To investigate the promoting effect and mechanism of exosomes derived from mesenchymal stem cells （MSCs） overexpressing periostin （POSTN） on liver regeneration.
Methods
A 70 % partial hepatectomy （PHx） model was established in mice， and mice were administered PBS （PBS group）， MSCs-derived exosomes （MSC-Exo group）， or POSTN- overexpressing MSCs-derived exosomes （MSC-ExoPOSTN group） via tail vein injection after operation.Liver tissues were collected 72 h after operation.Hepatocyte proliferation ability was assessed by immunofluorescence staining for Ki-67 and proliferating cell nuclear antigen（PCNA）was detected by Western blot to evaluate hepatocyte proliferation.mRNA and protein levels of pro- regenerative factors （e.g.， HGF， IL-6） in liver tissues were measured via RT-qPCR and Western blot.A co-culture system of MSC-ExoPOSTN with AML-12 hepatocytes was established， and the expression of PCNA， PI3K， p-PI3K， p65， p-p65 were detected by Western blot and RT-qPCR.The difference between two groups was compared by independent samples t-test and the difference among multi-group was compared by one-way ANOVA.LSD-t test was used for pairwise comparison between different groups.
Results
Compared with PBS group， liver-to-body weight ratio ［（3.63 ±0.06）% vs （2.96 ± 0.08）%］， PCNA protein levels （0.62 ± 0.02 vs 0.32 ± 0.03）， Ki-67-positive cell ratio ［（39.33 ± 2.87）%vs （21.03 ± 1.63）%］， HGF mRNA （1.66 ± 0.06 vs 0.99 ± 0.09） and protein levels （1.98 ± 0.12 vs 1.00 ± 0.19）， and IL-6 mRNA （1.87 ± 0.07 vs 1.09 ± 0.12） and protein levels （1.51 ± 0.04 vs 0.97 ± 0.07） were increased in MSC-ExoPOSTN group （all P ＜ 0.05）.Compared with MSC-Exo group， liver-to-body weight ratio ［（3.63 ± 0.06）% vs （3.28 ± 0.06）%］， PCNA protein levels （0.62 ± 0.02 vs 0.51 ± 0.02）， Ki-67-positive cell ratio ［（39.33 ± 2.87）% vs （30.33 ±1.70）%］， HGF mRNA （1.66 ± 0.06 vs 1.32 ± 0.10） and protein levels （1.98 ± 0.12 vs 1.50 ± 0.09），and IL-6 mRNA （1.87 ± 0.07 vs 0.77 ± 0.08） and protein levels （1.51 ± 0.04 vs 0.79 ± 0.06） were increased in the MSC-ExoPOSTN group （all P ＜ 0.05）.In the MSC-ExoPOSTN and AML-12 hepatocyte co-culture system， compared with PBS group， the expression levels of PCNA（0.81 ± 0.06 vs 0.32 ±0.03） and p-p65 protein （1.15 ± 0.10 vs 0.81 ± 0.14） were increased in MSC-ExoPOSTN group （both P ＜ 0.05）， while p-PI3K level remained unchanged.Compared with MSC-Exo group， the expression levels of PCNA （0.81 ± 0.06 vs 0.49 ± 0.04） and p-p65 protein （1.15 ± 0.10 vs 0.51 ± 0.07） were increased in MSC-ExoPOSTN group （both P ＜ 0.05）， while p-PI3K level remained unchanged.Compared to PBS treatment， the PCNA mRNA level was reduced after treatment with NF-κB inhibitor JSH23 （1.01 ± 0.09 vs 1.59 ± 0.07， P ＜ 0.05）.
Conclusion
MSC-ExoPOSTN enhances liver regeneration post- hepatectomy， potentially via activation of the NF-κB signaling pathway in hepatocytes.
To investigate the structural changes of the retina and the variations in inflammatory factors in a rat model simulating microgravity， and verify the effects of NETosis.
Methods
Forty rats were randomly divided into control group and tail-suspension group， with 20 rats in each group.Weightlessness model was established by tail-suspension method with a suspension duration of four weeks.HE staining was performed to observe changes in retinal structure， and OCTA was used to measure retinal perfusion area and thickness.Qualitative assessments were made of the retinal structures and NETosis-related regulatory factors［myeloperoxidase （MPO）， citrullinated histone H3 （CIT-H3）， and neutrophil elastase （NE）］.Quantitative evaluations were conducted on NETosis-related regulatory factors［MPO， peptidylarginine deiminase 4 （PAD4）， CIT-H3， and NE］，pro-inflammatory factors （IL-1， IL-6， and TNF-α）， and anti-inflammatory factors （IL-10） before and after simulating microgravity.Independent samples t-tests were used for statistical analysis of the quantitative results.
