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 effect of titanium particles on the proliferation, differentiation, and cytomorphology of osteoblasts, and to explore the possible internal relations and mechanism. MethodsCalvarial osteoblasts were separated from 10 newborn Sprague Dawley rats by repeated enzyme digestion, and were cultured in vitro. The cells were identified by alkaline phosphatase (ALP) staining and alizarin red staining. The cells at passage 3 were cultured with titanium particles culture medium at concentrations of 0.01, 0.05, 0.1, 0.5, and 1 mg/mL (0.01, 0.05, 0.1, 0.5, and 1 mg/mL groups). The absorbance (A) values were detected by cell counting kit 8 at 7 days after cultured to compare the effect of titanium particles at different concentrations on proliferation, and median lethal concentration was screened out. The expression of collagen type I was detected by ELISA to observe the effect of titanium particles on differentiation. The osteoblasts co-cultured with titanium particles of median lethal concentration (experimental group) for 7 days, and double fluorescence staining with FITC-phalloidine and propidium iodide was performed. The cytomorphology variation of osteoblasts after swallowing titanium particles was observed under laser scanning confocal microscope. The osteoblasts at passage 3 cultured with culture medium without titanium particles served as control group. ResultsThe cultured cells were identified as osteoblasts by ALP staining and alizarin red staining. Different concentrations of titanium particles could inhibit osteoblasts proliferation and differentiation in varying degrees, showing significant difference when compared with the control group at 7 days after culture (P<0.05). The cell proliferation and differentiation were decreased with increased titanium particles concentration; significant differences were found between the other groups (P<0.05) except 0.01 and 0.05 mg/mL groups (P>0.05). The median lethal concentration of titanium particles was 0.5 mg/mL. Laser scanning confocal microscope showed cellular shrinking, microfilaments distortion, pseudopodia contraction of osteoblasts that swallowed titanium particles in the experimental group. ConclusionTitanium particles can inhibit proliferation and differentiation of osteoblasts. The effect may be related to variation of cytomorphology after swallowing titanium particles.
ObjectiveTo investigate the effects of hypoxia inducible factor 1α (HIF-1α) overexpression on the differentiation of stem cells derived from human exfoliated deciduous teeth (SHED) into vascular endothelial cells.MethodsSHED was isolated from the retained primary teeth donated by healthy children by using collagenase digestion method. The third generation cells were identified by flow cytometry and alizarin red and alkaline phosphatase (ALP) staining after osteogenic differentiation culture. The SHED were divided into blank control group (SHED without any treatment), empty group (SHED infected with empty lentivirus), HIF-1α overexpression group (SHED infected with HIF-1α overexpression lentivirus), Wnt inhibitor group (SHED interfered by IWR-1), and combination group (HIF-1α overexpressed SHED interfered by IWR-1). Real-time fluorescence quantitative PCR (qRT-PCR) and Western blot were used to analyze the expressions of HIF-1α mRNA and protein in the SHED of blank control group, empty group, and HIF-1α overexpression group. Then the SHED in 5 groups were induced differentiation into vascular endothelial cells for 14 days. The expressions of cell surface marker molecule [von Willebrand factor (vWF) and CD31] were detected by flow cytometry. The mRNA expressions of vascular cell adhesion protein 1 (VCAM-1), KDR (Kinase-inserted domain containing receptor), and VE-cadherin (VE) were analyzed by qRT-PCR. The protein expressions of phosphate-glycogen synthasc kinase 3β (p-GSK3β) and β-catenin were analyzed by Western blot. The tube forming ability of induced cells was detected by Matrigel tube forming experiment. The ability of endothelial cells to phagocytic lipid after differentiation was detected by DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL) phagocytosis.ResultsAfter identification, the cells were SHED. After lentivirus transfection, compared with the blank control group and the empty group, the expressions of HIF-1α mRNA and protein in the HIF-1α overexpression group increased significantly (P<0.05). Compared with the blank control group and the empty group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly higher (P<0.05), the relative expression of p-GSK3β protein was significantly lower (P<0.05), the number of tubules formed and the ability to phagocytic lipids significantly increased (P<0.05) in the HIF-1α overexpression group; while the indicators in the Wnt inhibitor group were opposite to those in the HIF-1α overexpression group (P<0.05). Compared with the HIF-1α overexpression group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly lower (P<0.05), the relative expression of p-GSK3β protein was significantly higher, and the number of tubules formed and the ability of phagocytosis of lipids significantly reduced, showing significant differences between groups (P<0.05).ConclusionOverexpression of HIF-1α can promote SHED to differentiate into vascular endothelial cells by activating Wnt/β-catenin signaling pathway.
Objective To investigate the feasibility of Y27632 to induce transdifferentiation from human retinal pigment epithelial (hRPE) cells into neuron-like cells in vitro. Methods The third to sixth generation of primary hRPE cells were cultured with 2% fetal bovine serum + Dulbecco's modified eagle medium/F12 culture solution, with (experimental group) or without (control group) 10 mu;mol/L Y27632. At 3, 6 hours and 1, 3, 5, 7 days after induction, the morphologic changes of RPE cells were observed by inverted microscope. The expression rate of CK18, Map2, NF200 and Pax6 at 3 days after induction in the experimental and control group were detected by immunofluorescent staining. chi;2 test was employed for comparison between the two groups. Results 50.1% cells of the experimental group formed axon-like processes and interconnected each other with typical neuron-like appearance. The expression rates of CK18, Map2, NF200 and Pax6 in the experimental group were 43.88%, 31.90%, 57.45% and 65.79%, while the above indexes in the control group were 93.97%, 4.49%, 22.37% and 8.33% respectively. Compared the expression rate of CK18 (chi;2=64.763), Map2 (chi;2=23.634), NF200 (chi;2=21.261) and Pax6 (chi;2=25.946) between the two groups, the differences were significant (P＜0.01). Conclusion The hRPE cells can be trans-differentiated into neuron-like cells in vitro by Y27632.
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.
ObjectiveTo investigate the feasibility of small molecule compound XAV939 to induce mouse embryonic stem cells (mESC) to differentiate into cardiac myocytes. MethodsWe revived and cultured undifferentiated mESC growing confluently on trophoderm made of mouse embryonic inoblast cell. The mESCs were digested by trypsin to form embryoid bodies (EBs) by handing drop method. After plated, EBs were induced by XAV939 to differentiate into cardiac myocytes. We observed the cardiac myocytes with lightmicroscopy and identified it with immunofluorescence method. Result The XAV939 can effectively induce mESC into cardiac myocytes with the mean efficiency rate of 71.85%±1.05%. The differentiated cardiac myocytes shrinked spanteously and rhythmicly. The cardiac troponin T as the special marker of cardiac myocyte was positive. ConclusionThe small molecule compound XAV939 could effectively induce mES cells into cardiac myocytes.
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 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.