BACKGROUND: It is demonstrated that bone marrow stem cells (BMSCs) can generate neurosphere structures, which is similar to cloning sphere of neuron-specific enolase (NSE), in a specially induced system in vitro; therefore, BMSCs draw more and more attention as seed cells to repair central nerve injury.OBJECTIVE: To investigate the differentiation from BMSCs into neuron-like calls induced by fetal spinal cord tissue.DESIGN: Observational study.SETTING: Department of Spine Surgery, Second People's Hospital of Shenzhen.MATERIALS: SD rats (16 pregnant days old and 2 months old) were provided by Animal Center, Tongji Medical College,Huazhong University of Science and Technology. Single antibody of NSE, multi-antibody of glial fibrillary acidic protein (GFAP) and single antibody of neurofilament (NF200) were provided by Wuhan Boster Company.METHODS: The experiment was carried out in Immune Opening Laboratory and Central Laboratory, Basic Medical Department, Tongji Medical College, Huazhong University of Science and Technology in September 2006. Bones of lower extremities of rats were collected to centrifuge BMSCs. Fetal spinal cord tissue homogenate was extracted from 16 pregnant days old rats to make inducing solution and induce differentiation of BMSCs. Otherwise, embryo muscle tissue was used to make muscle tissue homogenate as the same way so as to regard as the controls.MAIN OUTCOME MEASURES: BMSCs underwent morphological observation after induction; in addition, anti-NSE, NF200 and anti-GFAP were used to label neurons and astrocytes, respectively. Ten non-overlapping sights were randomly selected from positive reactive-induced cells after immunohistochemical staining under optic microscope to calculate ratio of positive cells of NSE and NF200 counting for total numbers of cells.RESULTS: ① Morphological changes of BMSCs after induction: During early induction, optic microscope indicated that soma of partial cells rebounded; whose apophysis was long and thin; apophysis of differentiated cells grew gradually and intercrossed each other. It was similar to nerve cells and some branches were similar to dendrite branches. However,morphological changes of cells in the control group were not obvious. ② Expression of relevant antigens differentiated from BMSCs into neuron-like cells at one week after induction: Most cells in spinal cord homogenate group expressed as positive NSE and NF200; a few of cells expressed as GFAP. While, positive staining of nerve cell antibody was not observed in the control group; meanwhile, positive reaction of nerve cell antigen was not observed in the control group,too. Immunohistochemistry examination demonstrated that positive rates of NSE and NF200 expressions were (68±1.7)%and (76.2±2.9)%, respectively.CONCLUSION: Fetal spinal cord tissue homogenate can induce differentiation from BMSCs into neuron-like cells.

Objective To discuss the teaching outcomes of project draft design combined with PBL teaching method in TCM pharmaceutics technology.Methods Fifty students majoring in 2009 TCM Pharmaceutics Technolgy were divided into A and B groups. 25 students in group A were set as experimental group by using project draft design combined with PBL teaching method, while 25 students in group B were set as control group by using traditional teaching method. This study evaluated the effects of teaching methods through questionnaire and score analysis of the two groups.Results Students taught by project draft design combined with PBL teaching method generally believed that this teaching method can stimulate their learning interest, improve their ability of locating problems, solving problems, and comprehensively applying knowledge and enhance test scores.Conclusion Project draft design combined with PBL teaching method shows better teaching outcomes than traditional teaching method, which can cultivate students’ all-round ability and comprehensive quality.

Objective To compare the micromeritics properties and external dissolution rates of Sanhuang Powder in different particle sizes;To provide references for its direct use and application as raw materials for TCM preparation.Methods Particle size, bulk density, tap density, angle of repose, and hygroscopicity ofSanhuang micro-powder and common powder were investigated and evaluated. External dissolution rates ofSanhuang micro-powder and common powder were detected by ultraviolet spectrophotometry.Results The flowability of bothSanhuang micro-powder and common powder were not very well. With the sizes decreasing, the hygroscopicity of micro-powder became stronger. The external dissolution ofSanhuang micro-powder was more sufficient and much more quickly than common powder.Conclusion Properties ofSanhuang micro-powder and common powder are obviously different.Sanhuang micro-powder has stronger hygroscopicity and worse flowability compared with common powder. However, external dissolution ofSanhuang micro-powder is more sufficient and much more quickly than common powder. WhenSanhuang micro-powder is used directly and used as raw materials for TCM preparation, much more discretion should be considered.

