[Effects of exogenous p21 gene on cells cycle of human lens epithelial cells].
OBJECTIVE
To investigate the effects of transfected exogenous p21 gene on the cells cycle of HLE-B3 cells line. The feasibility of prevention of secondary cataract by gene therapy was evaluated.


METHODS
Total length of human p21 gene cDNA was cloned on the parent's plasmid pcDNA3 to construct the recombinant plasmids of pcDNA3/p21, a large amount of pcDNA3/p21 plasmid DNA was prepared by QIAGEN endofree maxi kit. After harvest of the plasmid DNA, the HLE-B3 cells line was transfected. The cell growth was observed and the cells cycle was analyzed by flow cytometry. The expression of p21 mRNA was detected by RT-PCR and the expression of p21 protein was detected by immunohistochemistry and western blot analysis.


RESULTS
Forty eight hours after transfection, the growth of transfected cells became slower, some cells floated and died; the control cells and blank plasmid (blank pcDNA3) transfected cells grew normally. Flow cytometry analysis revealed that the number of cells in G(1) phase increased markedly in transfected cells. The RT-PCR showed that the product of p21 in the transfected cells (dead or alive cells) was obviously higher than that of the controls. Immunohistochemical studies showed few positive cells in the controls, and very high positive signal was detected after transfection. Western blot showed a positive band at the level of 21 000 in transfected cells, no positive band could be found in the controls.


CONCLUSIONS
Exogenous p21 gene can transfect the HLE-B3 cells and can be expressed. The transfection can affect the cells cycle by G(1) arrest and induces cells death through the apoptosis process. This result suggests that it is possible to prevent the occurrence of secondary cataract by gene therapy.
Wu
Ming-xing
MX
Zhongshan Ophthalmic Center, Zhongshan University, Guangzhou 510060, China. wumx63@21cn.com
Li
Shao-zhen
SZ
Zeng
Jun-wen
JW
Liu
Yi-zhi
YZ
D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D004847:Epithelial Cells / Q000502:physiology*; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D014162:Transfection
D016283:Proto-Oncogene Proteins p21(ras)