[Expression, roles and therapy target values of CD24 in oral squamous cell carcinoma].

OBJECTIVE: To determine the expression profile and potential roles of CD24 in oral squamous cell carcinoma and explore the values of CD24 function as a potential target of clinical therapy. METHODS: Semi-quantitative immunohistochemistry was used to construct the expression profile of CD24 in 78 human oral tissues and 59 Hamster buccal pouch tissues. Real-time RT-PCR and Western blot were used to analyze the CD24 expression levels in oral DOK4 cells, oral cancer CAL-27 and WSU-HN6 cells. Then these two cancer cell lines were selected to evaluate the effect of all-trans retinoic acid (ATRA) and CD24 antibody on CD24 expression, and the proliferation and tumorsphere formation capacity of these two cell lines. RESULTS: CD24 expression was found significantly elevated in both human and animal tissues compared with normal and benign tissues (P<0.05), as well as in oral cancer CAL-27 and WSU-HN6 cells compared with DOK cells (P<0.05). CAL-27 and WSU-HN6 cells possess increased proliferative and specific tumorsphere formation capability compared with DOK cells (P<0.05). Both ATRA and CD24 antibody were able to effectively inhibit the proliferation and tumorsphere formation of CAL-27 and WSU-HN6 cells (P<0.05). Among them ATRA at least involved partially in the proliferation by down-regulating the CD24 expression (P<0.05), while CD24 antibody blocking had no effect on the CD24 expression. CONCLUSION: CD24 was upregulated in oral cancer and functioned as a potential factor that promoted the proliferation and tumorsphere formation of CAL-27 and WSU-HN6 cells. Both ATRA and CD24 antibody might effectively inhibit the proliferation and tumorsphere formation of CAL-27 and WSU-HN6 cells and function as a potential therapy target.

[Overexpression of high mobility group A2 and its correlation with microRNA let-7 family in serous ovarian cancers].

OBJECTIVE: To examine the expression of high mobility group A2 (HMGA2), P53 and let-7 family microRNA, to investigate the correlation of HMGA2 and let-7, and to compare the HMGA2 and P53 expressions in human serous ovarian cancer. METHODS: Immunohistochemistry assay was used to examine the expressions of HMGA2 and P53 in 50 paraffin-embedded tissue specimens of human serous ovarian cancer and 4 normal fallopian tube tissues. HMGA2 mRNA and let-7 family microRNA were detected by real time fluorescent quantitative reverse transcription polymerase chain reaction in the corresponding frozen tissues. RESULTS: HMGA2 and P53 were immuno-positive in 70% (35/50) and 78% (39/50) of the ovarian cancer tissues, respectively. HMGA2 was weakly expressed in the ciliated cells, but negative in the secretary cells of the fallopian tube. There was a tendency that the expression of HMGA2 increased with higher pathological grade of the ovarian cancer, but no correlation was observed between the HMGA2 overexpression and clinical stages. HMGA2 mRNA was detected in all the ovarian cancer samples, and its expression level was higher than that of the normal fallopian tube tissues in 72% (36/50) of the ovarian cancer samples. The expression of HMGA2 mRNA was much higher in more malignant SKOV3.ipl cells than in its corresponding SKOV3 cells. All let-7 family members were detectable in all ovarian cancer samples, and their expression were inversely correlated with HMGA2 mRNA expression (r=-0.305,P<0.05). CONCLUSION: HMGA2 can be a biomarker complement to P53, and its high expression has an inclination of more malignancy. The downregulation of let-7 is, but not the only mechanism of HMGA2 overexpression in serous ovarian cancer.

Efficacy and reliability of long-term implantation of multi-channel microelectrode arrays in the optical nerve sheath of rabbit eyes.

