RESUMEN
【Objective】 To establish an effective method for acquiring bone marrow-derived dendritic cells (DCs) from BALB/C mice in vitro and to establish a reservoir of DC precursor cells. 【Methods】 CD117+ hematopoietic stem cells (HSCs) were isolated and purified from bone marrow of BALB/C mice by immunomagnetic beads separation system (MACS), and then amplified in vitro with mouse stem cell factor (SCF) and interleukin-3 (IL-3). HSC was induced to differentiate into DCs by adding granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and IL-4. Different cytokines (tumor necrosis factor-alpha or IL-10) were added to control the maturity of dendritic cells. Then the morphology (electron microscopy), surface molecular markers (FACS method) and cytokine secretion level (ELISA method) were identified. 【Results】 ① The purity of CD117 + HSC isolated and purified by MACS system was over 95%. ② SCF plus IL-3 could effectively stimulate HSC amplification. ③ The morphology of mature DC (mDC) and immature DC (imDC) was significantly different under light and scanning electron microscopy. ④ In the expressions of surface markers CD40, CD80, CD86, I-A/I-E, there were significant differences between imDC group and mDC group (P<0.01). ⑤ After LPS stimulation, the secretion of IL-12 in imDC group did not change significantly (P=0.064), while the secretion of IL-12 in mDC group increased significantly (P=0.009). LPS and TNF-α had a synergistic effect in stimulating DC maturation. 【Conclusion】 Specific combinations of cytokines can effectively induce the differentiation of bone marrow HSCs into DCs in BALB/C mice, and can control the maturity of DCs. This study makes it possible to use gene modified dendritic cells in GD immunotherapy.
RESUMEN
Objective To observe the promoting effect of hypoxia on proliferation of human bone marrow mesenchymal stem cells (BMMSCs) and maintaining their potential of multi-directional differentiation in vitro.Methods BMMSCs were isolated from bone marrow blood samples of patients with bone fracture, and cultured under hypoxic (group A) or normoxic (group B) conditions.The morphology, proliferation and osteogenic and adipogenic potential of the BMMSCs were observed.Results BMMSCs in the group A showed a long spindle shape and a fish shoal-like distribution, and were well-grown, while the morphology of cells in the group B appeared polygonal or flat.The quantity and growth rate of BMMSCs in the group A were increased compared with the group B (P< 0.05), with an osteogenic and adipogenic potential.Conclusions Hypoxia can promote the proliferation of BMMSCs in vitro and maintain their multi-directional differentiation potential.
RESUMEN
Objective To evaluate the biological effect of tumor necrosis factor-α(TNF-α) on the proliferation and multi-directional differentiation of stem cells from rat apical papilla(SCAP).Methods SCAP was extracted by combining enzyme digestion method with tissue block method.The cells were divided into control group(TNF-α 0 ng/mL) and experimental group(TNF-α 5,10,20,50 ng/mL).The ability of proliferation of SCAP was measured by MTT method.The ability of osteogenic/dentinogenic differentiation of SCAP was measured by alizarin red staining and quantitative real-time PCR.The ability of adipogenic of SCAP was measured by oil red O staining.The expression of vascular related genes of SCAP was measured by quantitative real-time PCR.Results SCAP was consistent with the characteristics of mesenchymal stem cells and possessed the ability of multi-directional differentiation.The MTT results showed that experimental group promoted the proliferation of SCAP in comparison with the control group.The difference was statistically significant(P<0.05),and 10 ng/mL was the optimum concentration.The results of alizarin red staining showed that with the increase of the concentration of TNF-α,the mineralized nodules in the experimental group gradually became smaller,and the number of the formation decreased gradually.The results of quantitative real-time PCR showed that the expression of OC,DMP-1 and DSPP in the experimental group was significantly lower than that of the control group at 3 and 7 days,in which the expression of OC was statistically significant different(P<0.05);at 14 days,the expression of OC,DMP-1 in the experimental group was significantly lower than that of the control group(P<0.05).The result of Oil red O staining showed that with the increase of the concentration of TNF-α,the lipid droplets formation in the experimental group gradually decreased.The result of quantitative real-time PCR showed that the expression of ANGPT1,VEGFA,PECAM-1 in the experimental group was significantly lower than that of the control group(P<0.05).Conclusion TNF-α might promote the proliferation and inhibit the multi-directional differentiation of SCAP.
