Objective To construct three kinds of doxorubicin liposomes modified with cholesterol-galactose ligand by lipase-catalyzed method and compare their characteristic of pharmacokinetics and tissue distribution in vivo. Methods Three types of cholesterol-galactose ligands, CHS-C8-GalNAc, CHS-C8-GAL, and CHS-C8-LA were synthetized by lipase-catalyzed method in nonaqueous phase. The structure characterizations of products were obtained by MS and NMR. Conventional liposomes (CL DOX) and ligand-coupled liposomes (NGal-LP DOX, Gal-LP DOX, and LA-LP DOX) were prepared by thin film dispersion-ammonium sulphate gradient method. Structure-activity relationship between asialoglycoprotein receptor (ASGPr) and the chemical structure of the glycolipids was explored through the pharmacokinetics and tissue distribution parameters of ligand-coupled liposomes in vivo. Results The desired compounds with a high yield of above 90% were confirmed by MS and NMR. The liposomes average size was lower than 90 nm, polymer dispersity index was lower than 0.1, encapsulation efficiency was greater than 99%, leakage rat was lower than 5% with 24 h, and zeta potential closed to zero. The affinity of the three ligand molecules to liver was the following order: CHS-C8-GalNAc > CHS-C8-LA > CHS-C8-Gal. However, only the liposomes modified with CHS-C8-GalNAc could significantly be inhibited by the preinjection of asialofetuin for hepatic uptake rate (P < 0.01), but the liposomes modified with CHS-C8-LA and CHS-C8-Gal could not be inhibited by the preinjection of asialofetuin for hepatic uptake rate (P > 0.05). Conclusion The ligand with N-acetylgalactosamine residue showed high targeting efficiency for hepatocytes, while the ligand with D-galactose (Gal) or lactitol residue could competitive bind with Gal particle receptor on kupffer cells.

Objective: To synthesize brain targeting lipid material [(5-cholesten-3β-yl) (D-glucopyranose-6) sebacate, CHS-SE-GLU] by lipase as catalyst in nonaqueous phase and optimize the preparation technology and formulation of CHS-SE-GLU-modified liposomes. Methods: CHS-SE-GLU was synthesized from CHS-SE prepared in previous work and D-glucose using lipase Novozym 435 in acetone. The structure characterization of the products is obtained by MS and NMR. The CHS-SE-GLU-modified paclitaxel-loaded brain targeting liposomes (GLU-PTX-LP) were prepared by thin film dispersion method. Single factor evaluation was applied to optimizing its preparation technology and formulation. Results: CHS-SE-GLU was confirmed by MS and NMR as target products. The optimal formulation and technology of GLU-PTX-LP were as follows: HSPC as membrane material, the ratio of HSPC to PTX was 0.1, the ratio of CHS to HSPC was 0.5, the dosage of DSPG-Na was 2.5%, hydration time was 0.5 h, and hydration temperature was 50 ℃. Three batches of samples were prepared by optimum preparation process and the average encapsulation efficiency was (93.62±1.34)%, (93.75±1.77)%, (92.04±1.50)%; The average particle size was (89.56±1.35), (92.05±3.42), (104.91±3.71) nm; And the average Zeta potential was (-25.21±0.27), (-26.43±0.44), (-25.17±0.65) mV, respectively. Conclusion: Lipase-catalyzed method for the preparation of brain targeting lipid material is simple and environment friendly with high yield. The entrapment efficiency, particle size, and stability of brain targeting drug-loading liposomes modified by CHS-SE-GLU all meet the requirement, which shows good application prospect.

The CrdS protein responding to the acidic adaptation was prokaryotic-expressed in our Laboratory to explore the regulatory mechanism in the acidic adaptation of Helicobacter pylori (H. pylori). The whole genomic DNA of H. pylori strain 26695 was abstracted and set as the template firstly. And then the hp1364 gene coding CrdS protein was amplified via the PCR technique. Then the clonal recombinant plasmid pUCm-T-hp1364 and the prokaryotic expression plasmid pQE30-hp1364 were built and identified by the methods of PCR, cutting with two enzymes and sequencing. After that, the plasmid pQE30-hp1364 was transferred into the E. coli XL1 blue and induced with IPTG. Using western blot and SDS-PAGE, it can be analyzed that the expressed recombinant protein existed mainly in the form of the inclusion bodies and its relative molecular mass was about 46 kDa. The successfully attained recombinant protein CrdS will provide the material to explore the regulatory mechanism in the acidic adaptation of H. pylori and the new way to resist the infection of H. pylori.
Bacterial Proteins ; biosynthesis ; genetics ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; metabolism ; Helicobacter pylori ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; genetics

