BACKGROUND: Growth differentiation factor-5 (GDF-5) plays an important role in the development and formation of cartilage, extremities, and joints, which is a widely used joint development marker.OBJECTIVE: To express mature peptide of human GDF-5 in E. coil by the way of genetic engineering, and to explore the inductive activity of recombinant protein in vivo.DESIGN, TIME AND SETTING: The observation experiment based on gene was performed at the Analysis and Testing Center of Southern Medical University from January to June 2006.MATERIALS: Human fetus cartilage tissue was harvested from Department of Gynaecology and Obstetrics, and the consent was obtained from the family. Ten KM mice were purchased from experimental animal center of Southern Medical University, half male and half female, weighing 18-22 g, aged 6-8 weeks.METHODS: The hGDF-5 gene encoding mature peptide was gained by RT-PCR from the total RNA which was extracted from fetus cartilage tissues, and was inserted into the pET22b(+) vector to construct recombinant prokaryotic expression plasmid pET22b(+)-GDF5, which was transformed into E. coil BL-21 to be expressed after IPTG induction. Proteins of interest were purified with sepharose chelated with nickel ions (Ni2+) and then implanted in mouse hindlimb muscle to evaluate the biological activities by routine hematoxylin-eosin staining.MAIN OUTCOME MEASURES: The expression, sequencing of target gene was observed by agarose gel electrophoresis, and the protein expression was detected by SDS-PAGE electrophoresis, meanwhile, the GDF5-inducing activity was evaluate by histological observation.RESULTS: RT-PCR product was about 350 bp in length, which was confirmed by double enzyme digestion of the recombinant plasmid, sequencing result was in agreement with the reported hGDF-5 sequence in Genbank. SDS-PAGE analysis showed a conspicuous band representing a new foreign protein with relative molecular mass of approximately 14 KD after induced expressioin. Cartilage tissues were formed in the mouse muscle where the purified proteins were implanted. CONCLUSION: The integral human GDF-5 mature peptide gene was cloned successfully from human fetus cartilage tissue and a high-yield expression was achieved in E. coli, the pudfied protein has chondrogenic activities in vivo.

Objective To optimize the extraction process for the effective fraction of Bushen Yizhi ( BSYZ) formula. Methods Yield of the effective fraction and contents of two marker compounds 2,3,5,4 -tetrahydroxystilbene-2-O-β-D-glucoside and osthole assayed by high performance liquid chromatography (HPLC) were used as the optimizing indexes, and weighted coefficient of the indexes was analyzed by analytic hierarchy process ( AHP). Ratio of liquid-to-solid, extraction time, and extraction times were used to screen the optimal conditions by orthogonal test. Results The optimized extraction process of the effective fraction of BSYZ formula was as follows: extracting with 8-fold water for three times, and boiling for 1.5 hours each time. The verification test showed that the comprehensive scores of the optimized extractive process were higher than those obtained in the orthogonal test. Conclusion The optimized extraction process and conditions are practical in the manufacture of the effective fraction of BSYZ formula.

Objective To investigate the immunologic enhancement of Porphyra polysaccharide. Methods Porphyra polysaccharide in different dosages (0.025,0.050,0.150g?kg~(-1)) were injected intraperitoneally into the immunosuppressive mice induced by cyclophosphamide for 7d. On day 8,the cytotoxic activity of NK cells,the levels of interferon-?(IFN-?)and nitric oxide (NO)in the cultured supernatants of spleen cells were determided. Results The cytotoxic activity of NK cell,the levels of IFN-? and NO produced by cultured spleen cells from the group of mice treated with 0.150g?kg~(-1) of Porphyra polysaccharide were higher than those from model group. Conclusion Porphyra polysaccharide could enhance immunological functions to a certain degree in immunosuppressive mice.

Objective To optimize extraction technology of flavonesingredients from Herba Epimedii by uniform design. Methods Ultraviolet spectrophotometry was adopted to determine the content of total flavonoids. Content of epimedin A, epimedin B,epimedin C,icariin and baohuosideⅠ were determined by HPLC. U10?( 108 ) uniform design was used to conduct the multiple comprehensive evaluation of six ingredients and comprehensively analyze the influence of the concentration and amount of ethanol, extracting time on extraction of flavonesin gredients from Herba Epimedii. Results The uniform design experiment showed that 15-fold weight of 60% ethanol,extracting 2 times and each time with 165 min were the optimum extraction condition. Conclusion The method is easy and reasonable to handle,has stable and reliable results,and good repeatability and feasibility.It can be applied in industrial production.

