Exploring the Mechanism of CircRNA-vgll3 in Osteogenically Differentiated Human Bone Marrow Mesenchymal Stem Cells

Objective:To explore the mechanism of circRNA-vgll3 in osteogenic differentiation of human bone marrow mesenchymal stem cells.Methods:BMSCs cells were transfected with circRNA-vgll3,and divided into circRNA-vgll3 high-level group,circRNA-vgll3 low-level group,and negative control group(circRNA-vgll3 not transfected)according to the amount of transfection.The proliferation and apoptosis of BMSCs osteoblasts in each group were analyzed,and the alkaline phosphatase(ALP)activity,type I collagen gray value,bone morphogenetic protein 2(BMP-2),Runx2 protein,and mRNA expression levels were detected.Results:The circRNA-vgll3 low-level group had a significant inhibitory effect on the proliferation of BMSCs osteoblasts,and the apoptosis rate of the circRNA-vgll3 low-level group was significantly higher than that of the circRNA-vgll3 high-level group(P<0.05);ALP activity,type I collagen gray value,BMP-2,Runx2 protein,and mRNA expression levels in the high-level circRNA-vgll3 group were significantly higher than those in the low-level circRNA-vgll3 group,and the difference was statistically significant(P<0.05).Conclusion:Overexpression of circRNA-vgll3 can promote the osteogenic differentiation ability of BMSCs,while low expression of circRNA-vgll3 can inhibit the osteogenic differentiation ability of BMSCs.The main mechanism of action is that circRNA-vgll3 can affect osteogenic differentiation by regulating the Runx2 protein.

HNF-4α determines hepatic differentiation of human mesenchymal stem cells from bone marrow

AIM: To investigate the differentiation status and key factors to facilitate hepatic differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). METHODS: Human MSCs derived from bone marrow were induced into hepatocyte-like cells following a previously published protocol. The differentiation status of the hepatocyte-like cells was compared with various human hepatoma cell lines. Overexpression of hepatocyte nuclear factor (HNF)-4α was mediated by adenovirus infection of these hepatocyte-like cells. The expression of interesting genes was then examined by either re-verse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR methods. RESULTS: Our results demonstrated that the differentiation status of hepatocyte-like cells induced from human MSCs was relatively similar to poorly differentiated human hepatoma cell lines. Interestingly, the HNF-4 isoform in induced MSCs and poorly differentiated human hepatoma cell lines was identified as HNF4γ instead of HNF-4α. Overexpression of HNF-4α in induced MSCs significantly enhanced the expression level of hepatic-specific genes, liver-enriched transcription factors, and cytochrome P450 (P450) genes. CONCLUSION: Overexpression of HNF-4α improves the hepatic differentiation of human MSCs from bone marrow and is a simple way of providing better cell sources for clinical applications.

Cardiomyocyte-like differentiation of human bone marrow mesenchymal stem cells after exposure to 5-azacytidine in vitro

Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073g/mL Percoll and propagated in the right cell culturing medium as previously described. The phenotypes of hMSCs were characterized with the use of flow cytometry. The hMSCs were cultured in cell culture medium (as control) and medium mixed with 5-aza for cellular differentiation. We examined by immunohistochemistry at 21 days the inducement of desmin, cardiac-specific cardiac troponin I (cTnI), GATA 4 and connexin-43 respectively. Results The hMSCs are fibroblast-like morphology and express CD44+ CD29+ CD90+ / CD34- CD45- CD31- CD11a. After 5-aza treatment, 20-30% hMSCs connected with adjoining cells and coalesced into myotube structures after 14days. Twenty-one days after 5-aza treatment, immunofluorescence showed that some cells expressed desmin,GATA4, cTnI and connexin-43 in 5,10 μmol/L 5-aza groups, but no cardiac specific protein was found in neither 3μmol/L 5-aza group nor in the control group. The ratio of cTnI positively stained cells in 10 μmol/L group was higher than that in 5 μmol/L group (65.3 ± 4.7% vs 48.2 ± 5.4%, P ＜ 0.05). Electron microscopy revealed that myofilaments were formed. The induced cells expressed cardiac-myosin heavy chain (MyHC) gene by reverse transcription-polymerase chain reaction (RT-PCR). Conclusions Theses findings suggest that hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration. (J Geriatr Cardiol 2004;1(2) :101-107. )

The genomic landscapes of histone H3-Lys9 modifications of gene promoter regions and expression profiles in human bone marrow mesenchymal stem cells