Objective: To explore the possibility of constructing tissue engineered adipose by adipose tissue derived extracellular vesicles (hAT-EV) combined with decellularized adipose tissue (DAT) scaffolds, and to provide a new therapy for soft tissue defects. Methods: The adipose tissue voluntarily donated by the liposuction patient was divided into two parts, one of them was decellularized and observed by HE and Masson staining and scanning electron microscope (SEM). Immunohistochemical staining and Western blot detection for collagen type Ⅰ and Ⅳ and laminin were also employed. Another one was incubated with exosome-removed complete medium for 48 hours, then centrifuged to collect the medium and to obtain hAT-EV via ultracentrifugation. The morphology of hAT-EV was observed by transmission electron microscopy; the nanoparticle tracking analyzer (NanoSight) was used to analyze the size distribution; Western blot was used to analyse membrane surface protein of hAT-EV. Adipose derived stem cells (ADSCs) were co-cultured with PKH26 fluorescently labeled hAT-EV, confocal fluorescence microscopy was used to observe the uptake of hAT-EV by ADSCs. Oil red O staining was used to evaluate adipogenic differentiation after hAT-EV and ADSCs co-cultured for 15 days. The DAT was scissored and then injected into the bilateral backs of 8 C57 mice (6-week-old). In experimental group, 0.2 mL hAT-EV was injected weekly, and 0.2 mL PBS was injected weekly in control group. After 12 weeks, the mice were sacrificed, and the new fat organisms on both sides were weighed. The amount of new fat was evaluated by HE and peri-lipoprotein immunofluorescence staining to evaluate the ability of hAT-EV to induce adipogenesis in vivo. Results: After acellularization of adipose tissue, HE and Masson staining showed that DAT was mainly composed of loosely arranged collagen with no nucleus; SEM showed that no cells and cell fragments were found in DAT, and thick fibrous collagen bundles could be seen; immunohistochemical staining and Western blot detection showed that collagen type Ⅰ and Ⅳ and laminin were retained in DAT. It was found that hAT-EV exhibited a spherical shape of double-layer envelope, with high expressions of CD63, apoptosis-inducible factor 6 interacting protein antibody, tumor susceptibility gene 101, and the particle size of 97.9% hAT-EV ranged from 32.67 nmto 220.20 nm with a peak at 91.28 nm. Confocal fluorescence microscopy and oil red O staining showed that hAT-EV was absorbed by ADSCs and induced adipogenic differentiation. In vivo experiments showed that the wet weight of fat new organisms in the experimental group was significantly higher than that in the control group ( t=2.278, P=0.048). HE staining showed that the structure of lipid droplets in the experimental group was more than that in the control group, and the collagen content in the control group was higher than that in the experimental group. The proportion of new fat in the experimental group was significantly higher than that in the control group ( t=4.648, P=0.017). Conclusion: DAT carrying hAT-EV can be used as a new method to induce adipose tissue regeneration and has a potential application prospect in the repair of soft tissue defects.

BACKGROUND: Construction of seedless tissue-engineered adipose tissue from acellular adipose tissue is a hot research topic in soft tissue filling. OBJECTIVE: To investigate the effects of preparation methods of acellular adipose tissue on the induction of adipose regeneration after transplantation in recent years, and to look forward to its clinical application prospects. METHODS: A computer-based online search of PubMed and Elsevier databases was performed to retrieve papers regarding acellular adipose tissue preparation and transplantation published between January 1971 and December 2018 with the search terms “adipose tissue engineering; adipose tissue extracellular matrix; soft tissue repair; angiogenesis; adipogenic induction”. The retrieved papers were summarized from the perspectives of improvement in preparation methods of acellular adipose tissue, cross-linking cytokines and biomaterials. RESULTS AND CONCLUSION: Retrieved studies have shown that extracellular matrix of adipose tissue can act as an ideal scaffold material for soft tissue filling. Subcutaneous implantation of extracellular matrix of adipose tissue can recruit host stem cells and induce their proliferation and adipogenesis. However, existing acellular schemes can lead to the loss of extracellular matrix proteins and structures. This greatly affects the fat regeneration ability of acellular adipose tissue implanted in vivo. However, supercritical carbon dioxide deoiling, mechanical pretreatment, cross-linking cytokines or biomaterials can reduce the loss of extracellular matrix proteins and supplement the proteins that promote tissue regeneration during the preparation of acellular adipose tissue. This can ultimately enhance the angiogenesis and adipogenesis of acellular adipose tissue after transplantation. Acellular adipose tissue has strong application prospects in adipose tissue engineering because of its natural adipogenic induction ability. If the loss of extracellular matrix protein can be overcome during preparation of acellular adipose tissue or under the premise of safety and controllability, acellular adipose tissue is expected to become a suitable soft tissue filler that allows allogeneic injection and in situ adipogenesis.

