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Aim To observe the effects of microRNA-204/-211 deficiency on osteoarthritis(OA) induced by medial meniscus amputation (DMM) in mice. Methods 12 C57BL/6J wild-type (WT) mice were randomly divided into sham operation groups and DMM groups, namely WT-control group and WT + DMM group. And twelve microRNA-204/-211 gene knockout (miR-204/-211-dKO) mice were randomly divided into sham operation groups and DMM groups, namely dKO group, and dKO + DMM group. The pain sensitivity of mice was measured by the von Frey test before sacrificing. Three months after the operation, the mice were sacrificed. The knee joints and dorsal root ganglion (DRG) were taken for detection. The subchondral bone structure was detected by micro-CT. Sections of knee joint tissue were stained with toluidine blue, PCNA, type Ⅱ collagen and immunohistochemistry. DRG tissues were detected for related pain factors and inflammatory factors by RT-qPCR. Results Compared with the mice in the WT-Control group, mice in the WT + DMM group showed typical OA symptoms such as osteophyte formation, subchondral osteosclerosis, and decreased pain thresholds. The expression of collagen Ⅱ in cartilage significantly decreased, while the expression of MMP13 significantly increased. The expression of inflammatory and pain-related factors in DRG significantly increased. At the same time, the OA phenotypes of mice in dKO + DMM were more obvious than that of mice in the WT + DMM group, indicating that miR-204/-211 deficiency aggravated the OA induced by DMM in mice. In particular, DMM did not cause synovial hyperplasia and synovial inflammation in WT mice, which could not completely represent the pathological characteristics of OA patients in clinical practice. However, miR-204/-211 deficiency significantly promoted synovial hyperplasia and synovial inflammation of knee joints in DMM mice. Conclusions After DMM operation, miR-204/-211 deficient mice showed not only typical OA phenotypes such as osteophyte formation, subchondral osteosclerosis, cartilage destruction and lower pain threshold, but also synovial hyperplasia and synovitis, which could better represent the pathological characteristics of clinical OA patients. MiR-204/-211 deficient mice with DMM can be used as a new OA model and an ideal animal model for screening anti-OA drugs.
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Aim To observe the immune regulation and cartilage protection of dexamethasone-loaded thermosensitive hydrogel (DLTH) on rheumatoid arthritis (RA) rats via conceiving a newly injectable sustained DLTH with chitosan-glycerin-borax as carrier. Methods Thirty rats were randomly divided into three groups, namely control, model and DLTH group. RA model was established through immune induction in model and DLTH group. From the 12th day, rats of DLTH group were injected with 40 (jlL DLTH (1 mg · kg
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OBJECTIVE@#To observe the effect of electroacupuncture (EA) pretreatment on inflammatory reaction, apoptosis and expression of Yes-associated protein (YAP) of ischemic penumbra of cerebral cortex in cerebral ischemia reperfusion injury rats, and to explore the possible mechanism of its neuroprotection effect.@*METHODS@#A total of 84 SD rats were randomized into a sham operation group (12 rats), a model group (18 rats), an EA group (18 rats), an EA+YAP virus transfection group (18 rats) and an EA+virus control group (18 rats). Except for the sham operation group, thread embolization method was adopted to establish the middle cerebral artery occlusion (MCAO) model in rats of the other groups. EA was applied at "Baihui" (GV 20) and "Dazhui" (GV 14) for 30 min in the 3 EA intervention groups 2 h before model establishment, disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in intensity. Adenovirus transfection technique was used to induce gene silencing of YAP in the EA+YAP virus transfection group, and adenovirus vectors was injected as negative control in the EA+virus control group 4 d before model establishment. Twenty-four hours after model establishment, neurological function score was evaluated, the relative cerebral infarction area was observed by TTC staining, the apoptosis in the ischemic penumbra of cerebral cortex was detected by TUNEL staining, the levels of inflammatory factors IL-1β, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex was detected by ELISA method, the expression of YAP was detected by Western blot and immunofluorescence.@*RESULTS@#Compared with the sham operation group, the expression of YAP was increased in the model group (@*CONCLUSION@#Electroacupuncture pretreatment can effectively improve the ischemia reperfusion injury, its mechanism may be related to up-regulating the expression of YAP in the ischemic penumbra of cerebral cortex and relieving the apoptosis and inflammatory reaction.
Subject(s)
Animals , Rats , Brain Ischemia/therapy , Electroacupuncture , Infarction, Middle Cerebral Artery , Rats, Sprague-Dawley , Reperfusion Injury/therapyABSTRACT
Objective To develop a microfluidic device with the adjustable concentration and pressure gradient for 3D cell culture in hydrogel and set up an in vitro model with the capability to closely simulate in vivo microenvironment for cell growth. Methods The microfluidic chip, with a middle channel for 3D cell culture and two side channels for delivering cell culture medium, was designed and fabricated using standard soft lithography and replica molding techniques. Its capability to generate concentration gradient, interstitial flow and image cell in situ was demonstrated. Results A simple microfluidic chip for 3D cell culture in hydrogel with the capability to generate the concentration and pressure gradient was obtained. At a flow rate of 2 μL•min-1 in each side channel, the concentration gradients remained constant after 3 h. The interstitial flow across the gel scaffold was generated by a 100 Pa pressure difference between two-side channels with the pressure gradient of 0.11 Pa/μm. Human adult dermal microvascular endothelial cells (HMVEC) were maintained in 3D culture with collagen type I and observed with confocal microscopy. Conclusions The microfluidic chip is simple and easy to operate and it can simulate the complicated microenvironment in vivo. The chip also allows the multiparameter control of microenvironment, facilitating the better understanding of interaction between cells and microenvironment.
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<p><b>OBJECTIVE</b>To express the L1 protein of human papillomavirus type 16 (HPV16) in insect cell suspension culture system.</p><p><b>METHODS</b>Optimized the conditions of suspension culture, recombinant virus amplification and protein expression. Determined the virus tilter by plague analysis and detected the target protein by SDS-PAGE and Western blot; The formation of VLPs by HPV16 L1 protein was observed with TEM.</p><p><b>RESULTS</b>The Sf9 cells could grow better in suspension culture with seeding density of 5 x 10-5 cell/mL and the maximum expression quantity was obtained by infection of cells with rBacV/HPV16L1 (MOI =10) and harvesting after 72-84 h. HPV16L1 protein could assemble into VLPs in Sf9 cells observed with TEM.</p><p><b>CONCLUSION</b>The conditions of cell culture, virus amplification and protein expression were optimized. HPV16 L1 protein could assemble into VLPs in Sf9 cells, which would provide a foundation for further study of the vaccine and diagnosis kits.</p>
Subject(s)
Animals , Capsid Proteins , Cell Proliferation , Oncogene Proteins, Viral , Recombinant Proteins , Spodoptera , Suspensions , VirionABSTRACT
Objective:To analyze the relationship between hepatitis B virus(HBV)gene mutation at 1896 in precore region with genotype and replication of HBV and the liver function of patients.Methods:HBV precore 1896 site mutation,the genotype of HBV and serum content of HBV DNA were determined by PCR in 60 patients positive of HBV DNA.Chemiluminescence miacropaticle immunoassay(CMIA)was used for detection of serum HBeAg and HBeAb.Liver function parameters were ob- tained by routine biochemistry method.Results:The alanine aminotransferase(ALT)level in HBV with 1896 site mutation was significantly higher than that in the wildtype virus.Site mutation at 1896 had no correlation with HBeAg,HBV genotype and HBV DNA content.HBV DNA content in patient with genotype C was significantly higher than that with genotype B(P