Mesenchymal stem cells （MSCs） of nonembryonic origins possess the proliferation and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms could be critical for determining the fate of stem cells, and MSCs derived from different origins exhibited different expression profiles individually to a certain extent. In this study, ChiP-on-chip was used to generate genome-wide histone H3-Lys9 acetylation and dimethylation profiles at gene promoters in human bone marrow MSCs. We showed that modifications of histone H3-Lys9 at gene promoters correlated well with mRNA expression in human bone marrow MSCs. Functional analysis revealed that many key cellular pathways in human bone marrow MSC self-renewal, such as the canonical signaling pathways, cell cycle pathways and cytokine related pathways may be regulated by H3-Lys9 modifications. These data suggest that gene activation and silencing affected by H3-Lys9 acetylation and dimethylation, respectively, may be essential to the maintenance of human bone marrow MSC self-renewal and multi-potency.

Human bone marrow mesenchymal stem cell transplantation attenuates axonal injury in stroke rats

Previous studies have shown that transplantation of human bone marrow mesenchymal stem cells promotes neural functional recovery after stroke, but the neurorestorative mechanisms remain largely unknown. We hypothesized that functional recovery of myelinated axons may be one of underlying mechanisms. In this study, an ischemia/reperfusion rat model was established using the middle cerebral artery occlusion method. Rats were used to test the hypothesis that intravenous transplantation of human bone marrow mesenchyrnal stem cells through the femoral vein could exert neuroprotective effects against cerebral ischemia via a mechanism associated with the ability to attenuate axonal injury. The results of behavioral tests, infarction volume analysis and immunohistochemistry showed that cerebral ischemia caused severe damage to the myelin sheath and axons. After rats were intravenously transplanted with human bone marrow mesenchymal stem cells, the levels of axon and myelin sheath-related proteins, including microtubule-associated protein 2, myelin basic protein, and growth-associated protein 43, were elevated, infarct volume was decreased and neural function was improved in cerebral ischemic rats. These findings suggest that intravenously transplanted human bone marrow mesenchymal stem cells promote neural function. Possible mechanisms underlying these beneficial effects include resistance to demyelination after cerebral ischemia, prevention of axonal degeneration, and promotion of axonal regeneration.

Mesenchymal Stromal Cells Derived from Human Embryonic Stem Cells, Fetal Limb and Bone Marrow Share a Common Phenotype but Are Transcriptionally and Biologically Different

Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to determine the optimal source of MSCs. We investigated if this biological heterogeneity in MSCs from different sources results in different mechanisms for their differentiation. In this study, we compared the gene expression patterns of phenotypically defined MSCs derived from three ontogenically different sources: Embryonic stem cells (hES-MSCs), Fetal limb (Flb-MSCs) and Bone Marrow (BM-MSCs). Differentially expressed genes between differentiated cells and undifferentiated controls were compared across the three MSC sources. We found minimal overlap (5% - 16%) in differentially expressed gene sets among the three sources. Flb-MSCs were similar to BM-MSCs based on differential gene expression patterns. Pathway analysis of the differentially expressed genes using Ingenuity Pathway Analysis (IPA) revealed a large variation in the canonical pathways leading to MSC differentiation. The similar canonical pathways among the three sources were lineage specific. The Flb-MSCs showed maximum overlap of canonical pathways with the BM-MSCs, indicating that the Flb-MSCs are an intermediate source between the less specialised hES-MSC source and the more specialised BM-MSC source. The source specific pathways prove that MSCs from the three ontogenically different sources use different biological pathways to obtain similar differentiation outcomes. Thus our study advocates the understanding of biological pathways to obtain optimal sources of MSCs for various clinical applications.

Effects of transplantation of human bone marrow mesenchymal stem cells in rats with liver failure

Objective: To investigate the effect of hepatic differentiation of human bone marrow mesenchymal stem cells (HBMSCs) induced in vitro and transplanted into rats with liver failure via portal vein, and observe the changes of liver function and pathological tissue. Method:After passage to the 6th generation in vitro, the hepatic differentiation was induced by HGFand EGF inducible factors. CCL4 acute liver failure model in rats were established, and randomly divided into 5 groups transplanted with differentiated stem cells via portal vein. These five groups included HGF-differentiated HBMSCs transplantation, EGF-differentiated HBMSCs transplantation, EGF+HGF-differentiated HBMSCs transplantation, non-differentiated HBMSCs transplantation, and non-HBMSCs transplantation. Liver function and pathological changes were detected. Results: Rats models survival, serum albumin, aminotransferase and coagulation indexes were observed at 12 h, 72 h, 7 d, 1 month and 2 months after treatment. The results showed that the survival and albumin, aminotransferase and coagulation function of rats were improved significantly after treatment in HGF-differentiated, EGF-differentiated, EGF+HGF-differentiated and non-differentiated transplantation groups, compared tothe non-HBMSCstransplantation group(P<0.05), while no significance was observed in above four groups(P>0.05).Pathological changes was ameliorated in the liver of rat models in HGF-, EGF-, EGF+HGF- and non-differentiated transplantation groups, compared to the non-HBMSCs transplantation group. Conclusion: Liver-differentiated BMSCs transplanted into rats with liver failure could effectively improve liver function and survival rate.

