[In vitro study on expression of tumor stem cell biomarker and transdifferentiation towards endothelial cells of retinoblastoma cells under hypoxia condition].

OBJECTIVE: To investigate the capability of RB cells that represent characteristics of tumor stem cell and trans-differentiate towards endothelial cells under hypoxia microenvironment, as well as their mechanism. METHODS: Experimental research. RB cell line Y79 was cultured in hypoxia environment with or without rapamycin treatment. Morphological changes, expression of neuron specific enolase (NES) and ATP binding cassette transporters G2 (ABCG2), and expression of HIF-1α protein and mRNA were detected. After been induced toward endothelial cells, expression of CD31 and vWF, up-taking of Dil-acLDL, vasculogenic mimicry (VM) formation in 3D culture and expression of HIF-1α,EphA2 and PI3K were detected. The ANOVA test was performed to compare the differences among groups, and SNK-q test was performed to further comparison. RESULTS: Y79 cells in normal oxygen group were single or agminated suspending cells. In hypoxia group, part of Y79 cells became adherent. No adherent cells were observed in rapamycin fore-treated group. Positive rates of NES and ABCG2 had significant difference among these three groups (FNES = 698.45, FABCG2 = 864.48, all P < 0.01). Compared with normal group [(98.2 ± 2.5)%, (2.1 ± 2.1)%],NES positive rate [(35.1 ± 3.4)%] was significantly reduced and ABCG2 positive rate [(67.4 ± 3.6)%] was significantly enhanced in hypoxia group (q = 46.11, 50.89; both P < 0.01), no significant changes were observed in rapamycin fore-treated group(P > 0.05). Expressions of HIF-1α protein and mRNA showed significant difference among these three groups (Fprotein = 314.85, FmRNA = 132.01, all P < 0.01). Compared with normal oxygen group (0.165 ± 0.056,0.927 ± 0.715) , HIF-1α protein (1.094 ± 0.077) and mRNA (6.408 ± 0.686) were significantly increased in hypoxia group(q = 31.81, 18.40; both P < 0.01), HIF-1α mRNA (7.219 ± 0.591) was significantly increased but no increased protein expression (0.218 ± 0.061) was observed in rapamycin fore-treated group. After transdifferentiation induction,Y79 cell in hypoxia group expressed CD31 and vWF, acquired the abilities of acLDL up-taking and VM formation. Compared with normal group(0.327 ± 0.108, 0.194 ± 0.033, 0.402 ± 0.068), expression of HIF-1α,EphA2 and PI3K protein (1.440 ± 0.089,0.377 ± 0.056,0.762 ± 0.090) was significant increased in hypoxia group(q = 8.72-23.00, all P < 0.01). CONCLUSIONS: Under hypoxic condition, part of RB cells can express tumor stem cell markers, transdifferentiate towards endothelial cells and acquire part of endothelial cell function and VM formation capability. HIF-1α through EphA2/PI3K pathway may play an important role in VM formation.

[HIF-1alpha, HPSE and VEGF promote malignant progression of retinoblastoma].

OBJECTIVE: To investigate the expression of heparanase (HPSE), hypoxia-inducible factor (HIF-1alpha) and their correlation with expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD) in retinoblastoma. METHODS: HIF-1alpha and VEGF were detected by SP immunohistochemical method in 34 cases of retinoblastoma and 10 cases of normal retina tissues. MVD was measured by anti-CD34. Eighteen samples of retinoblastoma tissues and ten samples of normal retina were examined for HPSE mRNA, HIF-1alpha mRNA, VEGF mRNA expression by RT-PCR. The correlation between HPSE, HIF-1alpha and VEGF expression, MVD and clinical and pathological characters were analyzed. The statistical methods are the measurement data using grouped quantitative data t test, variance analysis and q test; enumeration data compared with chi(2) test, Fisher test and rank sum test; correlation analysis using Spearman rank correlation analysis and the kappa test. RESULTS: The positive expression rates of HIF-1alpha and VEGF were 58.8%, 64.7% higher in retinoblastoma than in normal retina's (chi(2) = 10.784, P < 0.05; chi(2) = 9.269, P < 0.05). Positive expression of HPSE, HIF-1alpha, VEGF mRNA were found in 55.6%, 44.4%, 72.2%. The expression of HIF-lalpha and VEGF in retinoblastoma were correlated with MVD (r = 0.664, P < 0.05; r = 0.590, P < 0.05). VEGF was correlated with HPSE and HIF-1alpha (Z = 2.350, P = 0.009; Z = 2.940, P = 0.002). CONCLUSIONS: HPSE and HIF-1alpha can influence the expression of VEGF, an important angiogenesis factor. HPSE, HIF-1alpha and VEGF play a role in tumor angiogenesis and promote malignant progress of retinoblastoma.

