Objective To summarize the role of N-myc downstream regulated gene 1 (NDRG1) and the advancement of it in tumor research. Methods Publications on line at home and abroad involving the roles of NDRG1 and the correlation between it and tumor were collected and reviewed. Results NDRG-1, has a number of important functions such as in organism responses in hypoxia, histological differentiation, and so on, especially plays a significant role in generation, metastasis, and invasion of cancer. Conclusion NDRG1 may be a candidate of metastasis relevant gene for cancer and may serve as a useful prognostic marker of carcinoma.
Objective To investigate the influence of Nogo extracellular peptide residues 1-40 (NEP1-40) gene modification on the survival and differentiation of the neural stem cells (NSCs) after transplantation. Methods NSCs were isolated from the cortex tissue of rat embryo at the age of 18 days and identified by Nestin immunofluorescence. The lentiviruses were transduced to NSCs to construct NEP1-40 gene modified NSCs. The spinal cords of 30 Sprague Dawley rats were hemisected at T9 level. The rats were randomly assigned to 3 groups: group B (spinal cord injury, SCI), group C (NSCs), and group D (NEP1-40 gene modified NSCs). Cell culture medium, NSCs, and NEP1-40 gene modified NSCs were transplanted into the lesion site in groups B, C, and D, respectively at 7 days after injury. An additional 10 rats served as sham-operation group (group A), which only received laminectomy. At 8 weeks of transplantation, the survival and differentiation of transplanted cells were detected with counting neurofilament 200 (NF-200), glial fibrillary acidic portein (GFAP), and myelin basic protein (MBP) positive cells via immunohistochemical method; the quantity of horseradish peroxidase (HRP) positive nerve fiber was detected via HRP neural tracer technology. Results At 8 weeks after transplantation, HRP nerve trace showed the number of HRP-positive nerve fibers of group A (85.17 ± 6.97) was significantly more than that of group D (59.25 ± 7.75), group C (33.58 ± 5.47), and group B (12.17 ± 2.79) (P lt; 0.01); the number of groups C and D were significantly higher than that of group B, and the number of group D was significantly higher than that of group C (P lt; 0.01). Immunofluorescent staining for Nestin showed no obvious fluorescence signal in group A, a few scattered fluorescent signal in group B, and b fluorescence signal in groups C and D. The number of NF-200-positive cells and MBP integral absorbance value from high to low can be arranged as an order of group A, group D, group C, and group B (P lt; 0.05); the order of GFAP-positive cells from high to low was group B, group D, group C, and group A (P lt; 0.05); no significant difference was found in the percentage of NF-200, MBP, and GFAP-positive cells between group C and group D (P gt; 0.05). Conclusion NEP1-40 gene modification can significantly improve the survival and differentiation of NSCs after transplantation, but has no induction on cell differentiation. It can provide a new idea and reliable experimental base for the study of NSCs transplantation for SCI.
【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.
Objective Bone marrow mesenchymal stem cells (BMSCs) are multi potent and thus are able to differentiate into a number of different cell types under certain culture condition. However, the effect of age on the differentiation remains unknown. To explore the effect of the microenvironment formed by Schwann cells (SCs) on BMSCs differentiation into neurons and ol igodendrocytes in rats at different ages in vitro. Methods SCs were extracted and purified from the distal sciatic nerves of neonatal Wistar rats. BMSCs were isolated from bone marrow of Wistar rats (aged 1 month, 6 months, and 12 months, respectively) and cultured in vitro. The cells were identified by immunofluorescent staining. The BMSCs at passage 2 were labeled by PKH26 and cocultured with SCs at passage 3 in equal proportions in two layer Petri dish. According to the BMSCs from the rats at different ages, experiment was divided into 3 groups: SCs were cocultured with 1-month-old rat BMSCs (group A), 6-month-old rat BMSCs (group B), and 12-month-old rat BMSCs (group C), respectively. The morphological changes of cocultured BMSCs were observed by inverted phase contrast microscope, the expressions of neuron-specific enolase (NSE) and myel in basic protein (MBP) in the cocultured BMSCs were tested by immunofluorescent staining, and the expression of neuregul in 1 (NRG1) was detected by ELISA method. Results SCs and BMSCs were isolated and cultured successfully. The identification of SCs showed positive expression of S-100 and BMSCs showed positive expressions of CD29, CD44, and CD90. At 7 days after coculture, the BMSCs in group A began retraction, and became round or tapered with the processes and had a nerve cells or ol igodendrocytes-l ike morphology, but most BMSCs in groups B and C showed no obvious morphological changes under inverted phase contrast microscope. Immunofluorescent staining showed that the positive expression rates of NSE in groups A, B, and C were 22.39% ± 2.86%, 12.89% ± 1.78%, and 2.69% ± 0.80%, respectively, and the positive expression rates of MBP in groups A, B, and C were 16.13% ± 2.39%, 6.33% ± 1.40%, and 0.92% ± 0.17%, respectively. There were significant differences in terms of NSE and MBP positive expression rates among 3 groups (P lt; 0.05). ELISA analysis showed that NRG1 in the supernatant of group A was increased after coculture in a time-dependent manner. At 6, 9, and 12 days of coculture, NRG1 content was higher in group A than in groups B and C, and in group B than in group C, showing significant differences (P lt; 0.05). Conclusion The microenvironment formed by SCs can promote BMSCs differentiation into neurons and ol igodendrocytes, but the differentiation capabil ity of BMSCs decreases with aging, and the variety of growth factors secreted by SCs is l ikely important factors that induce the differentiation of BMSCs into neurons and ol igodendrocytes.