Human embryonic stem cell-derived mesenchymal stem cells improved premature ovarian failure

BACKGROUND Premature ovarian failure(POF)affects many adult women less than 40 years of age and leads to infertility.According to previous reports,various tissue-specific stem cells can restore ovarian function and folliculogenesis in mice with chemotherapy-induced POF.Human embryonic stem cells(ES)provide an alternative source for mesenchymal stem cells(MSCs)because of their similarities in phenotype and immunomodulatory and anti-inflammatory characteristics.Embryonic stem cell-derived mesenchymal stem cells(ES-MSCs)are attractive candidates for regenerative medicine because of their high proliferation and lack of barriers for harvesting tissue-specific MSCs.However,possible therapeutic effects and underlying mechanisms of transplanted ES-MSCs on cyclophosphamide and busulfan-induced mouse ovarian damage have not been evaluated.AIM To evaluate ES-MSCs vs bone marrow-derived mesenchymal stem cells(BMMSCs)in restoring ovarian function in a mouse model of chemotherapy-induced premature ovarian failure.METHODS Female mice received intraperitoneal injections of different doses of cyclophosphamide and busulfan to induce POF.Either human ES-MSCs or BMMSCs were transplanted into these mice.Ten days after the mice were injected with cyclophosphamide and busulfan and 4 wk after transplantation of the ESMSCs and/or BM-MSCs,we evaluated body weight,estrous cyclicity,folliclestimulating hormone and estradiol hormone concentrations and follicle count were used to evaluate the POF model and cell transplantation.Moreover,terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling,real-time PCR,Western blot analysis and immunohistochemistry and mating was used to evaluate cell transplantation.Enzyme-linked immunosorbent assay was used to analyze vascular endothelial growth factor,insulin-like growth factor 2 and hepatocyte growth factor levels in ES-MSC condition medium in order to investigate the mechanisms that underlie their function.RESULTS The human ES-MSCs significantly restored hormone secretion,survival rate and reproductive function in POF mice,which was similar to the results obtained with BM-MSCs.Gene expression analysis and the terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling assay results indicated that the ES-MSCs and/or BM-MSCs reduced apoptosis in the follicles.Notably,the transplanted mice generated new offspring.The results of different analyses showed increases in antiapoptotic and trophic proteins and genes.CONCLUSION These results suggested that transplantation of human ES-MSCs were similar to BM-MSCs in that they could restore the structure of the injured ovarian tissue and its function in chemotherapy-induced damaged POF mice and rescue fertility.The possible mechanisms of human ES-MSC were related to promotion of follicular development,ovarian secretion,fertility via a paracrine effect and ovarian cell survival.

Conditions to improve expansion of human mesenchymal stem cells based on rat samples

AIM: To improve the isolation and expansion of human marrow-derived mesenchymal stem cells (MSCs) based on rat samples. METHODS: Based on the fact that rat MSCs are relatively easy to obtain from a small aspirate, bone marrow-derived MSCs from rat were cultured and characterized to set up the different protocols used in this study. Then, accordingly, almost the same protocols were performed on human healthy bone marrow samples, after obtaining approval of the ethics committee and gaining informed consent. We used different protocols and culture conditions, including the type of basal media and the culture composition. The MSCs were characterized by immunophenotyping and differentiation. RESULTS: There was no difference in morphology and proliferation capacity between different culture media at the first passage. During the 5-7th passages, the cells gradually lost their morphology and proliferation potential on Dulbecco’s modified Eagle’s medium (DMEM) high glucose and α modified Eagle’s medium. Although the cells expanded rapidly for up to 10 passages on DMEM low glucose containing 10% to 15% fetal calf serum (FCS), their proliferation was arrested without change in morphology and differentiation capacity at the third passage on 5% FCS. Flow cytometric analysis and functional tests confirmed that more than 90% of marrow cells which were isolated and expanded by our selective protocols were MSCs. CONCLUSION: We improved the isolation and expansion of human bone marrow derived MSCs, based on rat sample experiments, for further experimental and clinical use.

