Objective To observe the clinical features of congenital hypertrophy of retinal pigment epithelium （CHRPE）. Methods The clinical data of 13 CHRPE patients including visual acuity, slit-lamp microscope examination, indirect ophthalmoscope examination and fundus fluorescein angiography (FFA) were retrospectively analyzed. The patients, 9 males and 4 females, with the mean age of 27.8 years. Results All patients were unilateral, without systemic diseases and no subjective symptoms in majority. Only 30.77% of initial diagnosis was correct, other diagnosis include choroidal nevi, old chorioretinopathy or no diagnosis. The round or oval black lesion was found in ocular fundus of all patients, 7.69% was located on the optic disk, 46.15% was located on the inferior temporal retina, 30.77% was located on the superior temporal retina, 15.39％ was located on the inferior nasal retina. 92.31％ was pigmented CHRPE and 7.69％ was non-pigmented CHRPE. FFA showed blocked fluorescence and transmitted fluorescence in the lesion, few eyes were found dilated capillary vessel and fluorescent leakage on the late stage of FFA, most eyes had normal retinal vessels. Conclusion The isolated CHRPE is round or oval black lesion in ocular fundus which lack of subjective symptoms, mostly located on the peripheral retina; the FFA characteristics showed blocked fluorescence and transmitted fluorescence, and CHRPE often misdiagnosed as other disease, it should be combine the ocular fundus manifestation with the FFA to diagnose properly.
Objective To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation. Methods Firstly, hyaluronic acid was oxidized with NaIO4 and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young’s modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR). ResultsFTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young’s modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point (P<0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group (P<0.05). ConclusionThe MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.
Objective To evaluate the effect of Schwann cell (SC) on the proliferation of marrow mesenchymal stem cells (MSCs) and provide evidence for application of SC in construction of the tissue engineered vessels.Methods SC and MSCs were harvested from SD rats(weight 40 g). SC were verified immunohstochemically by the S-100 staining, and MSCs were verified by CD 44, CD 105, CD 34 and CD 45. The 3rd passages of both the cells were cocultured in the Transwell system and were amounted by the 3H-TDR integration technique at 1, 3, 5 and 7 days,respectively. The results were expressed by the CPM(counts per minute, CPM) values. However, MSCs on both the layers were served as the controls. The Westernblot was performed to assess the expression of the vascular endothelial growth factor (VEGF), its receptor Flk-1, and the associated receptor neuropilin 1(NRP-1) in SC, the trial cells, and the controls. Results SC had a spindle shape in the flasks, and more than 90% of SC had a positive reaction for the S-100 staining.MSCs expressed CD44 and CD105, and had a negativesignal in CD 34 and CD 45. The CPM values of MSCs in the trial groups were 2 411.00±270.84，3 016.17±241.57，6 570.83±2 848.27 and 6 375.8±1 431.28at 1, 3, 5 and 7 days, respectively. They were significantly higher in their values than the control group (2 142.17±531.63，2 603.33±389.64，2 707.50±328.55，2 389.00±908.01), especially at 5 days （P＜0.05）. The Western blot indicated that VEGF was expressedobviously in both the SC group and the cocultured MSCs grou,p and was less visible in the control cells. The expressions of Flk-1 and NRP-1 inthe cocultured MSCs were much ber than in the controls. Conclusion SC can significantly promote the proliferation of MSCs when they are cocultured. The peak time of the proliferation effect appeared at 5 days. This effect may be triggered by the up-regulation of VEGF in MSCs, which also leads to the upregulation of Flk-1 and NRP-1 .