[Paying attention to the research of apoptosis of retinal photoreceptor cells in retinal photochemical damage].

Many retinal degeneration diseases, such as retinitis pigmentosa and age-related macular degeneration, the main pathologic character of such retinal degeneration diseases is apoptosis of retinal photoreceptor cells. Recently, It is still short of effective treatment. Evidence from epidemiological studies and from experiments indicates that excessive light exposure may promote retinal degeneration, in which photoreceptor death by apoptosis leads to loss of vision. It is, therefore, of critical importance to pay more attention to further understanding of retinal photochemical damage and the apoptosis of retinal photoreceptor cells.

[Relationship between vasculogenic mimicry and clinical pathological characters in retinoblastoma].

OBJECTIVE: To explore if vasculogenic mimicry (VM) exists in retinoblastoma (Rb) and to explore the clinical significance of VM. METHODS: It was an experimental study. Sixty Rb specimens with complete clinical and prognostic data were collected. Periodic acid-Schif staining and immunohistochemical staining of CD34 were conducted to explore if VM exists in those Rb specimens. The expression of HIF-1alpha and VEGF were examined by immunohistochemical staining. The expression of CD34 endothelial antigen was used to label the neo-microvessels. Microvessel density (MVD) in retinoblastoma tissues was calculated. RESULTS: In HE staining slides, VM was present in Rb specimens and was formed by tumor cells but not endothelial cells. Red blood cells were present in the VM. VM existed in 18.33% (11/60) of the Rb specimens. Low R-E graded Rb specimens exhibited a higher VM positive rate than that in the high R-E graded Rb (chi(2) = 8.861, P < 0.05). The positive rate of VM was 4.34% in differentiated type of Rb and was 22.02% in undifferentiated type of Rb (chi(2) = 4.872, P < 0.05). HIF-1alpha and VEGF expressions in Rb with VM were significantly greater than those in Rb without VM (P = 0.001). The density of endothelial vessels correlated with VM. The mean MVD was 49.77 +/- 2.05 in Rb without VM and 36.53 +/- 1.15 in Rb with VM (P < 0.05). CONCLUSIONS: VM exists in Rb. Highly differentiated Rb exhibits more VM than that in less differentiated Rb. Expression of HIF-1alpha and VEGF is greater in Rb with VM, indicating that these factors may stimulate the occurrence of VM.

[The research of the protection of recombinant human erythropoietin in human RPE cells by light-induced injuries].