Bioactive materials from berberine-treated human bone marrow mesenchymal stem cells promote alveolar bone regeneration by regulating macrophage polarization

Alveolar bone regeneration has been strongly linked to macrophage polarization.M1 macrophages aggravate alveolar bone loss,whereas M2 macrophages reverse this process.Berberine(BBR),a natural alkaloid isolated and refined from Chinese medicinal plants,has shown therapeutic effects in treating metabolic disorders.In this study,we first discovered that culture supernatant(CS)collected from BBR-treated human bone marrow mesenchymal stem cells(HBMSCs)ameliorated periodontal alveolar bone loss.CS from the BBR-treated HBMSCs contained bioactive materials that suppressed the M1 polarization and induced the M2 polarization of macrophages in vivo and in vitro.To clarify the underlying mechanism,the bioactive materials were applied to different animal models.We discovered macrophage colony-stimulating factor(M-CSF),which regulates macrophage polarization and promotes bone formation,a key macromolecule in the CS.Injection of pure M-CSF attenuated experimental periodontal alveolar bone loss in rats.Colony-stimulating factor 1 receptor(CSF1R)inhibitor or anti-human M-CSF(M-CSF neutralizing antibody,Nab)abolished the therapeutic effects of the CS of BBR-treated HBMSCs.Moreover,AKT phosphorylation in macrophages was activated by the CS,and the AKT activator reversed the negative effect of the CSF1R inhibitor or Nab.These results suggest that the CS of BBR-treated HBMSCs modulates macrophage polarization via the M-CSF/AKT axis.Further studies also showed that CS of BBR-treated HBMSCs accelerated bone formation and M2 polarization in rat teeth extraction sockets.Overall,our findings established an essential role of BBR-treated HBMSCs CS and this might be the first report to show that the products of BBR-treated HBMSCs have active effects on alveolar bone regeneration.

Comparison of phenotypic markers and neural differentiation potential of multipotent adult progenitor cells and mesenchymal stem cells

AIM: To compare the phenotypic and neural differentiation potential of human bone marrow derived multipotent adult progenitor cells (MAPC) and mesenchymal stem cells (MSC). METHODS: Cultures of MAPC and MSC were established in parallel from same samples of human bone marrow (n = 5). Both stem cell types were evaluated for expression of pluripotency markers including Oct-4 and Nanog by immunocytochemistry and reversetranscription polymerase chain reaction (RT-PCR) and expression of standard mesenchymal markers including CD14, CD34, CD44, CD45, CD73, CD90, CD105 andhuman leukocyte antigen (HLA)-ABC by flow cytometry. After treatment with neural induction medium both MAPC and MSC were evaluated for expression of neural proteins [neuronal filament-200 (NF-200) and glial fibrillar acidic protein (GFAP)] by immunocytochemistry and Western blotting and neural genes [NF-200, GFAP, Tau, microtubule-associated protein (MAP)-1B, MAP-2, neuron-specific enolase (NSE) and oligodendrocyte-1 (Olig-1)] by quantitative real-time-PCR. RESULTS: MAPC had small trigonal shaped while MSC had elongated spindle-shaped morphology. The MAPC expressed Oct-4 and Nanog both at gene and protein levels, whereas MSC were negative for these pluripotent markers. MAPC were negative for HLA-ABC while MSC had high expression of HLA-ABC. In addition, MAPC as compared to MSC had significantly lower expression of CD44 (36.56% ± 1.92% vs 98.23% ± 0.51%), CD73 (15.11% ± 2.24% vs 98.53% ± 2.22%) and CD105 (13.81% ± 3.82%vs 95.12% ± 5.65%) (P < 0.001, for all) MAPC cultures compared to MSC cultures treated with neural induction medium had significantly higher fold change expression of NF-200 (0.64), GFAP (0.52), Tau (0.59), MAP-2 (0.72), Olig-1 (0.18) and NSE (0.29) proteins (P < 0.01 for Olig-1 and P < 0.001 for rest) as well as higher fold change expression of genes of NF-200 (1.34),GFAP (1.12),Tau (1.08),MAP-1B (0.92), MAP-2 (1.14) andNSE (0.4) (P < 0.001 for all). CONCLUSION: MAPC can be differentially characterized from MSC as Oct-4 and Nanog positive stem cells with no expression of HLA-ABC and low expression of mesenchymal markers CD44, CD73 and CD105 and when compared to MSC they possess greater predilection for differentiation into neuro-ectodermal lineage.

