Schwann cells are the seed cells of tissue engineedng in the regeneration of peripheral nerve.so whether we can harvest enough Schwann cells of high purity is very important in the tissue engineering.Culture and purification technology of Schwann cells has been improved day by day.The classical cultural methods include tissue-clump cultural method and enzyme digestion cultural method.On the basis of the classical cultural methods,someone uses new technologies to delete fibroblasts,such as addition of cytosine arabinoside,addition of antimitogenic agents.The latest purified methods include magnetic activated-cells separation,co-culture with three-dimensional scaffolds and laminin-coated wells purified method.The aim of the skills above is to provide enough Schwann cells for the tissue engineering repeir of degenerated peripheral nerves.There are many ways of Schwann cells culture and purification.and we have made a primary progress,but it still needs further studies before the entire achievemenL such as long cycle,low activity in cultured calls,and the instable biological characteristics during passage culture,etc.

BACKGROUND: Differentiation of neural stem calls (NSCs) was mediated by many environmental factors. Several factors can induce NSCs to differentiate into neurons in varying degrees and it is now a focus on the control of NSCs differentiation.OBJECTIVE: To study the effects of combination of basic fibroblast growth factor (bFGF) and brain-derived neurotrophic growth factor (BDNF) on the differentiation of NSCs into neurons.DESIGN, TIME AND SETTING: The in vitro cytology observation was performed at the Neurotomia Laboratory of China Medical University in May 2008.MATERIALS: Three adult male SD rats were provided by Experimental Animal Center of China Medical University.METHODS: Dispositions to the rats were consistent with ethical standards of animals. The rat brain hippocampus was removed sterilely. After trypsin digestion, NSCs were cultured in serum-free medium. Cell suspension was prepared and diluted when the diameter of the fourth passage of clone sphere was 200 μm by mixture of DMEM/F12 containing 2% B27, 20 μg/L of epidermal growth factor (EGF), and 20 μg/L bFGF. Monoclonal calls were passagad. NSCs were divided into blank control, bFGF, BDNF and bFGF+BDNF groups by different growth factors added into the media. Fetal bovine serum of 0.1 volume fraction was added in blank control group. The media in the other three groups were added bFGF, BDNF and bFGF+BDNF respectively for 1 week.The concentration of bFGF was 10 μg/L and the concentration of BDNF was 200 μg/L.MAIN OUTCOME MEASURES: Immunocytochemistry staining was used to identify NSCs as well as to detect the differentiation of NSCs into neurons.RESULTS: The monoclonal calls expressed nestin and the differentiated call expressed neuron specific enolase and glial fibrillary acidic protein. Compared to blank control group, the proportion NSCs into neurons in the bFGF group, BDNF group and bFGF+BDNF group were much higher (t=3.409-7.558, P < 0.05), with the highest in bFGF+BDNF group (t =7.558, P < 0.05).CONCLUSION: Combination of bFGF and BDNF can promote the differentiation of adult hippocampus NSCs into neurons.

Objective To observe the repairing effect of the acellular nerve allografts on the sciatic nerve gap of rat. Methods The acellular nerve allografts,treated by hypotonic-chemical detergent,were put on the 10 mm gap of the sciatic nerve in the rat.The action potential of the regenerated nerves was determined by the electrophysiologic method 13 weeks after operation.The morphology of the regenerated nerves was observed under light microscope and electron microscope,and the results were analyzed statistically. Results No inflammation and rejected reaction were found in the period of 13 weeks after operation in the operated and control groups.There was no significant difference in number of the regenerated nerve fibers,diameter of the axons,and the thickness of the regenerated myelinated nerve between the experimental group and control group.Conclusion The present results indicated that the acellular nerve allografts had good biocompatibility for the host rat in vivo and might as a bridge promote the regeneration of the injured sciatic nerve.;

