To construct retroviral vector carrying human interleukin-3 c0mplementary DNA(HuIL-3 cDNA) under control of human a-fetoprotein gene enhancer core sequence and human SV4O pro-moter. Methods and Results: HuIL-3 cDNA was inserted into polylinker site of retroviral vector pMNSMto construct retr0viral vector pMNS-IL-3, in which the transcription of HuIL-3 cDNA was drived by SV40early region promoter. Human Q-fetoprotein gene enhancer core sequence was released from plasmidpGEM. 7Zf-AFPe and inserted into the polylinker site of pMNSM. Then human interleukin-3 cDNA wasinserted int0 p0lylinker site to construct retroviral vector pMNSA-IL-3, in which HuIL-3 cDNA transcrip-tion was drived by SV40 early region promoter and enhanced by human a-fetoprotein enhancer core se-quence- Conclusion: The vectors are of significance for hepatoma-specific gene therapy.

OBJECTIVE To set up and evaluate the microscopic observation drug susceptibility assay(MODS)technology and use it to detect rapidly Mycobacterium tuberculosis(MTB).METHODS The 24-hole cell culture plate method of liquid culture were used to set up MODS technology.The MODS technology was used to detect MTB and non-M.tuberculosis(NMT)isolates comparing with Lowenstein-Jensen(L-J) method.RESULTS When bacterial concentration was 3?103 CFU/ml,the time of reading test-result was the seventh day by MODS;four kinds of NMT(M.phlei,M.kansasii,M.chelonaeand M.marinum) in the liquid medium in the observation was similar to the growth of cording,it was difficult to distinct from the cording of MTB in the liquid medium;When using the 4-Nitro-benzoic acid 800 ?g/ml,thiophene-2-carboxylic acid hydrazine 2.5 ?g/ml for testing conditions,the correct diagnosis can be improved;the test results of clinical isolates by MODS were highly concordance rate with the results of L-J.If the results of L-J was the golden standard,the sensitivity,specificity,positive and negative predictive value as well as accuracy by MODS was 95.7%,100%,100%,99.9% and 97% respectively.CONCLUSIONS The results of MODS in detection of MTB are highly concordance wih the results of L-J method;MODS assay can be used for rapid detection of tuberculosis,with a rapid,simple,inexpensive,and other advantages.

BACKGROUND:Recently, electrospun materials have been extensively applied in the drug delivery system. OBJECTIVE:To overview the application prospect of electrospun materials in drug delivery systems. METHODS:A computer-based search of PubMed and NCBI databases was performed for literatures about the research progress of electrospinning in tissue engineering and chemotherapy published within the past 10 years using the keywords of“electrospinning, drug delivery system, nanofibers, electrospun materials”.RESULTS AND CONCLUSION:Compared with traditional materials, electrospun stents hold good versatility and control able parameters, thus granting its unique advantage under various physiological conditions. Current drug-loaded materials composed of natural products, synthetic polymers and blended materials;as to drugs, there are antibiotics, chemotherapy medication, DNA and protein. Electrospun materials have been used in tissue engineering, cancer chemotherapy and wound healing. We focus on not only the application progress of electrospun materials in traditional treatments, but also its usage, condition-control ed drug release and living-cel carrying. Electrospun materials combined with various drug-loaded present a broad prospect.