人骨髓间充质干细胞通过YAP影响人脂肪肉瘤SW872细胞的生物学行为

目的:观察人骨髓间充质干细胞(hMSCs)条件培养基(CM)与人脂肪肉瘤SW872细胞共培养后对肿瘤细胞增殖和迁移能力的影响,探讨hMSCs CM对脂肪肉瘤细胞的作用及可能的作用机制。方法:体外培养hMSCs,采用慢病毒方法分别转染慢病毒空载体shNS(对照组)和慢病毒shRNA Yes相关蛋白(YAP)(shYAP-hMSCs组),采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测各组hMSCs中YAP mRNA和蛋白表达水平,提取CM。体外培养SW872细胞,分为对照组(正常培养)、hMSCs CM组和shYAP-hMSCs CM组。采用CCK-8法检测各组细胞增殖活性,流式细胞术检测各组细胞凋亡率,细胞划痕实验检测各组细胞划痕愈合率,Western blotting法检测各组细胞中YAP、基质金属蛋白酶9(MMP-9)和细胞周期蛋白D1(cyclin D1)蛋白表达水平。结果:与对照组比较,shYAP-hMSCs组hMSCs中YAP mRNA和蛋白表达水平降低(P<0.01),表明成功构建了shYAP-hMSCs稳定转染细胞株。CCK-8法,与对照组比较,hMSCs CM组SW872细胞增殖活性升高(P<0.05),shYAP-hMSCs CM组SW872细胞增殖活性降低(P<0.01);流式细胞术,与对照组比较,hMSCs CM组SW872细胞凋亡率无明显变化(P>0.05),shYAP-hMSCs CM组SW872细胞凋亡率升高(P<0.01);细胞划痕实验,与对照组比较,hMSCs CM组SW872细胞划痕愈合率升高(P<0.05),shYAP-hMSCs CM组SW872细胞划痕愈合率降低(P<0.01);Western blotting法,与对照组比较,hMSCs CM组SW872细胞中YAP、MMP-9和cyclin D1蛋白表达水平差异无统计学意义(P>0.05),shYAP-hMSCs组SW872细胞中YAP、MMP-9和cyclin D1蛋白表达水平降低(P<0.05或P<0.01)。结论:hMSCs参与调控人脂肪肉瘤SW872细胞增殖和迁移,其机制可能与YAP表达有关。

人甲床来源的脱细胞支架搭载骨髓间充质干细胞向指甲干细胞分化的研究

目的探究人甲床来源的脱细胞支架搭载骨髓间充质干细胞向指甲干细胞分化的作用。方法收集2022年至2023年在承德医学院附属医院手足外科进行截指术的15例患者的临床废弃甲床组织,制备成脱细胞甲床支架,使用试剂盒检测参照组(未脱细胞组)与脱细胞组(脱细胞甲床支架组)中总胶原蛋白含量、残留DNA含量;将脱细胞甲床支架与人骨髓间充质干细胞(hMSCs)进行共培养14 d。比较对照组(hMSCs组)与共培养组(脱细胞甲床支架+hMSCs组)体外细胞分化能力及指甲干细胞标志物[角蛋白15,角蛋白17,G蛋白偶联受体6(Lgr6)以及β-联蛋白(β-catenin)蛋白]含量。结果脱细胞组总胶原蛋白含量为(189.62±45.45)μg/mg,低于参照组[(196.02±41.93)μg/mg],但两组相比差异无统计学意义(P>0.05);脱细胞组中DNA含量为(0.41±0.15)μg/mg,明显低于参照组[(0.87±0.13)μg/mg],差异有统计学意义(P<0.05)。培养14 d后,共培养组吸光度值为1.09±0.07,对照组吸光度值为1.10±0.5,两组相比差异无统计学意义(P>0.05)。培养14 d后,共培养组角蛋白15、角蛋白17、Lgr6、β-catenin含量分别为(39.56±5.09)、(45.83±4.01)、(5.74±0.99)、(146.79±5.34)pg/mL,均明显高于对照组[(12.10±4.28)、(10.47±3.19)、(0.93±0.67)、(67.28±7.41)pg/mL],差异均有统计学意义(P<0.05)。结论脱细胞甲床支架中的DNA有被清除、保留胶原蛋白,与hMSCs共培养后细胞增殖能力正常,且可诱导hMSCs向指甲干细胞分化,为临床上治疗甲床缺损提供新思路。

Construction of a bicistronic recombinant adenoviral vector for human interleukin-10 and enhanced green fluorescent protein expression in bone marrow mesenchymal stem cells

