OBJECTIVE:To validate the microbial limit tests of Shuanghuanglian oral liquid.METHODS:The recovery rates of 4 kinds of test organisms including E.coli treated by Shuanghuanglian oral liquid were detected by routine method,culture medium dilution method,and membrane-filter procedure respectively.RESULTS:Shuanghuanglian oral liquid was found to be of bacteriostatic action.The contamination of solution couldn't be detected by routine method.The recovery rate of the test organisms detected by membrane-filter procedure was higher than by culture medium dilution method,but the recovery rates in both methods were all up to the requirement.CONCLUSION:Membrane-filter procedure should be considered preferentially in performing microbial limit test for Shuanghuanglian oral liquid.

Background At present, the treatment of silicosis is still limited, and no method is available to cure the disease. miRNAs are involved in the process of fibrosis at the transcriptional level by directly degrading target gene mRNA or inhibiting its translation. However, how miR-130a-3p regulates silicosis fibrosis has not been fully elucidated yet. Objective To investigate whether miR-130a-3p promotes epithelial-mesenchymal transition (EMT) by inhibiting peroxisome proliferators-activated receptors gamma (PPAR-γ), thereby pro-moting the process of silicotic fibrosis. To identify effective new targets for the treatment of silicotic fibrosis. Methods (1) Animal experiments: C57BL/6J mice were intratracheally injected with a one-time dose of 10 mg silica suspension (dissolved in 100 μL saline) as positive lung exposure. A silicosis model group was established 28 d after the exposure. A control group was injected with the same amount of normal saline into the trachea. Hematoxylin-eosin staining and Sirius red staining were used to observe the pathological changes and collagen deposition in lung tissues respectively. Realtime fluorescence-based quantitative polymerase chain reaction (RT-qPCR) was used to assay the expression of miR-130a-3p and PPAR-γ mRNA in lung tissues. Western blotting was used to detect the protein expression of PPAR-γ, transforming growth factor (TGF)-β1, E-cadherin, α-smooth muscle actin (α-SMA), and Collagen Ⅰ in lung tissues. (2) Cells experiments: Mouse lung epithelial cells (MLE-12) were induced with 5 µg·L⁻¹ TGF-β1 for different time (0, 12, 24, 48 h). RT-qPCR was used to detect the expression of miR-130a-3p and PPAR-γ mRNA in cells. The binding relationship between miR-130a-3p and PPAR-γ mRNA was verified by dual luciferase reporter gene assay. MLE-12 cells were stimulated by 5 µg·L⁻¹ TGF-β1 after transfection of miR-130a-3p inhibitor, and Western blotting was used to measure the protein expression of PPAR-γ, E-cadherin, and α-SMA in the TGF-β1-induced cells. Results In the silicosis model group, the alveolar septum was widened and the pulmonary nodules were formed. The Sirius red staining collagen deposition in pulmonary nodules indicated that a silicosis fibrosis model was successfully established. The expressions of TGF-β1, α-SMA, and Collagen Ⅰ proteins were increased, and the expressions of E-cadherin and PPAR-γ proteins were decreased in lung tissues of the silicosis group, compared with the control group (P<0.05 or P<0.01). The expression of miR-130a-3p was increased and the expression of PPAR-γ mRNA was decreased in lung tissues of the silicosis model (P<0.01). The expression of miR-130a-3p was significantly increased, while the expression of PPAR-γ mRNA was decreased in the TGF-β1 induced MLE-12 cells (P<0.05 or P<0.01). The dual luciferase reporter assay showed a direct relationship between miR-130a-3p and PPAR-γ mRNA in MLE-12 cells. The transfection of miR-130a-3p inhibitor in the TGF-β1 induced MLE-12 cells inhibited the decrease of PPAR-γ and E-cadherin proteins, and the increase of α-SMA protein in the MLE-12 cells induced by TGF-β1 (P<0.05 or P<0.01). Conclusion miR-130a-3p promotes the development of silicosis fibrosis by targeting PPAR-γ to increase pulmonary EMT.