Objective To establish the quality control method for Sanhuang Suppository. Methods Coptidis rhizoma, Scutellariae radix and Phellodendri chinensis cortex in Sanhuang Suppository were identified by TLC. The contents of baicalin and berberine hydrocholride were determined by HPLC which was performed on an Agilent Zorbaxsb C18 column (4.6 mm×250 mm, 0.45 μm) with a mobile phase of methanol-water-phosphoric acid (47∶53∶0.2), with flow rate of 1.0 mL/min. The wavelength of detector was set at 280 nm for baicalin and 365 nm for berberine hydrochloride, with column temperature of 30℃.Results Chromatographic characteristics of qualitative identification were evident. The linear range of baicalin was 0.248-2.48 μg (r=0.999 9) and the average recovery rate was 99.77% (RSD=1.05%). The linear range of berberine hydrocholride was 0.336-1.68 μg (r=0.999 9) and the average recovery rate was 97.74% (RSD=1.48%).Conclusion The method of qualitative and quantitative analysis is accurate, feasible and suitable for effective quality control of Sanhuang Suppository.

Objective To discuss the teaching outcomes of problem-based learning method in classroom teaching of TCM pharmaceutics.Methods Fifty students majoring in 2009 science of Chinese materia medica were taught by using traditional teaching method in teaching of TCM pharmaceutics, and 56 students majoring in 2009 TCM pharmaceutics by using of problem-based learning method. This study evaluated students’ adaption to the two teaching methods through score analysis and questionnaire of the two classes.Results Students taught by problem-based learning method generally believed that this teaching method can stimulate their learning interest, improve self-study ability, thinking ability, creativity, and language competence, and enhance team spirit.Conclusion Problem-based learning method shows better teaching outcomes than traditional teaching method. It can guide students to look for ways to tackle problems in the course of solving problems, and cultivate all-round ability and comprehensive quality of students.

Objective To optimize the extraction technology of total flavonoids from Lycii Fructus by response surface methodology (RSM).Methods On the basis of single-factor tests, enzyme concentration, enzymolysis time, and enzyme solution temperature were selected as influencing factors during extraction to conduct three-factor three-level center combination design. The effects of three factors on the yield of total flavonoids from Lycii Fructus were analyzed by RSM.Results The optimum conditions for extraction technology of total flavonoids from Lycii Fructus were enzyme concentration 0.30%, enzymolysis time 1.0 h, enzyme solution temperature 60℃. The predicted extraction yield of total flavonoids was 0.9492%.ConclusionUsing RSM to optimize the extraction technology of total flavonoids from Lycii Fructus is stable, feasible, and simple, which can provide references for further study and application.