OBJECTIVE: The aim of this study was to assess the protection of recombinant human erythropoietin (rhEPO) in light-induced injures in human retinal pigment epithelial (RPE) cells. METHODS: It was a experimental study. Cultured human RPE cells were exposed to light of 8w 2000 +/- 500 Lux for 12 hours. The 3-(4,5-dimethylthiazole-2y1)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay were used to assess the effects of rhEPO in light-induced injury on human RPE cells. The effect of inhibiting apoptosis of rhEPO was detected by AnnexinV-fluorescein isothiocyanate/Propidium iodium labeling and flow cytometry. The enzyme linked immunosorbant assay (ELISA) and immunocytochemical staining were used to assess the expressions of caspase-3 treated by different doses of rhEPO in light-induced injury on human RPE cells and examine the protective mechanism of rhEPO by treatment with AG490 (the special inhibitor of jak2). RESULTS: There was a significant increase of inhibiting apoptosis in every rhEPO group, and cell viability was the highest in 40 U/ml rhEPO group, the value was 4.93 +/- 1.45/ml. The decrease in expression of caspase-3 was the most obvious in 40 U/ml rhEPO group, in which the value was 0.125 +/- 0.029 ng/ml. There was a significant increased effect on inhibiting apoptosis in every rhEPO group, and it was the most conspicuous in 40 U/ml rhEPO group. But these increased cell viability and effect on inhibiting apoptosis in rhEPO group were restrained by AG490, in which the value of apoptosis was 11.29 +/- 2.11/ml and the density of caspase-3 increased to 0.362 +/- 0.042 ng/ml. CONCLUSIONS: It is suggested that rhEPO can protect human RPE cells from the light-induced injures. Its protective mechanism is principally mediated by the EPO-EPOR pathway, which subsequently leads to jak2 activation.

[Hydrogen peroxide induces expression and transcription of erythropoietin in the human retinal pigment epithelium cells].

OBJECTIVE: To investigate the expression of erythropoietin (EPO) in human fetal retinal pigment epithelium (hfRPE) cells exposed to oxidative stress induced by hydrogen peroxide (H(2)O(2)) and to study the mechanisms. METHODS: The hfRPE cells were isolated, cultured and identified. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) of the hfRPE cells were examined. The changes of level of mRNA and protein of EPO in hfRPE cells exposed to oxidative stress were measured by semi-quantitative RT-PCR and immunocytochemical staining techniques. RESULTS: H(2)O(2) could increase the content of MDA and inhibit the activity of SOD in hfRPE cells. RT-PCR detected a significant increase of EPO mRNA level in cultured hfRPE cells exposed to oxidative stress. The level of EPO mRNA in the hfRPE cells reached a peak when exposed to 600 micromol/L H(2)O(2), then decreased after exposing to 800 micromol/L H(2)O(2). The immunocytochemical study detected that the changes of the level of EPO protein were similar to that of EPO mRNA. CONCLUSION: Oxidation stress by exposed to H(2)O(2) induces significant increase of EPO expression of hfRPE cells. Expression of EPO may be related to the survival and tolerance of hfRPE cells.

[Emphasis on the research of apoptosis in retinal degeneration diseases].

Many ocular fundus diseases such as retinitis pigmentosa and age-related macular degeneration are retinal degeneration diseases, which may lead to visual loss. The main pathologic character of such retinal degeneration diseases is apoptosis of retinal cells. In recent years, some progresses have been achieved on the prevention of apoptosis of retinal cells. In the field of ophthalmologic clinical and basic research, it is important to explore the use of anti-apoptotic approaches as novel preventive and therapeutic procedures for retinal degeneration diseases.

[Clinical and pathological analysis of choroidal metastatic carcinoma].

OBJECTIVE: To study the clinical and pathological characteristics of choroidal metastatic carcinoma. METHODS: The clinical data, pathological character, primary tumor origin and histological classification of 18 patients with choroidal metastatic carcinoma were analyzed retrospectively. RESULTS: Most patients had severe visual impairment. Solid mass was seen in the posterior pole of the eyes in all 18 patients through ocular fundus examination, 8 cases had retinal detachment. B scan and CT examination found flat or irregular masses. MRI examination had been performed on 5 patients, high signal intensities on T1W and low signal intensities on T2W were found. Five patients were adenocarcinoma, 4 were squamous carcinoma and 3 were undifferentiated carcinoma through pathological examination. Primary tumor was lung carcinoma in 10 cases (55%) and breast carcinoma in 4 cases (22%). CONCLUSIONS: Rapid decrease of visual acuity, flat neoplasm in ocular fundus and secondary retinal detachment are the main clinical characteristics of choroidal metastatic carcinoma. The most common primary tumor is lung carcinoma and the most common histopathological classification is adenocarcinoma. Imaging examination is helpful for the diagnosis of choroidal metastatic carcinoma.