In addition to epiretinal and subretinal areas, the optic nerve (ON) is also a candidate location for implanting visual prosthesis to restore vision of patients with retinitis pigmentosa (RP). Since the ON receives all the signals from the retina, stimulating the ON may potentially evoke phosphenes over a wider range of visual field. In this study, we designed a 9-channel microelectrode array and implanted it between the dura mater and pia mater of rabbit ONs by lateral orbitotomy. We recorded the current thresholds and evaluated the efficacy of the array using electrically evoked potentials (EEPs). Spatial discrimination of approximately 20° was verified by EEP maps over visual cortex. A large area of the visual field (over 130° along horizontal meridian) could be activated by this microelectrode array. Visual evoked potentials (VEPs) and different pathological examinations were used to examine potential damage of ONs. One year post implantation, we did not notice significant damages to either the ONs or the microelectrode arrays. EEPs were successfully recorded up to 6months post implantations. However, further studies are still needed to reduce fibrous encapsulation of the microelectrode array, which resulted in a gradual elevation of current thresholds to elicit EEPs.

[Effect of Abl-interacting protein 1 overexpression upon human gastric cancer cell proliferation in vitro].

OBJECTIVE: To investigate the expression of Abl-interacting protein 1 (ABI1) in normal gastric mucosal cell line GES-1 and gastric cancer cell line AGS, and the effects of ABI1 gene overexpression upon the proliferation of human gastric cancer cell AGS in vitro. METHODS: Firstly the ABI1 expression in GES-1 and AGS cells were identified by immunohistochemistry, immunofluorescence, RT-PCR, real-time PCR and Western blot. Secondly human gastric cancer cell line AGS was cultured and transfected with recombinant MSCV-GFP-ABI1 plasmid or blank plasmid MSCV-GPF. Real-time PCR and Western blot were used to detect the mRNA and protein expression of ABI1. And lastly the cell proliferation was detected by CCK-8 assay. RESULTS: ABI1 was expressed both in normal gastric mucosal cell line GES-1 and in gastric cancer cell line AGS. Compared to GES-1 cells, the ABI1 expression in AGS cells was lowered significantly. There were no significant differences in the ABI1 mRNA and protein expression between the AGS and AGS-MSCV-GFP groups. Compared to those of the AGS group, the ABI1 mRNA expression levels of the AGS-MSCV-GFP-ABI1 group increased by 1.87 times (P = 0.002). The protein expression levels of the AGS-MSCV-GFP-ABI1 group were remarkably higher than those of the AGS and AGS-MSCV-GFP groups (P = 0.002). CCK-8 assay showed that there were no significant differences in the proliferation rates at different time points between the AGS and AGS-MSCV-GFP groups. However, the proliferation rates at the time points of 24, 48, 72 and 96 hours of the AGS-MSCV-GFP-ABI1 were 1.46 +/- 0.31, 4.75 +/- 0.12, 6.62 +/- 0.32 and 8.96 +/- 0.27 respectively. And they were significantly lower than the proliferation rates of the AGS and AGS-MSCV-GFP groups (P < 0.01). CONCLUSION: ABI1 gene is down-regulated in gastric cancer cells. The ABI1 overexpression effectively inhibits the proliferation in human gastric cancer cell lines. It suggests that ABI1 may be involved in gastric cancer pathogenesis by regulating the proliferation of gastric carcinomas cells.

Effects of the phosphatidylinositol 3-kinase inhibitor in a mouse model of retinal neovascularization.

PURPOSE: To determine the effects of LY294002, a phosphatidylinositol 3-kinase inhibitor, on suppressing experimental retinal neovascularization in an animal model of ischemic retinopathy. METHODS: The effect of LY294002 on the survival of RF/6A cells stimulated by vascular endothelial growth factor (VEGF) was investigated colorimetrically. The inhibitory activity of LY294002 on the migration of cells stimulated with VEGF was measured by cell counting. C57BL/6N mice at postnatal day (P) 7 were exposed to 75 +/- 2% oxygen for 5 days (P7-P11) and then returned to room air for 5 days (P12-P17) to induce retinal neovascularization. Beginning on P12, mice received daily intraperitoneal injections of LY294002 or dimethyl sulfoxide and phosphate-buffered saline (control) through P17. Retinal neovascularization was examined by adenosine diphosphatase staining after 5 days in room air and was quantitated histologically by counting the neovascular endothelial cell nuclei anterior to the inner limiting membrane. RESULTS: LY294002 significantly inhibited VEGF-induced survival and migration. LY294002-treated and control animals demonstrated no perfusion regions in the posterior retina. Retinas from control mice at P17 contained neovascular tufts at the junction between the perfused and nonperfused retina. The tufts contained numerous neovascular nuclei. Retinas from mice treated with LY294002 demonstrated a significant reduction in neovascular cell nuclei compared with control mice. CONCLUSIONS: LY294002 significantly inhibits retinal neovascularization in a mouse model of retinal neovascularization.

