Objective To observe the regulation effect of transforming growth factor alpha (TGFalpha;) on expression of glutamate transporter（GLAST）and ingestion activity of retinal Muuml;ller cells in mice. Methods To take the retinal tissue of Kunming mouse at postnatal 7～10 day, and then cultured Muuml;ller cells according to literature. The 3～4 generation cultured cells of the same primary cell were divided into two groups at random: ① TGFalpha; group: maintained in different concentrations of TGFalpha; as 50, 75, 125 and 150 ng/ml, 3 holes in each concentration;② Control group: cultured by Eagle culture medium which improved from Dulbeccon and contained 20% fetal calf serum. The influence of different concentrations TGFalpha; on GLAST activity in Muuml;ller cells were observed by L-3H-glutamate uptake detection; the expression of GLAST mRNA in Muuml;ller cells was determined by RT-PCR; the expression of GLAST protein was detected with immunocytochemical staining. Results With the increase of TGFalpha; concentration, both L3H glutamate uptake and GLAST mRNA expression were increased. The L-3H-glutamate accumulation had got to the maximum uptake at concentration of 125 ng/ml, which was 266% of that in control group, meanwhile, the expressions of GLAST mRNA also got to the maximum as 4 times of control group. Immunocytochemical staining indicated that the effect of 125ng/ml TGFalpha; on expression of GLAST protein was higher than that in the control group, the differences between two groups were statistically significant (Plt;0.05). Conclusion TGF-alpha; can increase GLAST activity through up-regulating the expression of GLAST mRNA and protein.
An experimental model of retinal acute photic injury was developed in Wister rats. Aninmls were exposed to white light in intensity of 20 000 lux under general anesthesia for 1 hr. Two rats were sacrificed at 24, 48hr, day 7, 14,21,28,35 after liht exposure respectively. The histopathologic study showed that the retinal acute photic injury initiated in the outer seSment characterized by disorganization and loss of the outer segment at the early stage, and then the inner segment and RPE were involved. The decrease of the thickness of the outer nuclear layer was seen in a few of our cases. Some of our samples showed recovery of the outer and inner segment from the light damage of certian degrees 2 or 3 weeks after light exposure, but others did not. One sample from 5 weeks after light exposure demonstrated that the outer nuclear layer, the outer and inner segment completely lost. Thc inflammatory responses were not observed in all of our samples inplicating that the retinal acute photic damage is a degeneration process. （Chin J Ocul Fundus Dis,1994,10:84-85）
Purpose To investigate the protective effect ofldquo;haw drink compoundrdquo;against carbon disulfide (CS2)toxic retinal damage． Methods Thirty healthy white adult New Zealand rabbits were divided at random into 3 groups:the normal control group,the exposure control group and the treatment group．The experimental rabbits were contaminated by inhaling CS2 for 3 continuoues hours in 6 consecutive days in a week for totaly 3 weeks．The rabbits in the treatment group were given haw drink compound before containation．After 3 weeks of the experiment,the retinal tissues of the rabbits were examined with light microscope and transmissing electronmicroscope(TEM). Results The ultrastructures of the retinal tissues of the exposure control group were more abnormal than those of the treatment group and the normal control group．TEM showed that every layer cell of the retinal in the exposure control group showed apparent degenerative changes ,but that in the treatment group showed apparent degenerative changes,but that in the treatment group was nomal．The light microscpic picture showed that the inner segments and the outer segments of photoreceptors construction were destroyed,the nerve fiber layer was loose and ganglion celles vacuolated in the retinat of the exposure control group．TEM showed that The photorecepter synapses were vacuolated,the postsynaptic space became wider and the synaptic sticks disappered． Conclusion How drink compound can improve the tolerance to CS2 toxicity in inducing the retinal damage of rabbits. (Chin J Ocul Fundus Dis, 1999, 15: )
Objective To evaluate the cytotoxicity of microdosis peracetic acid (PAA) so as to provide the evidence for making residual l imit of PAA steril ization. Methods Mouse fibroblasts (L929 cell l ine) cultured in vitro were observed to evaluate the influence of microdosis PAA including 1 × 10-6, 2 × 10-6, 3 × 10-6, 4 × 10-6, 5 × 10-6, and 10 × 10-6 (V/V). Theproliferation of cells was determined by MTT assay at 2, 4, and 7 days of culture. The growth curve and the relative growth rate (RGR) were obtained. The cytotoxicity of PAA at different concentrations was evaluated according to RGR. Results At 2, 4, and 7 days after culture, fibroblasts of 1 × 10-6 group grew with normal morphology analogous to control group, while the cell growth of other groups were poor. With the increase of PAA concentration, the absorbance (A) values decreased, which suggested that there was a significant negative correlation between cell prol iferation and PAA concentration. And the correlation coefficient was — 1.000 at 2 and 4 days, — 0.964 at 7 days. There was no significant difference in A value between 1 × 10-6 group and the control group (P gt; 0.05), while there were significant differences in A value between the control group and other concentration groups (P lt; 0.05). The growth curve of 1 × 10-6 group was similar to that of the control group, both had obvious phase of exponential growth. The growth curves of other groups had no obvious phase of exponential growth. The cytotoxicity of 1 × 10-6 group was classified as level 1, 2 × 10-6 group as level 2, 3 × 10-6 group as level 3, 4 × 10-6 group as level 3-4, 5 × 10-6 group and 10 × 10-6 group as level 4. Conclusion PAA of 1 × 10-6 had no obvious cytotoxicity. The residual l imit of PAA less than 1 × 10-6 was recommended.
