can be seeded directly on scaffolds to construct tissue-engineered ligament, and the short-term biocompatibility in vivo is good.

BACKGROUND: Presently, the biomaterial used in ligament tissue engineering such as collagen protein, polylactic acid, polyglycolic acid, small intestinal submucosa, glycan and nanomaterial are characterized by rapid degradation, resulting in inflammatory reaction after applying in host.OBJECTIVE: To investigate mechanical strength and in vitro degradation of silk scaffold and explore the reaction to macrophages.DESIGN: Controlled experiment.SETTING: Experiments were performed at the Department of Orthopaedics, Union Hospital, Huazhong University of Science and Technology from September 2004 to January 2005.MATERIALS: White raw Bombyx mori silkworm fibers of size 20/22 (according to the manufacturer) were obtained from the market. Bundles of 30 parallel fibers were prepared for a bundle of scaffold, which was put into fervens 5g/L Na2CO3for degumming. Ratio of Na2CO3 solution (Ml) to raw silk (g) was 1000.METHODS: In vitro degradation: 8cm long silk scaffold was weighed after drying. Subsequently, the silk scaffold was separately dipped into phosphate buffer saline (PBS) and 1.0g/L collagenase prepared with PBS. Twelve weeks later, silk scaffold was weighed to calculate weight loss rate. Simultaneously, tensile test was performed to detect the ultimate tensile strength (UTS) of samples. Culture of monocyte strain RAW264.7:2×108L-1 macrophage suspension (1mL) were separately added in a silk scaffold group, a control group and a lipopolysaccharide (LPS) group. At days 1 and 7, cell supernatant was collected from each group. Tumor necrosis factor-α(TNF-α) levels were measured by enzyme linked immunosorbent assay (ELISA).MAIN OUTCOME MEASURES: ① Changes in weight loss rate and UTS of the silk matrices after incubated with collagenase and the PBS. ②TNF-αlevels in the supernatant of each groups at days 1 and 7.RESULTS: Mass of silk matrices reduced by over 50% after incubated with collagenase for 8 weeks, but no change was found in PBS. UTS decreased by over 50% 8 weeks after incubated with collagenase, but no change was detected in PBS. At days 1 and 7, TNF-α levels in the supernatant was less in the silk scaffold group; TNF-α levels in the supernatant was significantly higher in the LPS group than in the silk scaffold group (P<0.01), but no significant difference in TNF-α levels was measured between the silk scaffold group and the control group (P>0.05).CONCLUSION: After 12-weeks degradation, silk scaffold still has good mechanical properties. Macrophages possess immunological inertia at days 1 and 7 after inoculated with macrophages.

Objective To analyze temporal and kinematic parameters of video fluoroscopic images of swallowing using a digital acquisition and analysis system and to verify the reliability of this method.Methods Eighteen patients with dysphagia were requested to completed six swallows (3 ml and 5 ml of thin liquid, thick liquid, and paste mixed with 600 kg/m3 barium sulfate suspension) in the natural sitting position.Video fluoroscopy was used to measure the oral transit times, soft palate elevation times, hyoid movement times, laryngeal closure times, cricopharyngeal muscle opening times, hyoid anterior movement (HAM) , hyoid superior movement (HSM) , cricopharyngeal muscle opening diameter and pharyngeal constriction rate.Each was extracted from the videos four times by two raters working separately with an interval of 4 weeks between the sets of evaluations.Results Reliability varied among the different observations.HAM and HSM showed inter-rater reliability between 0.41 and 0.60 and intra-rater reliability between 0.61 and 0.80.The other observations all demonstrated acceptable reliability.Conclusion The self-designed digital acquisition and analysis system tested showed acceptable reliability and could be applied to analyze swallowing function clinically.

In order to investigate the effect of Arg-Gly-Asp (RGD) peptide-modified silk biomaterial on the adhesion and proliferation of bone marrow-derived mesenchymal stem cells (MSCs), MSCs of third generation were seeded onto the surface of RGD-decorated silk (silk-RGD group), silk alone (silk group) or tissue culture plate (TCP group). After incubation for 4 or 12 h, MSCs were examined quantitatively by using precipitation method for cell attachment. The cell proliferation, which was defined as cell density, was compared among the three groups after culture for 1, 2, 3, and 4 days. Cell skeleton, which was labeled fluorescently, was observed under laser confocal microscope after 24 h of culture. The results showed that cell adhesion rate in silk-RGD group was higher than in silk group (P<0.05), but similar to that in TCP group after incubation for 4 or 12 h (P>0.05). There were no significant differences in the cell proliferation among the three groups at different time points (P>0.05 for all). Laser confocal microscopy revealed that in silk-RGD group, MSCs, strongly fluorescently stained, spread fully, with stress fibers clearly seen, while in silk group, actin filaments were sparsely aligned and less stress fibers were found. It was concluded that RGD peptide could improve the adhesion of MSCs to the silk scaffold, but had no impact on the proliferation of the cells.
Biocompatible Materials/*chemistry ; Bone Marrow Cells/cytology ; Cell Adhesion/drug effects ; Cell Proliferation/drug effects ; Mesenchymal Stem Cells/*cytology ; Oligopeptides/*chemistry ; *Silk/chemistry ; Tissue Scaffolds