Wound healing is a dynamic process which involves interaction of various types of cells, cytokines, and extracellular matrix. Among them, epithelial cells and mesenchymal cells are the key components which involve in wound healing and scar formation. Related scholars had done a great number of studies about the functions of epithelial cells and fibroblasts(Fbs) in wound healing and scar formation. The results showed that under the stimulation of complex microenvironment, epithelial cells would lose their epithelial characteristics and acquire the typical characteristics and migration ability of mesenchymal cells. At the same time, with the complex changes of cell structure and cell behavior, they would participate in the process of tissue wound repair, including normal or fibrotic repair, by covering the wound with migration. Fbs are the key cells for the wound fibrotic repair, and play important roles in the process of wound healing, including excessive wound healing or delayed wound healing. In the recent years, the researchers realized that the cross-talk between epithelial cells and Fbs in wound healing, which is referred to as epithelial-mesenchymal interaction, significantly changes the biological behaviors of these two cell types, which affects the dermal remodeling and re-epithelialization quality of wound. Epithelial-mesenchymal interaction plays an important role in skin morphogenesis during embryonic development and maintaining the structural integrity of adult skin. In the process of re-epithelialization, Fbs could promote the proliferation and migration of keratinocytes, meanwhile keratinocytes would receive the signals from Fbs to reconstruct functional epithelium, which has become a hot topic in the field of wound healing at present. In this paper, a comprehensive analysis of the literature on the role of epithelial-mesenchymal interaction in wound healing and scar formation at home and abroad in recent years is presented for the reference of relevant scholars.

Gene editing tools with nucleases as the main component have now implemented programmable targeted mutagenesis or insertion or deletion of mammalian genomes.From zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas system to safer and more accurate Cas9 fusion protein gene editing tools and other nuclease gene editing tools, this paper systematically describes the development and evolution of gene editing, with detailed introduction to the development and optimization of next-generation gene editing tools, and a prospect of the clinical application of and challenges for gene editing tools.

Objective:To investigate the feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice.Methods:(1) Ten eight-week-old C57BL/6 male mice were collected and full-thickness skin tissue with diameter of 1.0 cm on both sides of the midline of the back was taken with a perforator to make the normal skin tissue homogenate supernatant. At 48 h after the full-thickness skin defect wound was established, the wound tissue within 2 mm from the wound edge was taken to make inflammatory wound tissue homogenate supernatant. Two kinds of tissue homogenate supernatant were taken to adjust the total protein concentration to 1 mg/mL, and the tumor necrosis factor α (TNF-α) content was detected by enzyme-linked immunosorbent assay. The number of sample was 6. (2) The primary passage of human umbilical cord mesenchymal stem cells (hUCMSCs) were collected and cultured to the 3rd passage with the normal exosomes being extracted from the hUCMSCs after cultured for 48 h. Another batch of hUCMSCs in the 3rd passage was collected and stimulated with inflammatory wound tissue homogenate supernatant of 30, 50, and 100 μg/mL total protein and normal skin tissue homogenate supernatant of 30, 50, and 100 μg/mL total protein, respectively. After cultured for 48 h, the exosomes stimulated with normal protein of 30, 50, and 100 μg/mL and exosomes stimulated with inflammatory protein of 30, 50, and 100 μg/mL were extracted. Normal exosomes, exosomes stimulated with 30 μg/mL normal protein, and exosomes stimulated with 30 μg/mL inflammatory protein were collected, the morphology was observed by transmission electron microscope, the particle size was