Objective To establish a multiplex suspension array for simultaneously detecting five species of important arbovirus: dengue virus (including four serotypes), japanese B encephalitis virus, west nile virus, tick-borne encephalitis virus and yellow fever virus. Method All published genomic sequences of the above arboviruses were collected from GeneBank, and then, the genus universal primers and specific probes were designed according to the conservative and specific NS5 region of the genus Flavivirus. The amine-modified probes were covalently coupled to different carboxylated microspheres by carbodiimide methods. By combining general RT-PCR with virus-specific probe hybridization, a Luminex xMAP system-based suspension array, which could simultaneously differentiate five arboviruses and discriminate 1～4 serotypes of dengue virus, was successfully established. Results A cutoff value twice higher than that of the background fluorescence was judged as positive reaction. RT-PCR products of many arboviruses were used to evaluate the specificity and repeatability of the array, the results showed that the assay was highly specific and had good repeatability. Meanwhile, the coefficient of variability was less than 8% from inter- and intra- assay, implying that both the repeatability and stability of the array were good. Furthermore, several viruses which had been quantitated by plaque assay were used to determine the array’s sensitivity. The results showed that the array’s detection limits were about 1.4 PFU for JBEV and YFV, 14 PFU for WNV respectively. 55 clinical and cultured samples were detected by the established assay, 16 out of the 55 samples were confirmed to be positive. There was only one sample which was not detected contrasted with conventional RT-PCR. However, it had advantages in detecting complex samples rapidly. Conclusion A multiplex suspension array for simultaneously detecting and serotyping five species of important arboviruses has been successfully developed, and it may play an important role in diseases diagnosis and prevention as well as epidemiological studies.

BACKGROUND:There is notan effective method for inducing bone marrow-derived mesenchymal stem cells (BMSCs) differentiatied in to intervertebral disc cells.OBJECTIVE:To establish a model of human BMSCs (hBMSCs) differentiated in to nucleus pulposus-like cells in vitro.DESIGN,TIME AND SETTING:A single sample contrast.The experiment was performed at the Orthopaedics Institute and Central Laboratory,the Affiliated Hospital of Qingdao University Medical College,from August 2008 to March 2009.MATERIALS:Two New Zealand white rabbits with 8-week-old were used to culture nucleus pulposus cells.Human nucleus pulposus was obtained from a aged 16 years patient with T_(12)L_1,L_(1.2) congenital scoliosis.The informed consent was obtained from his patients,hBMSCs were purchased from Salye Biological Science and Technology Co.,Ltd.(Guangzhou,China).METHODS:hBMSCs and nucleus pulposus cells of rabbit were co-cultured in new-style culture model (in 6-well plates and inserts with track-etched membrane having 0.4 μm pores without arms) for 7 days,conventionally laminar culture,solitary hBMSCs and nucleus pulpesus cells culture were set up as controls at same time.MAIN OUTCOME MEASURES:The expressions of collagen Ⅱ and aggrecan mRNA were detected by RT-PCR and Western blot.RESULTS:hBMSCs in the conventionally laminar and single hBMSCs groups were negative to collagen Ⅱ and aggrecan.The collagen Ⅱ and aggrecan expression of hBMSCs were upregulated evidently in new-style culture group,which was similar to nucleus pulposus cells that from degenerate intervertebral disc of human,but these expression did not appear in conventionally laminar culture and solitary hBMSCs culture (P ＜ 0.01).The difference among new-style culture group and other groups had no significance (P ＞ 0.05).CONCLUSION:A new-style model of hBMSCs differentiation to nucleus pulposus-like cells is constructed in vitro,which can highly express collagen Ⅱ and aggrecan and provide a foundation for intervertebral disc degeneration cellular treatment.

AIM: To evaluate the cytotoxicity of a novel anticholinergic drug penehyclidine hydrochloride (PHC) and its four optical isomers R-1, R-2, S-1, and S-2. METHODS: Two in vitro assays, MTT assay and neutral red uptake assay, were used to evaluate the cytotoxicity following PHC and its isomers exposure to HepG2 cells at different concentrations. RESULTS: PHC and its isomers induced decreases of viability of HepG2 cells in a concentration-dependent manner. Comparison of the cytotoxicity of the five anticholinergic agents with 50% inhibitory concentration (IC50) values indicated that the order of potency was PHC>R-2>R-1>S-2>S-1 for MTT assay, and R-2>PHC≈R-1>S-2>S-1 for neutral red uptake assay. CONCLUSION: With respect to the cytotoxicity of the four isomers on HepG2 cells, the R configuration was more potent than the S configuration, and R-2 was the most potent isomer whereas S-1 was the least potent isomer among the four optical isomers.

