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)
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.
ObjectiveTo investigate the impact of L-Phenylalanine on the efficiency of retinal pigment epithelial (RPE) cell derivation from human embryonic stem cells (hESCs) and explore the underlying mechanisms. MethodsH1 hESCs were routinely cultured with mTeSR medium and divided into control and experimental groups. When cells reached over-confluence, spontaneous differentiation was triggered using 10% KSR differentiation medium without bFGF. L-Phenylalanine (0.2 mmol/L) was supplemented in the experimental group from the 3rd week. The expression of RPE markers and Wnt signaling components in the two groups was detected by Real time-RCR, Western blot and Flow cytometry analyses. Purified hESC-RPE cells and PBS were injected into the subretinal space of sodium iodine-induced retinal degeneration rats separately. Retinal function was assessed by ERG 6 weeks after the transplantation. ResultsOn the 7th week, much more pigment cell clumps appeared in the experimental group compared to the control group. Within these areas there were monolayer hexagonal RPE cells full of pigment granules. The experimental group showed significantly higher expression of Pax6, MITF, Tyrosinase, RPE65, Wnt3a, Lef1 and Tcf7 genes than the control group (P < 0.01). Higher expression level of MITF and RPE65 proteins and higher percentage of RPE65 (+) cells (P < 0.01) were detected in the experimental group. 6 weeks after sub-retinal transplantation of hESC-RPE cells, the amplitudes of a-b wave in the transplanted eyes were significantly higher than those in the control eyes (P < 0.01) at the stimulus intensity of 3.0 cd·s/m2. ConclusionsL-Phenylalanine effectively promoted the differentiation of embryonic stem cells into retinal pigment epithelial cells, and its impacts on the Wnt/β-catenin signaling pathway may partially explain the underlying mechanisms. Subretinal transplantation of hESC-RPE remarkably improved the retinal functions of retinal degenerative animal models.
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.
ObjectiveTo systematically evaluate the expression and clinical features of phospho-p70 ribosomal protein S6 kinase (p-p70S6K) in lung cancer. MethodsWe systematically searched the published researches about p-p70S6K expression and clinical features of lung cancer in Medline, EMbase, Chinese BioMedical Literature, China National Knowledge Infrastructure and WeiPu databases from their establishment to February 4th, 2014. According to the inclusion and exclusion criteria, the data were extracted and the Cochrane Review Manager 5 and Stata 12.0 were used for data analysis. ResultsEight studies including 953 patients were included in this systematic review. Analysis with random effects model showed that the positive expression rate of p-p70S6K in non-small cell lung cancer (NSCLC) tissues spread from 41% to 70%. In small cell lung cancer tissues, the positive expression rate of p-p70S6K ranged from 17% to 91%. The positive expression rate of p-p70S6K in NSCLC was significantly higher than adjacent normal tissues[OR=5.08, 95%CI (2.96, 8.71), P<0.00001]. Divided by status of cell differentiation, the positive expression rate of p-p70S6K between low differentiation and moderate-high differentiation groups had no statistically significant difference[OR=1.40, 95%CI (0.50, 3.92), P=0.53]. In addition, the positive expression rate of p-p70S6K was not related to lymph node metastasis[OR=1.11, 95%CI (0.56, 2.23), P=0.76]. ConclusionCompared with adjacent normal tissues, positive expression rate of p-p70S6K in NSCLC is significantly higher, indicating that p-p70S6K may be associated with the development of lung cancer. The positive expression rate of p-p70S6K in different kinds of lung cancer is still unclear, which needs further studies to explore.