detected by nanoparticle tracking analyzer, and the expressions of CD9 and CD63 were detected by Western blotting. (3) Twenty one-day-old C57BL/6 mice were taken to isolate the primary passage of fibroblasts (Fbs) and the 3rd passage of Fbs, whose morphology was observed under the inverted phase contrast microscope. The Fbs of 3rd passage were collected to observe the expression of vimentin by cell crawling method combined with immunofluorescence method at culture hour (CH) 2. (4) The Fbs of 3rd passage were divided into control group, normal exosome group, 30, 50, 100 μg/mL normal protein stimulating exosome group, and 30, 50, 100 μg/mL inflammatory protein stimulating exosome group according to the random number table, with 4 wells in each group. Cells in control group received no treatment, and cells in the other 7 groups were respectively added with normal exosomes, exosomes stimulated with normal protein of 30, 50, and 100 μg/mL, and exosomes stimulated with inflammatory protein of 30, 50, and 100 μg/mL prepared in experiment (2). The final mass concentration of exosomes was adjusted to 10 μg/mL. The cell viability was detected by cell count kit 8 at CH 48. (5) Two batches of Fbs in the 3rd passage were divided and treated as those in experiment (4), with 4 wells in each group, and the final mass concentration of exosomes was adjusted to 1 and 10 μg/mL, respectively. The cell mobility was detected by cell scratch test at CH 6, 12, and 24. (6) Two batches of the Fbs of 3rd passage were collected, divided, and treated as those in experiment (4) except with no control group, with 3 wells in each group, and the final mass concentration of exosomes was respectively adjusted to 1 and 10 μg/mL. The mRNA expression levels of transforming growth factor β 1 (TGF-β 1), TGF-β 3, and α smooth muscle actin (α-SMA) were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction at CH 48. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Bonferroni method. Results:(1) The content of TNF-α in inflammatory wound tissue homogenate supernatant of mice was (116±3) pg/mL, significantly higher than (97±5) pg/mL in normal skin tissue homogenate supernatant at post injury hour 48 ( t=3.306, P<0.05). (2) Normal exosomes, exosomes stimulated with 30 μg/mL normal protein, and exosomes stimulated with 30 μg/mL inflammatory protein of hUCMSCs showed the typical saucer-like shape. The particle sizes of the three exosomes of hUCMSCs were 30-150 nm, which were all within the normal particle size range of exosome. Three exosomes of hUCMSCs positively expressed CD9 and CD63. (3) The primary passage of cells were clearly defined and showed protruding spindle shape, irregular polygon shape, or slender strip shape. The morphology of the 3rd and the primary passage of cells is similar. At CH 2, vimentin in cells was positively expressed, and the cells were identified as Fbs. (4) At CH 48, the cell viability was (137.4±2.8)% in 30 μg/mL inflammatory protein stimulating exosome group, obviously higher than 100%, (107.5±2.4)%, (113.3±3.2)%, (104.0±2.0)%, and (101.9±1.5)% in control group, normal exosome group, 30 μg/mL normal protein stimulating exosome group, and 50 and 100 μg/mL inflammatory protein stimulating exosome groups, respectively ( P<0.01), and cell viability in 30 μg/mL normal protein stimulating exosome group was obviously higher than that in control group, normal exosome group, and 50 and 100 μg/mL normal protein stimulating exosome groups [(103.4±2.2)% and (102.5±1.4)%], respectively ( P<0.01). (5) At CH 6, 12, and 24, the mobility rate of cells in 30 μg/mL inflammatory protein stimulating exosome group was significantly higher than that in control group, normal exosome group, 30 μg/mL normal protein stimulating exosome group, and 50 and 100 μg/mL inflammatory protein stimulating exosome groups, respectively, when the final mass concentrations of exosome was 1 μg/mL ( P<0.05) . At CH 12, the mobility rate of cells in 30 μg/mL normal protein stimulating exosome group was obviously higher than that in control group, normal exosome group, and 50 and 100 μg/mL normal protein stimulating exosome groups, respectively, when the final mass concentration of exosome was 1 μg/mL ( P<0.05). At CH 6, the