RESUMEN
To explore the clinical ,pathological and immunohistochemical features of carcinoma with multi-directional differentiation derived from junction of bladder and prostate .We collected clinical data and tissue samples of three typical cases of carcinoma from our hospital .Routine preparation of slides and immunohistochem-ical methods combined with clinical data and pathological changes analysis were adopted .We found that all 3 ca-ses occurred in the elderly lesions involving the bladder and prostate .Dysuria was the main common symptom .All the cases had a history of chronic inflammation in urethral and prostate gland .The following up data showed that survival time in two of them were no more than 15 months.The third patient without chemotherapy who took drugs of bicalutamide,enantone and pamidronate still was alive after 28 months.The pathological changes of these cases had the common features with diversity .They all showed the structure with nests ,papillary,solid,adenoid,single or syncytial cells .The nuclear of cancer cell was enlarged with hyperchromatic feature .The mitotic figures were easily found.The metaplasia and atypical hyperplasia of prostate were all found .The immunohistochemistry results showed positive results for HCK,LCK,p53,p63,PSA,P504S and negative for vimentin and S -100.The average proliferation index of Ki-67 was 77%.
RESUMEN
Objective To observe the effect of the in vitro isolation, culture and identification of rabbit bone marrow-derived mesenchymal stem cells (BMSCs) and explore the differentiation potentials of BMSCs. Methods BMSCs were isolated and purified by density gradient centrifugation combined with attachment culture method. The biological characteristics of BMSCs were observed under an inverted microscope. The BMSCs were identified with HE staining and CD34/CD44 immunohistochemical staining. The biological activity of BMSCs was detected by osteogenic induction and steatogenic induction. Results BMSCs were isolated successfully in vitro and had good homogenicity. Immunohistochemical staining of CD34/CD44 showed strong positive expression of CD44 and negative expression of CD34. Fortis osteogenic ability and positive staining of ALP could be detected by osteogenic induction, and fortis steatogenic ability and positive staining of axungia could be detected by steatogenic induction. Conclusion BMSCs have the capability of multi-directional differentiation in vitro.
RESUMEN
@# As a multipotential stem cell, adipose-derived mesenchymal cell presents similar characters with bone marrow-derived menchymal stem cell in morphology and biology, and has the ability of directional differentiation to the all three germ layers. The adipose-derived mesenchymal stem cell could be harvested conveniently with slighter wound, and has a broad prospect in cell therapy and tissue engineering.
RESUMEN
Objective To induce directional differentiation from spinal cord-derived neural stem cells(SNSCs)into neurocytes in the simulated microenvironment of development of embryonic spinal cord of rat after isolation and culture of SNSCs and to investigate the proliferation and differentiation of SNSCs in vitro.Methods SNSCs were isolated mechanically from rat embryonic(E10 to 11d)spinal cord under microscope.SNSCs were cultured and maintained in serum-free medium.Directional differentiation from SNSCs to neurocytes was induced by adding N4 supplement.The morphologic features of the differentiated cells were noted.Cultured and differentiated cells were identified by immunochemistry stain and t he mean differentiating percentages of the positive cells were calculated.Resul ts SNSCs isolated by the mechanical method under microscope were vigorous and pr oliferative,and could differentiate into neurons,astrocytes and oligodendrocyt es.Conclusion The mechanical method under microscope of isolating SNSCs is simp le and efficient to obtain a high percentage of neurons.N4 supplement can induc e SNSCs to differentiate into neurocytes.