BACKGROUND: Injectable hydrogel-chitosan is the latest developed material for cartilage tissue engineering. It provides a novel cell attachment mechanism. OBJECTIVE: To investigate the effect of allologous chondrocytes in combination with chitosan in repairing cartilage defects. DESIGN, TIME AND SETTING: Randomized controlled animal trial was performed at the Central Laboratory of China Medical University from November 2004 to March 2006. MATERIALS: Thirty 2-week-old New Zealand rabbits weighing 0.3 kg were selected to model cartilage defect. METHODS: Thirty rabbits were randomly divided into three groups. In the experimental group, the defects were repaired by the injection of second passage of chondrocytes (2.5?1011 L-1) combined with chitosan into articular capsule; the cell suspension group was only injected with chondrocyte suspension (2.5?1011 L-1); the control group was not given any treatment. MAIN OUTCOME MEASURES: The animals were sacrificed at the 4th, 8th, and 12th weeks after operation, respectively to harvest the lateral condyle of knee joint. Hematine-eosine staining, gross and histological examinations, and electron microscopic evaluations were performed to observe the repair condition. RESULTS: The gross observation showed that there was no obvious swelling or other inflammatory responses in each rabbits. Histological examination suggested that in the experimental group, the cartilage defects were repaired by the hyaline cartilage tissue with smooth surface, which integrated smoothly with articular cartilage at 12 weeks after operation. No obvious defect repair was observed in the other groups. The transmission electron microscopic observation showed that in the new-formation tissues of the experimental group, there were round or oval chondrocytes, and abundant rough endoplasmic reticulum, ribosome and Golgi complex in cytoplasm. At 8 to 12 weeks, plenty of collagen fibers were found in cell suspension group. In the control group, only fibrous tissues were seen on the surface of defects. CONCLUSION: The articular cartilage defects of rabbits are repaired with allograft of chondrocytes seeded onto chitosan, indicating chitosan can serve as carrier in cell transplantation.

Objective Triple negative breast cancer(TNBC), a special breast cancer subtype, is lack of effective target therapy.The article aimed to investigate the role of lysophosphatidic acid (LPA) and Hippo Yes-associated protein (Hippo-YAP) signaling pathway in TNBC invasion and metastasis and the mechanisms.Methods The specific small interfering RNA (siRNA) of YAP was synthetized in vitro, and was transfected into MDA-MB-231 cells using liposome transfection.The experiment was divided into YAP-siRNA group, positive control group and blank control group.Each group is provided with 2 parallel holes.Evaluation was made on the effects of each group on Hippo-YAP, the mechanisms and regulation on upstream and downstream molecules of Hippo-YAP pathway.Results In experiment group, YAP content, the capacity of invasion and metastasis after transfection ([0.035±0.005], [2.200±1.000], [3.500±0.800]) significantly decreased compared with positive control group([0.343±0.012], [27.600±5.100], [22.300±5.000]) and blank control group([0.384±0.017], [26.500±4.800], [22.350±6.000]) (P<0.05).YAP expression levels at 60 min, 120 min, and 240 min in experiment group significantly decreased compared with positive control group and blank control group (P<0.05).YAP relative expression levels of 10, 20, 50 μmol/Lwere significantly lower than those of positive control group and blank control group (P<0.05).After respective interference of C3 transferase and Y27623, significant difference was found in the pYAP mRNA contents of experiment group([0.255±0.052], [0.326±0.017]), blank control group([0.048±0.032], [0.534±0.017]) and positive control group([0.052±0.021], [0.528±0.024])(P<0.05).The expression levels of YAP mNA and AREG mNA significantly increased in experiment group([0.176±0.032], [0.263±0.008]) compared with blank control group([0.043±0.013], [0.263±0.008]) and positive control group([0.049±0.025], [0.057±0.043])(P<0.05).Conclusion LPA induces breast cancer invasion and metastasis, which is YAP-dependent, time-dependent and concentration-dependent.LPA-Hippo-YAP singaling pathway may be one of the mechanisms promoting delayed metastasis of TNBC.