Background Human interleukin-10 （hlL-10） is a cytokine synthesis inhibitory factor,which is involved in various immune responses.The purpose of this study was to construct an adenoviral vector carrying the hlL-10 gene for expression of biologically active hlL-10 in rat bone marrow mesenchymal stem cells （rMSCs）.Methods A pSNAV2.0-hlL10 plasmid was used as a template to obtain a hlL-10 cDNA fragment that was subcloned by restriction enzyme digestion and ligation into a pDC316-IRES-EGFP-lacZ alpha plasmid carrying an enhanced green fluorescent protein （EGFP） marker gene.The pDC316-hlL-10-IRES-EGFP plasmid was linearized by Pmel digestion and used to transfect HEK293 packaging cells using the adenovirus packaging system AdMax.Virus particles were amplified by repeatedly infecting HEK293 cells with the seed virus and then purified by ion exchange.After the number of virus particles and titer was determined,rMSCs were infected with the adenoviral vector.The infection rate was determined by fluorescence microscopy and flow cytometry,and hlL-10 protein expression in rMSCs was measured by Western blotting.Results The virus particle concentration,OD260/280 value and virus titer of the amplified and purified recombinant adenovirus were 3.2×1011 VP/ml,approximately 2.0,and 1.1×1010 TCID50/ml,respectively.Bright green fluorescence was observed by fluorescence microscopy and flow cytometry in the recombinant adenovirus-infected rMSCs.GFP expression was considered the multiplicity of infection （MOI） and was time-dependent.The infection rate was 92.9% at 100 MOI.Conclusions A bicistrenic recombinant adenoviral vector for hlL-10 and EGFP gene expression were successfully constructed.The infection rate of rMSCs by the adenovirus was high （92.9% at 100 MOI） and the target gene hlL-10 was highly expressed in cells.The present study provides an experimental basis for further research of immunosuppressive therapy using hlL-10.The expression level of hlL-10 protein as detected by Western blotting was also MOI- and time-dependent.

Human bone marrow mesenchymal stem cell-derived extracellular vesicles inhibit shoulder stiffness via let-7a/Tgfbr1 axis

Shoulder stiffness(SS)is a common shoulder disease characterized by increasing pain and limited range of motion.SS is considered to be an inflammatory and fibrotic disorder pathologically.However,there is no consensus on the most effective conservative treatment for fibrosis.Given that human Bone Marrow Mesen-chymal Stem Cell-derived extracellular vesicles(BMSC-EVs)displayed promising therapeutic effects for various tissues,we investigated the therapeutic effect of BMSC-EVs on fibrosis in a mice immobilization model and two cell models.By conducting a series of experiments,we found that BMSC-EVs can significantly inhibit the fibrogenic process both in vitro and in vivo.In detail,BMSC-EVs suppressed the aberrant proliferation,high collagen production capacity,and activation of fibrotic pathways in TGF-β-stimulated fibroblasts in vitro.Besides,in vivo,BMSC-EVs reduced cell infiltration,reduced fibrotic tissue in the shoulder capsule,and improved shoulder mobility.In addition,via exosomal small RNA sequencing and qPCR analysis,let-7a-5p was verified to be the highest expressed miRNA with predicted antifibrotic capability in BMSC-EVs.The antifibrotic capacity of BMSC-EVs was significantly impaired after the knockdown of let-7a-5p.Moreover,we discovered that the mRNA of TGFBR1(the membrane receptor of transforming growth factorβ)was the target of let-7a-5p.Together,these findings elucidated the antifibrotic role of BMSC-EVs in shoulder capsular fibrosis.This study clarifies a new approach using stem cell-derived EVs therapy as an alternative to cell therapy,which may clinically benefit patients with SS in the future.

慢病毒沉默Piezo1蛋白与人骨髓间充质干细胞成骨分化及TAZ的表达

背景:Piezo1作为机械敏感蛋白与成骨分化密切相关,TAZ也被证明参与调节成骨分化,但Piezo1调控人骨髓间充质干细胞成骨分化时TAZ是否参与其中目前尚不明确,故研究其具体机制对防治股骨头坏死具有重要意义。目的:探讨Piezo1对人骨髓间充质干细胞成骨分化及TAZ表达的影响。方法:构建靶向Piezo1的siRNA,转染至293T细胞,通过RT-qPCR检测其沉默效率并筛选出Piezo1-Homo-2337进行包装,使用荧光染色检测其最佳感染复数值。Piezo1沉默重组慢病毒进一步被转染至人骨髓间充质干细胞中,通过RT-qPCR、Western blot检测其沉默效果;通过茜素红染色、碱性磷酸酶活性分析、免疫荧光染色、RT-qPCR及Western blot检测分析沉默Piezo1对人骨髓间充质干细胞成骨分化能力及TAZ表达的影响。结果与结论:①与正常组、阴性对照组比较,转染si-Piezo1后人骨髓间充质干细胞中Piezo1的mRNA及蛋白水平明显降低;②与阴性对照组比较,si-Piezo1组碱性磷酸酶活性明显降低,钙沉积明显减少;③与阴性对照组比较,si-Piezo1组成骨相关基因Runx2、OPN、DLX5、osteocalcin、β-catenin及TAZ的mRNA水平明显降低。si-Piezo1组TAZ、β-catenin的蛋白表达明显降低;相反,si-Piezo1组p-TAZ、p-β-catenin的蛋白表达明显增加;④与阴性对照组比较,免疫荧光染色结果提示si-Piezo1组人骨髓间充质干细胞中TAZ、β-catenin的表达较少;⑤结果表明Piezo1可促进人骨髓间充质干细胞的成骨分化,沉默Piezo1后人骨髓间充质干细胞的成骨能力明显降低,同时TAZ的表达也同样降低。

