Effects Of All Trans Retinoic Acid On Growth And Cx43 Gene Expression Of Retinoblastoma Cells

Part 1 Expression of connexin43 protein in normal retina and retinoblastoma(RB) tissue of humanObjective: To investigate the expression of connexin(Cx) 43 protein in human normal retina and retinoblastoma(RB) tissue and explore its effect on carcinogenesis and development of RB. A valuable index in RB diagnosis, prognosis and therapy wish to be found.Methods: Expression and localization of Cx43 protein in 5 cases of human normal retina and 22 cases of RB tissue were detected by using immunohistochemical method. The difference of expression were also compared among normal retina tissue, RB tissue and nonneoplastic tissue surrounding primary tumor; differentiated and undifferentiated tissue as well as localized and expanding period tissue respectively.Results: 1. In human normal retina tissue, Cx43 protein was expressed in nerve fiber layer, ganglion cell layer, inner nuclear layer, external limiting membrane, rods and cones layer and pigment epithelium. In nerve fiber layer, rods and cones layer, external limiting membrane and pigment epithelium, expression was full-thickness. In ganglion cell layer and inner nuclear layer, it was punctate and areatus. No expression was found in inner limiting membrane, inner plexiform layer, outer plexiform layer as well as outer plexiform layer.2. Comparison of staining scores:â‘ Score of RB tissue was lower than that of normal retina. There were significant difference between them(P＜0.01);â‘¡Scores of expanding period(Ⅳ～Ⅴstage) tissues were lower than those of localized period tissues(Ⅰ～Ⅲstage), which had significant difference between them(P＜0.05), but no difference among groups in each period(P＞0.05);â‘¢Score of differentiated tissue was higher than that of undifferentiated one. There was significant difference between them(P＜0.05);â‘£Score of normal retina was higher than that of nonneoplastic tissue surrounding primary tumor which was also lower than RB tissue(both P＜0.05).Conclusions: 1. There is inherent and extensive expression of Cx43 protein in human normal retina tissue.2. Cx43 protein expression in human RB tissue is decreased evidently, which also has negative relationship with differentiation, extension and invasion of the tumor but not with the size.3. The aberrant expression of Cx43 protein may play an important role in carcinogenesis and development of RB. Cx43 may be a valuable index for RB diagnosis, prognosis and therapy. Part 2 Effect of all trans retinoic acid on growth of RB cells in vitroObjective: To study the effect of all trans retinoic acid (ATRA) on biological behavior of RB cells in vitro and explore a new medication of RB.Methods: RB cells were divided into 5 groups randomly: A: black control; B: vehicle control, cells were treated by 0.1% alcolhol solvent; C: induced by 10^-5mol/L ATRA; D: induced by 10^-6mol/L ATRA; E: induced by 10^-7mol/L ATRA. Cells were collected when induced by ATRA for 48 hours, 96 hours and 144 hours. Survival rate was detected by methodology of tetrazolium-based colorimetric assay(MTT assay); cell cycle and apoptosis were assessed with flow cytometry.Results: 1. Induced by ATRA, Rb cells show decreased proliferation: compared with control, cells of group C, D and E displayed decreasing growth velocity and survival rate, both of which had negative correlation with concentration and action time of ATRA.2. Cell cycle and apoptosis of RB cells were altered after induction of ATRA: compared with control, in group C, D and E, population of cells in phase G₀/G₁ increased and that in phase G₂M and S decreased correspondingly. At the same time, apoptosis rates stepped up. Both of them had positive correlation with concentration and action time of ATRA.Conclusion: 1. ATRA can inhibit the proliferation of RB cells.2. ATRA inhibits proliferation of RB cells by blocking cell cycle at phase G₀/G₁ and contributing apoptosis. In given range, increasing drug concentration and action time can augment these effects. Part 3 Inductive effect of ATRA on expression of Cx43 gene and its protein in RB cellsObjctive: To investigate the effect of ATRA on expression of Cx43 gene and its protein in cultured RB cells and explore the machanism of ATRA on growth inhibition of RB cells.Methods: Protocol of experimental grouping and cells treatment was the same as that in Part2. Cells were collected when