OBJECTIVE:To develop a method for rapid,accurate and simultaneous determination of dexzopiclone,zolpidam and zaleplon in human plasma. METHODS:After the plasma sample was processed by liquid-liquid extraction,UPLC-MS/MS was established to detect plasma sample with carbamazepine as internal standard. The separation was performed on a Waters ACQUITY UPLC HSS T3 column with mobile phase composed of 0.1% ammonium solution-acetonitrile (gradient elution) at flow rate of 0.2 ml/min. The column temperature was set at 40 ℃,and sample size was 5 μl. The detection was performed by multiple reaction monitoring (MRM) via electrospray ionization (ESI) source in positive mode. The mass transition ion-pairs were as follows:m/z 308.2→263.1(zolpidem),m/z 389.3→245.0(dexzopiclone),m/z 306.2→236.1(zaleplon),m/z 237.3→151.2(internal standard). RE-SULTS:The linear range of zolpidem,dexzopiclone and zaleplon were 0.02-20.00,0.50-20.00,0.02-20.00 ng/ml,respectively (r=0.990 1,0.996 8,0.991 7). LLOQ of them were 0.02,0.50,0.02 ng/ml,and detection limit were 0.01,0.20,0.01 ng/ml. RS-Ds of intra-day and inter-day were all lower than 15% ;extraction recovery were 88.9% -106.5% ;matrix effect were 94.8%-106.3%. CONCLUSIONS:The method is simple,rapid,sensitive and specific,and it can be used for simultaneous deter-mination of zaleplon,zolpidem and dexzopiclone in human plasma.

Objective To evaluate the differential expression of lysophosphatidic acid receptor (LPAR) in breast cancer (BC), and its relationship with clinicopathological features of BC patients. Methods The qRT-PCR and immunohistochemical staining were used to detect the LPAR expression in 37 normal tissues, 55 benign disease tissues and 82 BC tissues, besides, the correlation of LPAR expression with clinicopathological data was also analyzed. Results The expression levels of LPAR2 and LPAR3 mRNA and protein in BC tissues were higher than those in normal benign tissues (all P<0.05). LPAR1 mRNA expression had no difference in three tissues (χ2 =1.908, P >0.05). LPAR1 expression was not associated with clinicopathological features in BC tissues (P>0.05). LPAR2 expression in postmenopausal patients was higher than that in premenopausal patients (χ2=4.821, P<0.05). LPAR3 expression was significantly associated with nodal metastasis, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in BC tissues (all P<0.05). Conclusion LPAR in BC tissues has differential expression, which is associated with nodal metastasis, ER, PR and HER2.

BACKGROUND:The key of stem cells for treating nervous tissue injury is the transplantation of stem coils that have regeneration capacity.The structure and function of central nervous system were re-established by multiple action mechanisms.OBJECTIVE:To explore the effects and mechanisms of bone marrow mesenchymal stem cells(BMSCs)locally transplanted into rats with spinal cord injury on neurological recovery.METHODS:BMSCs were separated with density gradient centrifugation and cell attachment.10 mg/L BrdU was used for labeling before cell transplantation.Adult female Wistar rats were used to establish spinal cord injury models using an aneurysm clip,and they were then randomly divided into control group,saline group and transplantation group.In the transplantation group,BMSCs were transplanted into the damaged spinal cord by stereotaxis at day 7 following damage.In the saline group,an equal volume of saline was utilized.In the control group,the rats were left intact.Basso,Beattie and Bresnahan(BBB)locomotor rating scale was used before and at 7,14,30,60 and 90 days following damage.Rats were sacrificed at day 90.BrdU-positive cells,Brdu+neuron specific enolase,Brdu+glial fibrillary acidic protein(GFAP),Brdu+basic fibroblast growth factor(bFGF),and Brdu+brain-derived nerve growth factor(BDNF)immunohistochemistry double-staining cells and simple staining positive calls were observed.RESULTS AND CONCLUSION:The recovery of BBB function score was better in the transplantation group than in the control group(P＜0.05).The recovery speed of BBB function score was slower in the saline group than in the control group at 30 days following damage(P＜0.05).No significant difference was determined at day 90 compared with the control group(P＞0.05).BrdU-positive cells and double-staining cells of immunohistochemistry could be found at the center of damage site and 1 cm from caudal end to damaged site in rats of the transplantation group.The number of NSE,GFAP,bFGF and BDNF simple staining cells was significantly greater in the transplantation group than in the control and saline groups(P＜0.05).Results indicated that BMSC transplantation can improve the recovery of nervous function of rats with spinal cord injury.Its mechanism may be correlated with the differentiation of transplanted cells into neuron-like and glial cell-like cells,secretion or promoting secretion of neurotrophic factors in host.