Objective To investigate the distribution range and depth of the apocrine sweat glands of the axillary fossa,in order to supply with anatomic and histopathologic basis in the treatment on axillarv osmidrosis.Methods From December 2008 to ()ctober 2010,2 biopsy samples(with axillary osmidrosis),8 biopsy samples(normal,without axillary osmidrosis),were employed into the axillarv anatomy study. 25 patients with severe axillary osmidrosis were observed both maerographicallv and microscopically by using of operation and histopathological methods.Results Secretory portion of apocrine sweat glands was seen clearly,it was pitchy millet-like granules on axillary osmidrosis corpse,and pink millet-like granules in vivo.Secretory portions distributed most within the armpit hair area,exceeded the edge of armpit hair line,but not surpassed the edge of armpit hair line 1.0 cm.The depth of the apocrine sweat glands located vertically at superficial fat tissues between the dermal reticular 1aver and superficial fascia layers which were dissected away easily.Trimming with scissors under dermaIlayer,the secretory portion of apocrine sweat glands was removed cleanly without harms to reticular laver of dermas.Secretory portions became ducts under reticular layer of dermas.White Drominence-like granules were proved to be the compomers of hair follicle and sebaceous glands through Dathological section.Conclusions In order to treat axillary osmidrosis effectively,the secretary portion should be removed away through cutting off the tissues between the dermal reticular layer and suDerficial fastia layers;the ducts of apocrine sweat glands should be handled with removing hair follicle under the reticular layer of dermas．0peration area should not exceed 1.0 cm off the edge.

BACKGROUND:Cadherin-11 which was used to modify bone marrow mesenchymal stem cells aimed to promote cell adhesion ability;while,core binding factor α1 (cbfα1) which was used to modify bone marrow mesenchymal stem cells aimed to realize ossification of multipotential stem cells and play a signalization role in cascade effect.OBJECTIVE:To construct cadherin-11 and cbfα1 gene adenovirus expression vectors so as to modify bone marrow mesenchymal stem cells,and observe the expressions of the two genes.DESIGN,TIME AND SETTING:Cell-genetics experiment was performed at Central Laboratory of Zhujiang Hospital in August 2008.MATERIALS:Bone marrow mesenchymal stem cells were extracted from a male patient with thoracic vertebral fracture re from Zhujiang Hospital.The patient provided the informed consent.Full length cadherin-11 cDNA plasmid was provided by Professor Takeichi,Riken Molecular Organism Center,Japan;full length cbfα1 gene plasmid was provided by Professor Ducy,Baylor Medical College,USA;pAdeasy adenovirus system was provided by Professor Bert,McKuslck-Nathans Institute of Genetic Medicine,USA.METHODS:Human bone marrow mesenchymal stem cells were separated using Percoll density gradient centrifugation combined with adherence method.Cadherin-11 and cbfα1 genes were obtained by PCR,and then they were inserted into shuttle vector of adenovirus;thereafter,the recombinant adenovirus plasmids were gained by homologous recombination.Finally,the two plasmids were re-packed to obtain recombinant adenovirus venom,and cadherin-11 and cbfα1 genes were transfected in bone marrow mesenchymal stem cells by adenovirus.MAIN OUTCOME MEASURES:The expressions of cadherin-11 and cbfα1 genes were detected by Western biotting.RESULTS:The adenovirus venom carrying the cadherin-11 and cbfα1 gene was successfully obtained.Western blotting showed that the expressions of the cadherin-11 and cbfα1 genes in bone marrow mesenchymal stem cells were remarkably increased by infection viral.CONCLUSION:The bone marrow mesenchymal stem cells may express high level of cedherin-11 and cbfα1 by gene modification of adenovirus.

Objective To develop electronic medical tag system on scientifically,rationally,orderly and efficiently.Methods By analyzing total goals of system development and application requirement of military medical logistics to design rational-ly system function,scan carefully and examine application technology,insist on the independent development and innova-tion,focus on the standardization of equipment and information.Results Through the various tests of the system,the per-formance of the whole system can be met the requirements of tactical specification and perfect effect.Conclusion The de-velopment of equipment must be grasped the military requirements and the technical and non-technical component ele-ment,focus on the technical breakthrough and follow standardization requirements,then the quality of equipment can be guaranteed,and the task of serving for medical support can be accomplished.