induced by ATRA for 48 hours, 96 hours and 144 hours. Western blot assay was employed to detect expression of Cx43 protein; reverse transcription-polymerase chain reaction(RT-PCR) was carried out to analyze Cx43 mRNA expression.Results: 1. Expression of Cx43 protein was increased in RB cellsinduced by ATRA: compared with control(group A and B), photodensity ratio(ODR) of each ATRA induced group was raised. There was significant difference comparing group A with group C after induced for 48 hours, with group D for 96 hours, or with group E for 144 hours(all P＜0.05). The value of ODR showed significative positive correlation with concentration and action time of ATRA.2. Expression of Cx43 mRNA was increased in RB cells induced by ATRA: compared with control, ODR of each ATRA treated group was raised, There was significant difference comparing group A with group C after treated for 48 hours, or with group D and E for 96 hours(all P＜0.05). Significative positive relationship was showed between ODR and concentration or action time of ATRA.3. Synchronism was discerned among increased expression of Cx43 mRNA and protein, inhibition of proliferation and development of apoptosis in RB cells in vitro.Conclusion: 1. Upregulation of Cx43 gene and its protein expression is an improtant mechanism of ATRA on proliferative inhibition and apoptosis induction to RB cells. In given range, increasing drug concentration and action time can augment these effects.2。Expression of Cx43 mRNA and its protein in RB cells is regulated by ATRA at genic transcription level.3. Cx43 gene and its protein are valuable targets in therapy of RB. Part 4 Establishment of intraocular RB model in athymic mice and study of inhibitory effect of ATRA on the growth of RB in vivoObjective: To test the effect of ATRA on the growth of RB in vivo by using athymic mice model and offer experimental evidence for clinical application of the drug.Methods: To establish animal models of RB by injecting HXO-RB44 cells into anterior chamber of athymic mice. Mice having developed tumors were divided into 3 groups randomly: A: black control(no treatment); B: vehicle control(pseudo-treament by anterior chamber injection with normal sodium including 0.5% anhydrous alcohol); C: treament group(anterior chamber injection with 10μg ATRA). Frequency of administer was once injection every 3 days and 3 weeks' therapy was carried out. Condition of tumor development in anterior chamber was observed everyday. When experiment completed, mice eyes were enucleated and made into paraffin sections. Characteristic of tumor in growth and pathematology was observed by using HE stain.Results: 1. Models of intraocular RB were established successfully by transplanting HXO-RB44 cells into ocular anterior chamber of BALB/C athymic mice. Survival rate of tumors was 73.3%.2. Tumors were developed 6～9 days after transplantation. In group A, growth of tumors was rapid and nearly occupied the whole space of anterior chamber 20 days after transplantation. To the end of observation (25～30 days after transplantation), operated eyeballs were swollen, corneas were edema and neovascularized obviously. In group C, tumors' growth was slower than that in group A. Twenty days after transplantation, volume of the biggest tumor was about one third of anterior chamber in group C. To the end of observation period, all of tumors in this group were less than three quarters of anterior chamber and control rate of tumor was 50%. In group B, development of tumors had no significant difference with group A and its tumor control rate was 0%.3. Pathologic observation: tumor tissues in group A and B were compact and RB cells could be observed in anterior chamber, posterior of len as well as vitreous cave. In group C, tumor tissues were loose and limited in anterior chamber. Only a small quantity of RB ceils was scattered at posterior of iris. There was no tumor cell been observed in posterior of len and vitreous cave. In histology, The characteristic of transplantation tumor was similar to that of human RB. There was no much difference been observed among groups.Conclusion: 1. RB model of BALB/C athymic mice established by implanting HXO-RB44 cells into anterior chamber has a high survival rate of tumor. It is an excellent model for RB tumor research.2. ATRA has inhibitory effect on growth of RB transplantation tumor in vivo. It may be a great potential medication of RB.