Effects Of Amifostine On Cochlear Hair Cells And Expressions Of Fas Protein From Cisplatin

IntroductionIn recent years, Cisplatin (CDDP) has been one of the antitumor agent in platinum - based anticancer drugs which widely used in the treatment of various malignant tumors including head and neck, ovarian, testicular and lung cancers. Unfortunately, CDDP has some dose - limiting side effects such as ototoxic-ity. Researchers groped for the differences aspects to study mechanism of damage , then to seek the most effective protect way. To test how Amifostine protects cochlear organ from Cisplatin toxicity. By culturing the cochlear organ of wistar rats in vitro, we study the effect of Amifostine on Cisplatin - induced cochlear hair cells apoptosis and Fas protein. Then approved amifostine on cissplatin - induced cells anti - damage or anti - apoptosis, decreaseing patients ototoxicity in treatment procedures and provided a theory evidence.Material and Method1. Material1. 1 Specimen: Wistar rats were decapitated on postnatal day 3 - 5.1. 2 Cochlear organotypic cultures reagent.1. 3 Fluorescence dyeing reagent.2. Method2. 1 Separate basement membrana and culture: the cochlear basement membrana was carefully dissected out, tiled on a 15 - ul drop of Type I rat - tail collagen, the middle turn of cochlear was placed on the collagen matrix and 1 ml of DMEM -F12 culture medium. The cochlear explants were incubated at 37℃,in 5% C02 for 24h. On the second day, the culture medium was exchanged with new culture medium then incubated for another 24h period. The cultures were fixed for 12h in 10%formalin in 0. 1MPBS, rinsed in 0. 1 PBS, and immersed in TRITC - labeled phaloidine in PBS for 30 min, rinsed again, specimens were mounted in EMS and examined under a fluorescence microscope.2.2 pre - experiment The first, to compare the left and right survival of cochlear hair cells in common culture conditions, and no difference in statistics (P >0.05) , which of used culture specimen in the same conditions;to compare the different concentration in CDDP on survival hair cells, select lOug/ml as the best concentration for induced damage;to used different dose of amifostine on hair cells and found the best protective concentration lOug/ml;The results support that adding amifostine in different time to survival hair cells has protect effect, so advance 30 min to adding amifostine has protect effects to hair cells.2. 3 Design of experiment: 40 Wistar rats on postnatal day 3-5 were randomly divided into four groups , 10 rats in each group. (20 ears)Normal control groupCDDP group: lOug/mlAmifostine group-. lOug/mlAmi + CDDP group: add Ami before adding CDDP 30 minutesThe different treatment factors culture medium were used, and specimens were labeled with TRITC -conjugated phaloidine and FITC (Fas protein) ,they were observed under fluorescence microscope, we can count survival hair cells and expressions of Fas protein.3. Statistical analysisResult1. There was a negative correlation between concentration of CDDP and survival rate, r = -0. 94 ( P <0. 05) , CDDP group destroyed most of the hair cells, and surviving hair cells were disarrayed or missing. There was significant difference of hair cells in different concentration CDDP groups (P < 0. 05);Hair cells incubated in the normal group and Amifostine group for 48 h remainedalmost complete viability, the survival numbers has no significant difference (P >0. 05 );add Amifostine before adding CDDP 30 minutes group has significantly higher survival numbers than CDDP alone(P <0. 05). Given different factors groups have significant difference to survival numbers( F =618. 39 > Fo 01(3 36) ,P2. There were a few green fluorescene granule in normal group and Amifostine group, weak expression of Fas protein. A large amount of Fas protein appeared on the cell membrane in CDDP group, Ami + CDDP group appeared few green fluorescene granules, expression of Fas protein.DiscussionIn recent years, CDDP - induced ototoxicity has been a problem in the tumor treatment. Through adding concentration in the animal body, more and more research have proved that platinum - based drugs induced ototoxicity has sth to do with inner ear hair cells apoptosis. The machnism lies in that under the effect of CDDP, a great number of oxygen free radical appeared in cochlear, which affect the ability of free radical clearance system, and lessen the activity of antioxidant in cochlear, therefore free radical lead to the damage or apoptosis of outer hair cells in cochlear. Various animal models and the super - micro examination of the stucture of humanbeing's temporal bone demonstrated that platinum - basec drugs ototoxicity damaged the principal place were in basis and middle turn of Corti 's outer hair cells, which are in accordance with the loss of high frequency hearing in clinical. By studying CDDP - induced damage to cochlear hair cells, we can conclude that different concentration CDDP damaged outer hair cells in different degree. CDDP destroyed most of the haor cells and the stereocilia on surviving hair cells were disarrayed or missing. The dose > 15ug/ml, a few stereocilia on IHCs were disarrayed and missing. The loss rate is significantly higher than normal control group. The higher the concentration, the more hair cells lost. There was a negative correlation between concentration of CDDP and survival rate . As the concentration of CDDP increased, IHCs were damaged (stereocilia were missing). Expression of apoptosis Fas proteinon cell membrana appeared weak positive after adding Amifostine, which is in significant contrast with that of CDDP alone group, then Fas protein is positive and high level. Therefore, it is further proved that Fas protein got involved in the process of CDDP - induced apoptosis. Amifostine can protect the normal tissue cells in the process of CDDP - induced cochlear hair cells Apoptosis, which is according to its own machnism to delay the Apoptosis.ConclusionAmifostine can protect the hair cells from CDDP ototoxicity to a certain degree. Fas protein express itself on a higher level in the process of CDDP - induced cochlear OHCs apoptosis. The group which were added Amifostine before adding CDDP inhibited expressions of Fas protein in the process of CDDP - induced apoptosis.