Effects Of Interleukin-2 And Estradiol On The C6 Glioma Cell Line

Interleukin-2(IL-2) is one of the cytokines produced by T cells in immune system. Results of research on neuroimniunoendocrinology show that IL-2 is shared in neuroimmunoendocrinologic network and it plays a significant role in central nervous system (CNS).The action of IL-2 is mediated by IL-2 receptor(IL-2R).IL-2R has 3 subunits a, P, y. P and y chains are critical to the signal transduction ,especially to the proliferation signal transduction of IL-2.IL-2R is expressed hi CNS broadly. There are distributed in different regions and with different densities in the brain ,so it can be supposed that IL-2 has physiological functions in these different regions.For instance , IL-2 has the nutrition function on the neurons and neuroglias , and affects the release of neurotransmitter. It has been tested that IL-2R can be expressed in oligodentrocytes and microglias.Estrogen is one of steriod hormones , which is mainly sythesized and secreted by the gonad. Estrogen exerts a variety of effects on neurons in the adult CNS. Estrogen Through binding specific estrogenDepartment of Physiology 7receptor(ER) in nucleus ,functions like a nucleus transcription factor and modulates expression of some genes .It has been found that there exist two subtypes of ER.There are a and p subunits which are different in their distribution , density and mediated action. ER can be expressed whether in nucleus or in cellular membrane . In addition to the classical genomic pathway through nucleus receptors, estrogen can cause to produce some cellular rapid membrane - mediated effects through nongenomic pathway.Astrocytes account the majority population in CNS, which has dramatic effects on the regularity of the physiological function of CNS.The experiment purpose is to confirm that there exist IL-2Rand ER in C6 glioma cell line whether at protein level or mRNA level, and explore the effects of IL-2 and estradiol on the cells as well as the possible mechnism of signal transduction of them .To attain the above goal , we fully examine the expression of 3 subunits of IL-2R and 2 subtypes of ER in C6 cells . Then I observe the effect of IL-2 and estradiol on the proliferation of C6 cells and the influence on the intracellular Ca2"1" level. The following methods are adopted in the experiment: 1 .The culture of C6 glioma cell line . 2.We used immunocytochemistry to explore the expression of IL-2Rand ER.protein3.Western blotting analysis was applied to test the expression of ER protein .Department of Physiology 84.The C6 cells were cultured for 3 days ,then total RNA was extractedfrom the cultured cells . 5.The cDNA was synthesized from the above-mentioned total RNA,then PCR was used . Through this method , IL-2mRNA andERmRNA were detected. 6.We adopted MTT assay to observe the effects of IL-2 and estradiolon OD value of the cells. 7.3H-TdR incorperation showed the stimulation of IL-2 and estradiolon the proliferation of C6 cells . 8.Laser scanning confocal cytometry was used to investigate theinfluence of IL-2 and estradiol on leval of the intracellular Ca2+.The results are as follows :l.The immunoreactive production of IL-2Ra,p,y chains were detectedin cytoplasm of C6 cells . 2. EL-2Ra,p,y mRNA were expressed in the cells 3.10U/ml,100U/ml,1000U/ml IL-2 stimulated dramatically theproliferation of the cells4. There existed ERp immunoreactive production in cytoplasm of C6 cells ,and no ERa immunoreactive production was detected.5. The result of western blotting analysis of ERP protein was positive. 6.C6 cells expressed ERpmRNA.7.10"7mol/L, lOVol/L, 10"5mol/L 17p-estradiol raised the viability ofC6 cells significantly ,but exerted no effects on cell proliferation. 8. 10~6mol/L17|3-estradiol significantly elevated the intracellularDepartment of Physiology 9level of Ca2+ with an increase of fluorescence intensity from 96.10?.12 to 126...