To mask the bitterness of azithromycin（AZI） and improve patient compliance, an AZI-loaded microsphere (AZI-EC MS) for oral administration was prepared by O/W emulsion solvent evaporation with ethylcellulose (EC) as carrier. The release profiles and taste-masking effect of AZI-EC MS were preliminarily assessed. Its physical properties and morphology were then investigated by differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). The results indicated that the polymer weight of EC could influence the drug release behavior. With a drug polymer ratio of 1∶1 and mixed EC (N22/T10, 7∶3) as carrier, the cumulative release of AZI-EC MS at 0.5 h was less than 40% and reached 90% at 8 h; the drug loading efficiency of microspheres was (48.95 ± 0.86)% with smooth spherical morphology. The AZI bitterness threshold is 9.93 μg/mL with a strong bitter taste, which indicated a better taste masking effect. Therefore, AZI-EC MS prepared in this study can mask AZI bitterness and improve patient compliance, setting the stage for the research of new AZI preparations.

Objective:To construct the molecular transmission network of human immunodeficiency virus type 1 (HIV-1) epidemic strains in northern Zhejiang Province (Jiaxing City and Huzhou City) and to explore the HIV-1 transmission characteristics in this region.Methods:A total of 371 newly diagnosed human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) patients in Jiaxing City and Huzhou City in 2017 were included as study subjects, and the blood samples were collected and the basic demographic and epidemiological information were obtained. RNA in plasma was extracted, and the pol region gene sequence was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (PCR) to construct phylogenetic tree for identifying subtypes. The pairwise genetic distances were calculated, and the optimal threshold of genetic distance was selected, and finally the molecular transmission network was constructed. Chi-square test was used for statistical analysis. Results:The pol region gene sequences of 336 samples were successfully obtained, and 11 subtypes were detected, mainly including circulating recombinant form (CRF)07_BC (40.8%, 137/336) and CRF01_AE (31.2%, 105/336). Based on the 1.0% genetic distance threshold, the molecular transmission network of HIV-1 was plotted. A total of 38 transmission clusters (cluster sizes ranging from two to 28) including 119 patients were found, with males predominantly (82.4%, 98/119) and most of the patients aged over 40 (include 40) years old (52.9%, 63/119), mainly infected with CRF07_BC subtype (57.1%, 68/119) and CRF01_AE (24.4%, 29/119). The clustering rate of CRF07_BC (49.6%, 68/137) was significantly higher than that of CRF01_AE (27.6%, 29/105), the difference was statistically significant ( χ2=5.27, P=0.022). Two large clusters C1 (28 cases) and C2 (11 cases) were identified, the majority of which were men who have sex with men (17 cases and seven cases, respectively). High-risk cases generally sought sexual partners in local or nearby cities through mobile phone dating software, of which the infected sequences mostly had high homology with other economic developed regions (Guangdong Province, Beijing City and Hangzhou City, etc.). Conclusions:The HIV-1 subtypes are diverse in Jiaxing City and Huzhou City, mainly CRF07_BC and CRF01_AE. The HIV-1 transmission networks are complex, among which high-risk cases may be the key factor leading to the HIV-1 epidemic in the region. Therefore, it is urgent to deepen the transmission network monitoring and formulate timely precise intervention and prevention strategies.

