| Objective:1. To observed the effect on migration and transdifferentiation of the human lens epithelial cell line HLE B-3 cultured in vitro induced by TGF-?2.2. To observed the effect of different concentrations of NF-?B p65 ASODN and different doses of transfection agent HiPerFect on the human lens epithelial cell line HLE B-3.3. To study the inhibition of NF-?B p65 ASODN on migration and transdifferentiation induced by TGF-? 2 in normal human lens epithelial cells in vitro.4. To investigate the inhibition of NF-?B p65 ASODN on the posterior capsular opacification of New Zealand white rabbits cataract model eye, which was transferred by anterior chamber injection method.Materials and Methods:1.Human lens epithelial cell line HLE B-3 was cultured in vitro stimulated by different concentrations of TGF-?2 for 6h, 12h. 24h and 48h to set up model of posterior capsular opacification, the morphology and migration distance of lens epithelial cell were observed and the a-SMA protein was detected by ELISA.2. NF-? B p65 ASODN was full phosphorothioate modificated,different concentrations of NF-? B p65 ASODN and different doses of transfection agents HiPerFect mixed, which were transfected into the lens epithelial cell line HLE B-3 in vitro, and to observed the suitable combination for best transfection efficiency without significant effect on the cells.3. Cells were synchronized cultured and divided into five groups: (1) control group(simple FSM culture), (2) negative control group (HiPerFect transfection added to the FSM),(3) TGF-?2 group (T group) (10ng / ml TGF-?2),(4) TGF-?2 plus ASODN group (T + A group) (TGF-?2, HiPerFect transfection and ASODN added to the FSM ), (5) TGF-?2 plus MSODN group (T + M group) (TGF-?2, HiPerFect transfection and MSODN added to the FSM).The content of the supernatant of five groups were collected at 6h, 12h, 24h, 48h four different points to detect the a-SMA,and using RT-PCR to detect the expression of NF-?B mRNA in cells.4. The 16 New Zealand white rabbits were conducted phacoemulsification cataract surgery and randomly divided into four groups after the surgery, four in each group.Group A: control group. four rabbits eight eyes: 100ul BSS solution was injected into the anterior chamber, Group B: negative control group, four rabbits eight eyes: 3ul HiPerFect and 100ul BSS solution was injected into the anterior chamber, group C:ASODN single injection group, four rabbits eight eyes: 3ul HiPerFect and lOnmol / L of NF-?B p65 ASODN of BSS solution 10Oul was injected into the anterior chamber,D group: ASODN twice injections groups, four rabbits eight eyes: 3ul HiPerFect and 10nmol / L of NF-?B p65 ASODN of BSS solution 100ul was injected into the anterior chamber for two times. The central thickness of the cornea was measured by the ultrasonic thickness measurement, and the anterior chamber inflammation and posterior capsule opacification was detected by the slit lamp microscope at 7,14,21,28 days after surgery respectively. The pathological changes of the membrane was observed after HE stained at the end of experiment, the expression of a-SMA and NF-? B p65 mRNA were detected by ELISA and RT-PCR respectively.Results1. The impact of TGF-?2 on the migration and transdifferentiation of lens epithelial cells showed significant time- and concentration-dependent, and the concentration of 10ng / ml performed strongest effect. With the TGF-?2 stimulating prolongation, the morphology of the HLE B-3cells became approximate elliptical or polygonal spindle-shaped gradually, with small projection formed and the distance of cell migration increased, and the expression of a-SMA protein increased too. The migration distance had increased following the increasing of TGF-?2 concentration after induced for 48h,and the concentration of 10ng / ml TGF-?2 being the strongest effect, meanwhile the migration distance had increased following the extension of time after induced by 10ng / ml TGF-P2, and the distance reached the peak in the 48h.The positive cell of a-SMA and the absorbance values of the cell climbing had increased following the increasing of TGF-(32 concentration after induced for 48h, and the concentration of 10ng / ml TGF-?2 being the strongest effect, meanwhile the positive cell of a-SMA and the absorbance values of the cell climbing had increased following the extension of time after induced by 10ng / ml TGF-?2, and the distance reached the peak in the 48h.2. The transfection rate of NF-?B p65 ASODN into the HLE B-3 cells increased with time, and which had been to more than 60% for 48h, and the activity of cells unaffected. With the increase of concentration, the transfection rate increased too,while the concentration of 10?g / ml performed strongest effect, and no significant effect on cell viability.The transfection efficiency of NF-? B p65 ASODN into the HLE B-3 cells increased with the increase of the transfection