Objective:This study investigates the effect of signal-to-noise ratio (SNR), frequency, and bandwidth on horizontal sound localization accuracy in normal-hearing young adults.Methods:From August 2022 to December 2022, a total of 20 normal-hearing young adults, including 7 males and 13 females, with an age range of 20 to 35 years and a mean age of 25.4 years, were selected to participate in horizontal azimuth recognition tests under both quiet and noisy conditions. Six narrowband filtered noise stimuli were used with central frequencies (CF) of 250, 2 000, and 4 000 Hz and bandwidths of 1/6 and 1 octave. Continuous broadband white noise was used as the background masker, and the signal-to-noise ratio (SNR) was 0, -3, and -12 dB. The root-mean-square error (RMS error) was used to measure sound localization accuracy, with smaller values indicating higher accuracy. Friedman test was used to compare the effects of SNR and CF on sound localization accuracy, and Wilcoxon signed-rank test was used to compare the impact of the two bandwidths on sound localization accuracy in noise.Results:In a quiet environment, the RMS error in horizontal azimuth in normal-hearing young adults ranged from 4.3 to 8.1 degrees. Sound localization accuracy decreased with decreasing SNR: at 0 dB SNR (range: 5.3-12.9 degrees), the difference from the quiet condition was not significant ( P>0.05); however, at -3 dB (range: 7.3-16.8 degrees) and -12 dB SNR (range: 9.4-41.2 degrees), sound localization accuracy significantly decreased compared to the quiet condition (all P<0.01). Under noisy conditions, there were differences in sound localization accuracy among stimuli with different frequencies and bandwidths, with higher frequencies performing the worst, followed by middle frequencies, and lower frequencies performing the best, with significant differences (all P<0.01). Sound localization accuracy for 1/6 octave stimuli was more susceptible to noise interference than 1 octave stimuli (all P<0.01). Conclusions:The ability of normal-hearing young adults to localize sound in the horizontal plane in the presence of noise is influenced by SNR, CF, and bandwidth. Noise with SNRs of ≥-3 dB can lead to decreased accuracy in narrowband sound localization. Higher CF signals and narrower bandwidths are more susceptible to noise interference.

Objective:This study aims to explore the potential effects of adipose-derived stem cell-extracellular vesicles(ASC-EVs) on TGFβ-Smad signaling pathway during myofibroblast trans-differentiation in vitro. Methods:ASCs were isolated from liposuction and flow cytometry was used to detect the surface protein markers. ASC-EVs were isolated from the supernatant of the third to fifth generation ASCs, and the microscopic morphology was observed by transmission electron microscope. The particle size distribution was detected by nano-particle tracking analyzer NanoSight and the membrane surface marker proteins CD63, Alix and TSG101 were detected by flow cytometry. The uptake of EVs by dermal fibroblasts co-cultured with PKH67 fluorescence labeled ASC-EVs was observed by confocal microscope. Dermal fibroblasts were continuously induced by TGFβ1 for five days, and ASC-EVs at the dose of 50 and 100 μg/ml were added. The expression of α-SMA and Smad-2/3/4 were detected by immunofluorescence staining, RT-PCR and Western Blot.Results:The results of flow cytometry showed that the surface markers CD73, CD49d, CD90 and CD105 of the third generation ASCs, were positive, and CD34 and CD45 were negative. Under transmission electron microscope, ASC-EVs was a round membranous vesicle with clear edge and surrounded by bilayer phospholipid membrane. The particle size of more than 95% of the ASC-EVs was distributed between 30 nm to 261 nm, with an average of (166.0±86.1)nm. The specific marker proteins of extracellular vesicle, CD63, Alix and TSG101 were highly expressed. Under confocal microscope, ASC-EVs with green fluorescence were uptake by dermal fibroblasts and distributed in the cytoplasm, and part of ASC-EVs was distributed around the nucleus.TGF-β1 induced a significant increase in the expression of α-SMA in dermal fibroblasts, and the addition of 50 or 100 μg/ml ASC-EVs reduced the expression of α-SMA genes and proteins, but did not show a dose-dependent manner. The gene and protein expression changes of Smad-2/3/4 were consistent with α-SMA. In addition, ASC-EVs could significantly reduced the content of type Ⅰ collagen in the supernatant, but had no significant effect on the secretion of type Ⅲ collagen.Conclusions:The mechanism of ASC-EVs inhibiting scar hyperplasia may be closely related to the suppression of TGF-β-Smad signaling pathway in dermal fibroblasts, inhibition of myofibroblast trans-differentiation and reduction of type Ⅰ collagen secretion.