Objective To evaluate the value of PET/CT in preoperative assessment and postoperative monitoring of ovarian cancer. Methods A retrospective analysis was conducted in 45 patients of ovarian neoplasm with clinical records underwent 18F-FDG PET/CT, including 10 patients underwent PET/CT before surgery and 35 patients after surgery. The clinical follow-up time was 6 months at least. The diagnosis based on pathology and clinical follow-up data. Results (1) The sensitivity, specificity and accuracy of PET/CT in detecting ovarian cancer were 94.6%,75.0%and 91.1%, respectively. (2) Ten patients before surgery were all detected tumor by PET/CT, but 2 of them were false positive based on pathologic results. (3) Two patients with non-standard surgery were detected tumor by PET/CT. In 33 patients after standard surgery, 6 patients were no tumor detected by PET/CT. In addition,4 patients with normal CA125 and no signs of recurrence and metastasis were detected tumor by PET/CT. The pathology and clinical follow-up data supported the results. 23 patients with higher CA125 were diagnosed recurrence and metastasis based on pathology and clinical follow-up data, 21 of them were detected tumor by PET/CT. Conclusion 18F-FDG PET/CT plays an important role in preoperative assessment, early diagnosis and accurate positioning of recurrent and metastasis of ovarian cancer. It can be used to guide the clinical treatment.

OBJECTIVETo cultivate human mesenchymal stem cells (MSC) derived from fetal bone marrow and examine their pluripotentiality.

METHODSBone marrow mononuclear cells from human fetal femur were collected by Percoll gradient centrifugation. The low density cells including MSCs were cultivated and expanded in MSCGM media. The characteristics of the multipotent MSCs were observed by implanting them into nude mice for 4 weeks.

RESULTSHuman fetal marrow MSCs were successfully cultivated and differentiated in vivo into many kinds of tissues such as bone, cartilage, adipose, skeletal muscle and tendon-like tissue.

CONCLUSIONHuman fetal marrow MSCs were multipotent stem cells.

Animals ; Bone Marrow Cells ; cytology ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cell Division ; Cell Transplantation ; methods ; Cells, Cultured ; Humans ; Mesoderm ; cytology ; Mice ; Mice, Nude ; Stem Cells ; cytology ; Transplantation, Heterologous

BACKGROUND:Clinical work shows that there are a large proportion of patients suffering from osteoerthritis(OA)and osteoporosis(OP),therefore establishing OA+OP models to simulate the clinical disease in postmenopausal women addreasing the basic characteristics of lesions,will offer better prevention and treatment of OA+OP in clinical practice.OBJECTIVE:To attempt to create OA+OP animal model.DESlGN,TIME AND SETTING:Randomized controlled animal expedment in terms of call pathology was perforrned between August 2008 and January 2009 in the Scientific Research Center and Traditional Chinese MediciRe Hospital of Xinjiang Medical University.MATERIALS:Forty 4-month-old female SD rats,weighing(210±10)g,were randomly divided into normal control group,OA group,OP group,OA+OP group.METHODS:In the OA+OP group,rats underwent abdominal incision 1.5 cm longitudinal on both sides of lumbar spine on back,followed by bilateral ovarian resection and ovarian artery ligation,to establish OP models.One month after the skin incision,left knee skin,subcutaneous tissue and fascia were cut,then joint capsule was given a vertical incision.Anterior cruciata ligament was cut off in orthophoria,meniscus was removed,followed by subcutaneous tissue and skin suture,OA model was prepared and placed under warm environment.antibiotic subcutaneous injection for 3 days,and the displacement of one month.OA group and OP group were produced in accordance with the above method of OA.OP model.Normal control rats received no treatment.MAIN OUTCOME MEASURES:When the rats were 6 months old,the left knee femoral condyle articular cartilage were examined by light microscopy and electron microscopy,left femur was used to measure proximal femoral bone mineral density.RESULTS:In three groups of model rats,articular cartilage become thinning and degeneration.In the OP model group and OA+OP model group,trabecular was sparse and arranged in disorder.In the OA model group and OA+OP model group,the incision layer was chiefly deleted,transferring layer was greatly injured,call hypertrophy and colony were observed,a large amount of blood capillary invaded into cartilage and calcification layer,even break through tidal line;in OP model group,incision layer of articular cartilage became thinning and appeared bilateral tidal line.In the OA model group and OA+OP model group,knee condylar number of special-shaped cartilage cells increased,manifested as irregular nuclei,reduced cell organelle,nuclear shnnkage,chromatin uneven distdbution,mitochondrial swelling,rough expansion of endoplasmic raticutum,accumutation of cytoplasmic microfilaments,showing lipid droplets and glycogen granules,gliel fibdllary fracture,disorder arrangement,a large number of cartilage calls were apoptotic.Three groups of model rats exhibited a dramatically decreased bone mineral density compared with control rats(P＜0.05).CONCLUSION:The animal modal of OA+OP was successfully established.