[The mechanism of accumulative oxidative injury by hydrogen peroxide on human retinal pigment epithelial cells].

OBJECTIVE: To explore the effects of oxidative injury induced by hydrogen peroxide on human retinal pigment epithelial (RPE) cells. METHODS: Cultured human RPE cells were treated by 600 micromol/L hydrogen peroxide (H2O2) for 1, 6, 12, 24 and 72 hours. Cell viability was assessed by the MTT assay. Apoptosis was assessed by Annexin V-fluorescein isothiocyanate/Propidium iodium (Annexin V-FITC/PI) staining. The expression of clusterin was assessed by Western blot. RESULTS: (1) The treatment of RPE cells with 600 micromol/L H2O2 caused a time-dependent decrease of cellular viability. (2) Apoptosis was detected in cultured human RPE cells treated with 600 micromol/L H2O2 for 6 hours. The number of apoptotic cells reached a maximum at 24th hour after being exposed to 600 micromol/L H2O2 (P < 0.05). (3) Western blot showed the expression of clusterin protein was demonstrated in 6 hours exposure to 600 micromol/L H2O2, a significantly increasing of clusterin expression was observed overtime (P < 0.01). Thereafter the expression of clusterin protein decreased at 24th hour after being exposed to 600 micromol/L H2O2. At 72nd hour, the expression of clusterin protein was quite weak. CONCLUSION: Hydrogen peroxide can inhibits RPE proliferation and induces apoptosis and aging gene expression;the result suggest that accumulative oxidative injury induced by hydrogen peroxide in RPE in vitro may be similar to the aging changes in vivo.

[The protective effect of hypoxic preconditioning against light-induced photoreceptor degeneration in mice].

OBJECTIVE: To study the protective roles of hypoxic preconditioning in light induced retinal injury in a mice model and investigate the possible mechanism of related gene regulation. METHODS: 54 BALB/c mice were randomly divided into simple light exposure group (SL), hypoxic pretreatment group (HP) and control group (CON). The mice of SL and HP were continually exposed to light for 3 h, which built model of light-induced damage. Morphologic changes of photoreceptor cells in different group were examined by light microscope and the apoptosis was detected by TdT-mediated dUTP nick end labeling. Different expressions of c-fos and caspase-1 gene were examined by immunohistochemical staining. RESULTS: In group SL, the photic injury appeared very obviously and early. Different changes appeared in the outer nuclear layer after light exposure. Photic injury was aggravated following the increased light exposure. Positive staining of c-fos and caspase-1 could be seen in the outer nuclear layer. In group HP, the changes of retinal morphology appeared slightly and lately. Compared with group SL, caspsae-1 expression was decreased obviously, while no difference was seen in the expression of c-fos. The retinal structures was normal in the mice of control group and c-fos and caspase-1 were stained negative. CONCLUSION: Hypoxic preconditioning has neuroprotective effect on photoreceptor cells by inhibiting the expression of apoptosis-related genes in photic injured mice model. caspase-1 may be involved in the protective mechanism.

[Erythropoietin administration protects retina against light-induced retinal degeneration].