Objective To observe the expression of genes related to hereditary retinal diseases (IRD) in human microglia (hMG). MethodsA experimental study. Efficient differentiation of human induced pluripotent stem cells (iPSC) into hMG. Identification of octamer-binding transcription factor 4 (OCT4), sex-determining transcription factor 2 (SOX2), Nanog homeobox (NANOG), stage-specific embryonic antigen-4 (SSEA4), alpha-fetoprotein (AFP), α-smooth muscle actin (α-SMA) as markers associated with iPSC dryness and pluripotency by immunofluorescence staining Glial fibrillary acidic protein (GFAP); hMG associated marker transmembrane protein 119 (TMEM119), purinergic receptor P2Y12 (P2RY12), and allograft inflammatory factor 1 (IBA1). The proportion of CD11b+ and CD45+ cells was detected by flow cytometry. Mature hMG was collected and stimulated with lipopolysaccharide for 0, 4, 8 and 12 h, and were divided into groups 0 h, 4 h, 8 h and 12 h, respectively. Total RNA samples from the 4 groups were extracted for transcriptome sequencing, and the persistently significant differentially expressed genes (DEG) were screened. Real-time quantitative polymerase chain reaction (qPCR) was used to verify and analyze the expression of DEG mRNA. The two-tailed Student t test was used for comparison between the two groups. ResultsiPSC expressed the dry related markers OCT4, SOX2, NANOG and SSEA4, and differentiated into endoderm, mesoderm and ectoderm, expressing the corresponding markers AFP, α-SMA and GFAP, respectively. iPSC formed embryoid bodies under specific culture conditions, and then differentiated into hMG, and hMG expressed related markers TMEM119, P2RY12 and IBA1 by immunofluorescence staining. The double positive ratio of CD11b+ and CD45+ was > 95%. Transcriptomic analysis showed that the expression of 18 DEG in hMG stimulated by LPS was changed. qPCR test results showed that compared with group 0 h, mRNA expressions of Toll-like receptor 4 (TLR4), phosphoglycerate kinase 1, disintegrin and metallopeptidase domain 9 (ADAM9) in LPS stimulated group 4 h were significantly increased (t=25.43, 15.54, 6.26; P＜0.01). The mRNA expression levels of MER proto-oncogene tyrosine kinase (MERTK), non-hydrolase domain containing lysophospholipase 12 (ABHD12), retinal dehydrogenase 11 (RDH11), DNA damage autophagic regulator 2 (DRAM2) decreased (t=5.94, 14.14, 8.21, 6.97; P＜0.01), and the differences were statistically significant. Compared with group 0 h, mRNA expressions of RDH11, MERTK, ABHD12, DRAM2 and ADAM9 in group 8 h stimulated by LPS were significantly decreased, with statistical significance (t=25.97, 5.47, 43.97, 38.40, 3.84; P＜0.05). Compared with the group 0 h, the mRNA expressions of TLR4, ADAM9, MERTK, ABHD12, RDH11 and DRAM2 in the 12 h stimulated group were significantly decreased, and the differences were statistically significant (t=6.39, 46.11, 5.34, 14.14, 25.97, 25.65; P＜0.05). ConclusionIRD-related genes may be involved in the occurrence and development of IRD by regulating the function of hMG.
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.
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 study the effects of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) on the proliferation and differentiation of human bone marrow mesenchymal stem cells (hBMSCs). MethodshBMSCs at passage 4 were divided into 4 groups according to different culture conditions:cells were treated with complete medium (α-MEM containing 10%FBS, group A), with complete medium containing 10 ng/mL LIF (group B), with complete medium containing 10 ng/mL bFGF (group C), and with complete medium containing 10 ng/mL LIF and 10 ng/mL bFGF (group D). The growth curves of hBMSCs at passage 4 in different groups were assayed by cell counting kit 8; cellular morphologic changes were observed under inverted phase contrast microscope; the surface markers of hBMSCs at passage 8 including CD44, CD90, CD19, and CD34 were detected by flow cytometry. ResultsThe cell growth curves of each group were similar to the S-shape; the cell proliferation rates in 4 groups were in sequence of group D > group C > group B > group A. Obvious senescence and differentiation were observed very early in group A, cells in group B maintained good cellular morphology at the early stage, with slow proliferation and late senescence; a few cells in group C differentiated into nerve-like cells, with quick proliferation; and the cells in group D grew quickly and maintained cellular morphology of hBMSCs. The expressions of CD44 and CD90 in groups A and C at passage 8 cells were lower than those of groups B and D; the expressions of CD19 and CD34 were negative in 4 groups, exhibiting no obvious difference between groups. ConclusionLIF combined with bFGF can not only maintain multiple differentiation potential of hBMSCs, but also promote proliferation of hBMSCs.