Objective To introduce the basic research and cl inical potential of the hair foll icle stem cells related signal transduction in prol iferation and differentiation. Methods The recent original articles about the hair foll icle stem cells were extensively reviewed. Results Many different signal pathways had been involved in the skin development and self-newals.The hair foll icle stem cells could play an important role in the skin self-renewal and regeneration which were modulated by several different signal pathways, which included bone morphogenetic protein/transforming growth factor β, Wnt, Notch and ectodysplasin A genes. Conclusion The hair foll icle stem cells may be a future approach to repair cutaneous wounds as a cell therapy．
Objective To investigate the division, prol iferation and differentiation abil ities of nestin+/GFAP+cell after spinal cord injury and to identify whether it has the characteristic of neural stem cells (NSCs). Methods Twelvemale SD rats, aged 8 weeks and weighing 200-250 g, were randomized into 2 groups (n=6 per group): model group inwhich the spinal cord injury model was establ ished by aneurysm cl ip compression method, and control group in which no processing was conducted. At 5 days after model ing, T8 spinal cord segment of rats in each group were obtained and the gray and the white substance of spinal cord outside the ependymal region around central tube were isolated to prepare single cellsuspension. Serum-free NSCs culture medium was adopted to culture and serum NSCs culture medium was appl ied to induce differentiation. Immunohistochemistry detection and flow cytometry were appl ied to observe and analyze the type of cells and their capabil ity of division, prol iferation and differentiation. Results At 3-7 days after injury, the model group witnessed a plenty of nestin+/GFAP+ cells in the single cell suspension, while the control group witnessed few. Cell count of the model and the control group was 5.15 ± 0.71 and 1.12 ± 0.38, respectively, indicating there was a significant difference between two groups (P lt; 0.01). Concerning cell cycle, the proportion of S-phase cell and prol iferation index of the model group (15.49% ± 3.04%, 15.88% ± 2.56%) were obviously higher than those of the control group (5.84% ± 0.28%, 6.47% ± 0.61%), indicating there were significant differences between two groups (P lt; 0.01). In the model group, primary cells gradually formed threedimensional cell clone spheres, which were small in size, smooth in margin, protruding in center and positive for nestin immunofluorescence staining, and large amounts of cell clone spheres were harvested after multi ple passages. While in the control group, no obvious cell clone spheres was observed in the primary and passage culture of single cell suspension. At 5 days after induced differentiation of cloned spheres in the model group, immunofluorescence staining showed there were a number of galactocerebroside (GaLC) -nestin+ cells; at 5-7 days, there were abundance of β-tubul in III-nestin+ and GFAP-nestin+ cells; and at 5-14 days, GaLC+ ol igodendrocyte, β-tubul in II+ neuron and GalC+ cell body and protruding were observed. Conclusion Nestin+/GFAP+ cells obtained by isolating the gray and the white substance of spinal cord outside the ependymal region around central tube after compressive spinal cord injury in adult rat has the abil ity of self-renewal and the potential of multi-polarization and may be a renewable source of NSCs in the central nervous system.
Objective To analyze MC3T3E1 cell morphology, prol iferation, and osteogenic differentiation in fibrin gel (FG) so as to lay a fundament for use of FG in tissue engneering. Methods MC3T3E1 cells were incubated in three concentrations (20, 10 and 5 mg/mL)of FG as the experimental groups (groups A, B and C) and in the common medium culture as the control group (group D). The cell morphology and distribution in FG were observed by inverted phase contrast microscope and confocal laser scanning microscope at different time. The cell prol iferation was assessed by fluorospectrophotometer. The alkal ine phosphatase (ALP) activity was detected by automatic biochemistry analyses and von Kossa staining was used to analyze calcium salts mineralization. RT-PCR was used to analyze the ALP and bone sialoprotein (BSP)mRNA expression at 14 and 21 days. Results In groups A, B and C, the MC3T3E1 cells had long processes which connected each other and formed network; but fusiform or cube cells were observed in group D at 21 days. The fluorescence intensity was increased gradually with time, was the highest at 14 days and the lowest at 28 days in group D; it was highest in groups A, B and C at 28 days, there were statistically significant differences when compared with group D (P lt; 0.05). The ALP activity was increased gradually with time, and it was the highest at 28 days in group D and at 21 days in groups A and B, there were significant differences (P lt; 0.05), no statistically significant differences compared with group D at other time points (P gt; 0.05). The mineral ization nodus were seen at 21 and 28 days in group A, but no mineral ization nodus was seen in group D at 28 days. The RT-PCR results showed the mRNA expressions of ALP and BSP were enhanced in group A when compared with group D (P lt; 0.05). Conclusion The osteogenic differentiation was most obvious and cell prol iferation was most active after 21 days of incubation in FG.
