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.
ObjectiveTo review the related studies on the application of nanomaterials in the treatment of osteomyelitis, and to provide new ideas for the research and clinical treatment of osteomyelitis.MethodsThe literature about the treatment of osteomyelitis with nanomaterials at home and abroad in recent years was reviewed and analyzed.ResultsAt present, surgical treatment and antibiotic application are the main treatment options for osteomyelitis. But there are many defects such as antibiotic resistance, residual bone defect, and low effective concentration of local drugs. The application of nanomaterials can make up for the above defects. In recent years, nanomaterials play an important role in the treatment of osteomyelitis by filling bone defects, establishing local drug delivery system, and self-antibacterial properties.ConclusionIt will provide a new idea and an important research direction for the treatment of osteomyelitis to fully study the related characteristics of nanomaterials and select beneficial materials to make drug delivery system or substitute drugs.
Dental pulp stem cells(DPSCs) are adult stem cells with strong proliferative ability, self-renewal ability and multidirectional differentiation potential. DPSCs have abundant source are easy to obtain, and do not have ethical problems. As seed cells, they played an important role and showed great potential in tissue engineering and regenerative medicine, making them potential ideal seed cells for repairation and regeneration of tissue and organ. Clinical application of DPSCs in bone regeneration has already been achieved, and studies on differentiation of DPSCs into other tissues are still at different levels of basic stage. In this paper, the research and application of directional differentiation potential such as tooth formation, osteogenesis, and nerve formation are reviewed in order to provide clues and ideas for further study on DPSCs in the field of tissue engineering and regenerative medicine.
Objective To investigate the histological origin, diagnosis, differential diagnosis and treatment of thyroid carcinoma showing thymus-like differentiation (CASTLE). Methods Five patients with thyroid CASTLE were adopted by surgical resection and postoperative radiotherapy, and the CD5, CD117, CK5/6, P63, thyroid transcription factor-1 (TTF-1), carcino-embryonic antigen (CEA), calcitonin (CT), Ki-67, chromogranin A (CgA), thyrobolulin (Tg), peroxisome proliferator activated receptorγ (PPAR-γ), sodium iodide symporter (NIS), and thyroid stimulating hormone receptor (TSHR) were detected in tumor tissues by immunohistochemistry S-P method and v-raf murine sarcoma viral oncogene homolog B1 (BRAF)V600E gene and telomerase reverse transcriptase (TERT) promoter mutations were detected by DNA sequencing. Eight cases of poorly differentiated thyroid carcinoma and 6 cases of anaplastic thyroid carcinoma were adopted by comprehensive comparative analysis. Results Thyroid CASTLE tumor cells showed the positive expression of CD5, CD117, CK5/6 and P63, and the negative expression of TTF-1, CT, CgA, Tg, PPAR-γ, NIS and TSHR. There were partly positive expression for CK5/6, P63, TTF-1, CgA, Tg, NIS and TSHR, and negative expression for CD5 and CD117 in the poorly differentiated thyroid carcinoma and anaplastic thyroid carcinoma. The BRAFV600E gene and TERT promoter mutations were not detected in thyroid CASTLE, and the BRAFV600E gene mutations were also not detected in the poorly differentiated thyroid carcinoma and anaplastic thyroid carcinoma. Four cases of poorly differentiated thyroid carcinoma showed the TERT promoter mutations (4/8) included 3 cases with C228T and 1 case with C250T. Two cases of anaplastic thyroid carcinoma showed the TERT promoter mutations (2/6) included 1 case with C228T and 1 case with C250T. There was no recurrence and metastasis after 3–47 months (an average of 25.6 months) of followed-up in thyroid CASTLE patients. Conclusions The histological origin of thyroid CASTLE may be not related to the thyroid. There is important clinical value to combined detection of CD5, CD117, P63, TTF-1, Tg, NIS, and TSHR for the diagnosis and differential diagnosis of thyroid CASTLE. The further study still need for the diagnosis and differential diagnosis of thyroid CASTLE according to the detection of BRAFV600E and TERT promoter mutations.
