Preparation of functional polyhydroxyalkanoate microspheres and their antibacterial activity and osteogenic effect evaluation / 中国修复重建外科杂志

OBJECTIVE@#To construct polyhydroxyalkanoate (PHA) microspheres loaded with bone morphogenetic protein 2 (BMP-2) and human β-defensin 3 (HBD3), and evaluate the antibacterial activity of microspheres and the effect of promoting osteogenic differentiation, aiming to provide a new option of material for bone tissue engineering.@*METHODS@#The soybean lecithin (SL)-BMP-2 and SL-HBD3 were prepared by SL-mediated introduction of growth factors into polyesters technology, and the functional microsphere (f-PMS) containing BMP-2 and HBD3 were prepared by microfluidic technology, while pure microsphere (p-PMS) was prepared by the same method as the control. The morphology of microspheres was observed by scanning electron microscopy and the water absorption was detected; the release curves of BMP-2 and HBD3 in f-PMS were detected by ELISA kit. The antibacterial effect of microspheres in Staphylococcus aureus and Escherichia coli was tested with the LIVE/DEADTM BacLightTM bacterial staining kit; the biocompatibility of microspheres was tested using Transwell and cell counting kit 8 (CCK-8). The effect of microspheres on osteogenic differentiation was determined by collagen type Ⅰ (COL-1) immunofluorescence staining and alkaline phosphatase (ALP) concentration.@*RESULTS@#In this experiment, the f-PMS and p-PMS were successfully constructed. Morphological characteristics showed that p-PMS surface was rough and distributed with micropores of 1-3 μm, while f-PMS surface was smooth and existed white granular material. There was no significant difference in water absorption between the two groups (P>0.05). The release curves of BMP-2 and HBD3 in the f-PMS and p-PMS were basically the same, showing both early sudden release and late slow release. The antibacterial activity of f-PMS was significantly higher than that of p-PMS in the test that against Staphylococcus aureus and Escherichia coli (P<0.05), but there was no significant difference in biocompatibility between the two groups (P>0.05). The results of osteogenic differentiation of human BMSCs showed that the fluorescence intensity of osteogenic specific protein COL-1 of f-PMS was significantly higher than that in p-PMS, and the activity of ALP in f-PMS was also significantly higher than that in p-PMS (P<0.05).@*CONCLUSION@#The p-PHA have good antibacterial activity and biocompatibility, and can effectively promote the osteogenic differentiation of human BMSCs, which is expected to be applied to bone tissue engineering in the future.

Role of human βdefensin 2 on preventing oxidized low-density lipoprotein induced monocyte foaming / 中华传染病杂志

Objective To clarify the role of human β-defensin2 ( hBD2) on preventing oxidized low-density lipoprotein (OX-LDL) induced human leukemic monocyte (THP-1) foaming.Methods The monocyte foaming model was established using THP-1 cell induced by OX-LDL and the model was identified by oil red staining.The hBD2 was overexpressed on THP-1 cells by using lentivirus system and the effect of hBD 2 overexpression on THP-1 cell foaming induced by OX-LDL was detected.The levels of inflammatory factors including tumor necrosis factor (TNF)-α, interleukin (IL)-1βand IL-6 in cell supernatant of each group were detected by enzyme-linked immunosorbent assay ( ELISA).Differences between the groups were compared by using the t test.Results The gene transfection efficiency of the cells was close to 100%at 72 h after infection. The hBD2 protein levels were 0.122 ±0.024 in the control group, 0.123 ±0.022 in Lv-control infection group and 0.981 ±0.183 in Lv-hBD2 infection group; and the level in control group was statistically higher than that in hBD-2 infection group (t＝-3.175, P＝0.007).The relative levels of hBD2 mRNA at 72 h after virus infection were 0.131 ±0.021 in control group, 0.128 ±0.022 in Lv-control group and 1.001 ±0.105 in Lv-hBD2 infection group; and the level in control group was statistically higher than that in hBD-2 infection group (t＝-7.213, P＝0.003).The results of oil red staining showed that OX-LDL inducing THP-1 cells for 72 h could significantly induce lipid accumulation in cells.Overexpression of hBD2 could effectively inhibit lipid accumulation in THP-1 cells induced by OX-LDL.The expression of hBD2 mRNA in THP-1 group was significantly higher than that in THP-1+OX-LDL group (t＝3.237, P＝0.004); and the difference was also significant when comparing THP-1+Lv-hBD2+OX-LDL group with THP-1+OX-LDL group (t＝-6.021, P＝0.003).The level of hBD2 protein in THP-1 group was significantly higher than that in THP-1+OX-LDL group (t＝0.314, P＝0.006); and the difference was also significant when comparing THP-1+Lv-hBD2+OX-LDL group with THP-1+OX-LDL group (t＝-4.061,P＝0.007).The levels of TNF-α, IL-1βand IL-6 in the supernatant of THP-1 cells induced by OX-LDL for 72 h were significantly increased compared with those in THP-1group (t＝-3.825,-2.017 and -3.551, respectively; P＝0.007, 0.004 and 0.005, respectively). The levels of TNF-α, IL-1βand IL-6 in THP-1+Lv-hBD2+OX-LDL group were significantly lower than those in THP-1+OX-LDL group ( t＝4.132, 3.681, and 2.991, respectively; P＝0.003, 0.002, and 0.007, respectively).Conclusions hBD2 can effectively inhibit THP-1 foaming induced by OX-LDL, which may be related to its inhibition of inflammatory response.