Purpose To investigate retinoic acid (RA) induced apoptosis in retinal pigment epithelial (RPE) cells. Methods 10-5、10-6、10-7 mol/L were added to cultured PRE cells.Aridine orange fluorescence and TdT-mediated dUTP nick end labelling(TUNEL) techniques were used to observe apoptotic changes. Resultss 10-5、10-6、10-7 mol/L RA induced apoptosis in RPE cells.Cell shringkage,chromatin condensation and nuclear DNA fragmentation of RPE cells were observed by TUNEL technique.When 10-7、10-6、10-5mol/L RA treated RPE cells for 5 days,apoptotic index(AI)was 36.9%、4409% and 61.4% respectively,and 48.0%、59.9%、74.2% for 6 days.At the same concentration of RA,AI increased when time prolonged．At the same day,AI increased when the concentration of RA rose.There was significant difference in the results(Plt;0.05). Conclusion Our results showed that RA-induced apoptosis in RPE cells was detected with a good dose and time response. (Chin J Ocul Fundus Dis,1998,14:153-155)
Objective To investigate the effect of human tooth bone graft materials on the proliferation, differentiation, and morphology of macrophages, and to understand the biocompatibility and cytotoxicity of human tooth bone graft materials. Methods Fresh human teeth were collected to prepare human tooth bone graft materials, the adhesion of mouse mononuclear macrophages RAW264.7 to human bone graft materials was observed under confocal microscopy. Scanning electron microscopy was used to observe the morphology of human tooth bone graft materials, OSTEONⅡ synthetic highly resorbable bone grafting materials, and untreated tooth powder (dental particles without preparation reagents). Different components of the extract were prepared in 4 groups: group A (DMEM medium containing 10% fetal bovine serum), group B (human tooth bone graft materials), group C (OSTEONⅡ synthetic highly resorbable bone grafting materials), group D (untreated tooth powder without preparation reagents). The 4 groups of extracts were co-cultured with the cells, and the cytotoxicity was qualitatively determined by observing the cell morphological changes by inverted microscope. The cell proliferation and differentiation results and cell relative proliferation rate were determined by MTT method to quantitatively determine cytotoxicity. The cell viability was detected by trypanosoma blue staining, and tumor necrosis factor α (TNF-α ) and interleukin 6 (IL-6) expressions were detected by ELISA. Results Scanning electron microscopy showed that the surface of the human tooth bone graft material and the OSTEONⅡ synthetic highly resorbable bone grafting materials had a uniform pore structure, while the untreated tooth particle collagen fiber structure and the demineralized dentin layer collapsed without specific structure. Confocal microscopy showed that the cells grew well on human tooth bone graft materials. After co-culture with the extract, the morphology and quantity of cells in groups A, B, and C were normal, and the toxic reaction grades were all grade 0, while group D was grade 3 reaction. MTT test showed that the cytotoxicity of groups B and C was grade 0 or 1 at each time point, indicating that the materials were qualified. The cytotoxicity was grade 2 in group D at 1 day after culture, and was grade 4 at 3, 5, and 7 days. Combined with cell morphology analysis, the materials were unqualified. The trypanosoma blue staining showed that the number of cells in groups A, B, and C was significantly higher than that in group D at each time point (P<0.05), but no significant difference was found among groups A, B, and C (P<0.05). ELISA test showed that the levels of TNF-α and IL-6 in groups A, B, and C were significantly lower than those in group D (P<0.05), but no significant difference was found among groups A, B, and C (P<0.05). Conclusion The human tooth bone graft materials is co-cultured with mice mononuclear macrophages without cytotoxicity. The extract has no significant effect on cell proliferation and differentiation, does not increase the expression of inflammatory factors, has good biocompatibility, and is expected to be used for clinical bone defect repair.
