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 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 .
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.
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.
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.
ObjectiveTo investigate the function and possible mechanism of microRNA-199b-5p (miR-199b-5p) in Ewing's sarcoma cell lines so as to provide theoretical basis for biological treatment in the future. MethodsA673 cells and TC252 cells were adopted as Ewing's sarcoma cell lines in vitro. miR-199b-5p oligonucleotide fragments (mimic) and scramble control (mimic control) were used to transfect TC252 cells and A673 cells, respectively. The expression of miR-199b-5p was measured in 2 cell lines by real-time quantitative-PCR and compared with that in mesenchymal stem cells (MSCs). The cell proliferation was examined by cell counting kit 8. The cell cycle and apoptosis were detected by flow cytometry. The possible targets of miR-199b-5p were determined by luciferase assays. The protein expressions of possible targets were measured by Western blot. ResultsThe expression of miR-199b-5p in control group was significantly down-regulated in A673 cells and TC252 cells when compared with that in MSCs (P<0.05); and the expression of miR-199b-5p in experimental group was significantly up-regulated when compared with that in control group (P<0.05). G1 phase cells increased obviously and S phase cells decreased significantly compared with corresponding cells in control group (P<0.05); but no significant difference was found in G2/M phase cells between 2 groups (P>0.05). The apoptosis rate increased significantly in experimental group when compared with that in control group (P<0.05). The possible targets of miR-199b-5p were cyclin-L1 (CCNL1) and c-Kit by luciferase assays. Western blot results showed that the CCNL1 and c-Kit protein expression levels in experimental group were significantly lower than those in control group (P<0.05). ConclusionmiR-199b-5p can suppress the cell proliferation, block the cell cycle, and promote the cell apoptosis, so miR-199b-5p inhibits Ewing's sarcoma cell lines by targeting CCNL1 and c-Kit.
Objective To investigate the influence of RNA interference targeting c-Jun gene on the proliferation of rat vascular smooth muscle cells (VSMCs). Methods The experiment was performed with c-Jun siRNA （c-Jun siRNA group）, control reverse sequence siRNA (control siRNA group) or no siRNA (control group). VSMCs were transfected with siRNA targeting c-Jun gene by liposome. Effects of c-Jun siRNA on mRNA and protein expressions of c-Jun were examined by RT-PCR analysis and Western blot respectively. MTT test and 3H-TdR incorporation were used to detect VSMCs proliferation. Cell cycle analysis of VSMCs in vitro was determined by flow cytometer. Results The expression levels of mRNA and protein of c-Jun in c-Jun siRNA group were significantly lower than those in control group （P＜0.05， P＜0.01). There was no significant difference between control group and control siRNA group （Pgt;0.05）. Proliferation activity of VSMCs decreased significantly in c-Jun siRNA group compared with that in control group （P＜0.05） and VSMCs was blocked in the G0/G1 phase of cell cycle significantly （P＜0.05）. There was no significant difference between control group and control siRNA group （Pgt;0.05）. Conclusion c-Jun gene silenced by RNA interference can inhibit VSMCs proliferation effectively in vitro.
Objective To investigate the relationship of the expression between heat shock protein (HSP) 70 and 90, and Survivin and its effects on the proliferative activity in retinoblastoma (RB) cells. Methods Expression of Survivin, HSP70 and 90, and Ki-67 in conventional paraffin samples from 43 patients with RB and 6 healthy people was detected by streptavidin-biotin peroxidase (SP) immunohistochemical method. Ki67 labeling index was used to evaluate the proliferative activity in RB. Results In 43 cases of RB, positive expression of HSP70 and 90 and Survivin was found in 28 (65.12%), 37 (86.05%) and 27 (62.79%) cases, respectively. None of the 6 normal retinal tissue expressed HSP70, HSP90 or Survivin. Positive expression of Survivin was more frequent in positive expressions of HSP90 than that in negative expressions of HSP90 (P＜0.05). Ki67 labeling index was higher in positive expressions of HSP90 and positive expressions of Survivin than that in their negative expressions respectively (P＜0.05). Meanwhile, higher Ki67 labeling index was found in positive HSP90Survivin expressions than that in negative HSP90Survivin expressions and those cases where only HSP90 or Survivin was found (P＜0.05). Expression of HSP70 did not correlate with that of Survivin, nor had any significant effect on Ki67 labeling index (P＞0.05). Expression of HSPs and Survivin and Ki67 labeling index did not correlate with histological types (P＞0.05). Conclusion Expression of HSP90 correlates with that of Survivin in RB. Co-existence of Survivin and HSP90 probably plays an important role in the genesis of RB.