Results
Compared with the control group， the retinal perfusion area ［0- 6mm：（26.93 ± 0.18） vs （24.96 ± 0.26）mm2］ and retinal thickness［0-6mm：（255.42 ± 3.37）vs（230.77 ±4.13）μm］ were increased in tail-suspension group （all P ＜ 0.05）with varying degrees of thickening as well as disrupted and disorganized arrangement of retinal cell layers.In addition， the expression levels of pro-inflammatory cytokines， including IL-1β［（10.59 ± 0.31） vs （8.25 ± 0.21）pg/（mg·pro）］，IL-6［（32.97 ± 1.11） vs（ 22.89 ± 0.87）pg/（mg·pro）］， and TNF-α［（102.47 ± 4.01） vs （65.84 ±3.48）pg/ （mg·pro）］ as well as NETosis-related regulatory factors MPO （0.68 ± 0.01 vs 0.27 ± 0.01），NE （0.50 ± 0.02 vs 0.18 ± 0.02）， CitH3 （0.89 ± 0.01 vs 0.31 ± 0.02）， and PAD4 （0.61 ± 0.01 vs 0.15 ± 0.02）were elevated， while the anti-inflammatory cytokine IL-10［（15.34 ± 0.35） vs （21.17 ±0.63）pg/（mg·pro）］ was reduced in tail-suspension group（all P＜ 0.05）.
Conclusion
Simulating a microgravity environment induces pathological changes in the retinal structure of rats， elevates inflammation levels， which maybe related with NETosis.
To explore the effects of circular RNA CCDC66 （Circ-CCDC66）on colorectal cancer progression through miR-618/phosphatase and tensin homolog deleted on chromosome ten （PTEN）.
Methods
Ten pairs of colorectal cancer tissues and adjacent normal tissues were collected.Bioinformatics methods were used to predict the targets of Circ-CCDC66， and miR-618 and PTEN were selected for subsequent experiments.Real- time fluorescence quantitative PCR （RT-qPCR） was used to detect the expression of Circ- CCDC66 in NCM620， SW480， SW620，and HCT116 cells.The expression of CCDC66 and Circ-CCDC66 in HCT116 as well as SW620 cells were detected after RNase R treatment.The expression of miR-618 in SW620 and HCT116 cells were detected after transfection with miR- 618 mimic or lentivirus vector transfection of Circ- CCDC66.Western blot was used to detect the expression levels of PTEN， AKT， and BCL- 2 proteins in cells.Dual-luciferase reporter gene assay was used to detect the targeted relationship between Circ- CCDC66 and miR-618， miR-618 and PTEN.Cell Counting Kit-8 （CCK- 8） assay was used to detect the changes in cell proliferation after different treatments， scratch assay and Transwell invasion assay were used to detect the invasion and migration of colorectal cancer cells after differenttreatments.Independent sample t test was used to compare the measurement data between the two groups， one-way analysis of variance was used for comparison among multiple groups， and LSD-t test was used for pairwise comparison between groups.
Results
Compared with the adjacent tissues of colorectal cancer， the expression of Circ-CCDC66 （0.69 ± 0.20 vs 1.00 ± 0.21） and PTEN mRNA （0.26 ± 0.13 vs 1.00 ± 0.48） and PTEN protein （0.67 ± 0.23 vs 1.00 ± 0.13） in colorectal cancer tissues were decreased （all P ＜ 0.05）.Compared with the normal colon epithelial cell line NCM460， the expression of Circ-CCDC66 （0.50 ± 0.03， 0.18 ± 0.02， 0.62 ± 0.05 vs 1.00 ± 0.10）was decreased in colorectal cancer cell lines HCT116， SW620， and SW480 （all P ＜ 0.01）， and the expression of Circ-CCDC66 in HCT116 and SW620 cells was lower than that in SW480 cells （all P ＜0.05）.Bioinformatics analysis and lentivirus transfection experiments indicated that miR- 618 was a downstream target of Circ-CCDC66.Dual-luciferase reporter assays showed that miR-618 targeted the PTEN gene in colorectal cancer cell lines.After overexpression of miR-618， the expression of PTEN proteins in colorectal cancer SW620 and HCT116 cells （0.49 ± 0.01 vs 1.00 ± 0.01， 0.52 ±0.00 vs 1.01 ± 0.00） were decreased （all P ＜ 0.001）.CCK-8， cell scratch， and Transwell assays demonstrated that overexpression of Circ-CCDC66 could inhibit the proliferation （0.48 ± 0.09 vs 0.83 ± 0.05， 0.41 ± 0.05 vs 0.68 ± 0.05）， migration ［（25.33 ± 2.08）% vs （41.00 ± 3.61）%， （28.00 ±2.00）% vs （40.00 ± 5.00）%］， and invasion ［（42.33 ± 4.93） vs （61.33 ± 4.16）cells， （25.00 ± 5.00）vs （41.00 ± 3.61） cells］ abilities of colorectal cancer SW620 and HCT116 cells at 48 h （all P ＜ 0.05）.However， miR-618 could reverse the inhibitory effects of Circ-CCDC66 overexpression ［proliferation（0.78 ± 0.05 vs 0.48 ± 0.09， 0.58 ± 0.05 vs 0.41 ± 0.05）； migration ［（35.00 ± 2.00）% vs （25.33 ±2.08）%， （40.00 ± 1.73）% vs （28.00 ± 2.00）%］； invasion ［（68.67 ± 5.03） vs （42.33 ± 4.93）cells， （39.33 ± 5.13） vs （25.00 ± 5.00） cells］ （all P ＜ 0.05）.