Influential factors of thresholds for electrically evoked potentials elicited by intraorbital electrical stimulation of the optic nerve in rabbit eyes.

Influential factors of electrical thresholds for electrically evoked potentials elicited by intraorbital stimulation of the optic nerve, including stimulation positions of the optic nerve, stimulating electrodes, frequency and duration of electrical pulses and pathological status of the optic nerve, were evaluated in 48 pigmented rabbit eyes. Intravenous injection of sodium iodate was used to induce transneuronal degeneration of the retinal ganglion layer subsequent to photoreceptor death. Two equations were derived to predict electrical thresholds needed to elicit cortical responses.

Relationship between hepatitis B virus DNA levels and liver histology in patients with chronic hepatitis B.

AIM: To study the relationship between hepatitis B virus (HBV) DNA levels and liver histology in patients with chronic hepatitis B (CHB) and to determine the prevalence and characteristics of hepatitis B e antigen (HBeAg) negative patients. METHODS: A total of 213 patients with CHB were studied, and serum HBV DNA levels were measured by the COBAS Amplicor HBV Monitor test. All patients were divided into two groups according to the HBeAg status. The correlation between serum HBV DNA levels and liver damage (liver histology and biochemistry) was explored. RESULTS: Of the 213 patients with serum HBV DNA levels higher than 10(5) copies/mL, 178 (83.6%) were HBeAg positive, 35 (16.4%) were HBeAg negative. The serum HBV DNA levels were not correlated to the age, history of CHB, histological grade and stage of liver disease in either HBeAg negative or HBeAg positive patients. There was no correlation between serum levels of HBV DNA and alanine aminotransferanse (ALT), aspartate aminotransferase (AST) in HBeAg positive patients. In HBeAg negative patients, there was no correlation between serum levels of HBV DNA and AST, while serum DNA levels correlated with ALT (r=0.351, P=0.042). The grade (G) of liver disease correlated with ALT and AST (P<0.05, r=0.205, 0.327 respectively) in HBeAg positive patients. In HBeAg negative patients, correlations were shown between ALT, AST and the G (P<0.01, and r=0.862, 0.802 respectively). HBeAg negative patients were older (35 +/- 9 years vs 30 +/- 9 years, P<0.05 ) and had a longer history of HBV infection (8 +/- 4 years vs 6 +/- 4 years, P<0.05) and a lower HBV DNA level than HBeAg positive patients (8.4 +/- 1.7 Log HBV DNA vs 9.8 +/- 1.3 Log HBV DNA, P<0.001). There were no significant differences in sex ratio, ALT and AST levels and liver histology between the two groups. CONCLUSION: Serum HBV DNA level is not correlated to histological grade or stage of liver disease in CHB patients with HBV DNA more than 10(5) copies/mL. Compared to HBeAg positive patients, HBeAg negative patients are older and have a lower HBV DNA level and a longer HBV infection history. There is no significant difference in sex ratio, ALT and AST levels and liver histology between the two groups.

Diffusion of macromolecule through retina after experimental branch retinal vein occlusion and estimate of intraretinal barrier.

The disposition and diffusion knowledge of intravitreally injected macromolecule drugs through retina in pathological condition is crucial but the related studies are absent. Retinal edema is a common pathological change of fundus diseases and retinal vein occlusion (RVO) pig model were established to emulate it. FITC-dextrans of various molecular weights were dissolved in RPMI-1640 solutions and the rate of transretinal diffusion was determined with a spectrophotometer. Theoretical maximum size of molecule (MSM) was calculated by extrapolating the trend-linear relationship with the diffusion rate. In separate experiments to determine the sites of barrier to diffusion, FITC-dextrans were applied to either the inner or outer retinal surface, processed as frozen sections, and viewed with a fluorescence microscope. Paired-Samples T test was used to compared the diffusion rate of dextrans of the both eyes of one pig. The MSM in RVO tissues and normal tissue was 6.5+/-0.39 nm and 6.18+/-0.54 nm respectively (t=4.143, P=0.0001). FITC-dextrans applying to inner retinal surface, 4.4 kDa dextran were largely arrested at inner nuclear layer (INL). The INL of the 19.6-71.2 kDa dextran diffusion retina section became dark and the nerve fiber layer (NFL) and inner plexiform layer got brighter. As for 150 kDa dextran, the NFL was bright and the other layers were dark. FITC-dextrans applying to outer retinal surface, most dextrans were blocked before outer nuclear layer (ONL). In summary, ONL and INL may act as bottle-neck barriers to diffusion of macromolecules. Compared with normal neuroretina, the MSM of fresh edema retina after RVO increased limitedly.