BACKGROUND: Schwann calls are seed cells for constructing tissue engineered peripheral nerves. But their in vitro isolation, culture and purification are difficult. Acellular nerve allografts (ANA) have a great effect on repairing peripheral nerve defects, and it can induce marrow stromal cells (MSCs) into Schwann-like calls. Accordingly, MSCs can be used as seed calls theoretically in constructing tissue engineered peripheral nerves, instead of Schwann cells OBJECTIVE: To observe the repairing effect of tissue engineered peripheral nerves constructed with MSCs on sciatic nerve defects and to assess the feasibility of repairing peripheral nerve defects with MSCs as seed calls. DESIGN, TIME AND SE'I-rlNG: A randomized control animal experiment was conducted in the laboratory of Basic Medicine Collage of Dali Collage from July to December in 2008.MATERIALS: A total of 30 SD rats were divided randomly into 3 groups, with 10 in each group. In MSCs+ ANA group, end-to-end anastomosis were performed with 10/0 non traumatic suture to broken ends of rat sciatic nerves and tissue engineered nerves cultured using MSCs combined with ANA; In ANA group, ANA were bridged to broken ends of sciatic nerves; In METHODS: The 10ram sciatic nerve defects in rats were repaired using MSCs-constructed tissue engineered peripheral nerves, whose repairing effects were evaluated at week 12 post transplant through sciatic function index (SFI), gastrocnemius wet weight recovery rate, S-100 immunohistochemical staining and electron microscope observation, etc. MAIN OUTCOME MEASURES: Morphological changes of calls were observed during the culture of compounds; SFI and gastrocnemius wet weight recovery rates were detected after transplantation; New myelinization, axon growth and nerve fiber distdbuUon were observed with toluidine blue staining; Schwann calls growth and nerve fiber regeneration were observed with transmission electron microscope combined with S-100 protein immunohistochemical staining method.RESULTS: The detection results showed that SFI and gastrocnemius wet weight recovery rate were higher in the MSCs+ ANA group than in the ANA group (P < 0.05). Compared the ANA group, the MSCs+ ANA group had more S-100 proteins expressed in its compounds obviously, and the number, the diameter of its myelinated nerve fibers as well as its myelin sheath thickness were all greater than those of the ANA group (P < 0.05). The repairing effect of the MSCs+ ANA group was close to that of the autotransplantation group.CONCLUSION: MSCs-constructed tissue engineered nerves have a better effect on repairing peripheral nerve defects than ANA, and MSCs as seed cells have a high value in peripheral nerve tissue engineering.

Objective The experiment was designed to investigate the effect of acellular nerve allografts on the functional recovery and reconstruction of the nerve-muscle structure of the sciatic nerve defect in rats. Methods Acellular nerve allograft was transferred into the defected rat sciatic nerve with 10mm long.The wet weight of tibialis anterior was weighed at 12 and 24 weeks postoperatively compared with control group.The conducted velocity of regenerated nerve and the effect of regenerated nerve on tibialis anterior were investigated by electrophysiologic test,and silver staining combined with AChE histochemical methods were used in the experiment separately. Results The wet weight of tibialis anterior and the conducted velocity of regenerated nerve in experimental group were similar to those in control group in 12 and 24 weeks after transplantation.The positive acetylcholinesterase(AChE)histochemical reaction was observed in the tibialis anterior at 12 weeks with deeper staining and located in the middle of tibialis anterior tidily at 24 weeks after operation.The regenerated nerve bundles and nerve terminals were found to grow into the motor end-plate of the tibialis anterior in silver staining combined with AChE staining in experiment group.Electromyogram showed that the regenerated nerve has innervated tibialis anterior already.Conclusion The results indicated that extracted nerve allografts as a bridge can promote the motor functional recovery and reconstruction of the nerve-muscle structure of the defected rat sciatic nerve.

Objective Set up virtual experimental teaching platform of medical molecular biology, and explore effective operating system of virtual experiment teaching. Methods 400 students of majored in clinical medicine in Kunming Medical University in Grade 2011 were randomly divided into the virtual experiment teaching group (n = 195 ) and the traditional experiment teaching group (n = 205 ). We realized the teaching effect by questionnaire survey, and analyzed the final exam results of two groups statistically. Results The experiment teaching way of virtual experiment has been widely accepted by students, and it could help students to understand and master experiment operations and theory knowledges.No statistical difference was found between two groups on the final exam. Conclusion Virtual experiment technology as a new teaching method has a lot of advantages, but it can't completely replace traditional experiments. We should use both the two kinds of teaching methods reasonally in the medical molecular biology experiment teaching.

Objective To investigate the risk factors for the occurrence of cerebral infarction in patients with capsular warning syndrome (CWS). Methods Consecutive patients with transient ischemic attack (TIA) meeting the CWS clinical manifestations were col ected retrospectively. They were divided into either a cerebral infarction group or a non-cerebral infarction group according to the brain diffusion weighted imaging findings. The independent risk factors for patients with CWS were identified through the comparison of demographic and baseline clinical data. Results A total of 39 patients were enrol ed, including 25 males (64. 1%) and 14 females (35. 9%), and their mean age was 58. 9 ± 10. 3 years. There were 21 patients in the cerebral infarction group and 18 in the non-cerebral infarction group. Compared with the non-cerebral infarction group, the age of patients in the cerebral infarction group was older (62. 5 ± 9. 3 years vs. 54. 8 ± 10. 2 years;t=2. 470, P=0. 018). The constituent ratio of the patients with a history of previous stroke or transient ischemic attack was higher (33. 3% vs. 5. 6%; P=0. 049), the fasting blood glucose level was higher (8. 2 ± 3. 2 mmol/L vs. 6. 0 ± 1. 3 mmol/L; t=2. 748, P=0. 009), and ABCD2 score was higher (5. 2 ± 1. 1 vs. 3. 5 ± 1. 1;t=4. 734, P<0. 001). Multivariate logistic regression analysis showed that the ABCD2 score was an independent risk factor for cerebral infarction in patients with CWS (odds ratio, 4. 529, 95% confidence interval 1. 233-16. 627;P=0. 023). Conclusions The higher ABCD2 score was an independent risk factor for the occurrence of cerebral infarction in patients with CWS. It can be used as an evaluation tool for predicting the risk of cerebral infarction in patients with CWS.