骨痹通消颗粒对激素型股骨头坏死人骨髓间充质干细胞成骨与成脂分化的影响

目的 探究骨痹通消颗粒对激素性股骨头坏死人骨髓间充质干细胞(h BMMSC)成骨与成脂分化的影响。方法 取激素性股骨头坏死患者骨髓,体外进行h BMMSC的培养,通过细胞形态学观察、成骨及成脂分化潜能来鉴定h BMMSC。以完培组(基础培养基+10%的胎牛血清)作为对照,采用CCK-8法测定1%、2%、5%、8%、10%体积分数的骨痹通消含药血清A组作用24 h后对细胞增殖的影响。细胞在96孔板的培养过程中,每孔加入地塞米松溶液0.52μl,以完培组(基础培养基+10%的胎牛血清)和损伤组(基础培养组+10%的胎牛血清+0.52μl地塞米松溶液)作为对照,采用CCK-8法测定2%、5%、8%体积分数的骨痹通消含药血清B组对激素环境中细胞活力的影响。以无激素诱导细胞作为对照组(基础培养基+10%的胎牛血清),将体外激素诱导的h BMMSC细胞分为模型组(基础培养基+10%的胎牛血清+10-5mol/L地塞米松溶液)、实验组(基础培养基+5%的骨痹通消含药血清+10-5mol/L地塞米松溶液)。茜素红染色试剂盒测定各组成骨分化后矿化结节的形成;油红O染色试剂盒测定各组成脂分化后脂滴的形成;RT-q PCR测定成脂相关标志基因PPARγ、C/EBP-α与Fabp4中m RNA的表达,Western blot检测成骨相关蛋白BMP-2、Runx2及β-catenin的蛋白表达含量。结果 原代细胞培养7 d后,可见梭形、纺锤形或多角形的贴壁细胞。第三代细胞生长均匀,平行排列,透光率好。成骨诱导21 d,细胞发生改变,局部细胞聚集,并有矿化结节产生,茜素红染色呈阳性;成脂诱导21 d,脂滴与油红O染液结合变为红色,油红O染色呈阳性。与完培组比较,10%体积分数的含药血清A组细胞活力下降(P<0.05)。与完培组比较,损伤组细胞活力下降(P<0.05);与损伤组比较,2%、5%、8%体积分数的含药血清B组细胞活力升高(P<0.05)。与对照组比较,模型组染色面积降低(P<0.05);与模型组比较,实验组中矿化结节与沉积升高,染色范围也更广(P<0.05)。与对照组比较,模型组细胞内脂质积累增多(P<0.05);与模型组比较,实验组较细胞内脂质积累降低(P<0.05)。与对照组比较,模型组PPARγ、C/EBP-α和Fabp4的表达量升高(P<0.01),与模型组比较,实验组PPARγ、C/EBP-α和Fabp4的表达量降低(P<0.05)。与对照组比较,模型组BMP-2、Runx2及β-catenin蛋白表达含量升高(P<0.01),与模型组比较,实验组BMP-2、Runx2及β-catenin蛋白表达含量降低(P<0.05)。结论 骨痹通消颗粒能够促进激素性股骨头坏死h BMMSC的增殖及其向成骨分化的能力,抑制成脂分化,为临床上防治激素性股骨头坏死提供了理论基础。