Effects of subretinal implant materials on the viability, apoptosis and barrier function of cultured RPE cells.

BACKGROUND: Subretinal microphotodiode array (MPDA) is a type of visual prosthesis used for the implantation in the subretinal space of patients with progressive photoreceptor cell loss. The present study aimed to evaluate the effect of materials for MPDA on the viability, apoptosis and barrier function of cultured pig retinal pigment epithelium (RPE) cells. METHODS: Primary culture of pig RPE cells was performed and 24 pig eyes were used to start RPE culture. The third passage of the cultures was plated on different materials for MPDA and MPDAs. The tetrazolium dye-reduction assay (MTT) was used to determine RPE cell viability. Flow cytometry was measured to indicate the apoptosis rates of RPE cells on different materials. RPE cells were also cultured on microporous filters, and the transepithelial resistance and permeability of the experimental molecule were measured to determine the barrier function. RESULTS: The data from all the methods indicated no significant difference between the materials groups and the control group, and the materials tested showed good biocompatibility. CONCLUSIONS: The materials for MPDA used in the present study had no direct toxicity to the RPE cells and did not release harmful soluble factors that affected the barrier function of RPE in vitro.

[The effects of hematopoietic stem cells on mice vascular endothelial cells of diabetic retinopathy].

OBJECTIVE: The purpose of the study was to determine whether hematopoietic stem cell (HSCs) mobilization can regulate early diabetic retinopathy in mice. METHODS: Mice were divided into four groups: control group, normal mice HSC-mobilized group, diabetic mice control group and diabetic mice HSC-mobilized group. Murine stem cell growth factor (SCF) and recombined human granulocyte colony stimulating factor (rhG-csf) were administered to the mice with diabetes and without diabetes for continuous 5 days to induce autologous HSCs mobilization, and subcutaneous injection of physiological saline was used as control. The changes associated with autologous HSCs mobilization were characterized using flow cytometry, Immunohistochemistry and semiquantitative RT-PCR. Evans blue quantitative test was used to measure the breakdown level of blood-retina barrier. RESULTS: HSCs were marked by CD34-/low and Sca1+ in this study. The acceleration of endothelial cell regeneration was observed. A decrease of VEGF expression due to autologous stem cell mobilization was found. HSCs could increase the content of VEGFR-2 in mouse retina and significantly downregulated the expression of VEGF and ang-2 in diabetic mice. CONCLUSIONS: The experiment suggest that autologous HSCs mobilization can be approach of therapeutic vascular reconstruction and functional restoration of blood-retina barrier in mice.

[Effect of heat stress on expression of gp96 in K562 cell line of the chronic myeloid leukemia and its significance].

This study was purposed to investigate the effect of different heat stress conditions on expression level of heat shock protein gp96 in K562 cell line of chronic myeloid leukemia in order to provide experiment basis for seeking optimal heat stress condition increasing extraction amount of gp96 from K562 cells. The expression changes of gp96 in K562 cell line was detected by immunocytochemistry under 38, 40, 42, 44, 46 and 48 degrees C for 30 minutes in water, by flow cytometry under 40, 44, 48 and 52 degrees C for 30 minutes in water, by Western blot under 40, 44, 48 and 52 degrees C for 30 minutes in water. Immunocytochemistry assay showed that gp96 existed mainly in cytoplasm. The peak of gp96 expression was at 30 minutes after 48 degrees C in water. The result of flow cytometry was consistent to immunocytochemistry detection results under temperatures 40, 44, 48 and 52 degrees C (P < 0.01). Western blot showed that detection result was the same as the immunocytochemistry and flow cytometry detections. In conclusion, the expression of gp96 in K562 cell line reached peak at 30 minutes after 48 degrees C in water. This condition may be an effective preparative condition for extraction of gp96 from K562 cells.

