Objective To isolate neural stem cells (NSCs) from rabbit retina and brain, and induce differentiation of those NSCs using different culture media. Methods Single-cell suspensions of retina and cerebral cortex were prepared from rabbit embryo, cultured in 5 types of different media to isolate the NSCs by continual passages. After 3 passages, NSCs were induced to differentiation in 2 types of different media for 8 to 10 days. NSCs and inducedretinal cells were examined by immunofluorescence and flow cytometry for the expression pattern of some specific antigens.Results Immunofluorescence showed that NSCs from retina and brain, cultured in serumfree media, both expressed Nestin partially. Flow cytometry showed that Nestin positive cells were significantly decreased while the Rhodopsin and Thy1.1 positive cells were increased after induction. Compared with the combined induction of alltrans retinoid acid (ATRA) and serum, 5%FBS (fetal bovine serum) led to higher expression of Rhodopsin（P＜0.01），but lower expression of Thy1.1（P=0.01）.Conclusion Serumfree media with N2, EGF, bFGF, LIF is the best for NSCs purification. Both induciton media can induce NSCs to differentiate.Retina NSCs have higher potentials to differentiate into retinal neuroepithelial cells than brain NSCs.
Objective To observe the differentiation effect of rabbit amnion-derived stem cells (ADSC) induced into neural cells.Methods ADSC of New Zealand female rabbits were isolated and cultured. Its mRNA level of Fibronectin, Nestin and Vimentin were detected by real-time quantitative polymerase chain reaction. The selfreplication ability of ADSC was confirmed by monoclonal formation experiments. These ADSC were further induced into neural cells in vitro. Five days after induced differentiation, the expression of -tubulin and glial fibrillary acidic protein (GFAP) were detected by immunofluorescent staining. Results ADSC were separated from amnion tissue gradually after 24 hours. There were polygonal cells gathered around the amnion tissue at 72 hours, and were distributed compactly around the amnion at 120 hours. The morphology of cleavage daughter cells was basically the same as parent cells. ADSC has the ability of self-replication. The Nestin, Vimentin, Fibronectin mRNA expressions in ADSC were 15.79, 1.91, 7.65 times those in spleen cells. The differences were statistically significant(Z=-5.243, -3.972, -2.524; P＜0.05). The beta;-tubulin expression was found in cytoplasm of most cells. The GFAP expression was found in cytoplasm in some cells. Conclusions ADSC has self-replication ability. It can be induced into neurons and neuroglial cells under the right conditions.
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)
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.
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.
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.
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.
ObjectiveTo investigate the effect of cytoskeleton modification on the adipogenic differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. After the 3rd passage cells were cultured with DMEM medium containing 15% fetal bovine serum and cytochalasin D (CYD) at the concentrations of 0, 50, 100, 500, and 1 000 ng/mL, the cell survival condition and morphology changes were observed by inverted phase contrast microscope, the cytoskeleton was observed through fibrous actin (F-actin) staining, and the ratio of F-actin/soluble globular actin (G-actin) was detected and calculated through Western blot. According to the above results, the effective concentration of CYD was selected and used for next experiments. After TSCs were cultured for 3 and 7 days respectively with adipogenic induction media (induction group), adipogenic induction media containing CYD (CYD+induction group), ordinary medium (ordinary group), and ordinary medium containing CYD (CYD+ordinary group), the real-time quantitative PCR (qRT-PCR) and Western blot were carried out to measure the mRNA and protein expressions of adipogenic differentiation-related markers, including peroxisome proliferator-activated receptor γ (PPARγ), 1ipoprotein lipase (LPL), and fatty acid binding protein (aP2). ResultsThe final CYD concentration of 100 ng/mL can inhibit effectively G-actin polymerization into F-actin, but could not affect TSCs survival, which was used for next experiments. qRT-PCR and Western blot suggested that the mRNA expressions of PPARγ, LPL, and aP2 and the protein expressions of PPARγ and aP2 were increased significantly in the CYD+induction group at 3 and 7 days when compared with the induction group (P<0.05). In the CYD+ordinary group, there still was a significant increase in the mRNA expressions of PPARγ, LPL, and aP2 when compared with the ordinary group (P<0.05). ConclusionInhibition of F-actin polymerization can increase adipogenic differentiation of rat Achilles-derived TSCs in vitro, and cytoskeleton modification is a pre-requisite for TSCs differentiation into adipocytes, which might have important implications for the mechanism research of tendinopathy.
ObjectiveTo evaluate the effect of leucocyte- and platelet-rich plasma (L-PRP) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in treating avascular necrosis of the femoral head (ANFH) in rabbits. MethodsTwenty-four New Zealand white rabbits (4-6 months old, both genders, weighing 2.0-3.0 kg) were used for the establishment of bilateral ANFH models and divided into 4 groups (n=6). BMSCs were isolated from the bone marrow of iliac crest, cultured and identified. L-PRP was prepared by Landesberg method. Core decompression only (group A), core decompression and L-PRP implantation (group B), core decompression and BMSCs implantation (group C), and core decompression and implantation of BMSCs and L-PRP were performed in 4 groups. To evaluate bone formation and remodeling of the defects, X-ray photography was taken at 2, 4, and 8 weeks postoperatively. The modified Lane-Sandhu scoring system was used to evaluate the bone formation. Two rabbits were sacrificed at 2, 4, 8 weeks after operation to harvest the specimens for histological observation, new blood vessel count and new bone area ratio. ResultsThe observations of radiology and histology displayed different degrees of bone regeneration at bone defect sites in each group. At 2, 4, and 8 weeks postoperatively, the results of Lane-Sandhu X-ray photography scoring, new blood vessel count, and new bone area ratio showed that groups C and D were significantly better than groups A and B, group D was significantly better than group C. and group B was significantly better than group A (P<0.05). ConclusionThese findings demonstrate that L-PRP can promote osteogenic differentiation of BMSCs in treating ANFH in rabbits, and core decompression associated with BMSCs and L-PRP is an effective and feasible method to treat ANFH.
Objective To investigate the feasibility of differentiation of invitro induced rat bone marrowderived mesenchymal stem cells（rMSCs） into retinal pigment epithelial （RPE） cells.Methods The rMSCs from BrwonNorway (BN) rats were isolated and cultured by adherent screening method. RPE cells lysate made by repeated freezethawing was put into the rMSCs culture system to identify whether the induced cells could express characteristic label cytokeratin（CK）and S-100 simultaneously or not.Results The growth rate of rMSCs induced by RPE cells lysate was slower and protuberant burr surrounded the fusiform cells. The results of immunoblotting and double immunofluorescence showed that partial induced cells expressed CK and S-100 simultaneously. The result of flow cytometry indicated that 14.1% induced cells expressed CK and S-100 simultaneously.Conclusion Induced by RPE cells lysate, rMSCs can differentiate into RPE cells.