Effects And Mechanism Of Kinetin On Rat Mammary

Subject: Study on the influence of kinetin (KT) on the growth and development,oxidative damage, cell structure and protein expression of mammary gland cells; theinhibition of KT on breast cancer cells in rats; the role of kinetin against the mammary glandhyperplasia. Evaluation the effect of kinetin and its derivatives against cell oxidative damageand anti-aging of mammary gland hyperplasia, and explore its mechanism.Methods:(1) The isolation of primary rat mammary epithelial cell, with stable growth,analyzed the impact of kinetin on its growth by morphological observation, MTT assay andrelated structural protein gene expression determination.(2) Rat mammary epithelial cellspretreated with kinetin protection, replication of rat mammary epithelial cells oxidative injurymodel in vitro, detected the kinetin on rat mammary epithelial cells antioxidant by HEstaining, scanning and transmission electron microscopy, apoptosis detection, the distributionof NADPH detection means; determine effect of kinetin on the process of oxidative damagein mammary epithelial cytoskeleton by observing cell microfilaments, indirectimmunofluorescence in microtubules; by semi-quantitative RT-PCR, detected the expressionof β-Actin, Tubulinα, type I procollagen, type III procollagen in the cells with oxidativedamage, and then analysis the influence of kinetin on rat mammary epithelial cells'antioxidant effects in vitro.(3) The rat mammary gland hyperplasia model, estradiol benzoate,progesterone by subcutaneous injection method, the rat model of mammary gland hyperplasiawere separated into different groups, different concentrations of kinetin were administeredorally for20consecutive days. then determination the blood hormone levels in rats; observedthe rat breast tissue under the microscope.(4) Obtained the rat breast cancer cells, culturedwith the kinetin and kinetin nucleoside, and then explored the influence of kinetin and kinetinnucleoside on the rat breast cancer cells.Results:(1) In the appropriate range for normal growth, especially in the proliferation ofrat mammary epithelial cells, kinetin has a significant promoting role.10mg/L to20mg/Lkinetin can promote the proliferation of rat mammary epithelial cells,20mg/L kinetin hadthe strongest effect. Therefore, this concentration used on the anti-oxidation mechanism isfeasible.40mg/L kinetin could significantly inhibited cell growth, which may be the cytotoxic effects.(2) In the process of oxidative damage, kinetin have a protective effect on cultured ratmammary epithelial cells, actin filaments, microtubules, which the protective effect of actinfilaments is relatively good. It can be seen in the process of oxidative damage kinetin had agood protective effect on the skeleton structure, especially good for the stress fibers, which isimportant for the maintance of the cell morphology during the oxidative damage.(3) In thetest against the mammary gland hyperplasia, in the high dose group, the kinetin significantlyimproved the shape of the diseased tissue, and can significantly reduce follicle hormone(FSH), serum estradiol (E2) and testosterone (T) levels, while improving prolactin (PRL) andserum luteinizing hormone (LH) levels, serum progesterone (P) changes a little.(4)Kinetinand kinetin riboside showed growth inhibition on rat breast cancer cells, and the IC50valuesof kinetin was six times to kinetin riboside. Kinetin and kinetin riboside can inhibit theproliferation of rat breast cancer cells in vitro, and there is a significant dose-dependentmanner, and this role is enhanced with the increase of both concentrations. However, whenthe two reaches a certain concentration, the inhibitory effect of the increase has slowed down,showing a certain degree of saturation. Correlation analysis results show that both inhibitionof cell proliferation rate and its concentration between the positive correlation, and kinetinriboside on cell inhibition is stronger than kinetin; and kinetin and kinetin riboside on ratbreast cancer cell proliferation inhibition the role of the obvious time effect: with time, bothinhibition of proliferation was significantly enhanced.Conclusion: Kinetin have significant role on oxidative damage, this effect can contributeto the proliferation of mammary epithelial cells in a certain concentration range, inhibition ofapoptosis and maintenance the integrity of cell membrane, cytoskeleton and other subcellularstructures during oxidative damage, maintenance the expression level of procollagen; Thekinetin, with the dose of2.5~40mg/(kg.d) can effectively prevent and therapy of ratsmammary gland hyperplasia. Kinetin and kinetin riboside showed growth inhibition on ratbreast cancer cells, and the inhibition showed a clear dose-effect and time-effect.