BACKGROUND: Pathophysiological mechanism of local microenvironment is complex after central nerve injury; especially,both inflammatory reaction at an acute phase and formation of secondary glial scar have tremendous effects on effective regeneration of axon, regeneration and arrangement of local nerve cells, proliferation and migration of local stem cells;therefore, it becomes a basic reason for blocking nerve repair in an early period. Thus, how to effectively resist inflammatory factors in injured region at an acute phase and how to optimize microenvironment of neural regeneration are the most important strategies for repairing spinal cord injury in recent years.OBJECTIVE: To design, establish and screen the best expression of interleukin-6 receptor (IL-6R) α to inhibit shRNA adenovirus expression vector by using spinal cord injury models.DESIGN: Duplicative measurement study.SETTING: Department of Spine Surgery, the Second People's Hospital of Shenzhen.MATERIALS: A total of 40 healthy Wistar rats, either gender, 8-10 weeks old, were selected in this study. Rabbit-anti-rat glial fibrillary acidic protein (GFAP) antibody Ⅰ was provided by Santa Cruz Compan; siRNA eukaryon expression plasmid pGenesil (cohtaining green fluorescent expression system) was provided by Wuhan Jingsai Bioengineering Company.METHODS: The experiment was carried out in ImmuneOpening Laboratory, Basic Medical Faulty, Tongji Medical College, Huazhong University of Science and Technology, and Medica Laboratory Center, the Second People's Hospital of Shenzhen in November 2006. Three pairs of shRNA template which composed of 19 bp reverse repeated motif of IL-6 receptor (IL-6R) α target sequence with 9 bp spacer were designed and synthesized, then the recombinant adenovirus expression vectors with green fluorescence protein were constructed in vitro respectively. The acute spinal cord injury models were completed, and the adenovirus recombinants were regionally injected post 12 hours after spinal cord injury;in addition, the inhibitory effect of RNA interference (RNAi) on expression of IL-6R in local region after spinal cord injury were detected by using real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot so as to screen adenovirus expression vector which had the best inhibitory effect on expression of IL-6R.MAIN OUTCOME MEASURES: Inhibitory effect of RNAi on expressions of IL-6R RNA and protein in local region after spinal cord injury.RESULTS: Sequence analysis showed that IL-6R-shRNA recombinant adenovirus expression vector was successfully constructed, and optimal IL-6R-shRNA recombinant adenovirus expression vector was screened by using real-time fluorescence quantitative RT-PCR and Western blot. The IL-6R expressions were 49% and 56% at the levels of mRNA and protein, respectively.CONCLUSION: The IL-6R--shRNA recombinant adenovirus expression vector is successfully constructed and screened.The gene expression of IL-6R can be highly inhibited after acute spinal cord injury.

BACKGROUND: Glial cell-derived neurotrophic factor (GDNF) and transforming growth factor-beta 1 (TGF-β1)co-subordinate to TGF-β family. Both of them play very important roles in the development and differentiation of central and peripheral nervous system, and regulation of cell cycle in mammals.OBJECTIVE: To observe the differentiation of spinal cord-derived neural stem cells(NSCs) induced by GDNF combined with TGF-β1, and make a comparison of differentiation results with GDNF or TGF-β1 culture fluid.DESIGN: Controlled observation.SETTING: Central Laboratory, Shenzhen Hospital Affiliated to Southern Medical University.MATERIALS: Ten SD rats of clean grade, which were at conception for 16 days, were provided by the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technplogy. Main reagents and materials:DMEM/F12,B27(GIBCO); basic fibroblast growth factor (bFGF), GDNF; TGF-β1(PeproTech);fetal bovine serum (FBS,Hyclone); nestin multiple antibody (Boster, Wuhan); glial fibrillary acidic protein (GFAP) multiple antibody; neurofilament protein (NF-200) monoclonal antibody (Sigma).METHODS: This experiment was carried out in the Central Laboratory, Shenzhen Hospital Affiliated to Southern Medcial University between October 2005 and September 2006. Under the aseptic condition, rat fetus was isolated for isolation and culture of spinal cord-derived neural stem cells. In this study, five groups were divided: basal medium group, control group, bFGF group, TGF-β1 group, GDNF+ TGF-β1 group. In the basal medium group, DMEM/F12 containing penicillin,streptomycin, amphotericin (AMPH) B and 0.02 volume fraction of B27 annex solution. At 1 week after primary culture, rat spinal cord-derived NSC clones proliferated in vitro stably were harvested. In the control group, 0.1 volume fraction of FBS was added into basal medium. In the later three groups, induced medium was exchanged, i.e. 20 μg/L bFGF, 2 μg/L TGF-β1, and 10 μg/L GDNF+2 μg/L TGF-β1 were added into the basal medium, respectively. ①The differentiation of spinal cord-derived NSCs induced by different factors were observed under the optical microscope. ②The expressions of neurons and astrocytes were detected by immunocytochemical staining labeling. ③ The differentiated cells were counted by sorting technique by means of fluorescence excitation flow cytometer, and the percentage of NSCs differentiating into neurons and astrocytes were detected under the different induction environments.MAIN OUTCOME MEASURES: ① Morphological feature of cell differentiation in each group. ② Immunohistochemical detection of NSCs in each group. ③ The percentage of NSCs differentiating into neurons and astrocytes in each group.RESULTS: ① Cell morphology during differentiation: At the early stage of differentiation, lots of cells creeped to all the directions, and one week later, most of the migrated cells adhered to the wall entirely. Neuron-like cells, astrocyte-like cells and oligodendrocyte-like cells could be identified in the low-density cell region. ②Immunohistochemical detection results: A lot of GFAP- positive astrocytes were found in the control group and TGF-β1 group; Many differentiated neurons and NF-200 staining positive were found in the bFGF group and GDNF+ TGF-β1 group. ③Percentage of stained neuron and astrocyte: at one week of induction, the percentage of stained neurons was higher in the GDNF+ TGF-β1 group than in the control group, bFGF group and TGF-β1 group (x2=24.15,19.56,25.32,P ＜ 0.05-0.01), and the percentage of stained astrocytes was lower in the GDNF+ TGF-β1 group than in the control group, bFGF group and TGF-β1 group (x2=24.45,23.79,P ＜ 0.01 ).CONCLUSION: The combined in vitro induction of GDNF and TGF-β1 contributes to the neuronal differentiation of spinal cord-derived NSCs, indicating that both of them have synergistic effect.