Conclusion
Circ-CCDC66 could influence the progression of colorectal cancer cells by binding to miR-618 and targeting PTEN
To investigate the effects of hydroxyapatite tricalcium phosphate（HA-TCP） scaffold complexes and periodontal ligament stem cells on periodontal tissue regeneration.
Methods
Healthy premolars from 15 children aged 16-18 who required corrective treatment were collected.The human periodontal ligament stem cells （hPDLSCs）， were isolated and identified.The self-replication ability of hPDLSCs was detected by using clone formation experiments.Flow cytometry was used to detect the expression of surface antigens CD90， CD44， CD34， and CD45 on hPDLSCs.Alizarin red and red oil O staining were used to detect the osteogenic and adipogenic abilities of hPDLSCs.Alkaline phosphatase （ALP）staining was used to detect alkaline phosphatase activity.hPDLSCs and HA-TCP complexes were prepared.Rat alveolar bone defect models were established.Thirty rats were divide into three groups randomly： NC group （no implantation），HA- TCP group （only HA-TCP implantation）， and hPDLSCs/HA-TCP group （hPDLSCs/HA- TCP composite implantation）.Pathological changes in alveolar bone tissues were detected by HE staining， and the expression of ALP， osteocalcin （OCN）， and osteopontin （OPN） proteins in alveolar bone tissue were detected by Western blot.
Results
Crystal violet staining shows that hPDLSCs can form clone colonies， and the clone colony status was observed under the microscope， indicating that hPDLSCs have self replication ability.The flow cytometry results showed high levels of mesenchymal stem cell markers CD90 and CD44 on the surface of hPDLSCs， while hematopoietic stem cell markers CD45 and CD34 were expressed at low levels， indicating that the extracted PDLSCs were mesenchymal stem cells.Alizarin red and red oil O staining revealed bone nodules， calcium mineralization nodules， and lipid droplet formation in hPDLSCs.The HE staining results showed that hPDLSCs/HA TCP promoted the formation of mature new bone， new blood vessels， and osteoblasts；ALP staining revealed an increase in the number of positive cells.Western blot results showed that compared with the NC group， the expression of ALP （0.63 ± 0.08， 1.21 ± 0.13 vs 0.32 ± 0.05）， OCN（0.61 ± 0.08， 1.10 ± 0.12 vs 0.23 ± 0.04）， and OPN protein （0.53 ± 0.07， 0.82 ± 0.10 vs 0.20 ± 0.04）in the alveolar bone tissue of the HA-TCP group and hPDLSCs/HA-TCP group were increased， and the expression of ALP， OCN， and OPN proteins in the alveolar bone tissue of the hPDLSCs/HA- TCP group was higher than that of the HA-TCP group （P ＜ 0.05）.
Conclusion
HA- TCP/PDLSCs complex can promote new bone formation， repairing alveolar bone loss and promoting bone tissue regeneration.
To explore the impacts and mechanisms of salvianolic acid A （SalA）on the biological behavior of cervical cancer cells.
Methods
Human cervical cancer SiHa cells were cultured in vitro and divided into different groups based on different treatment as and named control group； SalA-L group， SalA-M group， SalA-H group （untreated cervical cancer SiHa cells were treated with SalA at concentrations of 10， 25， and 50 μmol/L for 48 hours）； anti- miR- NC group， anti- miR-212-5p group， miR-NC group， miR-212-5p group （cells were transfected with anti- miR- NC， anti-miR-212-5p， miR-NC， and miR-212-5p respectively）； SalA-H+anti- miR-N group、SalA- H+anti- miR-212-5p group、SalA-H+anti-miR- 212-5p+si-PRR11 group （cells were transfected with anti-miR-NC， anti-miR-212-5p， and co-transfected with anti-miR-212-5p and si- PRR11， followed by treatment with 50 μmol/L SalA for 48 hours）.The expression levels of miRNA- 212- 5p （miR- 212-5p） and proline-rich protein 11 （PRR11） mRNA were detected by RT- qPCR.The expression level of PRR11 protein was detected by Western blot.The targeting relationship between miR-212-5p and PRR11 was verified using a dual-luciferase reporter assay.Cell proliferation activity was detected using the MTT method.Cell colony formation was assessed by cell colony formation assay.The apoptosis rate was determined by flow cytometry.Cell migration was evaluated by scratch assay.Cell invasion was assessed by Transwell assay.Comparisons between two groups were performed by t-test， comparisons among multiple groups were performed by one- way ANOVA， and pairwise comparisons between groups were performed by LSD-t test.
