Objective To study the method of inducing human marrow mesenchymal stem cells (MSCs) into osteoblasts directionally and to identify osteogenesis characteristics. Methods MSCs were isolated from adult marrow using density gradient separation method and were cultured in conditioned medium containing Dex 10 -8 mol/L，β-GP 10 mmol/L，and AA 50 μg/ml. The MSCs attachment formed soon and passage 3 cells were chosen to check osteogenesis characteristics, including alkaline phosphatase assay with modified calcium-cobalt staining method, type Ⅰ collagen assay with immunohistochemistry, osteopontin and osteonectin assay with in situ hybridization and calcium nodes assay with Von Kossa staining. Results Passage 3 MSCs had typical appearance of osteoblasts and could be passaged continuously till passage 10. The rate of ALP expression was 85%. The expressions of collagen type Ⅰ, osteopontin and osteonectin were positive and calcium nodeswere seen by Von Kossa staining. Conclusion We have successfully induced human MSCs into osteoblasts; the induced cells have typical osteogenesis characteristics.
ObjectiveTo observe the clinical efficacy of Xiao’er kang xian capsule added to anti-seizure medications (ASMs) in the treatment of children with refractory epilepsy and its influence on serum neuron-specific enolase (NSE) and cludter of differentiation 19+ (CD19+) levels. Methods A total of 60 children with refractory epilepsy were selected from the pediatric outpatient department and ward of Guangdong Provincial People's Hospital from February 2021 to June 2023. The study subjects were divided into two groups by numerical random method,with 30 cases in each group. The children with Xiao’er kang xian capsule added to the original treatment were the treatment group and the children without Xiao’er kang xian capsule added to the original treatment were the control group. The frequency, duration, EEG characteristics, adverse reactions and changes in serum NSE and CD19+ levels of the two groups were compared after treatment. Results Self-control before and after treatment in the treatment group: the frequency and duration of seizures were significantly reduced, with statistical difference (P<0.05). EEG discharge index in awake period and sleep period were significantly decreased, with statistical difference (P<0.05). After 6 months of treatment, comparison between the two groups of children: the seizure frequency of children in the treatment group was significantly decreased compared with the control group (P=0.03). There was a statistical difference (P<0.05), and the seizure duration in the treatment group was less than that in the control group (P=0.863), the clinical effective rate of treatment group 83.33% was higher than that of control group 63.33% (P=0.08), the effective rate of EEG in treatment group 80% was higher than that of control group 60% (P=0.091), serum NSE and CD19+ in treatment group were lower than that of control group, with no statistical difference (P>0.05). After 12 months of treatment, the frequency and duration of seizures in the treatment group were significantly decreased (P<0.05). The clinical efficacy and effectiveness of treatment group were significantly higher than that of control group (P=0.038). The incidence of adverse reactions in both groups was 16.67% (P>0.05). The effective rate of EEG in treatment group was significantly higher than that in control group (P=0.053). Serum NSE and CD19+ in treatment group were significantly lower than those in control group (P<0.05). ConclusionFor children with refractory epilepsy, the addition of Xiao’er kang xian capsule on the basis of the original treatment has obvious effect low adverse reaction and high safety. NSE and CD19+ can be used as monitoring indicators for the influence of the disease and prognosis evaluation during the treatment of children with epilepsy.