Objective To explore the feasibility and safety of early ambulation in patients with acute deep ve-nous thrombosis. Methods The literatures about early ambulation for acute venous thrombosis were collected and the quality of the literature was evaluated by two investigators independently. Results Thirteen published studies were recruited for systematic review,including 10 RCTs and 3 CCTs. The meta-analysis showed that compared with bed rest,early ambulation did not increase the incidence of secondary pulmonary embolism in patients with acute DVT[early activity:22/1408;bed rest:34/1417;RR=0.62,95%CI(0.37,1.03),and the incidence of DVT progression [early activity:48/1523;bed rest:45/1525;RR=0.85,95%CI(0.58,1.24)]. There were no statistically significant differ-ences in mortality,swelling,and pain relief in acute DVT patients. Conclusion Early ambulation did not increase the incidence of thrombus progression and secondary pulmonary embolism in patients with acute DVT compared with bed rest. Besides,early ambulation cannot relieve symptoms of swelling and pain in DVT patients. However,it can relieve the acute pain of DVT patients with moderate to severe pain.

Objective To investigate the role of BMSCg on enhancing the implant survival and bacheal epithelium regeneration. Methods After transplanted with cryopreserved 2 weeks and 6 weeks allocraft, PHK-26 labeled 3-5 passage BMSCs were injected into the recipient rats via tail vein. Rats in the control groups were injected with the same amount of PBS.We observed the histology of the transplanted trachea including epithelium growth and regeneration, and the PKH-26 fluorescence levels at the para-anastomotic trachea to evaluate the role of BMSC transplantation on the epithelium regeneration. Results Rats from BMSCs injection group survived a long period. Histological observation showed that the tracheal lumen was covered by psudo-striated ciliated columnar epithelium. The cartilage structure was intact. There are no signs of denaturation and necrosis. In the PBS injection group, epithelium regeneration is better in PBS-6-week group than PBS-2-week group. The longest survival time in PBS-6-week group was 32 days, whereas it was 10 days in PBS-2-week group. In BMSCs injection group, rats in BMSC-6-week groups survived longer than 8-week group(12 rats were terminated at 1 week, 4 weeks and 8weeks as planned). There was one rat who survived and were terminated at the designated 8 weeks time point (there were 8regenerated epithelium was similar in the two BMSC transplanted groups. PKH-26 labeled BMSCs migrated to the implant site and showed red fluorescence, with most red fluorescence shown at the anastomotic part. Conclusion BMSCs can migrate to the impaired tissue to repair it. BMSCs may exert their reparation function via enhancing epithelium regeneration.

Objective To establish a mini-pool nucleic acid testing (NAT) assay using multiplex RT-nested PCR for the detection of HIV RNA, and apply it in screening for acute HIV infection among MSM. Methods Frozen EDTA plasma samples collected between Oct. 2008 and Mar. 2009 from 3 HIV infectors during window-period, a total of 30 HIV chronically infected individuals and 97 healthy subjects were used to develop the NAT assay. Plasma samples from 10 cases were pooled into one tube and centrifuged at high speed for the collection of viruses. HIV RNA was extracted. Two pairs of primers were designed according to two conserved regions of HIV RNA ( HXB2 nt 5783-nt 6228 and nt 1235-nt 2012).Multiplex RT-PCR and nested PCR were performed. Individual NAT-reactive samples were confirmed by commercially available NAT assays. The sensitivity and performance efficacy were also evaluated. The assay was then applied to 1 005 plasma specimens from MSM with negative or uncertain HIV antibody test results.These were collected in the same period as the other samples. Results ( 1 ) Two fragments of HIV were amplified successfully with the low detection limit of 162 copies/ml plasma; (2) Results of the mini-pool HIV NAT validation with samples from 3 HIV infectors during window-period were consistent with the expected values; (3) All 30 plasma samples from MSM with positive HIV antibody, which were tested by multiplex RT nested PCR, were found to be HIV RNA positive; (4) One out of 1 005 plasma samples was found to be HIV RNA positive, for this case acute infection was followed-up and sero-conversion was found. Conclusion Mini-pool NAT has good sensitivity, and may be applied to screening HIV RNA among MSM during window-period.