Objective To investigate the effect of bone morphogenetic protein 2 (BMP-2) and dexamethason (DXM) on proliferation and differentiation of human dental pulp cellsin vitro. Methods Primary human dental pulp cells were cultured in vitro by tissue culture method. The 3rd generation cells were used to identify cell phenotype for vimentin and cytokeratin by immunocytochemistry staining. The 3-5 generations of human dental pulp cells were randomly divided into 4 groups: 100 ng/mL BMP-2 (group A), 1×10–8 mol/L DXM (group B), and both 100 ng/mL BMP-2 and 1×10–8 mol/L DXM (group C) were added; neither BMP-2 nor DXM was added in group D as control group. The cell growth curve was drawn at 1, 3, 5, and 7 days after culture. The expressions of osteo/dentanogenic genes including alkaline phosphatase (ALP), dentin sialophoshoprotein (DSPP), and dentin matrix protein 1 (DMP-1) were detected by RT-PCR analysis at 5 and 7 days after culture, the ratio between the positive staining area and the total area by ALP staining at 14 days, and absorbance (A) value at 562 nm by alizarin red staining at 21 days after culture. Results Human dental pulp cells were successfully isolated and cultured, which were long fusiform and showed a positive reaction for vimentin and a negative reaction for cytokeratin. The growth curve indicated that cells increased with the extending of incubation time, reached a peak at 5 days, then reduced at 7 days to the level at 3 days. At 5 days after culture, the cells were significantly more in groups A, B, and C than group D (P<0.05), in group C than group A (P<0.05), and in group A than group B (P<0.05). RT-PCR analysis showed that the mRNA expressions of ALP, DSPP, and DMP-1 at 5 days were significantly higher in groups A, B, and C than group D (P<0.05), and in group C than groups A and B (P<0.05), but no significant difference was found between groups A and B (P>0.05); the mRNA expression of DSPP in groups A, B, and C was significantly higher than that in group D (P<0.05), but there was no significant difference in mRNA expressions between other groups at 7 days (P>0.05). At 14 days, positive staining in varying degrees was observed in each group, especially in group C; the ratio between the positive staining area and the total area was significantly higher in group C than groups A, B, and D (P<0.05), and in groups A and B than group D (P<0.05), but there was no significant difference between groups A and B (P>0.05). At 21 days, there were a variety of mineralized nodules in groups A, B, and C in nonuniformly scattered or clustered distribution, but no mineralized nodules were observed in group D. TheA values of mineralized nodules showed significant difference between groups (P<0.05). Conclusion BMP-2 may be more effective in promoting proliferation of human dental pulp cells than DXM. Combined application of BMP-2 and DXM can remarkably promote the proliferation and differentiation of human dental pulp cells.
ObjectiveTo explore the expression of Ki-67 antigen in the breast cancer tissues and to evaluate the relationship of the expression to biology behavior as well as prognosis of breast cancer. MethodLiteratures about relation between Ki-67 and breast cancer were reviewed. ResultsThe expression of Ki-67 in the breast cancer tissue was obviously higher than that in the adjacent to cancer tissue or normal breast tissue. The Ki-67 positive expression rate was positively correlated with pathology classification and clinical stage of breast cancer, the correlation was not consistent about the expression of Ki-67 and axillary lymph node metastasis of breast cancer. ConclusionsKi-67 is a cell proliferation nuclear antigen related to cell cycle, and its expression changes along with the change of cell cycle, it has been employed as a reliable marker of cell proliferation. The expression of Ki-67 has an important significance in early diagnosis and guiding of neoadjuvant chemotherapy as well as prognosis of breast cancer.