Objective To evaluate the feasibility and validity of chondrogenic differentiation of marrow clot after microfracture of bone marrow stimulation combined with bone marrow mesenchymal stem cells (BMSCs)-derived extracellular matrix (ECM) scaffold in vitro. Methods BMSCs were obtained and isolated from 20 New Zealand white rabbits (5-6 months old). The 3rd passage cells were cultured and induced to osteoblasts, chondrocytes, and adipocytes in vitro, respectively. ECM scaffold was manufactured using the 3rd passage cells via a freeze-dying method. Microstructure was observed by scanning electron microscope (SEM). A full-thickness cartilage defect (6 mm in diameter) was established and 5 microholes (1 mm in diameter and 3 mm in depth) were created with a syringe needle in the trochlear groove of the femur of rabbits to get the marrow clots. Another 20 rabbits which were not punctured were randomly divided into groups A (n=10) and B (n=10): culture of the marrow clot alone (group A) and culture of the marrow clot with transforming growth factor β3 (TGF-β3) (group B). Twenty rabbits which were punctured were randomly divided into groups C (n=10) and D (n=10): culture of the ECM scaffold and marrow clot composite (group C) and culture of the ECM scaffold and marrow clot composite with TGF-β3 (group D). The cultured tissues were observed and evaluated by gross morphology, histology, immunohistochemistry, and biochemical composition at 1, 2, 4, and 8 weeks after culture. Results Cells were successfully induced into osteoblasts, chondrocytes, and adipocytes in vitro. Highly porous microstructure of the ECM scaffold was observed by SEM. The cultured tissue gradually reduced in size with time and disappeared at 8 weeks in group A. Soft and loose structure developed in group C during culturing. Chondroid tissue with smooth surface developed in groups B and D with time. The cultured tissue size of groups C and D were significantly larger than that of group B at 4 and 8 weeks (P lt; 0.05); group D was significantly larger than group C in size (P lt; 0.05). Few cells were seen, and no glycosaminoglycan (GAG) and collagen type II accumulated in groups A and C; many cartilage lacunas containing cells were observed and more GAG and collagen type II were synthesized in groups B and D. The contents of GAG and collagen increased gradually with time in groups B and D, especially in group D, and significant difference was found between groups B and D at 4 and 8 weeks (P lt; 0.05). Conclusion The BMSCs-derived ECM scaffold combined with the marrow clot after microfracture of bone marrow stimulation is effective in TGF-β3-induced chondrogenic differentiation in vitro.
To review the advance in the experimental studies and evaluate the potential therapeutic appl ication of the growth differentiation factor 5(GDF-5) and osteogenic protein 1 (OP-1) in intervertebral disc degeneration.Methods Relevant l iterature at home and abroad publ ished in recent years was searched and analyzedcomprehensively. Results The growth factor was one of the most potential proteins in curing the intervertebral discdegeneration. In vitro, exogenous GDF-5 or OP-1 increased the deoxyribonucleic acid and proteoglycan contents ofboth nucleus pulposus and annlus fibrosis cells types significantly. GDF-5 at 200 ng/mL or OP-1 significantly stimulatedproteoglycan synthesis and collagen synthesis. In vivo, the injection of GDF-5(100 μg) or OP-1(100 μg in 10 μL 5% lactose) resulted in a restoration of disc height, improvement of magnetic resonance imaging scores, and histologic grading scores had statistical significance. Conclusion A single injection of GDF-5 or OP-1 has a reparative capacity on intervertebral discs, presumably based on its effect to stimulate matrix metabol ism of intervertebral disc cells and enhance extracellular matrix production. A single injection of exogenous GDF-5 or OP-1 in the degenerated disc shows a good prospect.
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.
Objective To explore the in vitrodifferentiation of the rat mesenchymal stem cells (MSCs ) into the skeletal muscle cells induced by the myoblast differentiation factor (MyoD) and 5-azacytidine. Methods The MSCs were taken from the rat bone marrow and the suspension of MSCs was made and cultured in the homeothermia incubator which contained 5% CO2at 37℃. The cells were observed under the inverted phase contrast microscope daily. The cells spreading all the bottom of the culture bottle were defined as onepassage. The differentiation of the 3rd passage of MSCs was induced by the combination of 5-azacytidine, MyoD, transforming growth factor β1, and the insulin like growth factor 1. Nine days after the induction, the induced MSCs were collected, which were analyzed with the MTT chromatometry, theflow cytometry, and the immunohistochemistry. Results The primarily cultured MSCs grew as a colony on the walls of the culture bottle; after the culture for 5-7 days, the cells were shaped like the fibroblasts, the