[Dynamic changes in airway hyperresponsiveness as a result of allergen and therapeutic effects of budesonide in rats].

OBJECTIVE: To investigate the dynamic changes in airway hyperresponsiveness (AHR) in allergen-induced asthma in rats and the effects of budesonide. METHODS: Among 36 BALB/c female mice, 6 were randomized as negative control group (group C), the remaining mice were challenged with ovalbumin (OVA) to reproduce asthma. Kinetics of airway AHR of OVA-induced asthmatic mice was carried out as follows: on day 15, 18, 21 and 25 (A1, A2, A3, A4 groups), 6 mice were randomly chosen and sacrificed after measurement of airway AHR to investigate tidal volume (V(T)), airway resistance of expiring phase (R(A)), compliance of thorax and lung (C(T-L)) with different doses of methacholine chloride (MCH). In group B 6 mice were randomly chosen and treated with 1 mg budesonide aerosol once per day from day 15 to 17, then sacrificed on day 18. Their physiological and pathological changes were determined similarly to those of A2 group. RESULTS: (1) The increase of R(A) in group A1, A2 and A3 were significantly higher than that in group C when MCH reached the dose of 200 ng/g. (2) The decrease of C(T-L) in group A1 and A2 was significant with 100 ng/g and 200 ng/g MCH. (3) The value of V(T) markedly decreased in group A2 with 100 ng/g MCH and in all asthma model groups with 200 ng/g MCH than that in group C. (4) There was an eosinophil-dominant inflammatory infiltration in the asthma lungs, and the degree of infiltration peaked on day 15, and then alleviated afterwards. (5) With the dose of 200 ng/g MCH, the increase of R(A) in group B was significantly alleviated when compared with that in group A2, but there was no significant difference between group B and C. With the dose of 100 and 200 ng/g MCH, the decrease of C(T-L) in group B was significantly less marked compared with that in group A2 but there was no significant difference between group B and group C; the decrease of V(T) in group B was significantly lessened in degree when compared with that in group A2, while there was no statistical difference between group B and group C. The infiltration of inflammatory cells was obviously alleviated and the repair of airway epithelium was better in budesonide group. CONCLUSION: (1) Airway hyperresponsiveness increases greatly in mice challenged and sensitized with OVA, and it lasts for about 7 days and then declines afterwards. (2) Budesonide aerosol could effectively alleviate the eosinophil dominant inflammatory response and decrease AHR in the murine asthma model.

[Inhibitory effect of doxazosin on the growth of transplanted tumor of prostate cancer cell PC-3 in nude mice].

OBJECTIVE: To investigate the effects of alpha1 adrenoceptor antagonist doxazosin on the growth of androgen-independent prostate cancer cell PC-3 transplanted in nude mice. METHODS: PC-3 cells xenografts were transplanted (s.c.) in nude mice, and thirty xenografts were established successively. They were then randomly divided into 5 groups: A (control group), B (doxazosin 3 mg/kg), C (doxazosin 10 mg/kg), D (doxazosin 30 mg/kg), and E (doxazosin 100 mg/kg). Seven days after implantation, doxazosin was administered in sterile water by oral gavage, and the volumes of the transplanted tumors were measured during the therapy twice a week. All the mice were sacrificed after two weeks of doxazosin administration; the tumors were resected to do the following research. Immunohistochemistry of Ki67 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) was done to study the effects of doxazosin on the proliferation and apoptosis of prostate cancer cells; moreover, we also used Western blotting to study the protein expression of Smad-4 and IkappaB alpha. RESULTS: Nude mouse experiments showed that the in vivo doxazosin administration induced a notable decrease in the volumes of prostate cancer xenografts compared with the control group, and the tumor weights were also decreased. Interestingly enough, administration of doxazosin at higher concentrations (10, 30, 100 mg/kg) did not have further effect on tumor suppression. The percentage of PC-3 TUNEL positive cells was significantly higher than that of the control group; while the doxazosin treated groups and the control group did not have statistical difference on the percentage of Ki67 positive cells. In doxazosin treated groups Smad-4 and IkappaB alpha expressions were higher than that of the control group. CONCLUSION: Doxazosin can inhibit the growth of the prostate xenografts in the nude mice by inducing apoptosis without affecting the cell proliferation. Activation of transforming growth factor beta1 (TGF-beta1) signaling pathway may be the mechanism underlying doxazosin-mediated apoptosis.