Small scale restricted online course (SPOCs)+flipped classroomteaching is expected to become an important complement to the traditional teaching of dermatology. In this teaching mode, first of all knowledge nodes are divided separately according to the syllabus and for each node, 7 to 10 minutes of micro video or micro-lecture is made, which enables students to make best use of fragmented time through the network to master relevant knowledge independently before the trainee. In the flipped classroom, based on the difficulties and misunderstandings of learning reflected from the network monitor data, teacher can make full use of time on demonstrating typical cases, organizing discussion, grouping students to take diag-nostic and therapeutic exercise, answering targeted questions, teaching clinical thinking, experience and doctor-patient communication skills. Through this teaching mode, the new teaching idea can be imple-mented that students are in dominant position while teachers are in leading position, which contributes to deepening the understanding , absorption and application of knowledge and improving students' ability of communication, cooperation, diagnosis and treatment.

Objective To investigate the acid α glucosidase(GAA)gene mutations and clinical features of a Chinese patient exhibiting signs and symptoms of infantile glycogen storage disease type Ⅱ(GSD Ⅱ). Methods Clinical features of the patient were reviewed,and GAA activity in the patient＇s and her parents＇ whole leukocytes were measured. GAA coding regions were amplified by polymerase chain reaction(PCR),and analyzed by direct DNA sequencing. Results The patient showed feeding difficulties,generalized hypotonia and weakness starting at 2 months of age. Cardiomegaly and cardiomyopathy were found at 4 months. She died of cardiorespiratory failure at the age of 6 months. GAA activity in leukocytes was low in the patient(17.3% of the median normal range). Genotyping revealed the patient was a heterozygote for a novel nonsense mutation p.W738X and a previously reported nonsense mutation p.E888X. The reported pseudodeficiency allele c.1726G ＞ A;2065G ＞ Awas found in the patient and her mother. Conclusions Correct diagnosis was made for this patient by combination of GAA activity assay and genetic analysis. From the clinical course,this patient should be classified as infantile type of GSD Ⅱ,suggesting that the novel mutation p.W738X may have a damaging effect on the function of GAA. Pseudodeficiency allele found in this family highlights the importance of genetic analysis of GAA when performing diagnosis and prenatal diagnosis for the affected families,as this allele causes low GAA activity in normal individuals.