ObjectiveTo investigate the role and mechanism of S100A8/A9 in rat model of chronic obstructive pulmonary disease (COPD). Methods Twelve Wistar rats were randomly divided into a normal control group and a COPD group. The COPD model was established by exposing the rats to cigarette smoke and injected lipopolysaccharide (LPS) in bronchus for 1 month. The pathological changes of the lung tissue were observed under light microscope, and the emphysema indexes of pulmonary mean linear intercept (MLI), mean alveolar numbers (MAN) and pulmonary alveolar area (PAA) were analyzed by image analysis system. The concentrations of S100A8/A9 in serum and bronchoalveolar lavage fluid (BALF) were measured by enzyme linked immunosorbent assay. The mRNA expression levels of S100A8, S100A9, Toll-like receptor-4 (TLR4) and myeloid differentiation factor 88 (MyD88) of lung tissues were measured by real time polymerase chain reaction. The protein expressions of S100A8/A9, TLR4 and MyD88 of lung tissues were detected by immunohistochemistry. Results After cigarette smoking and LPS injection for 1 month, the rat lung tissue appeared in accordance with the typical pathological changes of COPD. The MLI, MAN and PAA had obvious difference compared with the normal control group (P<0.05). The concentrations of S100A8/A9 protein in BALF and serum of the COPD group were obviously higher than those of the normal control group (P<0.05). The levels of S100A8, S100A9, TLR4 and MyD88 mRNA of lung tissues in the COPD group were obviously higher than those in the normal control group (P<0.05), and the expression levels of S100A8 and S100A9 mRNA were positively correlated with the expression levels of TLR4 and MyD88 mRNA respectively (P<0.05). The levels of S100A8/A9, TLR4 and MyD88 protein of lung tissues in COPD group were obviously higher than those in normal control group (P<0.05), and the levels of S100A8/A9 protein were positively correlated with the levels of MyD88 and TLR4 protein (P<0.05). Conclusions As a new inflammatory mediator, S100A8/A9 may be involved in the occurrence and development of COPD. By up-regulating the expression of TLR4 and MyD88, the classical TLR4-MyD88 inflammatory pathway is activated, thus promotes the occurrence and development of COPD.
Previous studies have shown that growth arrest, dedifferentiation, and loss of original function occur in cells after multiple generations of culture, which are attributed to the lack of stress stimulation. To investigate the effects of multi-modal biomimetic stress (MMBS) on the biological function of human bladder smooth muscle cells (HBSMCs), a MMBS culture system was established to simulate the stress environment suffered by the bladder, and HBSMCs were loaded with different biomimetic stress for 24 h. Then, cell growth, proliferation and functional differentiation were detected. The results showed that MMBS promoted the growth and proliferation of HBSMCs, and 80 cm H2O pressure with 4% stretch stress were the most effective in promoting the growth and proliferation of HBSMCs and the expression level of α-smooth muscle actin and smooth muscle protein 22-α. These results suggest that the MMBS culture system will be beneficial in regulating the growth and functional differentiation of HBSMCs in the construction of tissue engineered bladder.
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 investigate the effect of Notch signaling pathway important target Hey1 expression on the differentiation and proliferation of C3H10T1/2 cells induced by bone morphogenetic protein 9 (BMP-9). MethodsHey1 lentivirus and Hey1 short hairpin RNA lentivirus were constructed and used to infect C3H10T1/2 cells to change the expression level of Hey1 in C3H10T1/2 cells. C3H10T1/2 cells infected with LV-Blank (empty plasmid) as control. The Hey1 expression levels of different groups were detected by fluorescence microscope, real-time fluorescence quantitative PCR, and Western blot. The C3H10T1/2 cells with different Hey1 expression level were induced by BMP-9 conditioned medium (BMP-9+C3H10T1/2 group, BMP-9+C3H10T1/2-Hey1 group, and BMP-9+C3H10T1/2-shHey1 group); the cells of control groups (C3H10T1/2 group and C3H10T1/2-Blank group) were cultured with normal medium. The mRNA and protein expression levels of osteogenesis related transcription factors (Runx2, osteopontin, and osteocalcin) were detected at 48 hours by real-time fluorescence quantitative PCR and Western blot assay. The cells proliferation and cycles were detected by MTT assay at 4, 5, 6, and 7 days and flow cytometry at 4, 5, and 10 days. The alkaline phosphatase (ALP) activity was analyzed by ELISA and observed by ALP staining at 4 and 7 days. ResultsC3H10T1/2 cell lines with different Hey1 expression levels were successfully established. In osteogenesis compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 enhanced the mRNA and protein expressions of transcription factors (Runx2, osteopontin, and osteocalcin), and the expression of osteogenic differentiation marker (ALP) (P < 0.05); however, inhibition of Hey1 expression significantly decreased the above indexes (P < 0.05). In cell proliferation activity compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 increased absorbance (A) value in MTT assay and pecentage of G2+S cells in cytometry assay, but inhibition of Hey1 expression significantly decreased the indexes (P < 0.05). ConclusionExpression of Hey1 is the important link in the osteogenic differentiation process of C3H10T1/2 cells induced by BMP-9, and plays an important role in the regulation of early cell proliferation.
