| Objective previous researchs were reported that concentration of transthyretin in serum and vitreum was different between normal people and patients with eye diseases,such as diabetic retinopathy and high myopia.Retinal microvascular endothelial cells and retinal pigment epithelial cells were fudemental for basic mechanism researchof ophthalmology.In our researchs,firstly,we improved the traditional isolation and cultivation methods of rat RVECs by semi suspension tissue culture.Proliferation,migration was detected by RVECs and RPECs,and then the relationship between angiogenesis-related factor(VEGF mRNA)expression and transthyretin(TTR)was examine on RVECs and RPECs.Finally in vitro,it was explored that whether TTR plays a role to biological behavior on RVECs and RPECs.From the above three aspects,it attempts to elaborate correlation between TTR and DR,it will provide a basis for further study of the mechanismMethods ?The retina isolated from a SPF SD rat was cut into pieces.4-5pieces were cultured with semi suspension tissue culture method.100ml·L-1 fetal bovine serum,recombinant bovine basic fibroblast growth factor,were composed into the endothelial cell culture medium,Cultured vascular endothelial cells were identified by Factor ? related antigen polyclonal antibody and preserved in the traditional cryopreservation and resuscitation method.? Human RMVECs was cultured in the concentrations of O?mol/L and 4?mol/L TTR.The proliferation,migration and healing abilities of human RMVECs with different concentrations of TTR were measured by methyl thiazol tetrazoliuM(MTT)assay,transwell assay,and scarification test.?Human RPECs was cultured with exogenous TTR to explore the effect of TTR on the proliferation of human RPECs.Silencing the expression of TTR of human RPECs by TTR specific small interfering RNA(siRNA)and the expression of TTR was detected by using Western blotting to identify the efficacy of TTR silence.The level of VEGF massage RNA(mRNA)was detected by using RT-PCR to identify the interaction between VEGF and TTR.The different proliferation and migration abilities of human RMVECs with different expression of TTR were measured by transwell system.Results ?The pieces of tissue fragments attached to the petri dish after 24 hours of Semi suspension.Then some RVECs travelled from them in 6 days,and the cells in primary culture began to clone,and a cell monolayer was seen in the 16th day after culture.The RVECs showed positive response to Factor ?.The rat RVECs could be maintained and passaged.?MTT assay shows that human RMVECs with the concentrations of 4?mol/L TTR glows faster than with the concentrations of Op?mol/L TTR(t=15.47,P=0.0001).The transwell assay shows human RMVECs with the concentration of 4p?mol/L TTR migrants faster than human RMVECs with the concentration of 0?mol/L(t=5.44,P=0.006),The scarification test shows that the human RMVECs with the concentration of 4?mol/L TTR heals faster than the human RMVECs with the concentration of 0?mol/L TTR(t=8.25,P=0.0012).?MTT assay showed that RPEC with the concentrations of 0?pmol/L TTR glowed faster than with the concentrations of 4?mol/L TTR(t=18.08,P<0.0001).The expression level of TTR was decreased in the siRNA group as compared with the negative control group(P<0.05).RT-PCR assay showed no differential expression of VEGF after the silencing of TTR(P>0.05).The transwell assay showed human RMVECs with the silence of TTR proliferated slower than human RMVECs without the treatment(t=4.901,P=0.0012),and migranted slower(t=4.213,P=0.0029),as well.Conclusions ?With the improved method,we can isolate rat RVECs more conveniently and effectively.The cells with perfect activity can be used for further research.?TTR can promote the proliferation,migration and healing abilities of human RMVECs,the mechanism may be worth to further study.?TTR can inhibit the proliferation of human RPECs.Silencing the expression of TTR can inhibit the proliferation and migration of human RMVECs without the help of VEGF. |
PDF Full Text Request