mobility rate of cells in 30 μg/mL inflammatory protein stimulating exosome group was significantly higher than that in control group and normal exosome group ( P<0.05), and the mobility rate of cells in 30 μg/mL normal protein stimulating exosome group was significantly higher than that in 50 and 100 μg/mL normal protein stimulating exosome groups, respectively, when the final mass concentration of exosome was 10 μg/mL ( P<0.05). At CH 12 and 24, the mobility rate of cells in 30 μg/mL inflammatory protein stimulating exosome group was significantly higher than that in control group, normal exosome group, and 50 and 100 μg/mL inflammatory protein stimulating exosome groups ( P<0.05), and the mobility rate of cells in 30 μg/mL normal protein stimulating exosome group was significantly higher than that in control group, normal exosome group, and 50 and 100 μg/mL normal protein stimulating exosome groups, respectively, when the final mass concentration of exosome was 10 μg/mL ( P<0.05). (6) There were no statistically significant differences in mRNA expression levels of TGF-β 1, TGF-β 3, and α-SMA of cells among the 7 groups at CH 48 when the final mass concentration of exosome was 1 μg/mL ( F=1.123, 1.537, 1.653, P>0.05). There were no statistically significant differences in mRNA expression levels of TGF-β 1 and α-SMA of cells among the 7 groups at CH 48 when the final mass concentration of exosome was 10 μg/mL ( F=1.487, 1.308, P>0.05), and mRNA expression level of TGF-β 3 of cells in 50 μg/mL inflammatory protein stimulating exosome group at CH 48 was significantly higher than that in normal exosome group, 50 μg/mL normal protein stimulating exosome group, and 30 and 100 μg/mL inflammatory protein stimulating exosome groups when the final mass concentration of exosome was 10 μg/mL ( P<0.05). Conclusions:The pretreatment with inflammatory wound tissue homogenate supernatant of mice has no significant effect on the total protein of hUCMSCs exosomes. The hUCMSCs exosomes stimulated by low concentration inflammatory wound tissue homogenate supernatant can significantly promote the proliferation and migration ability of Fbs. The content of inflammatory mediators in the wound tissue homogenate supernatant during the inflammatory phase is extremely low, which may be the reason that the anti-inflammation and tissue repair paracrine effects of mesenchymal stem cell cannot be effectively started.

Objective:To explore the influence of human amniotic mesenchymal stem cells (hAMSCs) on the in vivo and in vitro regulation of macrophage phenotypes and inflammatory factors associated with wound healing of full-thickness skin wounds in mice.Methods:Fresh amniotic membrane discarded from full-term delivery by 5 healthy pregnant women in the Department of Obstetrics and Gynecology of the Affiliated Hospital of Zunyi Medical University was used for the isolation and culture of hAMSCs by enzyme digestion method. The third passage of cells was used for identification of adipogenic and osteogenic differentiation. The fourth passage of cells was used for identification of hAMSCs surface markers. Ten C57BL/6 mice (all male, aged 6 to 8 weeks, the same gender and age below) were selected for extracting mouse peritoneal macrophages by intraperitoneal lavage, and M1-type macrophages were induced by Dulbecco′s modified eagle medium (DMEM) medium containing interferon-γ. The M1-type macrophages were divided into hAMSCs+ macrophage group and macrophage alone group. Then 1×10 4 hAMSCs/per well of fourth passage were added to macrophage in hAMSCs+ macrophage group and cultured in 2 mL DMEM medium for routine culture. In macrophage alone group, each well was only added with 2 mL DMEM medium for routine culture. On day 1 and 7 in culture, the content of interleukin-12 (IL-12), arginase 1, and IL-10 in the cell culture supernatant of the 2 groups were detected by enzyme-linked immunosorbent assay with sample number of 6/per group. (2) Full-thickness skin wound model was reproduced in the back of 56 C57BL/6 mice, which were divided into hAMSCs group and phosphate buffer solution (PBS) group using the random number table, with 28 mice in each group. Mice in hAMSCs group were subcutaneously injected with 100 μL of