【Abstract】 Objective To explore the interventional effect of platelet lysate (PL) on osteogenic differentiation ofBMSCs by induction in rats in vitro. Methods Twenty-four clean-grade adult Wistar rats, weighing from 250 g to 300 g, maleor female, were included in this study. PL was obtained through three times of centrifugation and repeated freeze-thaw for the blood aspirated from cardiac cavities in 16 Wistar rats. ELISA assay was conducted to detect the concentration of growth factors PDGF, TGF-β1, IGF-1 and VEGF in PL. The BMSCs harvested by flushing femurs of 8 adult Wistar rats were isolated, cultivated and expanded in vitro. The cells at the 4 passage were performed for osteogenic differentiation by induction in three groups of A (5% PL of final concentration in basic induction medium), B (1% PL of final concentration in basic induction medium), and C (no presence of PL in basic induction medium as a control). The morphological changes of the cells were dynamically observed with inverted phase contrast microscope during the whole period. At different time-points, ALP staining (7 days) and ALP/TP (2, 8, 12 days) of the cells were detected to evaluate ALP activity, and the mineral formation in extracellular martrix was examined with Al izarin red staining which provided quantitative analysis of mineral deposits. Results ELISA assay showed that the content of PDGF, TGF-β1, IGF-1 and VEGF in PL reached (300 ± 30), (140 ± 25), (80 ± 35), (70 ± 20) pg/mL, respectively. Morphological observation displayed BMSCs in group A or B gradually turned from spindle-shape to square- or polygon-shape as the morphorlogical type of osteoblast-l ike cells at 7 days. The cells in group A showed slower shape changesbut higher prol iferation than that in group B or C. Moreover, at the 20 days, the cells in group A still displayed dense gro wth and produced obviously decreased amount of mineral deposits in ECM when compared with group B or C. At the 7 days, the cells ofgroup A showed smaller amount of granules positive for ALP staining in cytoplasm when compared with groups B and C, and displayed marked reduction in ALP activity assay at the 2, 8, and 10 days compared with that of groups B and C (P lt; 0.05). At the 20 days, Al izarin red staining showed the number of mineral deposits in groups A, B and C were 7.67 ± 1.10, 12.87 ± 0.81 and 15.59 ± 0.25, respectively, while the area of mineral deposits were (161 778.70 ± 44 550.80), (337 349.70 ± 56 083.24), and (415 921.70 ± 71 725.39) pixels, respectively. The number of mineral deposits and the area of mineral deposits in group A were smaller than those in groups B and C (P lt;0.05). But there was no statistically significant difference between groups B and C (P gt; 0.05). Conclusion PL is a kind of system carrying various growth factors. Exposure of PL inhibits both ALP activity and mineral formation of BMCs in a dose-dependent way under the osteogenic induction environment.
Objective To observe the expression of miR-204 and 211 human embryonic stem cells (hESCs) differentiated into retinal pigment epithelial (RPE) cells. Methods RPE cells were derived from hESCs by natural differentiation method, and were identified. miRNA expression profiles and real-time polymerase chain reaction (RT-PCR) of miR-204 and 211 were generated from the following groups: hESCs, hESCs-derived cells containing pigmented foci, hESCs-derived RPE cells and human fetal RPE (hfRPE) cells. Results miRNA-204 was continuously upregulated throughout the entire differentiation process of hESCs to RPE cells. It increased 5.026 times in hESCs-derived cells containing pigmented foci compared to hfRPE cells; it was increased 3.337 times in hESCs-derived RPE cells compared to hESCs-derived cells containing pigmented foci; it increased 13.574 times in hfRPE cells compared to hESCs-derived RPE cells. miR-211 does not change during differentiation from hESC to RPE, but it increased 44.333 times in hESCderived RPE cells compared to hfRPE cells. miR-211 was the biggest difference in the miRNA expression pattern. In four cell types of hESCs, hESCs-derived cells containing pigmented foci, hESCs-derived RPE cells and hfRPE cells, RT-PCR showed the levels of miR-204 were 91.81plusmn;4.43, 2263.09plusmn;206.39, 5996.80plusmn;235.42, and 171676.45plusmn;999.82 respectively. miR-204 was significantly increased during the whole course (t=18.22, 20.66, 279.38;P＜0.001). The levels of miR-211 were 2.23plusmn;0.31, 129.33plusmn;3.75, 125.7592plusmn;4.78, and 16682.00plusmn;352.97 respectively. miR-211 was significantly increased from hESCs to cells containing pigmented foci and from hESCs-derived RPE cells to hfRPE (t=58.58, 81.24; P＜0.001). Conclusion There is a continuous change of miR-204 and 211 in differentiation of RPE cells from hESCs.