agent HiPerFect when the dose less than or equal to 3ul, and when the dose being of more than 4ul exhibited cytotoxicity and the transfection efficiency did not increase obviously. The best transfection condition was 3ul HiPerFect and 10?g / ml NF-? B p65ASODN, while the viability of cells being impacted little (80.4%) and the transfection efficiency up to 70.8%.3. Compared with the control group, the concentration of a-SMA in the supernatant of the negative control group, the T group, the T + A group, and the T + M group was significantly increased at the four different time points, and the difference was statistically significant (P <0.01). Compared with the negative control group, the concentration of a-SMA in the supernatant of the T group and the T + M group was higher and the difference was statistically significant (P <0.01). The difference of the concentration of a-SMA in the supernatant between the negative control group and the T + A group was not statistically significant (P> 0.05). Compared with the T + A group, the concentration of a-SMA in the supernatant of the T group, and the T + M group was higher and the difference was statistically significant (P <0.01). The difference of the concentration of a-SMA in the supernatant between the T group and the T +M group was not statistically significant (P> 0.05). NF-? B p65 mRNA appeared at 364 bp. Compared with the control group, the content of NF-? B p65 mRNA of the negative control group,the T group, the T + A group,and the T + M group was significantly increased at the four different time points, and the difference was statistically significant (P <0.01). Compared with the negative control group, the content of NF-? B p65 mRNA of the T group and the T + M group was higher and the difference was statistically significant (P <0.01). The difference of the content of NF-? B p65 mRNA between the negative control group and the T + A group was not statistically significant (P> 0.05). Compared with the T + A group, the content of NF-? B p65 mRNA of the T group, and the T + M group was higher and the difference was statistically significant (P <0.01). The difference of the content of NF-? B p65 mRNA between the T group and the T +M group was not statistically significant (P> 0.05).4. The inflammation of anterior segment had disappeared within a week, and there were no significant difference between groups. The posterior capsular opacification of the A group and the B group began about one week after the surgery, and became heavier gradually with time expand. The posterior capsular opacification of the C group and the D group was later than the A group and the B group, and less serious,while the difference was statistically significant (P < 0.05) . The difference of the posterior capsular opacification between the C group and the D group was not statistically significant (P> 0.05).Multilayer fibroblast cells arranged more closely on the capsular surfaces of the A group and the B group, and cortical hyperplasia appeared on the peripheral portion of the pouch, and cellulose-like material deposited clearly. The capsular surfaces of the C group and the D group were relatively clean, with a small amount of cell proliferated and the cell arrangement being more loose. Compared with the C group and the D group, the amount of a-SMA expression was significantly higher in the A group and the B group, the difference was statistically significant. The difference between the A group and the B group was not statistically significant (P> 0.05). The difference between the C group and the D group was not statistically significant (P>0.05). Compared with the C group and the D group, the content of NF-? B p65 mRNA detected by RT-PCR was significantly higher in the A group and the B group,the difference was statistically significant. The difference between the A group and the B group was not statistically significant (P> 0.05). The difference between the C group and the D group was not statistically significant (P> 0.05).Conclusion1. TGF-?2 could stimulate the migration distance of HLE B-3 cell increased, and the a-SMA who standing for mesenchymal transdifferentiation increased, which could be successfully establish the cell model of posterior capsular opacification.2. The best transfection condition was 3ul HiPerFect and 10?g / ml NF-?B p65ASODN, while the viability of cells being impacted little (80.4%) and the transfection efficiency up to 70.8%.3. NF-? B p65 ASODN could lower the expression of NF-? B p65 mRNA and the a-SMA protein who standing for mesenchymal transdifferentiation induced by TGF-?2.4. NF-? B p65 ASODN transfered by the anterior chamber injection could significantly inhibited the posterior capsular opacification of New Zealand rabbits models of eyes in vivo, which without increasing the inflammatory response and the impact on the cornea. |
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