cell suspension containing 1×10 7 hAMSCs per mL in PBS suspension along the wound edge. While mice in PBS group were only subcutaneously injected with 100 μL PBS along the wound edge. On post injection day (PID) 1, 3, 7, and 14, 7 mice in the two groups were sacrificed respectively. Histopathological observation was performed with hematoxylin-eosin staining. The expressions of macrophage surface markers [CD68 and inducible nitric oxide synthase (iNOS) double positive cells and CD68 and arginase 1 double positive] in the wounds were detected by immunofluorescent staining. The mRNA expressions of IL-10, macrophage inflammatory protein 1α (MIP-1α), and MIP-2 in the wounds were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were statistically analyzed with analysis of variance for factorial design, t test, and Bonferroni correction. Results:(1) On day 1 in culture, the content of IL-12 and arginase 1 in the cell culture supernatant of the two groups were similar ( t=0.448, 0.536, P>0.05), and the content of IL-10 in the cell culture supernatant of hAMSCs+ macrophage group was significantly lower than that in macrophage alone group ( t=14.722, P<0.01). On day 7 in culture, the content of IL-12 in the cell culture supernatant of hAMSCs+ macrophage group was significantly lower than that in macrophage alone group ( t=13.226, P<0.01), and the content of arginase 1 and IL-10 was significantly higher than that in macrophage alone group ( t=30.172, 31.406, P<0.01). (2) On PID 1, a large number of inflammatory cells infiltration were observed in the skin wounds of both groups. On PID 3, the inflammatory cells infiltration in the skin wounds increased in both groups, and the inflammatory cells infiltration in hAMSCs group was less than that in the PBS group. On PID 7, the inflammatory cells infiltration in the wounds decreased in both groups, and the inflammatory cells infiltration in hAMSCs group was less than that in the PBS group. On PID 14, no obvious inflammatory cells infiltration was observed in the wounds in the two groups. (3) On PID 1 and 14, the percentages of CD68 and iNOS double positive cells and CD68 and arginase 1 double positive cells in the wounds were similar in the two groups ( t1 d=0.134, 0.693, t14 d=1.146, 2.585, P>0.05). On PID 3 and 7, the percentages of CD68 and iNOS double positive cells in the wounds in hAMSCs group were significantly lower than those of PBS group ( t=6.396, 4.787, P<0.01), while the percentages of CD68 and arginase 1 double positive cells were significantly higher than those of PBS group ( t=3.928, 4.473, P<0.01). (4) On PID 1, the mRNA expressions of IL-10 in the wounds of mice in the two groups were similar ( t=2.005, P>0.05). On PID 3, 7, and 14, the mRNA expressions of IL-10 in the wounds of mice in hAMSCs group were significantly higher than those of PBS group ( t=7.758, 124.355, 80.823, P<0.01). On PID 1, 3, 7, and 14, the mRNA expressions of MIP-1α and MIP-2 in the wounds of mice in hAMSCs group (0.341±0.212, 0.648±0.004, 0.611±0.106, 0.763±0.049, 1.377±0.099, 1.841±0.042, 1.181±0.035, 0.553±0.028) were significantly lower than those of PBS group (3.853±0.035, 6.914±0.163, 3.648±0.113, 2.250±0.046, 11.119±0.495, 8.634±0.092, 5.722±0.021, 4.862±0.036, t=43.198, 101.904, 51.845, 58.231, 51.074, 177.501, 291.752, 251.614, P<0.01). Conclusions:hAMSCs demonstrates biological effects of promoting the transformation of M1-type macrophages into M2-type macrophages in full-thickness skin wounds of mice. They can up-regulate the expression of anti-inflammatory and anti-fibrotic factor IL-10, and down-regulate the expression of important inflammation mediated factors MIP-1α and MIP-2.

Bioluminescence is a widespread phenomenon in nature, and luminescent organisms can be found both on land and in the ocean. Among them, luciferase based bioluminescence systems have been widely studied, inspiring the exploration of genetic and epigenetic aspects and the development of a series of related assays for in vivo and in vitro studies. This paper summarizes the recent developments of luciferase based bioluminescence assays in terms of bioluminescence systems, types of luciferases, and the development and application of luciferase bioluminescence assays.

