Objective Dexamethasone is one of the basic agents which could induce osteogenic differentiation of mesenchymal stem cells. To investigate the optimal concentration of dexamethasone in osteogenic differentiation of adiposederivedstem cells (ADSCs) so as to provide the theoretical basis for further bone tissue engineering researches. Methods FiveNew Zealand rabbits (2-3 kg) of clean grade, aged 3 months and male or female, were obtained. ADSCs were isolated from the subcutaneous adipose tissue of inguinal region, and cultured with collagenase digestion, then were detected and identified by CD44, CD106 immunofluorescence staining and adi pogenic differentiation. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 105 cells/mL, then the cells were treated with common cultural medium (group A) and osteogenic induced medium containing 0 (group B), 1 × 10-9 (group C), 1 × 10-8 (group D), 1 × 10-7 (group E), 1 × 10-6 (group F), and 1 × 10-5 mol/ L (group G) dexamethasone, respectively. The cell prol iferation and the mRNA expressions of osteocalcin (OC) and core binding factor α1 (Cbfα1) were detected by MTT and RT-PCR, respectively. The activity of alkal ine phosphatase (ALP) was measured, and the percentage of mineral area was calculated. The mineral nodules were also detected by al izarin red staining. Results ADSCs mostly presented fusiform and polygon shape with positive expression of CD44 and negative expression of CD106. The result of oil red O staining was positive after ADSCs treated with adipogenic induced medium. The result of MTT revealed that the absorbance (A) value decl ined with the ascending of the concentration of dexamethasone, and there was significant difference in A value between groups D and E at 5 and 7 days after osteogenic induction (P lt; 0.05). The mRNA expressions of OC and Cbfα1 reached the peak in groups E and D at 7 days after osteogenic induction, respectively. The activity of ALP and the percentage of mineral area had the maximum value in group D at 14 days, then decl ined gradually. There was no significant difference in the mRNA expressions of OC and Cbfα1, the activity of ALP, and the percentage ofmineral area between groups D and E (P gt; 0.05), but significant differences were found between groups D and E and other groups (P lt; 0.05). After 14 days, the cells of group G died, and the result of al izarin red staining was positive in groups B, C, D, E, and F. Conclusion When the concentration of dexamethasone in osteogenic medium is 1 × 10-8 mol/L, it could not only reduce the inhibitive effect on cells prol iferation, but also induce osteogenic differentiation of ADSCs more efficiently.
ObjectiveTo investigate the multi-directional differentiation potential and other biological characteristics of chicken umbilical cord mesenchymal stem cells (UMSC), as well as their reparative effects on bleomycin (BLM)-induced lung injury in mice. MethodsAn acute lung injury model in mice was established by injecting BLM into the bronchus. UMSC were then transplanted via the tail vein. The reparative effects of UMSC on lung injury were evaluated through pathological section observation, survival and differentiation of transplanted cells in mice, and detection of hydroxyproline (HYP) content, among other indicators. ResultsThe UMSC successfully isolated in this study positively expressed specific surface markers CD29, CD44, CD90, and CD166, while the expression of CD34 and CD45 was negative. Induced UMSC could differentiate into adipocytes, osteocytes, chondrocytes, and alveolar epithelial cells. Animal experiments revealed that BLM-treated mice exhibited damaged alveolar structures, significant inflammatory cell infiltration, abnormal collagen deposition, and pulmonary fibrosis. However, after UMSC transplantation, the extent and severity of lung damage were reduced, and the HYP content in lung tissue decreased but remained higher than that of the control group. ConclusionUMSC can continuously proliferate and maintain their biological characteristics under in vitro culture conditions. They possess the ability to migrate to damaged sites and undergo directional differentiation, demonstrating a certain reparative effect on BLM-induced acute lung injury in mice.
ObjectiveTo study the effect of transforming growth factor β3 (TGF-β3), bone morphogenetic protein 2 (BMP-2), and dexamethasone (DEX) on the chondrogenic differentiation of rabbit synovial mesenchymal stem cells (SMSCs).
MethodsSMSCs were isolated from the knee joints of 5 rabbits (weighing, 1.8-2.5 kg), and were identified by morphogenetic observation, flow cytometry detection for cell surface antigen, and adipogenic and osteogenic differentiations. The SMSCs were cultured in the PELLET system for chondrogenic differentiation. The cell pellets were divided into 8 groups: TGF-β3 was added in group A, BMP-2 in group B, DEX in group C, TGF-β3+BMP-2 in group C, TGF-β3+DEX in group E, BMP-2+DEX in group F, and TGF-β3+BMP-2+DEX in group G; group H served as control group. The diameter, weight, collagen type II (immuohistochemistry staining), proteoglycan (toluidine blue staining), and expression of cartilage related genes [real time quantitative PCR (RT-qPCR) technique] were compared to evaluate the effect of cytokines on the chondrogenic differentiation of SMSCs. Meanwhile, the DNA content of cell pellets was tested to assess the relationship between the increase weight of cell pellets and the cell proliferation.
