Chao Liu1, Xiao-Li Wu2, Xin-Yi Wu3, Zhen-Hua Zhang4, Xiao-Hua Liu5. 1. Department of Ophthalmology, Qilu Hospital, Shandong University, Jinan 250012, Shandong Province, China; Department of Ophthalmology, Central Hospital of Qingdao, Qingdao 266042, Shandong Province, China. 2. Department of Ophthalmology, Qilu Hospital, Shandong University, Jinan 250012, Shandong Province, China; Department of Ophthalmology, Rongjun Hospital of Shandong, Jinan 250013, Shandong Province, China. 3. Department of Ophthalmology, Qilu Hospital, Shandong University, Jinan 250012, Shandong Province, China. 4. Department of Ophthalmology, Central Hospital of Qingdao, Qingdao 266042, Shandong Province, China. 5. Department of Stomatology, Central Hospital of Qingdao, Qingdao 266042, Shandong Province, China.
Abstract
AIM: To study the inhibition of nuclear factor kappa-B p65 (NF-κB p65) antisense oligodeoxynucleotide (ASODN) on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2 (TGF-β2) in vitro. METHODS: NF-κB p65 ASODN and NF-κB p65 missense oligodeoxynucleotide (MSODN) were designed and synthesized. Human lens epithelial cell line (HLE B-3) cells were prepared for study and divided into 7 groups. Control group was HLE B-3 cells cultured in vitro in dulbecco's modified eagle medium (DMEM). T1, T2, and T3 group were HLE B-3 cells cultured in vitro in DMEM with 10 ng/mL TGF-β2 for 6h, 12h, 24h respectively. A+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65 ASODN for 24h. M+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h. The negative control group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after cultured with transfer agent (HiPerFect) for 24h. Cell morphology was observed at different time points using an inverted microscope. The expression of NF-κB p65 mRNA was detected with reverse transcription-polymerase chain reaction (RT-PCR), and the expression of α-smooth muscle actin (α-SMA) protein was assayed with ELISA. RESULTS: With the TGF-β2 stimulation prolongation, the expression of NF-κB p65 mRNA and α-SMA protein increased in T1, T2, T3 groups compared with the control group, and the difference was statistically significant (P<0.05). NF-κB p65 ASODN lowered the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2. NF-κB p65 MSODN and HiPerFect did not lower the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2. The difference between control group and A+T group was not statistically significant (P>0.05), but the difference among A+T group and other groups was statistically significant (P<0.05). CONCLUSION: NF-κB p65 ASODN could lower the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2, and antagonized TGF-β2-induced transdifferentiation of HLE B-3 in vitro. NF-κB p65 ASODN could be used as a new biological therapeutic target of posterior capsular opacification.
AIM: To study the inhibition of nuclear factor kappa-B p65 (NF-κB p65) antisense oligodeoxynucleotide (ASODN) on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2 (TGF-β2) in vitro. METHODS: NF-κB p65ASODN and NF-κB p65 missense oligodeoxynucleotide (MSODN) were designed and synthesized. Human lens epithelial cell line (HLE B-3) cells were prepared for study and divided into 7 groups. Control group was HLE B-3 cells cultured in vitro in dulbecco's modified eagle medium (DMEM). T1, T2, and T3 group were HLE B-3 cells cultured in vitro in DMEM with 10 ng/mL TGF-β2 for 6h, 12h, 24h respectively. A+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65ASODN for 24h. M+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65MSODN for 24h. The negative control group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after cultured with transfer agent (HiPerFect) for 24h. Cell morphology was observed at different time points using an inverted microscope. The expression of NF-κB p65 mRNA was detected with reverse transcription-polymerase chain reaction (RT-PCR), and the expression of α-smooth muscle actin (α-SMA) protein was assayed with ELISA. RESULTS: With the TGF-β2 stimulation prolongation, the expression of NF-κB p65 mRNA and α-SMA protein increased in T1, T2, T3 groups compared with the control group, and the difference was statistically significant (P<0.05). NF-κB p65ASODN lowered the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2. NF-κB p65MSODN and HiPerFect did not lower the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2. The difference between control group and A+T group was not statistically significant (P>0.05), but the difference among A+T group and other groups was statistically significant (P<0.05). CONCLUSION: NF-κB p65ASODN could lower the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2, and antagonized TGF-β2-induced transdifferentiation of HLE B-3 in vitro. NF-κB p65ASODN could be used as a new biological therapeutic target of posterior capsular opacification.
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