Antifungal effects of synthetic human β-defensin 3-C15 peptide

OBJECTIVES: The purpose of this ex vivo study was to compare the antifungal activity of a synthetic peptide consisting of 15 amino acids at the C-terminus of human β-defensin 3 (HBD3-C15) with calcium hydroxide (CH) and Nystatin (Nys) against Candida albicans (C. albicans) biofilm. MATERIALS AND METHODS: C. albicans were grown on cover glass bottom dishes or human dentin disks for 48 hr, and then treated with HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, and 300 µg/mL), CH (100 µg/mL), and Nys (20 µg/mL) for 7 days at 37℃. On cover glass, live and dead cells in the biomass were measured by the FilmTracer Biofilm viability assay, and observed by confocal laser scanning microscopy (CLSM). On dentin, normal, diminished and ruptured cells were observed by field-emission scanning electron microscopy (FE-SEM). The results were subjected to a two-tailed t-test, a one way analysis variance and a post hoc test at a significance level of p = 0.05. RESULTS: C. albicans survival on dentin was inhibited by HBD3-C15 in a dose-dependent manner. There were fewer aggregations of C. albicans in the groups of Nys and HBD3-C15 (≥ 100 µg/mL). CLSM showed C. albicans survival was reduced by HBD3-C15 in a dose dependent manner. Nys and HBD3-C15 (≥ 100 µg/mL) showed significant fungicidal activity compared to CH group (p < 0.05). CONCLUSIONS: Synthetic HBD3-C15 peptide (≥ 100 µg/mL) and Nys exhibited significantly higher antifungal activity than CH against C. albicans by inhibiting cell survival and biofilm.

Construction of shRNA lentiviral vectors targeting humanβ-COP and detection of their interference effect / 医学研究生学报

[Abstract ] Objective The purpose of this study was to construct a short hairpin RNA (shRNA) interference lentiviral vector targeting the humanβ-COP gene and to evaluate its inhibitory effect on β-COP in THP-1 cells. Methods We designed and synthesized 4 humanβ-COP-specific oligonucleotide sequences and inserted them into the pGMLV-SC1 vector to construct a recombinant vector fol-lowed by transfection of HEK 293T cells with the recombinant vector and Lenti-HG Mix to produce lentiviruses and detect the viral con-tent.After infecting the THP-1 cells with the packaged lentiviruses , we analyzed the inhibitory effect of β-COP-shRNA on the β-COP gene by quantitative PCR and Western blot . Results Sequencing confirmed that the β-COP-specific oligonucleotide sequences were in-serted into the lentiviral vector and the lentiviruses were packaged in the transfected HEK 293T cells, with the final viral content of 1 × 109 TU/mL.Quantitative PCR showed that the 4 β-COP-shRNA vectors significantly decreased the mRNA expression of β-COP (P<0.01), with interference rates of 16.9 %,32.5%, 74.0%, and 50.3%, respectively.Western blot also indicated their inhibitory effect on the protein expression of β-COP, with an inhibition rate of 76.4% onβ-COP-shRNA3. Conclusion Lentiviral shRNA interference vectors targeting human β-COP were constructed successfully , which could suppress the expression of the human β-COP gene.