big flat polygonal cells, the medium sized polygonal cells, and the small triangle cells; after the culture for 12 days, the cells were found to be fused, spreadingall over the bottle bottom, but MSCs were unchanged too much in shape. After the induction by 5-azacytidine, some of the cells died, and the cells grew slowly. However, after the culture for 7 days, the cells grew remarkably, the cell volume increased gradually in a form of ellipse, fusiform or irregularity. After theculture for 14 days, the proliferated fusiform cells began to increase in a great amount. After the culture for 18-22 days, the myotubes increased in number and volume, with the nucleus increased in number, and the newly formed myotubes and the fusiform myoblst grew parallelly and separately. The immunohistochemistry for MSCs revealed that CD44 was positive in reaction, with the cytoplasm ina form of brown granules. And the nucleus had an obvious border，and CD34 was negative. The induced MSCs were found to be positive for desmin and specific myoglobulin of the skeletal muscle. The flow cytometry showed that most of the MSCs and the induced MSCs were in the stages of G0/G1，accounting for 79.4％ and 62.9％，respectively; however, the cells in the stages of G2/S accounted for 20.6％ and 36.1％. The growth curve was drawn based on MTT，which showed that MSCs weregreater in the growth speed than the induced MSCs. The two kinds of cells did not reach the platform stage，having a tendency to continuously proliferate.ConclusionIn vitro,the rat MSCs can be differentiated into the skeletal muscle cells with an induction by MyoD and 5-azacytidine, with a positive reaction for the desmin and the myoglobulin of the skeletal muscle. After the induction, the proliferation stage of MSCs can be increased, with a higher degree of the differentiation into the skeletal muscle.
Objective To investigate the experimental condition and mechanism of differentiation of human umbilical cord blood derived mesenchymal stem cells（hUCB-MSC）into neuron-like cells induced by recombined human epidermal growth factor （rhEGF） and taurine in vitro.Methods hUCB-MSC were primary cultured in Dulbeccoprime;s modified Eagle's medium/F12 （DMEM/F-12）which supplemented with 105U/L penicillin G, 100 mg/L streptomycin sulfate, 10% fetal bovine serum (FBS),5% autologous plasma,4 mmol Lglutamine, 30 ng/ml rhEGF.The DMEM/F-12 medium was replaced by taurine medium after 3 passages.The expression of surface antigen CD90,CD29,CD34,CD44 and CD45 were detected by flow cytometry；the expression of neuron specific enolase,rhodopsin and nestin were investigated by immunocytochemistry. The statistical method was chi square test.Results Morphologically similar to bonemarrow MSC,hUCB-MSC became attached cells after the first 5 to 7 days in culture，and reached 80% to 90% confluent after 3 to 4 weeks. Growth accelerated after passage. hUCB-MSC were positive for CD29,CD44 and CD90 but negative for CD34 and CD45. After taurine induction, 2515/3120 cells expressed NSE, 1168/3175 cells expressed rhodopsin and 903/3050 cells expressed nestin while only 234/2965 cells expressed NSE in the control group(P＜0.01).Conclusion rhEGF and taurine can induce hUCB-MSC differentiating into neuronlike or rhodopsin positive cells.
Objective To investigate the effects of the recombinanthuman bone morphogenetic protein 2 (rhBMP-2) and/or the osteogenic agents on proliferation and expression of the osteoblast phenotype differentiation of the SD rat mesenchymal stem cells（MSCs）. Methods The rat MSCs were cultured in vitro and were randomly divided into the experimental groups(Groups A-I) and the control group. In the experimental group, MSCs were induced by rhBMP2 in different doses (10, 50, 100 and 200 μg/L) in Groups BE, the osteogenic agent alone (Group A) and by the combined use of rhBMP-2 ［in different doses (10,50, 100 and 200 μg/L)］ and the osteogenic agent in Groups F-I. The MTT colorimetric assay was used to evaluate the proliferation, and the activities of alkaline phosphatase (ALP) and osteocalcin (OC) were observed at 3, 6, 9, 12 days, respectively. Results The inverted phase contrast microscopy showed that MSCs by primary culture for 12 hours were adhibited, with a fusiform shape at 48 hours. At 4 days they were polygonal or atractoid, and were spread gyrately or radiately at 6 days. At 10 days, they were spread at the bottom of the bottle.The statistical analysis showed that the expression of the osteoblast phenotype differentiation of MSCs could be induced in the experimental groups. The proliferation of MSCs could be enhanced in a dosedependent manner in GroupsB-E. The expression of the osteoblast phenotype differentiation, which was tested by the activities of ALP and OC, was significantly higher in Groups F-I than in Groups A-E. Conclusion The combined use of rhBMP-2 and the osteogenic agents can enhance the MSC proliferation and induce an expressionand maintenance of the osteoblast phenotype differentiation of the rat MSCs.