ResultsSMSCs were isolated from the knee joints of rabbits successfully and the findings indicated that the rabbit synovium-derived cells had characteristics of mesenchymal stem cells. The diameter, weight, collagen type II, proteoglycan, and expression of cartilage related genes of pellets in groups A-F were significantly lower than those of group G (P<0.05). RT-qPCR detection results showed that the relative expressions of cartilage related genes (SOX-9, Aggrecan, collagen type II, collagen type X, and BMP receptor II) in group G were significantly higher than those in the other groups (P<0.01). Meanwhile, with the increase of the volume of pellet, the DNA content reduced about 70% at 7 days, about 80% at 14 days, and about 88% at 21 days.
ConclusionThe combination of TGF-β3, BMP-2, and DEX can make the capacity of chondrogenesis of SMSCs maximized. The increase of the pellet volume is caused by the extracellular matrix rather than by cell proliferation.
Objective To evaluate the clinical relationship between serum carcinoembryonic antigen (CEA) and mortality of anti-melanoma differentiation associated gene 5 (MDA5) antibody positive dermatomyositis with interstitial lung disease (ILD). MethodsThe consecutive clinical data of 214 patients with anti MDA5 antibody positive dermatomyositis from West China Hospital of Sichuan University from February 2017 to September 2019 were collected retrospectively, including demographic, laboratory examination and imaging examination data. Patients were divided into CEA elevated group (CEA≥4.63 ng/mL) and CEA normal group (CEA<4.63 ng/mL) according to CEA level. R4.1.2 software was used for statistical analysis of all data, and Kaplan Meier method was used to draw the survival curve. Cox proportional hazard model was used to analyze the survival of patients with ILD, and to explore the risk factors associated with the survival of patients with anti-MDA5 antibody positive dermatomyositis with ILD. Results There were 180 patients with ILD who met the inclusion and exclusion criteria, 57 patients with rapidly progressive pulmonary interstitial fibrosis (RPILD), and 123 patients without RPILD; 121 women and 59 men, with an average age of 50.2±10.7 years; The average follow-up was 23.5 months, and 52 patients died. Univariable analysis suggested that CEA≥4.63 ng/mL, smoking, RPILD, lactate dehydrogenase (LDH) ≥321 IU/L, albumin<30 g/L and dyspnea were risk factors associated with death in patients with anti MDA5 dermatomyositis combined with ILD. Multivariable Cox regression analysis showed that CEA≥4.63 ng/mL [hazard ratio (HR) =3.01, 95% confidence interval (CI) 1.23 - 7.32, P=0.015], RPILD (HR=3.87, 95%CI 2.09 - 7.19, P<0.001), smoking (HR=2.37, 95%CI 1.25 - 4.47, P=0.008), LDH≥321 IU/L (HR=2.47, 95%CI 1.23 - 4.96, P=0.011), albumin<30 g/L (HR=2.57, 95%CI 1.38 - 4.78, P=0.003) were independent predictors for mortality. ConclusionsSerum CEA level can be used as a clinical prognostic predictor in patients with anti-MDA5 positive dermatomyositis and ILD. RPILD, smoking, LDH≥321 IU/L, and albumin<30 g/L are independent predictors for mortality.
ObjectiveTo summarize the mechanism of long non-coding RNA (lncRNA) in signal pathways related to osteogenic differentiation. Methods Relevant domestic and foreign researches in recent years were consulted. The characteristics and biological functions of lncRNA were introduced, and the specific mechanism of lncRNA regulating related signal pathways in osteogenic differentiation was elaborated. Results The exertion and maintenance of normal function of bone requires the closed coordination of transcription networks and signal pathways. However, most of these signal pathways or networks are dysregulated under pathological conditions that affect bone homeostasis. lncRNA can regulate the differentiation of various bone cells by activating or inhibiting signal pathways to achieve the balance of bone homeostasis, thereby reversing the pathological state of bones and achieving the purpose of treating bone metabolic diseases. Conclusion At present, the research on the mechanism of lncRNA regulating various osteogenic differentiation pathways is still in the early stage. Its in-depth regulator mechanism, especially the cross-talk of complex signal pathways needs to be further studied. And how to apply these molecular targets to clinical treatment is also a big challenge.