OBJECTIVE: To study whether recombinant human erythropoietin can pass through mice blood-retina barrier and the protective role in light-induced damage in retina. METHODS: After the injection of rHEPO, the content of rHEPO in 24 BALB/c mice retina was examined by enzyme linked immunosorbent assay (ELISA). 24 BALB/c mice were used to establish a light-induced damaged model, the difference of retina in rHEPO group and control group was compared using light microscope and TdT-mediated dUTP nick end labeling (TUNEL). RESULTS: The amount of retinal rHEPO in four deferent time points was (0.68 +/- 0.24) mU, (1.87 +/- 0.37) mU, (0.96 +/- 0.24) mU, (0.47 +/- 0.13) mU in 100 microg retinal total protein respectively by ELISA assay, there were statistical significances among groups. The density of rHEPO in the retina reached its peak at 4th hour after injection. Histology analysis: rHEPO group, at the 12th hour after light exposure the inner segment became condensed and disorganized. At the 36th hour the retina disorganized and vesiculated were seen in outer segments. At the 72nd hour the inner and outer segments were damaged more seriously and the outer nuclear layer became thinner and denser. On the 7th day, the retinal outer nuclear layer became thinner and condenses. rHEPO group showed a minimal damage in every time points but outer nuclear layer disorganized and vesiculated in inner and outer segments. No obvious changes in retinal thickness. The apoptotic cells were detected by TUNEL. At the 12th hour after light exposure, there were the apoptotic cells in outer nuclear layer near outer plexiform layer. At 36th hour the numbers of apoptotic cells were increased, however at the 72nd it was decreased obviously, only a few scattering apoptotic cells were revealed in the outer nuclear layer. Numbers of apoptotic cells between the rHEPO group and control group in outer nuclear layer were statistical significance (P < 0.01). CONCLUSIONS: rHEPO can pass through the mice blood-retina barrier and rHEPO has neuroprotective effect on mice retina. rHEPO may be used to treat degenerative retinal diseases.

Therapeutic effect of bFGF on retina ischemia-reperfusion injury.

BACKGROUND: Basic fibroblast growth factor (bFGF) plays important roles in retina degeneration, light injury, mechanical injury, especially in retina ischemia-reperfusion injury (RIRI). This study was to investigate the therapeutical effect of bFGF on RIRI and its mechanisms. METHODS: Experimental RIRI was induced by increasing intraocular pressure (IOP) in the eyes of 48 rats. These rats were divided into normal control, ischemia-reperfusion and bFGF-treated groups. Histological and ultrastructural changes of in the retina of different groups were observed, and the number of retinal ganglion cells (RGCs) was quantitatively analyzed under microscopy. Apoptotic cells were detected using the TdT-dUTP terminal nick-end labeling (TUNEL) method. The expression of caspase-3 was determined by streptavidin peroxidase (SP) immunohistochemistry. Atomic absorption spectrum method was used to evaluate the intracellular calcium changes. RESULTS: At the early stage of retinal ischemia-reperfusion injury, retina edema in the treated group was significantly eliminated compared with the untreated ischemic animals. RGCs in the bFGF-treated group was more than those in the untreated ischemic group during the post-reperfusion stages. In ischemic group, apoptotic cells could be found at 6th hour after reperfusion and reached the peak at 24 hours. At 72nd hour no apoptotic cells could be found.The changes in caspase-3 expression had a similar manner. The intracellular calcium of rat retina began to increase at 1st hour, reached the peak at 24 hours, and began to decrease at 72 hours. The change of the three markers in the treatment group showed a similar pattern, but they were all relatively less obvious. CONCLUSION: Apoptosis may play a vital role in RIRI. bFGF may has therapeutical effects on RIRI by inhibiting the increase of intracellular calcium and caspase-3 expression.

[The expression of cluster of differentiation 44 variant 6 and fibronectin in lacrimal gland tumors].

OBJECTIVE: To investigate the expression of cluster of differentiation 44 variant 6 (CD44V6) and fibronectin (FN) in lacrimal gland tumors. METHODS: Strept Avidin-Biotin Complex (SABC) immunohistochemistry method was used to study the expression of CD44V6 and FN in 49 cases of lacrimal gland tumors. RESULTS: In benign lacrimal gland tumors, the CD44V6 and FN positive rates were 30.8% (8/26) and 80.8% (21/26) respectively. In malignant lacrimal gland tumors, the CD44V6 and FN positive rates were 60.9% (14/23) and 34.8% (8/23). The positive expression rates of CD44V6 and FN in malignance were significantly different from benign and control tissue. There is also a significant difference in the expression of CD44V6 and FN in the cases of recurrence and non recurrence (P <0.05). CONCLUSIONS: The expression of CD44V6 and FN was correlated with the progress and recurrence of lacrimal gland tumors. The positive staining of CD44V6 and FN was useful for differentiating malignance from benignity and evaluating the progress and recurrence.