Objective To explore the effect of bone morphogenetic protein 4 (BMP4) on the glycolysis level of human retinal microvascular endothelial cells (hRMECs). MethodsA experimental study. hRMECs cultured in vitro were divided into normal group, 4-hydroxynonenal (HNE) group (4-HNE group) and 4-HNE+BMP4 treatment group (BMP4 group). 4-HNE group cell culture medium was added with 10 μmmol/L 4-HNE; BMP4 group cell culture medium was added with recombinant human BMP4 100 ng/ml after 6 h stimulation with 10 μmol/L 4-HNE. The levels of intracellular reactive oxygen species (ROS) were detected by flow cytometry. The effect of 4-HNE on the viability of cells was detected by thiazole blue colorimetry. Cell scratch test and Transwell cell method were used to determine the effect of 4-HNE on cell migration. The relative expression of BMP4 and SMAD9 mRNA and protein in normal group and 4-HNE group were detected by real-time quantitative polymerase chain reaction and Western blot. Seahorse XFe96 cell energy metabolism analyzer was used to determine the level of intracellular glycolysis metabolism in normal group, 4-HNE group and BMP4 group. One-way analysis of variance was used for comparison between groups. ResultsThe ROS levels in hRMECs of normal group, 4-HNE group and BMP4 group were 21±1, 815±5, 810±7, respectively. Compared with the normal group, the levels of ROS in the 4-HNE group and the BMP4 group were significantly increased, and the difference was statistically significant (F=53.40, 50.30; P＜0.001). The cell viability in the normal group and 4-HNE group was 1.05±0.05 and 1.28±0.05, respectively; the migration rates were (0.148±0.005)%, (0.376±0.015)%; the number of cells passing through the pores were 109.0±9.6, 318.0±6.4, respectively. Compared with the normal group, the 4-HNE group had significantly higher cell viability, cell migration rate, and the number of cells passing through the pores, and the differences were statistically significant (F=54.35, 52.84, 84.35; P＜0.05). The relative expression levels of BMP4 and SMAD9 mRNA in the cells of the 4-HEN group were 1.680±0.039 and 1.760±0.011, respectively; compared with the normal group, the difference was statistically significant (F=53.66, 83.54; P＜0.05). The relative expression levels of BMP4 and SMAD9 proteins in the cells of the normal group and 4-HEN group were 0.620±0.045, 0.860±0.190, 0.166±0.049, 0.309±0.038, respectively; compared with the normal group, the differences were statistically significant (F=24.87, 53.84; P＜0.05). The levels of intracellular glycolysis, glycolytic capacity and glycolytic reserve in normal group, 4-HNE group and BMP4 group were 1.21±0.12, 2.84±0.24, 1.78±0.36, 2.59±0.11, 5.34±0.32, 2.78±0.45 and 2.64±0.13, 5.20±0.28, 2.66±0.33. Compared with the normal group, the differences were statistically significant (4-HNE group: F=86.34, 69.75, 58.45; P＜0.001; BMP4 group: F=56.87, 59.35, 58.35; P＜0.05). There was no significant difference in intracellular glycolysis, glycolysis capacity and glycolysis reserve level between 4-HNE group and BMP4 group (F=48.32, 56.33, 55.01; P＞0.05). ConclusionBMP4 induces the proliferation and migration of hRMECs through glycolysis.