Rabdosia (Bl.) Hassk is a medicinal plant rich in enantiomers such as 15α-acetoxyl-6, 11α-epoxy-6α-hydroxy-20-oxo-6, 7-seco-ent-kaur-16-en-1, 7-olide and 15α-dihydroxy-6β-methoxy-6, 7-seco-6, 20-epoxy-1, 7-olide-ent-kaur-16-ene. It has high medicinal value and huge market potential, but the yield from wild Rabdosia (Bl.) Hassk species cannot meet the needs of marketization. Carrying out pollution-free cultivation of Rabdosia rubescens (Hemsl.) Hara and Rabdosia serra (Maxim.) Hara has become the most direct and effective measure to solve this problem in order to ensure the sustainable use of the resources of Rabdosia. This paper summarized a set of non- pollution cultivation system of R. rubescens and R. serra to achieve the"safe, effective, stable and controllable"target.

This study sought to assess the therapeutic effect of celecoxib (CEL)-loaded polylactic acid-glycolic acid copolymer (PLGA) microspheres on rheumatoid arthritis in rats after intra-articular injection.The celecoxib-loaded microspheres (CEL-MS) were prepared by the O/W solvent volatilization method with PLGA as carrier.In order to investigate the therapeutic effect of CEL-MS on rheumatoid arthritis in rats after intra-articular injection, a rat model of adjuvant arthritis (AA) was constructed by complete Freund''s adjuvant, and the evaluation indicators of the therapeutic effect were rat paw swelling, arthritis index,spleen index and joint synovial histopathological examination. The results showed that the microspheres had a smooth spherical morphology with a particle size of (2.1 ± 0.3) μm and a drug loading efficiency of (20.8 ± 0.6)%.The results of the in vivo efficacy test showed that intra-articular injection of CEL-MS compared to the CEL suspension oral and the celecoxib suspension intra-articular injection in adjuvant arthritis rat model can significantly reduce joint swelling and arthritis index, thus effectively inhibiting synovial inflammation.The above results indicate that intra-articular injection of CEL-MS has a good therapeutic effect on rheumatoid arthritis in rats.

Objective To understand the characteristics of distribution on HIV-1 subtypes and the transmission clusters in Yiwu in Zhejiang province.Methods A cross-sectional study of molecular epidemiology was carried out on newly reported H1V/AIDS cases in Yiwu.RNA was extracted from 168 plasma samples,followed by RT-PCR and nest-PCR for pol gene amplification,sequencing,phylogenetic tree construction used for analyzing the subtypes and transmission clusters.Mutations on drug resistance was analyzed by CPR 6.0 online tool.Results Subjects were mainly males (86.3％,145/168),with average age as (39.1 ± 13.4) years old and most of them were migrants (66.7％,112/168).The major routes of transmission included homosexual (51.2％,86/168) and heterosexual (48.8％,82/168) contacts.The rate of success for sequence acquisition was 89.9％ (151/168).The dominant subtypes showed as CRF01_AE (74,49.0％) and CRF07_BC (64,42.4％),followed by CRF08_BC (5,3.3％),CRF55_01B (3,2.0％),each case of subtype B,CRF45_cpx,CRF59_01B,CRF85_BC and URF (B/C).CRF45_cpx and CRF85_BC were discovered the first time in Zhejiang province.Twenty-six transmission clusters involving 65 cases were found,with the total clustered rate as 43.0％ (65/151),in which the CRF01_AE clustered rate appeared as 54.1％ (40/74),higher than that of CRF07_BC (21/64,32.8％).The average size of cluster was 2.5 cases/cluster,with average size of cluster in CRF01_AE patients infected through heterosexual transmission as the largest (3.5 cases/cluster).The prevalence of transmitted drug resistance was 4.6％ (7/151).Seven cases with surveillance drug resistant mutations (SDRM) were found,including 5 cases of M46L (3.3％),and one case of F77L or Y181C.Conclusion HIV genetic diversity and a variety of transmission clusters had been noticed in this study area (Yiwu).Programs on monitoring the subtypes and transmission clusters should be continued and strengthened.