Abstract: The differential expression and subcellular localization of single-cell proteins are closely related to the physiological state and pathological mechanisms of the body. The development of single-cell protein in situ imaging methods provides powerful tools for spatial single-cell proteomics research and single-cell protein profiling. This article summarizes the single-cell protein imaging methods developed in recent years, including the circulating immunofluorescence imaging methods based on ordered multi-round antibody incubation, mass spectrometry imaging based on metal element labeled antibodies, fluorescence imaging based on DNA-barcoded antibody, gene encoded fluorescence protein imaging and spectral imaging based on Raman spectroscopy or X-ray spectroscopy, with brief explanation of the imaging principles of these methods. It focuses on the multiple performance, imaging resolution and signal amplification performance of these methods, and analyzes their application characteristics in practical scientific research and clinical work, in the hope of providing some reference for the development of more revolutionary single-cell imaging methods, and promoting the development of biomedical and precision medicine.

Objective:To evaluate the clinical value of Xing's ureteroileal anastomosis technique in radical cystectomy.Methods:The data of 38 patients who underwent radical cystectomy with Xing's ureteroileal anastomosis technique at Cancer Hospital, Chinese Academy of Medical Sciences and Beijing Chaoyang Hospital from July 2013 to June 2021 were retrospectively reviewed. There were 30 males and 8 females. The mean age was 61.6±15.1 years old. The mean body mass index (BMI) was 25.1±2.7 kg/m 2. The American Society of Anesthesiology (ASA) graded 25 cases as grade 1, 10 cases as grade 2 and 3 cases as grade 3. There were 35 cases with stage cT 2N 0M 0 and 3 cases with cT 3N 0M 0. All patients underwent radical cystectomy and ileal conduit, and the ureteroileal anastomosis was performed using the Xing's ureteroileal anastomosis technique. Afferent loop entry was divided equally into two lumens. After 1.5 cm-long lengthwise incisions, each ureter was directly and end-to-end anastomosed to the aforementioned lumens. Postoperative information was recorded, including ureteric stricture, ureteric reflux, hydronephrosis, anastomotic leakage, renal calculus, urinary tract infection, and pyelonephritis. Results:Ureteroileal anastomosis was performed successfully in 38 cases with 76 units. The median follow-up time was 35.6 (17.0, 46.3) months. Three patients developed unilateral anastomotic stenosis after operation. Five patients had unilateral ureteral reflux. Two patients had unilateral hydronephrosis. No anastomotic leakage, urinary tract infection, or pyelonephritis occurred after the operation. Renal calculus appeared in 3 cases, all on the left unit.Conclusions:Xing's ureteroileal anastomosis technique is a simple method with few postoperative and good functional outcomes.

OBJECTIVE To explore the effects of Citrus medica before and after being steamed on blood metabolism and dryness indexes in rats. METHODS Twenty-four SD rats were randomly divided into blank group， C. medica group （2.4 g/kg）， processed C. medica group （2.4 g/kg）， Citrus aurantium group （positive control， 1.2 g/kg）， with 6 rats in each group. After 5 weeks of continuous administration， the body weight， food intake， water intake and urine volume were observed dynamically. The content of aquaporin 3 （AQP3） in serum was detected. The metabolic profile of rat serum samples was analyzed by liquid chromatography-mass spectrometry. Principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA） were used to screen differential metabolites， and screened differential metabolites were mapped to the KEGG database for pathway analysis. RESULTS The body weight of rats in each group increased slowly； the food intake and water intake of processed C. medica group tended to be higher than those in C. medica group and C. aurantium group， but there was no significant difference among those groups （P＞0.05）. On the 21st day of administration， compared with C. aurantium group， the urine volume of processed C. medica group was significantly increased （P＜0.05）， the serum AQP3 content of processed C. medica group was significantly decreased （P＜0.05）， and were close to those of blank group. According to the metabonomic study， C. medica mainly affected the lipids metabolism， and processed C. medica mainly affected the amino acid metabolism. The differential metabolites pathway between C. medica and processed C. medica involved the metabolism of cysteine and methionine， metabolism of glycine， serine and threonine， and metabolism of taurine and hypotaurine. CONCLUSIONS Processed C. medica might relieve the dryness of C. medica by affecting adenosine phosphate-protein kinase A. The content of AQP3 in serum can be used as the 52 evaluation index for the dryness of C. medica before and after processing. The effects of C. medica and processed C. medica on endogenous metabolites in rat serum are quite different，especially in the aspect of lipid metabolism.