Objective:This study aims to explore the potential effects of adipose-derived stem cell-extracellular vesicles(ASC-EVs) on TGFβ-Smad signaling pathway during myofibroblast trans-differentiation in vitro. Methods:ASCs were isolated from liposuction and flow cytometry was used to detect the surface protein markers. ASC-EVs were isolated from the supernatant of the third to fifth generation ASCs, and the microscopic morphology was observed by transmission electron microscope. The particle size distribution was detected by nano-particle tracking analyzer NanoSight and the membrane surface marker proteins CD63, Alix and TSG101 were detected by flow cytometry. The uptake of EVs by dermal fibroblasts co-cultured with PKH67 fluorescence labeled ASC-EVs was observed by confocal microscope. Dermal fibroblasts were continuously induced by TGFβ1 for five days, and ASC-EVs at the dose of 50 and 100 μg/ml were added. The expression of α-SMA and Smad-2/3/4 were detected by immunofluorescence staining, RT-PCR and Western Blot.Results:The results of flow cytometry showed that the surface markers CD73, CD49d, CD90 and CD105 of the third generation ASCs, were positive, and CD34 and CD45 were negative. Under transmission electron microscope, ASC-EVs was a round membranous vesicle with clear edge and surrounded by bilayer phospholipid membrane. The particle size of more than 95% of the ASC-EVs was distributed between 30 nm to 261 nm, with an average of (166.0±86.1)nm. The specific marker proteins of extracellular vesicle, CD63, Alix and TSG101 were highly expressed. Under confocal microscope, ASC-EVs with green fluorescence were uptake by dermal fibroblasts and distributed in the cytoplasm, and part of ASC-EVs was distributed around the nucleus.TGF-β1 induced a significant increase in the expression of α-SMA in dermal fibroblasts, and the addition of 50 or 100 μg/ml ASC-EVs reduced the expression of α-SMA genes and proteins, but did not show a dose-dependent manner. The gene and protein expression changes of Smad-2/3/4 were consistent with α-SMA. In addition, ASC-EVs could significantly reduced the content of type Ⅰ collagen in the supernatant, but had no significant effect on the secretion of type Ⅲ collagen.Conclusions:The mechanism of ASC-EVs inhibiting scar hyperplasia may be closely related to the suppression of TGF-β-Smad signaling pathway in dermal fibroblasts, inhibition of myofibroblast trans-differentiation and reduction of type Ⅰ collagen secretion.

Objective:To evaluate auditory spatial discrimination capabilities in patients with mild to moderately severe symmetrical sensorineural hearing loss (SNHL) and to compare the impact of different psychophysical testing methods on Minimum Audible Angle (MAA) and test duration.Methods:A total of 105 symmetrical SNHL patients aged from 18 to 60 years old were enrolled from April to July 2023, including 56 males and 49 females. They were divided into three groups based on PTA: mild, moderate, and moderately severe hearing loss, with 35 individuals in each group. Additionally, a control group of 35 individuals with normal hearing was tested, including 18 males and 17 females. Participants underwent four distinct psychophysical discrimination tests: the block up-down, 1-up/1-down, 1-up/2-down, and 1-up/3-down procedures. We recorded the MAA and test duration for each. We employed repeated measures of ANOVA to compare the MAA and test duration across different methods and groups, and Pearson′s correlation to assess the relationship between MAA and degree of hearing loss.Results:MAA of sound localization in patients with symmetrical SNHL was significantly positively correlated with the degree of hearing loss ( r=0.59, P<0.01). Significant deterioration in MAA was observed as hearing loss progressed to the moderate level (PTA≥35 dBHL, P<0.01). The testing methods significantly influenced MAA and testing duration ( F=24.02, P<0.01; F=75.56, P<0.01) and the 1-up/1-down method was the quickest, averaging only (0.69±0.32) mins. Conclusions:The horizontal plane auditory spatial discrimination abilities in patients with symmetrical SNHL is impaired progressively with increasing hearing loss, notably beyond moderate hearing loss levels. Different psychophysical methods influence both MAA and test duration, the quicker 1-up/1-down method is recommended for assessing MAA in symmetrical SNHL patients.