Objective To investigate the expression of SAPCD2 in the lung adenocarcinoma cells, and to study the effect of SAPCD2 regulating Hippo signaling pathway on the proliferation, invasion, migration and apoptosis of the lung adenocarcinoma cells and its mechanism. Methods Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression levels of SAPCD2 mRNA and protein in four types of lung cancer cells (HCC827, H1650, SK-MES-1, A549) and human normal lung epithelial cells (BESA-2B), respectively. Then, lung cancer cells with relatively high levels of SAPCD2 expression were selected for subsequent experiments. The experiment cells were divided into a normal control group (NC group), a si-SAPCD2 group, and a pathway inhibitor group (si-SAPCD2+XMU-MP-1 group). Firstly, SAPCD2 mRNA was silenced using small interfering RNA (siRNA) technology, and then qRT-PCR was used to detect the expression of SAPCD2 in transfected lung cancer cells; using clone plate assay to detect the proliferation of lung cancer cells after silencing; using flow cytometry to detect the apoptosis of lung cancer cells after silencing; observe the number of lung cancer cells at different stages through cell cycle experiments; then Transwell experiment was used to analyze the effect of silencing SAPCD2 on the migration and invasion of lung cancer cell migration. Finally, Western blot was used to detect the expression of ki-67, Bcl-2, Caspase-3, NF2, P-MST1, P-LATS1, P-YAP, YAP, and TAZ proteins.Results SAPCD2 had the highest expression level in lung adenocarcinoma A549 cells (P<0.01). Silencing SAPCD2 significantly decreased the proliferation ability of A549 cells (P<0.01), inhibited their migration (P<0.05) and invasion (P<0.01), and promoted A549 cell apoptosis (P<0.01); more than half of the cells remained in the G0/G1 phase. Compared with the NC group, A549 cells showed a significant increase in G0/G1 phase cells (P<0.01), a significant decrease in G2/M and S phase cells (P<0.01), and a significant increase in the proportion of early apoptotic cells (P<0.01). Western blot results showed that silencing SAPCD2 down-regulated the expression of ki-67, Bcl-2, YAP, and TAZ proteins compared to the NC group (P<0.01), and up-regulated the expression of Caspase-3, NF2, P-MST1, P-LATS1, and P-YAP proteins (P<0.01). Conclusions The expression of SAPCD2 in lung adenocarcinoma A549 cells is significantly higher than that in normal lung epithelial cells (BESA-2B), which promotes the proliferation, migration and invasion of A549 cells and inhibits apoptosis. The mechanism may be related to the inhibition of Hippo signaling pathway.
ObjectiveTo evaluate the expression of miR-338-5p in colorectal cancer tissues and study its role in colon cancer cell proliferation, apoptosis, and cell cycle. MethodsThe expression of miR-338-5p was detected by real-time PCR in the colorectal cancer tissues and corresponding adjacent to cancer tissue samples. The miR-338-5p-mimics was transfected into the colon cancer cell lines HCT116 and SW620 to investigate its role in cell proliferation, apoptosis, and cell cycle. The cell proliferation and apoptosis were measured by CCK-8 and flow cytometry, respectively. The cell cycle was also analyzed by flow cytometry. Results①miR-338-5p expression was significantly downregulated in the colorectal cancer tissues as compared with corresponding adjacent to cancer tissue samples(P < 0.01). 2 Compared with the transfected negative control cells, the proliferation ability of colon cancer cell HCT116 or SW620 was significantly decreased(P < 0.01), cell apoptosis was significantly increased[HCT116 cell:(11.43±0.67)% versus(7.98±0.36)%, P < 0.01;SW620 cell:(10.5±0.2)% versus(7.93±0.5)%, P < 0.01), and cell G1 was arrested[HCT116 cell:(80.41±1.34)% versus (64.87±1.83)%, P < 0.01;SW620 cell:(68.76±0.41)% versus(54.89±0.78)%, P < 0.01) after transfecting miR-338-5p-mimics cells. ConclusionmiR-338-5p may act as an anti-oncogene in colorectal cancer through regulation of cell proliferation, apoptosis, and cell cycle.
To study the potential molecular mechanism of tumor angiogenesis in its microenvironment, we investigated the effects of HepG2 conditioned medium on the proliferation of vascular endothelial cell and vascular angiogenesis in our laboratory. Human umbilical vein endothelial EA.hy926 cells were co-cultured with HepG2 conditioned medium in vitro. The proliferation and the tubulogenesis of EA.hy926 cells were detected by teramethylazo salt azole (MTT) and tube formation assay, respectively. The results showed that the survival rate of the EA.hy926 cells was significantly increased under the co-culture condition. HepG2 conditioned medium also enhanced the angiogenesis ability of EA.hy926 cells. In addition, the expressions of intracellular VEGF and extracellular VEGFR (Flk-1) were regulated upward in a time-dependent manner. In conclusion, the proliferation of vascular endothelial cells and Vascula angiogenesis were improved under the condition of indirect co-culture.
