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.

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 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.

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 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.

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: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 investigate the correlation of position movement of primary tumor with interested organs and skin markers,and to investigate the correlation of volume variation of primary tumors and lungs during different respiration phases for patients with lung cancer at free breath condition scanned by four-dimensional CT (4DCT) simulation.Methods 16 patients with lung cancer were scanned at free breath condition by simulation 4DCT which connected to a respiration-monitoring system.A coordinate system was created based on image of T5 phase,gross tumor volume (GTV) and normal tissue structures of 10 phases were contoured.The three dimensional position variation of them were measured and their correlation were analyzed,and the same for the volume variation of GTV and lungs of 10 respiratory phases.Results Movement range of lung cancer in different lobe differed extinct:0.8 - 5.0 mm in upper lobe,5.7 -5.9 mm in middle lobe and 10.2 - 13.7 mm in lower lobe,respectively.Movement range of lung cancer in three dimensional direction was different:z-axis 4.3 mm ± 4.3 mm＞ y-axis 2.2 mm ± 1.0 mm ＞ x-axis 1.7 mm ± 1.5 mm ( x2 =16.22,P =0.000),respectively.There was no statistical significant correlation for movement vector of GTV and interested structures (r =-0.50 - -0.01,P =0.058 - -0.961 ),nor for volume variation of tumor and lung ( r =0.23,P =0.520 ).Conclusions Based on 4DCT,statistically significant differences of GTV centroid movement are observed at different pulmonary lobes and in three dimensional directions.So individual 4DCT measurement is necessary for definition of internal target volume margin for lung cancer.

ObjectiveTo investigate the effect of the displacement of the selected silver clip in the different respiratory state achieved by active breathing control ( ABC ) system on the displacement of the geometry constituted by all of the silver clips at the border of the cavity in external-beam partial breast irradiation (EB-PBI).MethodsTwo sets of CT images in state of moderate deep inspiratory breathing hold (mDIBH),deep expiratory breathing hold (DEBH),and free breath (FB) were acquired in the same CT simulation assisted by ABC system for each of the 27 patients after breast conservative surgery.All silver clips in the cavity were delineated based on each set of CT images.Thereafter,the irregular geometry based on the silver clips as the vertices was automatically formed.Four selected clips located at the top,bottom,lateral border and medial border of the cavity were correspondingly manually registered based on automatic registration of the CT images acquired in the same or different state of respiration including mDIBH,FB,and DEBH.The displacement of center of the geometry in the direction of right-left (RL),anterior-posterior (AP),and superior-inferior (SI) separately based on automatic registration and manual registration was evaluated.The difference of the displacement was analyzed by Kruskal-Wallis H-test and Kolmogorov-Smirnov Z-test.Results When registered between mDIBH and mDIBH,FB and FB,DEBH and DEBH,the differences of the displacement of the center of geometry were not statistically significant (H =0.00 - 1.76,P=0.184-0.954). When registered between mDIBH and DEBH,the differences were statistically significant ( Z =11.31 - 23.00,P =0.000 - 0.001 ).There were statistically significant differences in the displacement of geometry center based on the selected silver clip between different registration forms in AP and SI directions (Z=4.76-25.54,P=0.000-0.029).ConclusionsThe difference of intrafraction displacement of the geometry constituted by the clips between the same respiratory states in the three dimensional direction is not statistically significant,but the difference is statistically significant between the different respiratory states in AP and SI directions.

Objective To investigate the effects of gene transfection with human growth/differentiation factor 5(GDF5)on the growth and difierentiation of bone nlarrow stromal stem cells (BMSCs).Methods GDF5 gene was trans fected into BMSCs by liposome method. Then cell proliferation and cycles were examined by MTT and flow cytometry respectively. Cell morphology was observed under light microscope and electron microscope (EM).GDF5 and Collagen Ⅱ were detected at the level of mRNA and protein by RT-PCR and immunocytochemistry. Alkaline phosphate activity was examined by lead citrate method. Osteocalcin mRNA expression was determined bv RT-PCR. ResulIs GDF5 gene was transfected into BMSCs successfully and the transfected cells still maintained their natural growth and proliferation features. Stable expression of GDF5 gene in BMSCs was obtained. The trans fected ceils had basically the same proliferation ability and cell cycles as the untransfccted ones. After transfection comparatively more polygonal cells could be seen in light microscope, showing irregular arrangement mode. Plenty organells were observed and cell nucleus showed irregular shape under EM. The expressions of Collagen Ⅱ mRNA and protein were positive. But osteocalcin mRNA expression was negative. Conclusion Since BMSCs can be induced by GDF5 to differentiate into chondrogenic cells. GDF5 gene-modified BMSCs may be used as candidate seed cells of cartilage tissue engineering.