[Distribution and expression of basement membrane proteins in malignant eyelid tumors and its clinical significance].

OBJECTIVE: To study the distribution and expression of type IV collagen (Col IV), laminin (LN) and fibronectin (FN) in the basement membrane of malignant eyelid tumors so as to explore the relationships between the basement membrane and the degree of differentiation, metastasis and prognosis of malignant tumors. METHODS: Eighty-five paraffin-embedded specimens of malignant eyelid tumors including basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and sebaceous cell carcinoma were studied to identify the Col IV, LN and FN with immunohistochemical SABC techniques. RESULTS: A continuous intact basement membrane (BM) could be identified in all of the normal tissues and benign tumor specimens. In the malignant tumor specimens, the BM was defective or absent. The positive expression rate of Col IV in BCC, SCC and sebaceous cell carcinoma was 65% (24/40), 56% (16/25) and 40% (8/20). The positive expression rate of LN was 58% (23/40), 40% (10/25) and 35.% (7/20), respectively. The positive expression rate of FN was 33% (13/40), 36% (9/25) and 30% (6/20), respectively. The positive expression rate of these extracellular matrices was correlated with the degree of differentiation of the tumor. The difference of positive rate between highly-differentiated and poorly-differentiated tumors was significant (P < 0.05). In a short term follow-up (6-18 months), a statistically significant correlation (P < 0.05) could be observed between tumor recurrence and BM integrity. A higher recurrence rate and early recurrence were found in patients with interrupted BM. CONCLUSION: The absence of expression of Col IV, LN and FN was related to the invasive capacity and recurrence of the tumor. This result can be used as an index for clinical judgment of prognosis of the eyelid tumors.

[An experimental study of the therapeutical effect of bFGF in retinal ischemia/reperfusion injury].

OBJECTIVE: To investigate the therapeutical effect of basic fibroblast growth factor (bFGF) on retina ischemia/reperfusion injury. METHOD: Experimental retinal ischemia/reperfusion injury was induced by increasing intraocular pressure of rats eyes. 48 rats were divided into groups of control, ischemia/reperfusion and bFGF-treated, randomly. Apoptotic cells were detected using the TdT-dUTP terminal nick-end labeling at 1 hour, 6 hours, 12 hours, 24 hours, and 72 hours after reperfusion. The expression of caspase-3 at specified times was determined by Streptavidin Peroxidase immunohistochemistry. Atomic absorption spectrum method was used to evaluate the intracellular calcium changes of retinal tissues. RESULTS: In ischemia group, apoptotic cells began to appear at 6th hour after reperfusion and increased progressively with time. The number of apoptotic cells reached the peak 24 hour after reperfusion, and no apoptotic cells could be found at 72 hours. Changes in caspase-3 expression followed a similar trend. The intracellular calcium level of rat retina began to increase at 1 hour after reperfusion, and continued to increase with the reperfusion time. At 24 hours after reperfusion the intracellular calcium level reached the peak, and decline thereafter up to 72 hours. The patterns of change of the three markers of treatment group were similar to the above. However, the magnitude of changes was relatively lower. A statistically significant difference (P < 0.05) between the ischemia group and treatment group at 6th, 12th and 24th after reperfusion was observed. CONCLUSION: Apoptosis may play a vital role in ischemia-reperfusion injury of the retina. bFGF may have a therapeutical effect on ischemia-reperfusion injury by inhibiting the increase of retinal intracellular calcium stores and caspase-3 protein expression.