Objective To study the mechanical properties and degradation behavior of biodegradable silicon-covered magnesium alloy stent in vitro,to investigate the technical feasibility of its implantation into rabbit esophagus and to observe the tissue reaction in vivo.Methods The mechanical compression recovery properties and the degradation behavior of biodegradable silicon-covered magnesium alloy stent were tested in vitro.A total of 30 healthy Holland rabbits were randomly divided into silicon-covered magnesium alloy stent group (n=15) and control group (n=15).For rabbits in the silicon-covered magnesium alloy stent group fluoroscopy-guided insertion of the stent into the lower third segment of esophagus was conducted,while for rabbits in the control group no intervention was adopted.One,2 and 4 weeks after the implantation of the stent,esophagography was performed for all rabbits of both groups,and each time every 5 rabbits from both groups were sacrificed,the specimens were collected and sent for histological examinations.Results In vitro test indicated that biodegradable silicon-covered magnesium alloy stent had good flexibility and elasticity,and in phosphate-buffered saline with pH 4.0 or pH 7.4 it degraded more slowly than bare magnesium alloy stent.In vivo test showed that the stent implantation could be well tolerated by all experimental rabbits.Before stent insertion the esophageal diameter was(9.2±0.8) mm,and at one,2 and 4 weeks after stent insertion the esophageal diameters were (9.7±0.7) mm,(9.6±0.8) mm and (9.6±0.5) mm respectively (P＞0.05).In the silicon-covered magnesium alloy stent group,stent displacement occurred in 6 rabbits in one week (n=l),2weeks (n=1) and 4 weeks (n=4).After stent implantation,the tissue reactions such as esophageal wall injury,collagen deposition,etc.were not obviously different from those in the control group (P＞0.05).Conclusion It is technically feasible to insert silicon-covered magnesium alloy stent into the rabbit's esophagus,the stent can provide sufficient support for at least 2 weeks,the stent displacement rate is low and acceptable,and no severe esophageal wall injury and collagen deposition are observed.

Objective·To construct a self-healing injectable hyaluronic acid(HA)-based hydrogel(HAPD-Cu)and investigate the effects of different copper ions on the properties of the hydrogel and its vasogenic effiicacy to evaluate its feasibility for clinical wound healing.Methods·Bisphosphonated hyaluronic acid(HAPD)was prepared via a blue-light mediated thiol-ene click reaction between thiolated hyaluronic acid(HASH)and acrylated bisphosphonate(Ac-PD)in the presence of photoinitiator 2959.Then,HAPD was further interacted with Cu2+through metal coordination to prepare HAPD-Cu hydrogels with different Cu2+concentrations,i.e.HAPD-Cu1,HAPD-Cu2,HAPD-Cu3 and HAPD-Cu4.The molecular structures of HASH,Ac-PD,HAPD and HAPD-Cu were verified with 1HNMR and FTIR.Microscopic morphology of HAPD-Cu was observed under SEM.The shear-thinning and self-healing properties of HAPD-Cu were verified by rheometer.The Cu2+release from HAPD-Cu was determined with ICP.Live-dead staining and CCK-8 assay were applied to evaluate the biocompatibility of HAPD-Cu.The in vitro vasculogenic activity of HAPD-Cu was determined by a tubule-forming assay with human umbilical vein vascular endothelial cells and the in vivo vasculogenic activity of HAPD-Cu was assessed by CD31 tissue staining.A rat wound defect model was established in vitro to evaluate its actual repair effect.Results·The preparation of the materials was demonstrated through chemical qualitative and quantitative analytical means.In vitro studies showed that all HAPD-Cu with a loose porous internal structure exhibited outstanding self-healing,injectability and degradability,with a one-week degradation cycle and abrupt release behavior,which can meet the needs of wound healing cycle.All HAPD-Cu showed good biocompatibility except HAPD-Cu4,due to its high Cu2+concentrations.Moreover,its angiogenic effect in vitro or in vivo was enhanced with increasing Cu2+concentrations within the permissible Cu2+concentration range.In vitro wound model experiments also showed that the HAPD-Cu hydrogel significantly promoted wound healing compared with the control group.Conclusion·HAPD-Cu hydrogel constructed via the metal coordination shows excellent shape plasticity,allowing the filling of defective sites in a minimally invasive form,and the release of Cu2+greatly facilitates the establishment of early vascular networks,with giant potential for use in the repair of clinically irregular wounds.