Research progress and prospect of human β-defensin 3 and its derivatives in ophthalmology / 中华实验眼科杂志

Currently,antimicrobial resistance has become an urgent global public health problem.Human defensin,a family of innate immune peptides,plays an important role in the control of infectious disease because of its low resistance to microbiological infection.However,so far there is seldom report on industrial production of bioactive defensin.Human β-defensin 3 (HBD3) is a member of human innate antimicrobial peptides.It has broad-spectrum antimicrobial activity and function in a variety of inflammation diseases.In addition,HBD3 is considered as a good candidate for antibiotics,because it has a strong killing activity to antibiotics-resistant microbes and resistance to highsalt environment,it is especially valuable for the treatment of ocular surface inflammatory diseases.This review covers the in vitro research on the production of HBD3,optimization of structure-activity and its antimicrobial potential for ocular surface infection,which may provide a supportive evidence for the commercial production of HBD3 and other antimicrobial peptides in biopharmaceutical area.

Expressions of human β defensin-2 in eutopic and ectopic endometrium of women with adenomyosis / 中国综合临床

Objective To investigate the expression of human β defensin-2(hβD-2)in the eutopic and ectopic endometrial tissue in patients with adenomyosis and in women with normal endometrium.Methods Twenty-five hysteromyoma patients with adenomyosis(AM) and 25 hysteromyoma patients without endometriosis (EMS) were selected and divided into three groups:AM ectopic endometrium group,AM eutopic endometrium group and control group( endometrial tissue in patients with hysteromyoma).The level of mRNA expressions of hβD-2,interleukin-1β ( IL-1β ),interleukin-6 ( IL-6 ) and tumor necrosis factor-α (TNF-α) was investigated quantitatively using Real Time PCR (RT-PCR) and the corresponding protein level was detected by immunohistochemistry.Results Comparing the expression of hβD-2,IL-1β,IL-6,TNF-α genes among the three groups,there was no significant difference between ectopic endometrium group and eutopic endometrium group and there was also no significant difference between eutopic endometrium group and the control group.( P ＞ 0.05 ).The expression of hβD-2 and IL-1β were 0.0320 (0.0095 ～ 0.0690 ) and 0.0427 ( 0.0038 ～ 0.0975 ) in the ectopic endometrium group,and they were 0.0034(0.0025 ～0.0424) and 0.0080(0.0040 ～0.0251 ) in the control group,respectively.They were both significantly higher in ectopic endometrium group than in the control group (P ＜ 0.05 ).In the ectopic endometrium group hβD-2 expression was positively correlated with the level of TNF-α ( r =0.857,P =0.014 ),and it had no correlation with both IL-1β and IL-6 ( r =0.750,P =0.052 ; r =0.464,P =0.464; respectively)Conclusion HβD-2 might not play an important role in the formation of adenomyosis.It may be related to no significant up-regulation of inflammatory factors in ectopic lesion tissue.

Anti-infection effect of human beta-defensin-2 gene therapy in a rat urinary tract infection model / 中华泌尿外科杂志

Objective To assess the human β-defensin-2 ( hBD2 ) gene therapeutic efficacy in a rat urinary tract infection (UTI) model via intravesical liposome-mediated gene transfer.Methods Fifty-six female Wistar rats (class SPF) weighting 1 80 -220 were randomly divided into an experiment group and a control group ( each n =28 ).The animals were administered either 250 μl recombinant pCAGG-hBD2 or control vector pCAGG intravesically.After 48 h,rats in both groups were infected via intravesical inoculation with 200 μl of the bacterial suspension of UTI89 ( 1 × 108 CFU/ml).The rats were sacrificed at 4,24,48,and 72 h post-inoculation.The bladders were aseptically removed and bisected.One half was fixed in neutral buffered formalin for histological analysis.The remaining half-bladders were homogenized and titered for surviving bacteria.The clean-catch urine sample from each rat was collected in sterile before they were killed for bacterial titers determined and WBC counted.Results Numbers of bacterial colony-forming unit in urine and bladders from hBD2 gene treated UTI rats were significantly lower than those from the control vector administered UTI rats at 24,36,and 72 h after infection ( P ＜ 0.05 ).The amount of WBC in urine was significantly less in the defensin group than in the control group.In addition,in vivo expression of hBD2 could reduce mucosa damage,interstitium edema and inflammatory cells infiltration in UTI animals.Conclusions The successful inhibition of UTI progression could be obtained with hBD2 gene therapy.Recombinant beta-defensin-2 could kill UTI89 in vivo and suppress the subsequent infection-induced inflammatory responses.