Results
Compared with the control group， the number of the invasive cells， cell colony formation，OD values， wound healing rates， and the expression levels of PRR11 mRNA and protein （0.49 ± 0.04，0.35 ± 0.03， 0.21 ± 0.03 vs 0.61 ± 0.05， P ＜ 0.05） were reduced in the SalA-L， SalA-M， and SalA-H groups （at 24， 48， and 72 hours）， while the apoptosis rate and expression level of miR-212-5p were increased （1.75 ± 0.14， 2.89 ± 0.23， 4.12 ± 0.35 vs 1.00 ± 0.06， P ＜ 0.05）.Compared with the control group and the miR-NC group， the expression levels of PRR11 protein （0.23 ± 0.03 vs 0.57 ± 0.05，0.58 ± 0.06， P ＜ 0.05）， the number of invasive cells， cell colony formation， OD values （at 24， 48， and 72 hours）， and wound healing rates were decreased in the miR-212-5p group， while the apoptosis rate was increased.Compared with the SalA-H group and the SalA-H+anti-miR-NC group， the expression levels of PRR11 protein （0.68 ± 0.07 vs 0.20 ± 0.02， 0.22 ± 0.03， P ＜ 0.05）， cell colony formation，OD values （at 24， 48， and 72 hours）， the number of invasive cells， and wound healing rates in SalA- H+anti- miR- 212-5p group were increased， while the apoptosis rate was decreased； Compared with the SalA-H+anti-miR-212- 5p group， the PRR11 protein expression levels （0.29 ± 0.03 vs 0.68 ±0.07， P ＜ 0.05）， cell colony formation， OD values （at 24， 48， and 72 hours）， invasive cell numbers，and cell scratch healing rates in SalA-H+anti-miR-212-5p+si-PRR11 group were reduced， while the apoptosis rate was increased.
Conclusion
SalA may regulate the biological behavior of cervical cancer SiHa cells through the miR-212-5p/PRR11 pathway.
Diabetic foot ulcers（ DFUs） are common complications in patients with diabetes，which has been a focus of medical attention due to their difficult treatment and high recurrence rates.In recent years， with the development of regenerative medicine， mesenchymal stem cell- derived exosomes（MSC-Exosomes） have gained significant attention for their unique biological properties and therapeutic potential.As a novel type of bioactive molecule， they possess various biological functions such as promoting cell proliferation， differentiation， migration， and angiogenesis， providing new ideas and methods for the treatment of diabetic wounds.However， the limited stability and delivery efficiency of exosomes in vivo restrict their clinical application.Hydrogels， a kind of biocompatible and biodegradable material， can provide a stable microenvironment for stem cell exosomes and facilitate their release to the wound site.Further optimized the preparation process and modification methods of hydrogels could enhance their drug loading capacity and stability， boosting their therapeutic efficacy.Additionally， the adhesiveness and plasticity properties of hydrogels allow them to closely adhere to the wound surface， reducing the risk of infection and accelerating the wound healing process.This article reviews the research progress of hydrogel loaded with stem cell exosomes in the treatment of DFUs， including the mechanisms underlying the difficulty of healing DFUs， the functions of stem cell-derived exosomes， the characteristics and functions of hydrogels， as well as the reserch progress on exosome-loaded hydrogels for promoting DFUs healing.
Stem cell therapy， as representatives of new quality productive forces in the field of biomedical， have high development potential and broad application prospects.In view of the particularity and complexity of stem cell therapy， governments and regulatory authorities of various countries have strengthened their supervision over the field of stem cell therapy， striving to promote the healthy development of the field while ensuring patient safety.In this context， it is theoretical and practical significant to study and discuss the domestic and international regulatory policies and technical specifications for stem cell therapy.This article comprehensively reviews the current regulatory policies in major countries and regions worldwide， systematically analyze relevant guidelines， discusses the challenges faced in stem cell therapy regulation，and proposes recommendations for research on regulatory system.This paper aims to provide valuable references for the safe， effective and compliant application of stem cell therapy.