LncRNA NEAT1抑制细胞焦亡促进人骨髓间充质干细胞的成骨分化

目的探讨长链非编码RNA(lncRNA)核旁斑组装转录本1(NEAT1)通过调节细胞焦亡调控人骨髓间充质干细胞(hBMSCs)成骨分化的作用。方法培养hBMSCs 7 d诱导细胞成骨分化,并分为对照组(常规培养)、成骨分化组(成骨分化诱导)、pcD-NEAT1组(转染NEAT1过表达质粒)、pcD-null组(转染NEAT1过表达质粒阴性对照)、成骨分化+CML组[成骨分化诱导联合NOD样受体家族蛋白3(NLRP3)炎性小体激活剂(Nε)-羧甲基赖氨酸(CML)处理]、成骨分化+CML+pcD-NEAT1组(成骨分化诱导联合pcD-NEAT1与CML处理)。经茜素红染色检测细胞矿化程度;碱性磷酸酶(ALP)活性检测试剂盒检测ALP活性;CCK-8检测各组细胞存活率,高倍镜下观察细胞形态变化。TUNEL实验检测细胞凋亡率。qRT-PCR检测NEAT1的表达。Western blot检测IL-1β、IL-18、NLRP3、cleaved-caspase 1(cleaved-CASP1)、gasdermin D、Runt-相关转录因子2(RUNX2)、ALP、骨桥蛋白(OPN)的表达。结果与对照组比较,成骨分化组细胞的矿化程度增加,ALP活性升高(P<0.05),NEAT1和RUNX2、ALP、OPN蛋白表达均上调(P<0.05)。与pcD-null组比较,pcD-NEAT1组细胞的矿化程度增加,ALP活性升高(P<0.05),NEAT1和RUNX2、ALP、OPN蛋白表达均上调(P<0.05)。与成骨分化组比较,成骨分化+CML组细胞矿化程度减轻,ALP活性降低(P<0.05),细胞存活率降低(P<0.05),细胞凋亡率增加(P<0.05),细胞膜出现破裂,细胞膨大变形,NLRP3和IL-1β、IL-18、cleaved-CASP1、gasdermin D蛋白表达均显著上调(P<0.05),而RUNX2、ALP、OPN蛋白表达均显著下调(P<0.05)。与成骨分化+CML组比较,成骨分化+CML+pcD-NEAT1组细胞矿化程度增加,ALP活性升高(P<0.05),RUNX2、ALP、OPN蛋白表达均显著上调(P<0.05),细胞存活率增加(P<0.05),细胞凋亡率降低(P<0.05),细胞膜较完整且细胞形态正常,NLRP3和IL-1β、IL-18、cleaved-CASP1、gasdermin D蛋白表达均显著下调(P<0.05)。结论NEAT1过表达通过抑制NLRP3炎性小体介导的细胞焦亡促进hBMSCs的成骨分化。

人骨髓间充质干细胞及其外泌体在肝移植术后肝缺血再灌注损伤中潜在治疗作用的研究进展

肝缺血再灌注损伤(HIRI)是传统肝移植术中不可避免的病理生理过程,其严重程度受到来自供体和受体的多方面因素影响,最严重者可导致术后早期移植肝无功能。人骨髓间充质干细胞(BMSCs)及其外泌体已在临床试验中被证实具有促进组织修复与免疫调节的能力,这一治疗作用在HIRI的防治中具有较好的应用前景。该文旨在综述BMSCs及其外泌体在移植肝HIRI防治中潜在作用机制的研究进展及未来研究方向。

白藜芦醇对人骨髓间充质干细胞自发衰老的抑制作用

目的:观察白藜芦醇(RSV)对人骨髓间充质干细胞(MSC)的自发衰老是否具有抑制作用。方法:将MSC传至p13和p15代,建立MSC的自发衰老模型组;用5 nmol/L RSV分别处理p13和p15代MSC 48 h,建立RSV处理组。通过SA-β-Gal染色法检测细胞衰老,MTT法检测细胞增殖,RT-PCR检测细胞衰老相关端粒酶活性,Western blot检测与衰老相关的磷酸化mTOR的水平。结果:SA-β-Gal染色显示,RSV处理组MSC的衰老细胞数量明显少于模型组(p13代MSC,RSV组vs模型组,P<0.05;p15代MSC,RSV组vs模型组,P<0.01)。MTT结果显示,RSV处理组MSC的增殖能力高于模型组,其中p13代MSC处理组与模型组在72 h相比有显著差异(P<0.05)。RT-PCR结果显示,RSV处理组中MSC的hTERT mRNA表达量高于模型组,其中p13代MSC的RSV处理组与模型组相比有显著差异(P<0.05)。Western blot结果显示,RSV处理组中MSC的磷酸化(Ser2448)mTOR水平低于模型组,其中p13代MSC的处理组与模型组相比有显著差异(P<0.05)。结论:RSV可通过调节mTOR活性抑制人MSC的自发衰老。