Objective To investigate the biocompatibility of true bone ceramic (TBC) and provide experimental basis for clinic application. Methods TBC was prepared from healthy adult bovine cancellous bone by deproteinization and high temperature calcinations. Mouse fibroblast cell line (L929 cells) were cultured with the leaching liquor of TBC in vitro, and the cytotoxicity was evaluated at 2nd, 4th, and 7th days. L929 cells were inoculated into the TBC and cultured for 4 days. The cell adhesion and proliferation on the surface of the TBC were observed by scanning electron microscopy, and evaluated the cell compatibility of TBC. Ten New Zealand white rabbits were divided into 2 groups, and drilled holes at the tibia of both hind limbs. TBC and hydroxyapatite (HA) were implanted into the left side (experimental group) and the right side (control group), respectively. And the biocompatibility of TBC was evaluated by general observation and histological observation at 4 and 26 weeks after implantation. Results Cytotoxicity test showed that the cytotoxicity level of leaching liquor of TBC was grade 0-1. Cell compatibility experiments showed that the L929 cells adhered well on the surface of TBC and migrated into the pores. The implantation test in vivo showed that experimental group and control group both had mild or moderate inflammatory response at 4 weeks, and new bone formation occurred. At 26 weeks, there was no inflammatory reaction observed in both groups, and new bone formation was observed in varying degrees. Conclusion TBC have good biocompatibility and can be used to repair bone defect in clinic.
Objective To observe the effects of structure and function of cornea, chamber angle and retina of varying doses of Bevacizumab which was injected intravitreally in rabbits. Methods Twenty-four New Zealand albino rabbits were divided into three groups randomly, the right eyes in three groups received int ravitreal injection Avastin at dose 1.25 mg，2.5 mg and 5 mg respectively as experimental eye, the left eyes accepted intravitreal injection 0.9% normal saline at the same volume as a control eye. The anterior segment of eye and ocular fundus were examined and intraocular pressure was measured by slit-lamp microscope and direct ophthalmoscope before and after injection. It was tested by Electroretino gram (ERG) before and after injection 1, 4, 8 weeks. At the 8th week, it carried out corneal endothelium counting; then enucleated eyes to observe by the light microscope and transmission electron microscope. Results No statistically significant difference regard to IOP,corneal endothelium counting, a-and b-waves of ERG at any stage of study in every group（P＞0.05）. No obvious change at cornea, chamber angle, retinal structure and retinal ultrastructure in every group under light microscope. Conclusion This study indicated that there is no obvio us toxicity of intravitreal injection with Avastin 1.25~5.0 mg in normal rabbit eyes. （Chin J Ocul Fundus Dis，2008，24：189-192）
Objective To inverstingate the effect of perfluorohexyloctane(F6H8)to the retina of rabbit eyes. Methods Fifteen vitrectomized New Zealand white rabbits were injectedF6H8(experiment group,12 rabbits ) and BSS(control group,3 rabbits) into vitreous cavity.Slit-lamp biomicroscopy and indirect ophthalmoscopy were performed pre- and postoperatively in all the eyes.Histopathological examination was done after the rabbits were sacrificed at the end of the study. Results A large clear balb was formed after intravitreal injection of theF6H8 in the vitreous was injected and no retinal detachment and cataract were found.The OPL was edematous and then thinned out in 4th week in experimental group.Degenerating cells was found in inner and outer nuclear layers.Cellular vaculoar degeneration was present in TEM. ConclusionF6H8 in vitreous cavity may cause significant side effects on retina,we could not recommend it to be used as an intraocular temponade.
ObjectiveTo investigate the effect of expressions of nucleoside transporters subtype (hENT1 and hENT2) on 5-fluorouracil(5-FU) cytotoxicity in breast cancer cell lines(MDA-MB-231, MDA-MB-468, SK-BR-3, MCF-7). MethodsFour breast cancer cell lines were chosen to detect the mRNA expressions of hENT1 and hENT2 by RT-PCR. Cells were incubated in the medium with a serial concentrations of 5-FU from 1.28×104 ng/L to 2.00×108 ng/L for 48 h. Then the cell proliferation in each cell line was measured by MTT assay and the IC50 was evaluated. Results①The mRNA expressions of hENT1 and hENT2 in the MDA-MB-231, MDA-MB-468, or SK-BR-3 cells were significantly higher than thoes in the MCF-7 cells(P < 0.05). The mRNA expression of hENT2 was detected in the MDA-MB-231, MDA-MB-468, or SK-BR-3 cells, not detected in the MCF-7 cells. 2MTT showed that IC50 of 5-FU in the MDAMB-231, MDA-MB-468, or SK-BR-3 cells was significantly lower than that in the MCF-7 cells(P < 0.05). There was no statistical difference of IC50 among the three lines(MDA-MB-231, MDA-MB-468, and SK-BR-3)(P > 0.05).③The three lines(MDA-MB-231, MDA-MB-468, and SK-BR-3) with lower IC50 of 5-FU highly expressed hENTs, and MCF-7 cell with the higher IC50 of 5-FU expressed less hENTs. ConclusionsThe expressions of hENTs in breast cancer cell lines can significantly influence 5-FU cytotoxic effect. It is implicated that the hENTs expressions might be the clue to the choice of nucleoside anticancer drugs in clinic.