OBJECTIVE: To prepare a novel hyaluronic acid methacrylate (HAMA) hydrogel microspheres loaded polyhedral oligomeric silsesquioxane-diclofenac sodium (POSS-DS) patricles, then investigate its physicochemical characteristics and in vitro and in vivo biological properties. METHODS: Using sulfhydryl POSS (POSS-SH) as a nano-construction platform, polyethylene glycol and DS were chemically linked through the "click chemistry" method to construct functional nanoparticle POSS-DS. The composition was analyzed by nuclear magnetic resonance spectroscopy and the morphology was characterized by transmission electron microscopy. In order to achieve drug sustained release, POSS-DS was encapsulated in HAMA, and hybrid hydrogel microspheres were prepared by microfluidic technology, namely HAMA@POSS-DS. The morphology of the hybrid hydrogel microspheres was characterized by optical microscope and scanning electron microscope. The in vitro degradation and drug release efficiency were observed. Cell counting kit 8 (CCK-8) and live/dead staining were used to detect the effect on chondrocyte proliferation. Moreover, a chondrocyte inflammation model was constructed and cultured with HAMA@POSS-DS. The relevant inflammatory indicators, including collagen type Ⅱ, aggrecan (AGG), matrix metalloproteinase 13 (MMP-13), recombinant A disintegrin and metalloproteinase with thrombospondin 5 (Adamts5), and recombinant tachykinin precursor 1 (TAC1) were detected by immunofluorescence staining and real-time fluorescence quantitative PCR, with normal cultured chondrocytes and the chondrocyte inflammation model without treatment as control group and blank group respectively to further evaluate their anti-inflammatory activity. Finally, by constructing a rat model of knee osteoarthritis, the effectiveness of HAMA@POSS-DS on osteoarthritis was evaluated by X-ray film and Micro-CT examination. RESULTS: The overall particle size of POSS-DS nanoparticles was uniform with a diameter of about 100 nm. HAMA@POSS-DS hydrogel microspheres were opaque spheres with a diameter of about 100 μm and a spherical porous structure. The degradation period was 9 weeks, during which the loaded POSS-DS nanoparticles were slowly released. CCK-8 and live/dead staining showed no obvious cytotoxicity at HAMA@POSS-DS, and POSS-DS released by HAMA@POSS-DS significantly promoted cell proliferation (P<0.05). In the chondrocyte anti-inflammatory experiment, the relative expression of collagen type Ⅱ mRNA in HAMA@POSS-DS group was significantly higher than that in control group and blank group (P<0.05). The relative expression level of AGG mRNA was significantly higher than that of blank group (P<0.05). The relative expressions of MMP-13, Adamts5, and TAC1 mRNA in HAMA@POSS-DS group were significantly lower than those in blank group (P<0.05). In vivo experiments showed that the joint space width decreased after operation in rats with osteoarthritis, but HAMA@POSS-DS delayed the process of joint space narrowing and significantly improved the periarticular osteophytosis (P<0.05). CONCLUSION HAMA@POSS-DS can effectively regulate the local inflammatory microenvironment and significantly promote chondrocyte proliferation, which is conducive to promoting cartilage regeneration and repair in osteoarthritis.
Animals ; Rats ; Matrix Metalloproteinase 13 ; Microspheres ; Hydrogels ; Collagen Type II ; Diclofenac ; Inflammation ; Osteoarthritis, Knee/drug therapy* ; Hyaluronic Acid ; Aggrecans

Central nervous system (CNS) injuries, including stroke, traumatic brain injury, and spinal cord injury, are essential causes of death and long-term disability and are difficult to cure, mainly due to the limited neuron regeneration and the glial scar formation. Herein, we apply extracellular vesicles (EVs) secreted by M2 microglia to improve the differentiation of neural stem cells (NSCs) at the injured site, and simultaneously modify them with the injured vascular targeting peptide (DA7R) and the stem cell recruiting factor (SDF-1) on their surface via copper-free click chemistry to recruit NSCs, inducing their neuronal differentiation, and serving as the nanocarriers at the injured site (Dual-EV). Results prove that the Dual-EV could target human umbilical vascular endothelial cells (HUVECs), recruit NSCs, and promote the neuronal differentiation of NSCs in vitro. Furthermore, 10 miRNAs are found to be upregulated in Dual-M2-EVs compared to Dual-M0-EVs via bioinformatic analysis, and further NSC differentiation experiment by flow cytometry reveals that among these miRNAs, miR30b-3p, miR-222-3p, miR-129-5p, and miR-155-5p may exert effect of inducing NSC to differentiate into neurons. In vivo experiments show that Dual-EV nanocarriers achieve improved accumulation in the ischemic area of stroke model mice, potentiate NSCs recruitment, and increase neurogenesis. This work provides new insights for the treatment of neuronal regeneration after CNS injuries as well as endogenous stem cells, and the click chemistry EV/peptide/chemokine and related nanocarriers for improving human health.