BACKGROUND: Mesenchymal stem cells-derived exosomes have been applied in the diagnosis and treatment of various diseases due to its many virtues. Therefore, its accurate and effective detection with low cost is critical for disease diagnosis and treatment. OBJECTIVE: To introduce the detection methods and newest progress of mesenchymal stem cells-derived exosomes. METHODS: WanFang, Baidu Scholar, VIP, CNKI, PubMed, Web of Science, Sinomed, Embase, Cochrane and Web of Knowledge databases were retrieved for the articles concerning the detection methods of mesenchymal stem cells-derived exosomes published between June 1997 and June 2019. The keywords were “mesenchymal stem cells, exosomes, detection methods” in Chinese and English, respectively. The duplicated and poorly correlated articles were excluded, and finally 60 eligible articles were included for analysis. RESULTS AND CONCLUSION: (1) The definition and biological characteristics of mesenchymal stem cells are summarized, and mesenchymal stem cells have been found to treat diseases by paracrine approach. (2) The definition and biological characteristics of mesenchymal stem cells-derived exosomes and its potential clinical application are summarized, including immunoregulation in vivo and promotion of tissue regeneration. (3) The commonly used detection methods of mesenchymal stem cells-derived exosomes and the newest progress are reviewed. (4) This review provides experimental basis for the clinical application of mesenchymal stem cells-derived exosomes regarding disease treatment and tissue repair.

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.

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.

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 feasibility and clinical effect of laparoscopic radical cystectomy with intracorporeal Xing's orthotopic neobladder.Methods Forty-one patients who underwent laparoscopic radical cystectomy with intracorporeal Xing's orthotopic neobladder from July 2013 to August 2019.There were 31 cases performed in Beijing Chaoyang hospital and 10 cases in National Cancer Center.Mean age was 59 (range 44-78) years,mean BMI was 25.3 (range 20.1-34.7) kg/m2,and mean CCI was 3 (range 2-6).No urethral stricture or urinary incontinence was found by preoperative examination.No distant metastasis was identified by bone scans,chest X-ray and sonography.Cystoscopy or TURBT was performed on all patients and biopsy was taken to confirm the diagnosis.Preoperative pathology showed 30 cases (73.2％) of MIBC,9 cases of NMIBC (22.0％) and 2 cases (4.9％) of in-situ cancer.Laparoscopic radical cystectomy and lymphadenectomy were performed under general anesthesia.Urinary diversion was completed in the peritoneal cavity,by intercepting the terminal ileum about 60 cm,and taking the proximal ileum 10 cm as input loop on the right side with proximal to distal way,and the middle 40 cm ileum was detubated.After u-shaped suture,the ileum was folded back and stitched into a sphere building a novd orthotopic neobladder with bilateral isoperistaltic afferent limbs.The prognosis of perioperative data and postoperative satisfaction regarding continence were analyzed,continence was defined as 0-1 pad/day.The 41 patients were divided into two groups to compare the difference in term of operation time and blood loss between the first 21 patients and the last 20 patients.Results Mean total operative time was 324.9 mins (range 210-480) mins,and mean estimated blood loss was 177.6(range 50-700) ml.There were significant statistical differences in term of total operation time,construction time and blood loss between the first 21 patients and the next 20 patients (P ＜ 0.05).Postoperative pathological results were urothelial carcinoma in 40 cases (2 in situ carcinoma) and small cell carcinoma in 1 case.Mean number of dissected lymph nodes was 19 (range 11-58),with 7 cases(17.1％)of positive lymph nodes,and 3 cases(7.3％) had positive surgical margin.At a mean follow up of 17.6 (range 2-64) months,36 patients (87.8％) survived,including 2 patients (4.9％) with metastasis and 1 patient (2.4％) with recurrence,and 5 cases (12.2％)died.All patients were able to urinate without catheterization.Thirty-seven patients (90.2％) were satisfied with voiding control during the daytime (0-1 urinal pad),and 29 patients (70.7％) were satisfied with voiding control at nighttime (0-1 urinal pad) by the follow-up 12 months after the operation.Conclusions Total laparoscopic radical cystectomy combined with Xing's orthotopic ileum neobladder is a simple method with fewer postoperative complications and a satisfactory continence rate.

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.

Fluorescence-guided surgery (FGS) with tumor-targeted imaging agents, particularly those using the near-infrared wavelength, has emerged as a real-time technique to highlight the tumor location and margins during a surgical procedure. For accurate visualization of prostate cancer (PCa) boundary and lymphatic metastasis, we developed a new approach involving an efficient self-quenched near-infrared fluorescence probe, Cy-KUE-OA, with dual PCa-membrane affinity. Cy-KUE-OA specifically targeted the prostate-specific membrane antigen (PSMA), anchored into the phospholipids of the cell membrane of PCa cells and consequently showed a strong Cy7-de-quenching effect. This dual-membrane-targeting probe allowed us to detect PSMA-expressing PCa cells both in vitro and in vivo and enabled clear visualization of the tumor boundary during fluorescence-guided laparoscopic surgery in PCa mouse models. Furthermore, the high PCa preference of Cy-KUE-OA was confirmed on surgically resected patient specimens of healthy tissues, PCa, and lymph node metastases. Taken together, our results serve as a bridge between preclinical and clinical research in FGS of PCa and lay a solid foundation for further clinical research.