Role Of Smad₄, P27～(kipl) In Proliferation Of Lens Epithelial Cell

PrefaceAs early as the beginning of last century, it was clear that the mechanism of aftercataract was the exorbitant proliferation and differentiation of lens epithelial cells ( LEG ) , and during this process LEG could secrete basement membrance -like products. Researchers tried to prevent or postpone the process of aftercataract by minimizing operation trauma, clear out residue LEG, implanting of intraocular lens (IOL) , administering of anti - metabolism or mitosis inhibitor drugs, but the LEG couldnt be completely eliminated and extensive application of drugs was also limited because of their toxicity. LEG is the most active cell in lens and plays an important role in the growth, differentiation and injury repair of lens. It is impossible to clear out all LEG during operation, thus LEG inevitably proliferates, moves, differentiates new lens fiber and transforms into fibro-blast. It is important for prevent aftercataract to elucidate the molecular mechanism of LEG proliferation and prevention the proliferation of residue LEG in view of cell cycle.LECs, just like other cells, proliferate according to cell cycle and are regulated by positive cell cycle regulatory substances, such as cyclin, cyclin dependent kinase (CDKs) , and negative mediating substances, such as cyclin dependent kinase inhibitors (CKIs). P27Klpl is a kind of cyclin dependent kinase inhibitor and inhibits cell proliferation by inhibiting the activity of CDK or Cyclin -CDK complex. In the meantime P27Klpl can mediate the conduction of extracellular negative cell signal ( such as TGF - (3 etc) so as to accomplish their effect of inhibiting cell proliferation.Lens epithelial cells can express Transforming Growth Factor β( TGF -β)and receptor of Transforming Growth Factor (3. Transforming Growth Factor can mediate Lens epithelial cells proliferation. Smad4 is the key cytoplasm medium in signal conduction pathway in TGF - p family and plays an important role in the signal transmission between cell membrane and cell nucleus. It conjugates with other transcription factors or DNA, regulates the process of transcription and inhibits the activity of CDKs and Cyclins, so inhibit the cell proliferation. There are no reports about the role of Smad4 and P27kip in lens epithelial cells proliferation. This research is to detect the change of Smad4, P27Kip1, CDK4, CyclinDj, compare with Proliferating Cell Nuclear Antigen ( PCNA, a proliferation index) during different period after rabbit lens cortex absorption and investigate the molecular mechanism of lens epithelial cells proliferation in view of cell cycle regulation, provide theoretic and experimental evidence for elucidating the molecular mechanism of LEG proliferation and further studying of inhibiting the LEG proliferation in the view of cell cycle.Material and MethodThis research includes four parts.I8' part; animal model and specimen preparationRandomize 80 white healthy rabbits (weight ranged from 0. 9 to 1. 1kg, gender not be limited) into two groups, test group (70) and control group (10). In test group, 70 rabbits anesthetized by Ketamine (15mg/kg, im) were operated crystal lens cortex absorption by the same surgeon. 10 rabbits were killed in 1st, 3rd, 1st week, 1st month, 2nd month, 3rd month and 5th month respectively after operation and lens were extirpate for test. Rabbits in control group without any treatment were also killed and prepare the lens for test. 14 lens of 7 rabbits in each group (112 in all) were fixed by 4% formaldehydum poly-merisatum and embedded by paraffin wax. Equatorial portion and subcapsular, which were obtained within 2mm of equator from the other 6 lens of 3 rabbits in each group (all 48) , were frozen in -70℃.2nd part: detection of lens epithelial cells proliferationThe expression of PCNA, CDK4 and CyclinD1 in equator and subcapsularlens epithelial cells were detected by immunohistochemistry method ( SABC) and RT - PCR. The number of PCNA positive cells in unit area and average absorption optical density ( A valu...