Gene transfer of kringle 5 of plasminogen by electroporation inhibits corneal neovascularization.

PURPOSE: To test the efficacy of naked plasmid that expresses human kringle 5 of plasminogen (K5) in suppressing experimental corneal neovascularization in a rat model. METHODS: A eukaryotic expression plasmid encoding human K5 (pSecK5) was constructed. COS cells were transiently transfected with pSecK5 using a lipid-based transfection reagent. K5 secretion was confirmed by Western blot analysis. The effect of the secreted K5 on the proliferation of human umbilical vein endothelial cells (HUVECs) was investigated colorimetrically. Forty-three Sprague-Dawley rats were used for a corneal neovascularization suppression experiment. Corneal injury was induced by placing a disk of filter paper (immersed in 1 mol/l NaOH, 3.0 mm in diameter) on the corneal surface for 2 min. The cornea was immediately washed with saline. pSecK5 and empty plasmids were injected subconjunctivally, and square-wave electric pulses were immediately applied to the eyes. The expression of K5 was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The extent of corneal neovascularization was evaluated by scores. RESULTS: The constructed plasmid could express itself in COS cells. Conditioned medium from K5-transfected COS cells apparently inhibited HUVEC proliferation, compared with conditioned medium from COS cells transfected with empty plasmid or nontransfected cells. RT-PCR and immunohistochemistry confirmed the expression of K5 in the conjunctiva and cornea. Corneal neovascularization was significantly suppressed by K5 gene transfer in rats' eyes. CONCLUSION: In a rat model, K5 gene transfer by subconjunctival injection and electroporation can effectively inhibit corneal neovascularization induced by an alkali burn.

[The effect of murine interferon-gamma transgene expression on bleomycin-induced pulmonary fibrosis in mice].

OBJECTIVE: To investigate the effect of murine interferon-gamma (IFN-gamma) transgene expression on bleomycin-induced pulmonary fibrosis in mice. METHODS: 6 - 8 week aged pathogen-free male ICR mice were treated with bleomycin (3 mg/kg weight in 70 micro l) by intranasal instillation on day 0 in the model group. Adenoviral vector with murine IFN-gamma cDNA (AdCMVmIFN-gamma) 5 x 10(8) plague forming unit (pfu) was administrated via nastril instillation 48 h before bleomycin treatment in the preventive therapeutic group, but 72 h after bleomycin treatment in the therapeutic group. Mice treated with a same volume of normal saline (NS) and a same dosage of sham recombinant adenoviruses (AdCMVNull) served as controls. The animals were weighted on day 0, 7, 14, and sacrificed on day 14. Bronchial alveolar lavage fluid (BALF) and lungs were recovered. The right lung was stained with either hematoxylin-eosin or masson. The left lung was weighted, and its content of hydroxyproline (HYP) was measured by HCl acid hydrosis method. The IFN-gamma concentration in BALF was determined with enzyme-linked immunosorbent assay. RESULTS: The concentrations of IFN-gamma in BALF from the AdCMVmIFN-gamma treated groups were remarkably increased, (21,250 +/- 6,497) pg/ml and (21,000 +/- 5,451) pg/ml in the IFN-gamma transgenetic preventive therapeutic group and the therapeutic group respectively. IFN-gamma was undetectable in BALF from other groups. Mice in the two groups treated with AdCMVmIFN-gamma had statistically significant weight loss (P < 0.05), higher HYP content (P < 0.05), and tended to have more severe alveolitis and pulmonary fibrosis (P = 0.07) as compared with those in other groups. CONCLUSIONS: (1) mIFN-gamma could be overexpressed in airway and alveolar epithelium by locally administrated AdCMVmIFN-gamma. (2) Early mIFN-gamma transgene expression via adenoviral vector in this bleomycin model aggravated alveolitis and fibrosis to some degree.