Objective To investigate the effect of berbamine (BBM) on the proliferation and apoptosis of retinoblastoma (RB) HXO-RB44 cells and its possible mechanism in vitro.Methods RB cells in logarithmic growth phase were divided into BBM treated group and control group. RB cells in BBM treated group were cultured with different concentrations of BBM (2,4,8,16 and 32 mg/L) for 24,48 and 72 hours, respectively. The proliferation was assayed by methyl Thiazolyl tetrazolium (MTT). RB cells were cultured with different concentrations of BBM (4,8 and 16 mg/L) for 24 hours. The early apoptotic rates were detected by flow cytometry; the expression of bcl-2 and Bax were measured by enzyme-linked immunosorbent assay (ELISA) and the activity of Caspase-3 was detected by colorimetric assay.Results BBM could obviously inhibit the proliferation of RB cells in a time and dose dependent manner (24 hours: F=70.547,P＜0.01; 48 hours: F=603.438,P＜0.01; 72 hours: F=577.521,P＜0.01). The IC50 value at 24,48 and 72 hours were 25.26, 10.94 and 6.25 mg/L, respectively. Necrosis rates of control group and BBM treated group were (1.25plusmn;0.45)%, (4.10plusmn;2.95)%, (4.39plusmn;0.21)% and (10.54plusmn;4.38)% respectively; the difference between two groups was statistically significant (F=6.527,P＜0.05). Apoptotic and necrosis rates in advanced stage of control group and BBM treated group were (2.13plusmn;0.71)%, (5.45plusmn;2.31)%, (9.86plusmn;3.18)% and (11.10plusmn;1.70)%, respectively. The difference between two groups was statistically significant (F=10.845,P＜0.05). Early apoptotic rates of control group and BBM treated group were (0.51plusmn;0.26)%, (2.68plusmn;0.35)%, (5.97plusmn;0.50)% and (11.22plusmn;1.17)%, respectively. The difference between two groups was statistically significant (F=144.976,P＜0.01). In addition, BBM dose-dependently reduced bcl-2 level and increased Bax expression, causing the reduction of the bcl-2/Bax protein ratio as well as increased the Caspase-3 activity in RB cells remarkably (bcl-2: F=835.726,P＜0.01; bax: F=111.963, P＜0.01;Caspase-3:F=298.058,P＜0.01).Conclusions BBM can inhibit the proliferation and induce apoptosis or necrosis of RB cells in vitro, down regulating the expression of bcl-2, up regulating the expression of Bax. Along with increased Caspase-3 activity these may be the apoptotic mechanisms.
Objective To observe the inhibition effect of curcumin on the proliferation of rabbit retinal pigment epithelial (RPE) cells and investigate its mechanism. Methods The 4th generation of RPE cells were selected and divided into curcumin group and blank control group. The concentration of curcumin included 10, 15, and 20 mu;g/ml. The MTT assay was used to evaluate the inhibition effect on the proliferation of RPE cells at the 24th, 48th, 72nd and 96th hour after cultured with curcumin (10, 15, and 20 mu;g/ml). The IC50 value of curcumin at different time points were calculated by Linear Regression. Flow cytometry was used to detect the effect on the cell cycle at the 72nd hour after cultured with curcumin (15 mu;g/ml); the expression and apoptosis of proliferating cell nuclear antigen (PCNA) were also determined at the 24th,48th, and 72nd hour after cultured with curcumin (15 mu;g/ml) respectively. The configuration of RPE cells were observed by transmission electron microscope. Results The IC50 value of curcumin at the 24th,48th, 72nd and 96th hour was 29.31, 17.50, 13.24, and 10.99 mu;g/ml respectively. Cell cycel analysis indicated that curcumin blocked cells in G0/G1 phase. At the 24th, 48th, and 72nd hour after cultured with curcumin (15 mu;g/ml), the expression of PCNA of RPE cells were 565.04plusmn;23.60, 473.61plusmn;36.88, and 396.15plusmn;32.45; the apoptosisrate were （12.83plusmn;0.13）%，（32.27plusmn;4.51）%，（56.81plusmn;8.67）%, respectively. The differeces of curcumin groups compared with the control group were significant (P＜0.05). Apoptosis of RPE cells was observed under transmission electron microscope. Conclusions Curcumin can inhibite the proliferation of RPE cells by inhibit the synthesization of PCNA and inducing the apoptosis of RPE cells. Curcumin may become a potential drug to prevent and treat PVR.