Background and purpose:The pro-oncogene Bmi-1 is a member of the polycomb-group family, can regulation of the proliferation and self-renewal of normal and tumor stem cells. In recent years, Bmi-1 has been found that it is overexpressed in varieties of human malignant tumors. The study aimed to observe the effects of Bmi-1-siRNA on the growth capacity of lung cancer cell line A549 in vivo and in vivo, and explore its mechanism. Methods:The most effective one as a target sequence was chosen from four Bmi-1 siRNA sequences which were designed by our lab, and one random sequence was chosen as a negative control. In short, the chemically synthesized siRNA and control sequences were connected to a retrovirus expressing vector, pSUPERretro-Neo plasmid, and then transfected into A549 cells. The stably transfected cells were cultured and passed. The level of mRNA and protein of Bmi-1 in A549 cells were assessed by RT-PCR and Western blot respectively. The proliferations of A549 cells in vivo was analyzed with MTT, trypan blue exclusion and plate colony forming methods. Flow cytometry was used for cell cycle analysis. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of A549 cells. The expressions of cyclin D, p21/27, p-AKT and PTEN were analyzed by Western blot. Results:Compared to A549-ctr and A549-wt cells, Bmi-1 mRNA and protein levels all signiifcantly reduced in A549-Bmi-1-siRNA cells. Bmi-1-siRNA inhibited the growth, colony formation in vitro and tumorigenesis in vitro of A549 cells, and the interference cells cell cycle arrested in G1 phase. In A549-Bmi-1-siRNA cells, p-AKT and cyclinD1 expression were down-regulated while p21/p27 and PTEN were up-regulated. Conclusion:Silencing Bmi-1 gene inhibits the proliferation of A549 cells through G1 phase arrest, which involves the downregulation of cyclin D/p-AKT and upregulation of p21/p27/PTEN.

0.05).CONCLUSION:The excellent mechanical properties of composite ligament can meet the mechanical requirements of appropriate ligament tissue engineering scaffolds.

Objective To monitor the immediate effect of Passi-Muir speaking valve (PMV) on patients with tracheostomy after acquired brain damage.Methods Twelve patients with tracheostomy after acquired brain damage were recruited.All of them underwent the high resolution manometry to measure the pressure in pharynx and upper esophagus during swallowing before and immediately after wearing PMV.The parameters including the pharyngeal peak pressure,increasing rate of the pharyngeal pressure,pharyngeal pressure duration,upper esophageal sphincter (UES) residual pressure and UES relaxation duration were recorded.Results No significant differences were found in all the measurements before and immediately after wearing PMV (P＞0.05).Conclusion PMV has no instant impact on the swallowing function of patients with tracheostomy after acquired brain damage.

Objective To evaluate the characteristics of dysphagia after brain stem infarction,and to determine the mechanism of aspiration.Methods The fluoroscopic videos of 12 dysphagia patients who had suffered brain stem infarction and 10 healthy counterparts were analyzed quantitatively using a digital analysis system.Each participant was requested to twice swallow 5ml of thick liquid.The observations included the oral transit time (OTT),the swallow response time (SRT),the hyoid movement time (HMT),the upper esophageal sphincter opening time (UOT) and the laryngeal closure time (LCT).An 8-point penetration-aspiration scale (PAS) was used to evaluate the severity of aspiration,and the results were correlated with the other 5 quantitative observations.Results The average OTT [(3.091±1.803)s],HMT [(1.498±0.550)s] and LCT [(0.651±0.186)s] of the brain stem infarction patients were all significantly longer than those of the healthy controls.However,no significant differences were found between the patients and the healthy volunteers in terms of SRT or UOT.Aspiration severity was significantly correlated with SRT but not with LCT.Conclusion Dysphagia after brain stem infarction involves both the oral and pharyngeal phases.OTT,HMT and LCT can be used to quantify dysphagia after brain stem infarction,while SRT is a predictor of aspiration.