BACKGROUND:Restoration of neurological functions after the damaged peripheral nerve is reconstructed is a hot topic in existing research. Within a short term fol owing peripheral nerve injury, nerve and muscle begin to develop irreversible degeneration. Restoration of the damaged nerve requires delayed degeneration and basic microenvironment. OBJECTIVE:To investigate the protective effect of cardiotrophin-1 on PC12 cells and Schwann cells. METHODS:Schwann cells and PC12 cells were obtained and cultured in complete medium, serum-free medium and 50 ℃ medium, respectively. cells in cardiotrophin-1 group were treated with exogenous cardiotrophin-1 solvent, while those in the control group were treated with equivalent Dulbecco’s modified Eagle’s medium, for 24 hours. The survival rate for PC12 cells and Schwann cells was determined using cellCounting Kit-8 colorimetric method. The lactate dehydrogenase activity in supernatant was detected by lactate dehydrogenase kit, and the malondialdehyde content and superoxide dismutase activity were measured by thiobarbituricaicd and xanthine oxidese method respectively. RESULTS AND CONCLUSION:The survival rate of PC12 cells and Schwann cells in cardiotrophin-1 group was obviously increased, lactate dehydrogenase releasing and malondialdehyde content were obviously decreased, superoxide dismutase activity was dramatical y improved compared with control group. Exogenous cardiotrophin-1 reduces the injury caused by ischemia and heat stress stimulation for PC12 cells and Schwann cells. The mechanism of protection may be related to the expression of anti-apoptosis protein activated by the combination of cardiotrophin-1 and its receptors.

OBJECTIVE: To evaluate the therapeutic effect of transplantation of autologous bone marrow mononuclear cells combined with traditional Chinese medicine for the treatment of limb ischemia. METHODS: Twenty-three patients with limb ischemia were treated. G-CSF was used to stimulate the bone marrow. The mononuclear cells were separated from the aspirated bone marrow fluid in the stem cell studio. The cell amount was above 1x10(9). The transplantation was performed by the way of intra-muscular multi-injection. Traditional Chinese medicine for replenishing qi to activate blood was prescribed from the first day after operation. The pain, poikilothermia, ulcer or necrosis and ankle/brachial index (ABI) of the ischemic limb were evaluated before and after the treatment. RESULTS: The pain score and poikilothermia score decreased one month after the transplantation, with distinct differences as compared with the scores before the treatment (P<0.05). The ABI increased gradually after the treatment, and one month after the treatment, it was 0.15 higher than that before the treatment. CONCLUSION: Transplantation of autologous bone marrow mononuclear cells combined with traditional Chinese medicine can decrease the symptoms and signs of severe lower limb ischemia effectively, and improve the circulation of the ischemic area.

Objective To investigate the action mechanism of an anti-photosensitivity mixture on skin photodamage. Methods Twenty-eight BLAB/c mice were divided into 4 groups, i.e., normal control group,treatment group, negative and positive control groups; the last three groups were irradiated with a single dose of UVB at 300 mJ/cm2 after 7-day pretreatment with sodium chloride physiological solution, anti-photosensitivity mixture, and hydroxychloroquine, respectively. Twenty-four hours after the irradiation, mice were killed and skin tissue samples were obtained at the irradiated sites. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and immunohistochemical staining were carried out to detect cell apoptosis,Fas and Caspase-3 protein expressions respectively. Results An increase was observed in the expression level of Fas and Caspase-3 and in the apoptotic index in keratinocytes from UV-irradiated mice compared with unirradiated control mice (all P ＜ 0.01 ). In comparison with sodium chloride physiological solution, the antiphotosensitivity mixture suppressed the UV irradiation-induced increase in the expression intensity of Fas and Caspase-3 and apoptotic index in keratinocytes (P ＜ 0.05 or 0.01 ). Conclusions The anti-photosensitivity mixture could alleviate UV-induced inflammatory damage to and apoptosis in epidermal keratinocytes, likely by regulating cell apoptosis and Caspase-3 pathway.

Objective To retrospectively evaluate and analyze the clinical effect of posterior pedicle subtraction osteotomy in treating chronic, posttraumatic thoracolumbar kyphosis. Methods Nineteen patients (11 males and 8 females) with chronic, posttraumatic thoracolumbar kyphosis were corrected surgically. The patients were at age range of 29-61 years (mean 42 years). Preoperative kyphosis Cobb angle ranged from 31° to 63° (mean 47°) and trauma history ranged from 8 months to 63 months (mean 29 months). All patients were treated with pedicle subtraction osteotomy according to the size of Cobb angle, extent of spinal stenosis and source of compression. Results Sagittal alignment was improved to average 40.2°, with a correction rate of 85.8%. Two patients developed postoperative leakage of cerebrospinal fluid. Among them, one was combined with encephalic infection and cured with active treatment, and the other developed postoperative wound infection, which were treated conservatively with antibiotics and local wound care. There were no other severe complications. The average follow-up period was 15 months (range 6-41 months). At the last follow-up, clinical symptoms and neurological function were improved significantly. Neither loss of correction nor failure of internal fixators was observed. X-ray and dynamic X-ray films showed a 100% fusion in all patients. Conclusions The single-stage posterior pedicle subtraction osteotomy is a safe and effective procedure for correction of posttraumatic thoracolumbar kyphosis. It is possible and safe to obtain a correction within 55° on single segment by this technique.