The Study Of Apoptosis Of Guinea Pig By Lens Induction And Roles Of BCL-2,BFGF In Scleral Remolding

Objective To observe the changes of refractive state and the sensitivities of guinea pigs to defocus . To determine the existence of the apoptosis cell in posterior sclera and to evaluate apoptotic and the proliferative changes in lens induction myopia. To determine the existence of the B-cell lymphoma/leukemia 2 and basic fibroblast growth factor in lens-inducted myopic sclera of guinea pigs. Moreover, to investigate effects of B-cell lymphoma/leukemia 2 and basic fibroblast growth factor on apoptosis in cultured guinea pig scleral fibroblasts, and to study the mechanism of the two genes on preventing myopia.Methods Four-week-old pigmented guinea pigs(n=45) were divided in 3 groups randomly, including group Ⅰ (the right eye wearing the -20D concave for two weeks),group Ⅱ (the right eye wearing the -10D concave for two weeks ),group Ⅲ(no interference).Before and after experiment, refraction was measured using retinoscopy and the ocular axial were determined by A-scan ultrasonography. At the end of experiment,animals were given over dose of pentobarbital and dead. The eyes were enucleated and residual orbital tissue carefully removed. And the scleras near posterior poles were observed by TUNEL(terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) and immunohistochemistry methods.The proliferative index (PI ), apoptotic index(AI) in scleral fibroblast were calculated and contrasted to controlled eyes. Morphologic alterations of apoptotic fibroblast were observed by transmission electric microscope. The expression of thevalue of PI and AI was determined by quantitative analysis .45 scleras near posterior of guinea pigs in the three groups were observed by immunohistochernistry methods. The positive rates of bcl-2 and bFGF in scleral fibroblast cytoplasma and the area percentage ratio of positive cells were calculated and contrasted to controlled eyes.Four-week-old pigmented guinea pigs (n=5) were used in this study and primary cultures were established using the sclera. The cultured cells was determined to be fibroblasts using immunofluorescence methods. The primary and the 7rd passage cells were collected when they became conflunt. Cultured guinea pig scleral fibroblasts were incubated with various concentration of bFGF for 24h.Cultured fibroblasts which added different degree bFGF were exposed to ultraviolet radiation for 60 minutes.We divided cultured scleral fibroblasts into 5 groups, including control group(group A),UVA group(group B),10ug/l bFGF preconditioned with UVA(group C) ,20ug/l bFGF preconditioned with UVA(group D), 40ug/l bFGF preconditioned with UVA(group E). The total cellular RNA was extracted from the cells , and the expression of mRNA of bcl-2 was analyzed by RT-PCR. At the same time, the protein was extracted from the cells , and the expression of bcl-2 protein was detected by Western-blot. Effects of bFGF on apoptosis was measured by flow cytometry and the apoptotic morphologic alterations were observed by TUNEL which labeled by FITC. Results Myopias of -3.45±0.21D and-3.57±0.78D were induced in group I and group II,and the axial lengths prolonged to 0.41±0.21mm and0.45±0.37mm in these two groups. There is significant difference in refraction , axial length between the two groups and the control. After the dectection by TUNEL, the value of AI in group I > II was 15.61±0.40,10.98±0.74 . There was no apoptotic cell in group III by TUNEL methods.The value of AI in lens-inducted eye is significantly higher than in control group.And the value of PI in group III (28.83±0.05)is higher than group I (10.54±0.06^ II(13.78±0.15)significantly. The ultrastructual alternation in inducted eyes (group I , II) appeared: vesiculated and degeneration Lysis was founded in apoptosis cytoplasma , mitochondrion vacuoles ,some cells appeard shrinkage and karyopyknosis ,apoptotic body appeard and cytoapoptosis changes inlate .There was no apoptotic changes in control group.We used immunhistochemical method to dye guinea pigs posterior sclera of above 3 groups. The results were that the positive rate of bFGF was 20% (group I ),40%(group II ),the two groups'positive rate were lower than contrast eyes(73.3%)significantly. And the positive rate of bcl-2 was 13.3%in group I and 26.6% in group II, lower than contrast group( 60% )significantly. Cultured cells began to grow out of the sclera after 1 -2 weeks, and grew to confluent after 3-4 weeks. The cultured cells was determined to be fibroblasts using immunofluorescence methods.Reverse-transcription poiymerase chain reaction shows that there is expression of mRNA of bcl-2 in cultured guinea pig scleral fibroblasts.Image analysis shows that the product of mRNA of bcl-2 is group A(23.3l±5.67),group B(l5.18±2.34),C(23.76±5.82), D(35.83±4.77),E(44.92±3.59). Compared with the group B, a significantly decreasing of bcl-2 mRNA was observed in group C,group D,E(P <0.01). The bcl-2 m-RNA in group D and group E is higher than that of the group A.It indicated that the average product of mRNA of bcl-2 rose(such as bFGF 20ug/l) compared to control group.The changes in expression of bcl-2 mRNA paralleled to the changes in its protein. The average value of bcl-2 protein was 1.3±0.51,0.8±0.48,1.5±0.64,2.14±1.78,2.59±0.58 respectively.There was significant difference between group A and B,the group C and D, group D and E (P<0.01) .It indicated that the bcl-2 protein increased significantly with the preconditioned bFGF degree increased (P<0.05) .Being preconditioned by bFGF, the apoptotic cell percentage rose by use of TUNEL labeling with FITC, and the value of five groups was 0, 56.23±4.12%, 43.46±3.78%, 23.16±2.87%, 14.62±2.69% respectively. Compared with the group A, the apoptotic rate was lower than the other group significantly (P<0.01) . Moreover, the results which detected by FCM(0.06%,56.20%,33.44%,25.61%,14.64%) paralleled to those of TUNEL.Conclusion:The negative lens can successfully induce guinea pig eye to form the model of myopia. The dioper pf induced lens has positive correlation with post-experiment diopter, and the higher the lens-induced diopter, the better the result.With the decreasing of the proliferation, the apoptotic cell increased in the posterior scleral fibroblasts in lens-induced myopia eye. At the same time,there showed that bFGF protein and bcl-2 protein decreased in the posterior sclera of guinea pig when the diopter increased. It indicated that the two genes took part in regulating mechanism in myopic scleral changes.The mRNA and protein of bcl-2 increased with the degree of bFGF raised in cultured guinea pigs scleral fibroblasts which exposed to ultraviolet radiation. The ultraviolet can induce apoptosis in cultured fibroblasts of guinea pig scleraThe extraneous bFGF can inhibit the occurrence of apoptosis of cultured scleral fibroblasts through upregulating the bcl-2 family.In lens-induced experimental myopias of guinea pigs, decreasing of bFGF can cause fibroblasts apoptosis increasing in sclera through downregulating the bcl-2 gene.