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 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.
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 investigate the role of bone morphogenetic protein 2 (BMP-2) combined with hypoxic microenvironment in chondrogenic phenotype differentiation of bone marrow mesenchymal stem cells (BMSCs) of rat in vitro. Methods BMSCs were harvested from 4-week-old female Sprague Dawley rats. BMSCs at passage 2 were divided into 4 groups according different culture conditions: normoxia control group (group A), normoxia and BMP-2 group (group B), hypoxia control group (3% oxygen, group C), and hypoxia and BMP-2 group (group D). Then the cellular morphology was observed under inverted phase contrast microscope. Alcian blue immunohistochemical staining was used to detect the glycosaminoglycans (GAG), Western blot to detect collagen type II and hypoxia-inducible factor 1α (HIF-1α), and RT-PCRto detect the expressions of chondrogenic related genes, osteogenic related genes, and hypoxia related genes. Results At 21 days after induction of BMP-2 and hypoxia (group D), BMSCs became round, cell density was significantly reduced, and lacuna-l ike cells were wrapped in cell matrix, while the changes were not observed in groups A, B, and C. Alcian blue staining in group D was significantly bluer than that in other groups, and staining became darker with induction time, and the cells were stained into pieces of deeply-stained blue at 21 days. Light staining was observed in the other groups at each time point. The expression level of collagen type II protein in group D was significantly higher than those in other groups (P lt; 0.05). HIF-1α protein expression levels of groups C and D were significantly higher than those of groups A and B (P lt; 0.05). The expressions of collagen II α1 (COL2 α1) and aggrecan mRNA (chondrogenic related genes) were highest in group D, while the expressions of COL1 α1, alkaline phosphatase, and runt-related transcri ption factor 2 mRNA (osteogenic related genes) were the highest in group B (P lt; 0.05). Compared with groups A and B, HIF-1α (hypoxic related genes) in groups C and D significantly increased (P lt; 0.05). Conclusion BMP-2 combined with hypoxia can induce differentiation of BMSCs into the chondrogenic phenotype, and inhibit osteoblast phenotype differentiation. HIF-1α is an important signaling molecule which is involved in the possible mechanism to promote chondrogenic differentiation process.
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 probe the relationship of differentiation degree with spread or survival prognosis in retinoblastoma (RB). Methods Clinical data, follow up status and eyeball specimens in 156 RB cases were investigated retrospectively. The tumors were divided into differentiated and undifferentiated groups. Conditions of the tumor invasion of ocular or surrounding tissues were reviewed. The fatality rate was obtained from the follow-up materials of 82 cases of RB. The fatality rate and the invasion rate between the two types were compared statistically by Chi-square test. In addition, the relation between the tumor invasion and death ,and the average survival time for dead people after surgery were explored. Results Local invasion of tumor cell was found in 8 eyes among 17 eyes with differentiated RB (47.06%),and in 66 eyes among 139 eyes with undifferentiated RB (47.48%).There was no significant difference with regards to the local invasion between the two types ( The fatality rate of cases of differentiated RB was 27.27%,and 22.54% in undifferent iated RB, and there was no statistical difference between the two types .The fat ality rate for patients with orbital and scleral extension was 100%, optic nerve invasion (grade Ⅳ) was 62.50%,and uveal invasion was 22.22%.The survival time for the dead victims were from 5 months to 41 months and averaged to 21.92 months. Conclusion There was no significant differ ence both in survival prognosis and local invasion between the two types. The survival prognosis of metastatic RB was dependent on the degree of spread and the efforts of treatment and regardless of the types of differentiation of RB cells. (Chin J Ocul Fundus Dis, 2001,17:18-20)
Objective To investigate the possibility of commitment differentiation of embryonic stem cells induced by the medium of cultured retinal neurons of SD rats. Methods The medium from cultured retinal neurons of SD rats were collected, sterilized and mixed with DMEM medium according to 2∶3 proportion, ES cells were cultured with these mixed medium and were observed under the phase contrast microscope daily, the induced cells were identified by NF immunohistochemistry methods. Results The ES cells cultured with these mixed medium can differentiate into neuron-like structure, and the induced cells were positive in NF immunofluorescence staining. Conclusion The medium from cultured retinal neurons of SD rats can induce ES cells commitment differentiation into neuron-like structure. (Chin J Ocul Fundus Dis, 2002, 18: 134-136)
Objective To investigate the method and conditions of isolation，proliferation of multipotent mesenchymal stem cells（MSCs）from human umbilical cord blood in vitro, and to induce osteogenic and adipogenic differentiation directly for identification. Methods Human umbilical cord blood was collected in asepsis condition, isolated by density gradient centrifugation,or sedimented red cell with methylcellulose, and then the same centrifugation was done, or obtained by negative immunodepletion of CD34+. These isolated mononuclear cells were used to carry on plastic adherent culture. To obtain single cellderived colonies, these cells were proliferated clonally in medium which consists of L-DMEM orMesencultTM medium and 10% fetal calf serum（FCS） respectively, then their differentiation potentiality to osteoblasts and lipoblasts was tested. Results The mononuclear cells isolated by sedimented and centrifugated way cultured in MesencultTM medium and 10%FCS were most available. These adhesive cells could become obviously short rodshape or shuttle-shape cells after 5-7 days.The colonies form well in 3rdpassage cells. The mononuclear cells obtained by onlycentrifugalized in density gradient were hard to form colony, isolated by immunomagnetic beads were hard to culture. The surface antigens of these colonies cells presented CD29, CD59, CD71 but not CD34,CD45 and HLADR etc. The colony cells differentiating into osteoblasts that produce mineralized matrices, stained by alizarin red, and differentiating into adipocytes that accumulate lipid vacuoles, stained by oil red. Conclusion MSCs can be isolated from human umbilical cord blood and proliferate it in vitro. The way that mononuclear cells are sedimented red cell by methylcellulose and cultured by MesencultTM medium and 10% FCS is the valid method of isolation. Proliferation colonies cells present matrix cell immunophenotypes, and candifferentiate into osteoblasts and adipocytes.