The purpose of this study was to identify specific microRNAs (miRNAs) during differentiation and maturation of interneurons and to predict their possible functions by analyzing the expression of miRNAs during in vitro differentiation of the rat interneuron precursor cell line GE6. In the experiment, the interneuron precursor cell line GE6 was cultured under three different conditions, i.e. the first was that had not added growth factors and the normal differentiation cultured for 4 days (Ge6_4d); the second was that cultured with bone morphogenetic protein-2 (BMP2) for 4 days (Ge6_bmp2); and the third was that cultured with sonic hedgehog (SHH) for 4 days (Ge6_shh). In addition, another group of undifferentiated GE6 (Ge6_u) was applied as a control. We found in this study that the expression levels of a large number of miRNAs changed significantly during GE6 differentiation. The expression levels of miR-710, miR-290-5p and miR-3473 increased in the GE6 cells with secreted factor BMP2. These miRNAs may play important regulatory roles during interneuron differentiation.
Objective
To review the latest development of amniotic fluid-derived stem cells (AFSCs) in regenerative medicine, and to discuss issues related to the studies in the field of AFSCs.
Methods
The recent articles about AFSCs were extensively reviewed. The important knowledge of AFSCs was introduced in the field of regenerative medicine, and the basic and clinical researches of AFSCs were summarized and discussed.
Results
Currently, it is confirmed that AFSCs have a multi-directional differentiation capacity, therefore, they have a wide application prospect in regenerative medicine, anti-tumor, and other fields.
Conclusion
AFSCs will become one of the ideal seed cells in the field of regenerative medicine with extensive research value because of the advantages of easy amniotic fluid sampling, little maternal and child trauma, no tumorigenesis, and no ethical restrictions.
Objective To clarify the trends of expression levels of several up-regulated micro RNA (miRNA) in tissues of atrophic bone nonunion and mRNAs and proteins of their related target genes in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and to explore their biological functions. Methods The hBMSCs were isolated from bone marrow of il iac bone by gradient centrifugation, and cultured. Osteogenic culture medium was used for osteogenic differentiation of the 4th generation of hBMSCs. The changes of corresponding miRNAs, mRNA and protein expression levels of related target genes were observed at 0 hour, 12 hours, 1 day, 2 days, 4 days, 7 days, and 14 days, by quantitative real-time PCR and Western blot. Results In the process of hBMSCs osteogenic differentiation, the mRNA and protein expression levels of osteoblastic target genes [alkal ine phosphatase l iver/bone/kidney (ALPL), bone morphogeneticprotein 2 (BMP-2), and platelet-derived factor alpha polypeptide (PDGF-A)] at most time points increased significantly whencompared with the values at 0 hour except that of BMP-2 decreased at 12 hours and 1 day, with maximum changes at 1 to 7 days. The miRNA expression levels, mRNA and protein expression levels changed significantly at different time points, while the trends of hsa-miRNA-149 and hsa-miRNA-654-5p changes were negatively correlated with the trends of ALPL and BMP-2 mRNA and protein expression changes respectively (P lt; 0.05). There was no obviously negative correlation between the trends of hsa-miRNA-221 change and PDGF-A change (P gt; 0.05). Conclusion In the osteogenic differentiation process of hBMSCs, hsa-miRNA-149 and hsa-miRNA-654-5p are closely related with the mRNA and protein regulation of ALPL and BMP-2, respectively.
ObjectiveTo explore the effect and mechanism of miR-21 down-regulated which was induced by H2O2 on osteogenic differentiation of MC3T3-E1 cells.MethodsMC3T3-E1 cells were cultured and passaged, and the 7th generation cells were harvested to use in experiment. The MC3T3-E1 cells were treated with different concentrations (0, 40, 80, 160, and 320 μmol/L) of H2O2. The expression of miR-21 was detected by real-time quantitative PCR (RT-PCR) and the cell viability was determined by MTS. Then the appropriate concentration of H2O2 was obtained. To analyze the effect of H2O2 on osteogenic differentiation of MC3T3-E1 cells, the MC3T3-E1 cells were divided into blank control group (group A), H2O2 group (group B), osteogenic induction group (group C), and H2O2+osteogenic induction group (group D). The expression of miR-21 and the osteogenesis related genes expressions of Runx2, osteopontin (OPN), and collagen type Ⅰ alpha 1 (Col1a1) were detected by RT-PCR. The expression of phosphatase and tensin homolog (PTEN) was detected by Western blot. The extracellular calcium deposition was detected by alizarin red staining. To analyze the effect on osteogenic differentiation of MC3T3-E1 cells after the transfection of miR-21 inhibitor and siRNA-PTEN, the MC3T3-E1 cells were divided