Study on the mechanism of human-β-defensin-2 expression in human vaginal epithelial cells induced by Lactobacillus cell wall extract / 中华微生物学和免疫学杂志

Objective To investigate the molecular and cell signal transduction mechanisms by Lactobacillus cell wall extract(LCWE)inducing human-β-defensin-2(hBD-2)expression in human vaginal epithelial cells.Methods The induction of hBD-2 in human vaginal epithelial cells(WZV-1)by LCWE was observed using real-time PCR and Western blot.After stimulating WZV-1.the activation of NF-κB and p38MAPK signaling pathways were determined by Western blot.The induction of hBD-2 in WZV-1 cells by LCWE was observed with signaling pathways inhibitors of NF-κB and p38MAPK using real-time PCR and Western blot.Results The results showed that LCWE significantly upregulated hBD-2 expression in the time and dose-dependent manner.The maximal stimulatory effect of LCWE on the expression of hBD-2mRNA in WZV-1 cells were observed at the concentration of 50μg/ml after treatment for 8 h.After stimulation by 50μg/ml LCWE,Western blot analysis demonstrated that the phosphorylation of p38MAPK increased at 0.5 h significantly,peaked at 1 h,moreover the concentration of NF-κB in nucleus increased at 0.5 h significantly(P<0.05),peaked at 2 h.Blocking with inhibitor of NF-κB and(or)p38MAPK pathways results in decreased levels of HBD-2 expression.Conclusion These findings suggest that p38MAPK and NF-κB pathways play the important roles in induction of hBD-2 expression by LCWE in human vagihal epithelial cells.

Regulation of Human Beta-Defensin 3(hBD-3) in Human Keratinocyte(HaCaT) Cell Lines

BACKGROUND: The large surfaces of the skin are often initial site of contact between microorganism and human. The skin are coated with epidermis and epithelial cells can recognize microorganism and mount a fast defense through the production of various inducible antibiotic peptides. This leads to chracteristic broad spectrum of antimicrobial activity against bacteria, fungi, and viruses. Recent studies introduce us new peptides with antimicrobial activity such as P,-defensins and cathelicidins. They are expressed on the epithelia and polymorphonuclear leukocytes, which are first lines of defence from various invasive environments. Futhermore, they are considered very interesting and important endogenous antibiotics. Our previous study has shown that the expression of human defensin(hBD-2) mRNA, which is potent antibiotic peptide against Gram-negative bacteria(P. aeruginosa), was upregulated with ultraviolet(UV) irradiation, tumor necrosis factor-α(TNF-α) and lipopolysaccharide(LPS) in HaCaT cells. A novel hBD-3, 5-kDa, nonhemolytic antimicrobial peptide, was demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes in especially, multiresistant S. aureus. We have analyzed the expression patterns of hBD-3 in HaCaT cell lines. OBJECTIVE: This research have done in order to evaluate the expression and regulation of hBD-3 mRNA in human keratinocyte cell lines. METHODS: HaCaT cell lines were used to all culture experiments. Cultured human keratinocytes were stimulated with UV irradiation or TNF-α or LPS to determine whether hBD-3 mRNA production occurred. Reverse transcription-polymerase chain reaction (RT-PCR) was per-formed to amplify hBD-3 cDNA from stimulated keratinocytes in a time dependant manner, and densitometry was used to verify the specificity of RT-PCR amplication products. RESULTS: Expression of hBD-3 was upregulated with UV irradiation, TNF-α and LPS in Ha-CaT cells compared to control CONCLUSIONS: Human keratinocytes are capable to induce hBD-3 mRNA, as well as hBD-2, in response to UV irradiation, TNF-α and LPS. suggesting that these cells could play an important role against the bacterial infection and UV light damage in human skin.