ObjectiveTo investigate the microRNA (miRNA) expression profile during chondrogenic differentiation of human adipose-derived stem cells (hADSCs), and assess the roles of involved miRNAs during chondrogenesis. MethodshADSCs were harvested and cultured from donors who underwent elective liposuction or other abdominal surgery. When the cells were passaged to P3, chondrogenic induction medium was used for chondrogenic differentiation. The morphology of the cells was observed by inverted phase contrast microscopy. Alcian blue staining was carried out at 21 days after induction to access the chondrogenic status. The expressions of chondrogenic proteins were detected by ELISA at 0, 7, 14, and 21 days. The miRNA expression profiles at pre- and post-chondrogenic induction were obtained by microarray assay, and differentially expressed miRNAs were verified by real-time quantitative PCR (qRT-PCR). The targets of the miRNAs were predicted by online software programs. ResultshADSCs were cultured successfully and induced with chondrogenic medium. At 21 days after chondrogenic induction, the cells were stained positively for alcian blue staining. At 7, 14, and 21 days after chondrogenic induction, the levels of collogen type Ⅱ, Col2a1, aggrecan, Col10a1, and chondroitin sulfate in induced hADSCs were significantly higher than those in noninduced hADSCs (P<0.05). Eleven differentially expressed miRNAs were found, including seven up-regulated and four down-regulated. Predicted target genes of the differentially expressed miRNAs were based on the overlap from three public prediction algorithms, with the known functions of regulating chondrogenic differentiation of stem cells, selfrenewal, signal transduction, intracellular signaling cascade, and cell cycle control. ConclusionA group of miRNAs and their target genes are identified, which may play important roles in regulating chondrogenic differentiation of hADSCs. These results will facilitate the initial understanding of the molecular mechanism of chondrogenic differentiation in hADSCs and subsequently control hADSCs differentiation, and provide high performance seed cells for cartilage tissue engineering.
ObjectiveTo observe whether interleukin-27 (IL-27) intervention could diminish allergic airway inflammation of mouse asthma induced by ovalbumin (OVA) and to investigate the related molecular mechanisms. MethodsSixty female C57/6J mice were randomly divided into six groups, a control group, an asthma group, two IL-27 prevention groups and two IL-27 treatment groups. Based on being sensitized and challenged with OVA in the asthma model, two kinds of IL-27 intervention asthma models were set up, one of which was low-dose multiple prevention model, the other was high-dose few times treatment model. HE stain and inflammation score were done for the lungs. CD4+ T cells were purified from mice spleen and cultured under Th2 medium with/without IL-27. Interleukin-4 (IL-4) was measured by ELISA. CD4+ T cells were cultured under different stringent Th2 medium and stimulated by IL-27. The level of total signal transducer and activator of transcription-1 (STAT1) protein and phos-STAT1 were tested by Western blot. ResultsIn low-dose multiple prevention group, IL-27 inhibited inflammation around bronchial and vascular obviously, the inflammation score was lower than the asthma group (P < 0.05), while the treatment group had no obvious statistical significance (P > 0.05). IL-27 repressed Th2 differentiation of naïve CD4+ T cells which was independent of interferon-γand IL-10. This effect was via STAT1 signaling pathway. CD4+ T cells from asthma mice or cultured under high-IL-4 inducing medium were found impairment of STAT1 phosphorylation. ConclusionsIL-27 could inhibit Th2 differentiation of naïve CD4+ T cells, but not in already committed Th2-CD4+ T cells. The inhibition effect of IL-27 for airway inflammation is obvious in prevention group, while the treatment group shows obviously resistance to inhibitory effect of IL-27. Already committed Th2-CD4+ T cells existed in asthma airway might be the reason for IL-27 resistance.