into H2O2 group (group A1), H2O2+osteogenic induction group (group B1), H2O2+osteogenic induction+miR-21 inhibitor group (group C1), and H2O2+osteogenic induction+miR-21 inhibitor negative control group (group D1); and H2O2 group (group A2), H2O2+osteogenic induction group (group B2), H2O2+osteogenic induction+siRNA-PTEN negative control group (group C2), and H2O2+osteogenic induction+siRNA-PTEN group (group D2). The osteogenesis related genes were detected by RT-PCR and the extracellular calcium deposition was detected by alizarin red staining.ResultsThe results of MTS and RT-PCR showed that the appropriate concentration of H2O2 was 160 μmol/L. The expression of miR-21 was significantly lower in group B than in group A at 1 and 2 weeks (P<0.05). The expression of miR-21 was significantly lower in group D than in group C at 1 and 2 weeks (P<0.05). The expression of PTEN protein was significantly lower in group C than in groups A and D (P<0.05). The mRNA expressions of Runx2, OPN, and Col1a1 were significantly lower in group D than in group C at 1 and 2 weeks (P<0.05). The extracellular calcium deposition in group D was obviously less than that in group C. The expression of PTEN protein was significantly higher in group C1 than in group D1 (P<0.05). The mRNA expressions of Runx2 and OPN were significantly lower in group C1 than in groups B1 and D1 at 1 and 2 weeks (P<0.05). The mRNA expression of Col1a1 was significantly lower in group C1 than in groups B1 and D1 at 2 weeks (P<0.05). The extracellular calcium deposition in group C1 was obviously less than those in groups B1 and D1. The mRNA expressions of OPN and Col1a1 were significantly higher in group D2 than in groups B2 and C2 at 1 week (P<0.05). The extracellular calcium deposition in group D2 was obviously more than those in groups B2 and C2.ConclusionH2O2 inhibits the osteogenic differentiation of MC3T3-E1 cells, which may be induced by down-regulating the expression of miR-21.
Objective To observe the expression of genes related to hereditary retinal diseases (IRD) in human microglia (hMG). MethodsA experimental study. Efficient differentiation of human induced pluripotent stem cells (iPSC) into hMG. Identification of octamer-binding transcription factor 4 (OCT4), sex-determining transcription factor 2 (SOX2), Nanog homeobox (NANOG), stage-specific embryonic antigen-4 (SSEA4), alpha-fetoprotein (AFP), α-smooth muscle actin (α-SMA) as markers associated with iPSC dryness and pluripotency by immunofluorescence staining Glial fibrillary acidic protein (GFAP); hMG associated marker transmembrane protein 119 (TMEM119), purinergic receptor P2Y12 (P2RY12), and allograft inflammatory factor 1 (IBA1). The proportion of CD11b+ and CD45+ cells was detected by flow cytometry. Mature hMG was collected and stimulated with lipopolysaccharide for 0, 4, 8 and 12 h, and were divided into groups 0 h, 4 h, 8 h and 12 h, respectively. Total RNA samples from the 4 groups were extracted for transcriptome sequencing, and the persistently significant differentially expressed genes (DEG) were screened. Real-time quantitative polymerase chain reaction (qPCR) was used to verify and analyze the expression of DEG mRNA. The two-tailed Student t test was used for comparison between the two groups. ResultsiPSC expressed the dry related markers OCT4, SOX2, NANOG and SSEA4, and differentiated into endoderm, mesoderm and ectoderm, expressing the corresponding markers AFP, α-SMA and GFAP, respectively. iPSC formed embryoid bodies under specific culture conditions, and then differentiated into hMG, and hMG expressed related markers TMEM119, P2RY12 and IBA1 by immunofluorescence staining. The double positive ratio of CD11b+ and CD45+ was > 95%. Transcriptomic analysis showed that the expression of 18 DEG in hMG stimulated by LPS was changed. qPCR test results showed that compared with group 0 h, mRNA expressions of Toll-like receptor 4 (TLR4), phosphoglycerate kinase 1, disintegrin and metallopeptidase domain 9 (ADAM9) in LPS stimulated group 4 h were significantly increased (t=25.43, 15.54, 6.26; P＜0.01). The mRNA expression levels of MER proto-oncogene tyrosine kinase (MERTK), non-hydrolase domain containing lysophospholipase 12 (ABHD12), retinal dehydrogenase 11 (RDH11), DNA damage autophagic regulator 2 (DRAM2) decreased (t=5.94, 14.14, 8.21, 6.97; P＜0.01), and the differences were statistically significant. Compared with group 0 h, mRNA expressions of RDH11, MERTK, ABHD12, DRAM2 and ADAM9 in group 8 h stimulated by LPS were significantly decreased, with statistical significance (t=25.97, 5.47, 43.97, 38.40, 3.84; P＜0.05). Compared with the group 0 h, the mRNA expressions of TLR4, ADAM9, MERTK, ABHD12, RDH11 and DRAM2 in the 12 h stimulated group were significantly decreased, and the differences were statistically significant (t=6.39, 46.11, 5.34, 14.14, 25.97, 25.65; P＜0.05). ConclusionIRD-related genes may be involved in the occurrence and development of IRD by regulating the function of hMG.