Objective To investigate the correlation between down-regulation of miR-381-3p and inhibition of osteogenic differentiation of mouse embryonic palatal mesenchymal (MEPM) cells in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cleft palate of fetal mice. Methods Thirty-two pregnant mice were randomly divided into TCDD group and control group, 16 in each group. On embryonic day 10.5 (E10.5), the pregnant mice in TCDD group were orally administrated with TCDD at dosage of 28 μg/kg, while the pregnant mice in control group received equivalent corn oil. The pregnant mice in each group were sacrificed on E13.5 and E14.5, fetal palates were collected for analysis. The expression of miR-381-3p was detected by real-time fluorescent quantitative PCR and the protein expressions of runt- related transcription factor 2 (RUNX2) and osteopontin (OPN) were detected by Western blot. MEPM cells were extracted from fetal palates on E14.5 in control group and passaged. The 3rd passage cells were cultured with TCDD at dosage of 10 nmol/L for 0, 0.5, 1, 2, and 3 days. The expression of miR-381-3p was detected after 0, 0.5, 1, 2, and 3 days and the protein expressions of RUNX2 and OPN were detected after 0, 1, 2, and 3 days. Then, the 3rd passage cells were divided into 4 groups. The MEPM cells were transfected with miR-381-3p inhibitor (inhibitor group), NC inhibitor (NC inhibitor group) and miR-381-3p mimics (mimics group), NC mimics (NC mimics group) for 48 hours, respectively. And the expressions of miR-381-3p and the protein expressions of RUNX2 and OPN were detected. Results On E13.5 and E14.5, 96 fetal mice in control group and 92 in TCDD group were obtained. The bilateral palates contacted in control group on E14.5, and a gap between the bilateral palates existed in TCDD group. On E13.5 and E14.5, the relative expressions of miR-381-3p and RUNX2 and OPN proteins were significant lower in TCDD group than in control group (P<0.05). The relative expression of miR-381-3p at 0.5 and 1 day after TCDD treatment of MEPM cells were significantly lower than that at 0 day (P<0.05); then, the relative expressions at 2 and 3 days significantly increased, showing no significant difference when compared with that at 0 day (P>0.05). The relative expressions of RUNX2 and OPN proteins at 1, 2, and 3 days were significantly lower than that at 0 day (P<0.05). The relative expressions of miR-381-3p and RUNX2 and OPN proteins significantly lower in inhibitor group than in NC inhibitor group (P<0.05) and higher in mimics group than in NC mimics group (P<0.05). Conclusion Down-regulation of miR-381-3p expression may be associated with inhibition of osteogenic differentiation of MEPM cells in TCDD-induced cleft palate of fetal mice.
ObjectiveTo investigate the effect of tissue interface stiffness change on the spreading, proliferation, and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs), and to find the suitable stiffness range for stem cell differentiation. MethodsBone marrow of male Sprague Dawley rats (4 weeks old) were selected to isolate and culture BMSCs by whole bone marrow cell adherent method. The third generation BMSCs (1×105 cells/mL) were inoculated into the ordinary culture dishes covered with polyacrylamide hydrophilic gel (PA) which elastic modulus was 1, 4, 10, 40, and 80 kPa (cells seeded on PA), and ordinary culture dish (75 MPa extreme high elastic modulus) as control. Spreading of cells in different stiffness of PA was observed under light microscope. The elastic modulus values of 4, 10, and 40 kPa PA were selected as groups A, B, and C respectively; the ordinary culture dish (75 MPa extreme high elastic modulus) was used as control group (group D). Cell counts was used to detect the growth conditions of BMSCs, alkaline phosphatase (ALP) kit to detect the concentration of ALP, alizarin red staining technique to detect calcium deposition status, and real-time quatitative PCR technique to detect the expressions of bone gla protein (BGP), Runx2, and collagen type I mRNA. ResultsWith increased PA stiffness, BMSCs spreading area gradually increased, especially in 10 kPa and 40 kPa. At 1 and 2 days after culture, the growth rate showed no significant difference between groups (P > 0.05); at 3-5 days, the growth rate of groups B and C was significantly faster than that of groups A and D (P < 0.05), but difference was not statistically significant between groups A and D (P < 0.05); at 5 days, the proliferation of group C was significantly higher than that of group B (P < 0.05). ALP concentrations were (53.69±0.89), (97.30±1.57), (126.60±14.54), and (12.93±0.58) U/gprot in groups A, B, C, and D respectively; groups A, B, and C were significantly higher than group D, and group C was significantly higher than groups A and B (P < 0.05). Alizarin red staining showed that the percentages of calcium nodules was 20.07%±4.24% in group C; group C was significantly higher than groups A, B, and D (P < 0.05). The expression levels of BGP and collagen type I mRNA were significantly higher in groups A, B, and C than group D, and in group C than groups A and B (P < 0.05). The expression level of Runx2 mRNA was significantly higher in groups B and C than group D, and in group C than group B (P < 0.05), but no significant difference was found between groups A and D (P > 0.05). ConclusionPA elastic modulus of 10-40 kPa can promote the proliferation and osteogenic differentiation of BMSCs, and the higher the stiffness, the stronger the promoting effect.