Objective To investigate the feasibil ity and effect of inducing adi pose-derived stem cells (ADSCs) treated with growth differentiation factor 5 (GDF-5) to undergo chondrogenic differentiation in vitro. Methods Six healthy Japanese rabbits aged 3 months (2-3 kg) of clean grade were chosen, irrespective of sex. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by vimentin immunohistochemistry and CD44, CD49d, CD106immunofluorescence staining. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 106/mL, then the ADSCs were treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium, respectively. The morphology changes of the induced ADSCs were observed by inverted contrast phase microscope and their growth state were detected by MTT. The mRNA quantities of Col II and proteoglycan expressed by the induced ADSCs were detected with RT-PCR. The Col II proteoglycan synthesized by the induced ADSCs were detected with alcian blue staining, toluidine blue staining, immunohistochemistry staining, and Western blot method. Results ADSCs mostly presented small sphere, fusiform and polygon shape with positive expression of CD44 and CD49d and negative expression of CD106 and vimentin. The ADSCs treated with 100 ng/mL GDF-5 presented sphere or sphere-l ike change and vigorous prol iferation. The mRNA quantities of Col II and proteoglycan synthesized by the induced ADSCs treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium increased in a dose-dependent manner at 7 days. There were significant differences among all the groups (P lt; 0.05), except that no significant difference was evident between the 0 ng/mL group and the 10 ng/mL group (P gt; 0.05). When ADSCs were treated with 100 ng/mL GDF-5 for 14 days, the Col II and the mRNA and protein quantities of ptoteoglycan reached the peak, and the results of alcian blue, toluidine blue and Col IIimmunohistochemistry staining were positive. Conclusion ADSCs treated with certain concentration of GDF-5 have higher expression of Col II and proteoglycan and possess partial biological function of chondrocyte.
【Abstract】 Objective To investigate the biocharacteristics of c-kit+ human amniotic fluid-derived mesenchymal stemcells (HAFMSCs) and its capacity to differentiate into cardiomyocytes in vitro. Methods Fifty samples of human amnioticfluid were obtained from amniocenteses or voluntary termination of pregnancy and were expanded in vitro. c-kit+ HAFMSCs were sorted by flow cytometry and were recultured in the same media. c-kit+ HAFMSCs were amplified and identified, then exposed to osteogenic , adi pogenic, and myogenic media. The flow cytometry, and immunocytochemistry were used for identifying the cell phenotype, von Kossa staining for osteogenic differentiation, oil red O staining for adi pogenic differentiation, and real-time fluorescent quantitative PCR for the expressions of NKx2.5, Tbx5, GATA-4, and α-MHC genes. Results After the selection procedure, the percentage of c-kit+ HAFMSCs was 3.07% ± 1.03% of the total adherent cells. The cells expressed MSCs markers (CD29, CD44, CD73, CD90, CD105), and did not express hematopoietic stem cells markers (CD34, CD45). The cells were positive for human leukocyte antigen (HLA)-ABC, and negative for HLA-DR. They also expressed Oct-4, which characterized the undifferentiated stem cell state. The growth curves of c-kit+ HAMFSCs at passages 5, 10, and 15 were similar. Li pid droplet was observed by oil red O staining and calcium deposition by von Kossa staining in the cells at 21 days after adi pogenic and osteogenic induction. The myocardium special gene expressions of Tbx5, Nkx2.5, GATA-4, and α-MHC were significantly ber after myogenic induction than those before myogenic induction (P lt; 0.05). Conclusion Selected c-kit+ HAFMSCs by flow cytometry is a group of pure MSCs, which has potential to differentiate into cardiomyocytes and can be used as seeding cells for myocardium regeneration treatment.