Construction and identification of small hairpin RNA expression plasmid targeting opticin gene in rat / 中华眼底病杂志

Objective To construct small hairpin RNA (shRNA) expression plasmid targeting rat opticin gene. Methods Four pairs of opticin oligonucleotides were synthesized and inserted into the plasmid vector, resulting into four plasmids: shRNA-1, shRNA-2, shRNA-3 and shRNA-4. Then the four constructed shRNA expression vectors and empty vector were transfected into rat ciliary non-pigment epithelium (NPE) cells by lipofectmaine 2000. Non-transfected NPE cells were set as control group. The expression of opticin mRNA and protein were measured by Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot respectively. Results The opticin mRNA expression of the shRNA-1,shRNA-2, shRNA-3, shRNA-4 group were decreased compared with the control group (F = 10. 239, P = 0. 000);the inhibitory rate were 85.7% ,62. 87% ,54.87% and 48.77% respectively. The opticin protein expression of the shRNA-1,shRNA-2, shRNA-3, shRNA-4 group were also decreased compared with the control group (F=17.870, P= 0.000);the inhibitory rate were 78.7%, 34.6%, 31.1% and 16.8% respectively.Conclusions The shRNA-1 expression plasmid has most potent inhibitory effect on opticin expression in rat ciliary NPE cells.

Expression of opticin in the eyes of diabetic mice / 中华眼底病杂志

Objective To observe the opticin expression in the eyes of non-obese diabetes (NOD) mice and non-diabetic NOD mice . Methods Twenty NOD mice were divided into diabetic group (experimental group) and non-diabetic group (control group). All the mice were killed by cervical dislocation method. The eyes were harvested, and the vitreous, retina and sclera were separately collected. Western blot and real-time reverse transcription-polymerase chain reaction(RT-PCR)were respectively used to determine opticin protein and OPTC-mRNA levels. Results The opticin protein level in the vitreous and retina was lower in the experimental group(t = 4.42,4.58; P = 0. 002,0. 002), but is same in the sclera between the 2 groups (t = 0. 27, P = 0. 794). OPTC-mRNA level was vitreous> retina> selera. OPTC-mRNA levels of vitreous and retina in diabetic group were significantly Iower(t = 3.30,2. 48; P= 0. 01, 0.04); there was no statistical significant on OPTC mRNA of sclera between two groups(t = 0. 27, P = 0. 80). Conclusion Expression of opticin was suppressed in retina and vitreous of diabetic mice.