Objective To determine the persistence time of genotype 4 hepatitis E (HE) viremia after the onset of clinical symptoms in HE patients and provide essential data for study on HE epidemiologieal transmission, so that to evaluate potential contagiousness of HE patients after clinical stage. Methods The first serum samples from 162 HE patients after hospitalized in Eastern China were collected and tested for hepatitis E virus (HEV) RNA by nested reversed transcription- polymerase chain reaction (RT-PCR). The persistence time of HEV viremia after the onset of clinical symptoms was estimated with Kaplan-Meier survival analysis. Results HEV RNA was detectable in 101 out of 162 serum samples with positive rate of 62.35%, which was all grouped to genotype 4 by homology analysis. Furthermore, HEV RNA was detectable in 74 (64.91%) out of 114 male and 27 (56.25%) out of 48 female, which was not significantly different (χ2 = 1.08, P=0. 30). Kaplan-Meier survival analysis showed that the median persistence time of HEV genotype 4 viremia was 24 days after the onset of clinical symptoms (95% CI: 18-30 days), which meant that the viremia of 50% HE patients remaining detectable up to 24 days after the onset. The 75% and 25% percentiles were 14 days and 31 days, respectively. There was no significant difference of viremia persistence time between male and female (Breslow test: P=0.98, Tarone-Ware test: P=0.91). Conclusions The viremia of 75% patients with HEV genotype 4 infection could persistent until 2 weeks after the onset of clinical symptoms and that of some patients could persistent over 1 month. It is indicated that the viremia is still persistent and HE patient could be a reservoir even after the clinical symptoms disappeared and biochemical marks normalized.

DNA barcoding is a technique in which species identification and discovery are performed by using short and standard fragments of DNA sequences. In this study, eleven species of Paris, including seven varieties, were sampled. Five chloroplast sequences, psbA-trnH, rpoB, rpoC1, rbcL, matK, and one nuclear marker, the second internal transcribed spacer (ITS2) of ribosomal DNA, were amplified and sequenced. The PCR amplification and sequencing efficiency, intra- and inter-specific divergence and barcoding gap were used to evaluate different loci, and the identification efficiency was assessed using BLAST1 and Nearest Distance methods. The ITS2 sequences in the studied samples of Paris were amplified and sequenced successfully using primers designed by our group, while matK showed low level in the amplification and psbA-trnH was difficult for sequencing because of over 800 bp and poly (A) structure. Analysis of the intra- and inter-specific divergence and barcoding gap showed ITS2 was superior to other loci. The ITS2 showed a much higher percentage of success (100%) in identification than other five loci, none of which indicated more than 50% except matK (52.9%). The 2-locus combination of rbcL+matK didn't improve ability of authentication. In addition, the rate of successful identification with ITS2 kept 100% when the samples were expanded to 67 samples of 29 species. In conclusion, ITS2 can be used to correctly identify medicinal plants of Paris, and it will be a potential DNA barcode for identifying medicinal plants of other taxa.

BACKGROUND:Calcium phosphate bone cement has been applied to clinical surgery because of its good biocompatibility and osteoconduction. However poor mechanical properties and lack of osteoinductivity limit its wide application.
OBJECTIVE:To develop calcium phosphate cement incorporated with N-acetylcysteine (NAC) loaded silk fibroin microspheres (SFM), which is a kind of new injectable bone graft material with slow-release function, and evaluate its physical and chemical properties and cel compatibility.
METHODS: Empty SFMs were prepared with emulsion solvent evaporation to absorb NAC solution of different concentrations by NAC-SFM and the concentration of NAC at the maximum drug loading ratio was determined. Then, NAC-SFM was loaded into calcium phosphate bone cement to test the drug release propertiesin vitro. MC3T3-E1 osteoblasts were cultured on the surface of NAC-SFM calcium phosphate bone cement and cel attachment and growth were observed by scanning electron microscope. Additionaly, MC3T3-E1 cels were cultured with three kinds of bone cement extracts (calcium phosphate cement, SFM-calcium phosphate cement, NAC-SFM-calcium phosphate cement, as wel as cultured in theα-minimum essential medium containing a volume fraction of 10% fetal bovine serum and 1% penicilin-streptomycin double antibody as the control. MTS assay was used to evaluate cel proliferation.
RESULTS AND CONCLUSION:Microspheres in the composite bone cement presented with smooth surface, same size, diffused distribution and no obvious destroy. Thus, the SFM could remain stable in the reaction process of the composite bone cement. The double slow release system which contained silk fibroin microspheres and calcium phosphate bone cement showed a significant decrease in the cumulative release percentage of NAC within the first 24 hours compared with the control group (P < 0.05). In the next 28 days, the release speed of NAC was significantly lower in the NAC-SFM-calcium phosphate cement group than the calcium phosphate cement group (P< 0.05). In addition, different extracts had no significant cytotoxicity to the growth of MC3TC-E1 cels. Thus, the NAC-SFM-calcium phosphate cement has good cytocompatibility, which provide a new insight into the development of bone repair biomaterials.