Effects Of Lithium Chloride On The Apoptosis And Express Of HSP70 Of Hcaf Cells And Effects Of LiCl, HSP70 On Function Of Inhibiting Tumor Of 615 Mouse

PrefaceHeat shock proteins(HSPs),previously called stress proteins,are a group of proteins produced following different kinds of stresses.They exist ubiquitously across eukaryotic and prokaryotic organism with highly conservation.To date, they can be divided into following four families:HSP90,HSP70.HSP60 and small HSP60.HSP70 is generally existing in normal cells mostly intra -cellular, which has low expression in normal ceils but significantly increases under stress conditions.Further studies have shown that HSP70 has over - expression in many tumor cells.Since that HSP70 has no expression on surface of normal cells,it's considered that HSP70 is a specific structure on surface of tumor cells. There are studies shown that the component evoked tumor immune response is HSP70,but the HSP70 sequence of tumor cells is almost the same as that of normal tissue cells.Further studies have discovered the key to achieve tumor immune response is HSP70 combined small peptides.Therefore,it's considered the tumor peptides derived of HSP70 of tumor cells have tumor immune functions.Lithium salts are mainly used for treatment of mental disorders,they can also increase levels of tumor necrosis factor(TNF) in peripheral blood,which promotes cells sensitive to apoptosis and necrosis of TNF.Lithium salts possess relatively widespread safety margin,so they can up- regulate levels of TNF without obvious toxicity.Safety,generally without adverse reaction is the characteristic. Since HSP70 is over- expressed in tumor cells which is essential to proliferation and survival of tumor cells,so the proliferation of tumor can be suppressed by inhibition of expression of HSP70 to induce apoptosis of tumor cells. HSP70 can achieve the purpose of inhibiting proliferation of tumor cells and killing tumor cells through stimulating monocytes to produce cytokines(TNF -α) or inducing over - expression of T cells in vivo when combining with surface of human monocytes.Lithium salts can increase levels of cytokines like TNF -αand directly affect expression of proto -oncogene like c -fos;HSP70 -associated tumor peptides of Hcaf cells will stimulate tumor immunity of organisms and increase levels of cytokines like IL -2 and TNF -αto obtain the purpose of inhibiting tumor growth.This research studied Hcaf tumor cells derived from pure line of 615 mouse,achieving tumor origin of HSP70 by ultrasonication,chromatography, gradient elution,purification and identification;we also studied the function and effect of different doses of LiCl interference with cultivated Hcaf cells in vitro and HSP70 of Hcaf cells;additionally,we established tumor modals of 615 mouse through application of LiCl,HSP70 of Hcaf cells and HSP70 of normal mouse tissues interfering with Hcaf cells to observe change of TNF -αin peripheral blood and effect of anti -tumor.Materials and MethodsThe primary course of the experiment is as follows:by use of Hcaf cells, following the procedures of ultrasonication,ultracentrifugation of 20000r/min for one hour,chromatography of ConA - sephrose,dialysis of combining parts, chromatography of DEAE collumn,gradient elution with FPLC - system of combining parts on DEAE column,we collected protein samples of all peaks.We proved the sample of C peak was proteins with molecular weight of 70KD by SDS - PAGE and this sample was identified as HSP70 by Western - blot.After purification, freezing and filtration sterilization,the sample was conserved at 70℃for anti -tumor research of HSP70.The secondary course of the experiment is using tumor modals of 615 mouse through application of LICl,HSP70 of Hcaf cells and HSP70 of normal mouse tissues interfering with Hcaf cells to observe change of TNF -αin peripheral blood and effect of anti - tumor.The tertiary course is as follows:through different doses of LiCl interfering with Hcaf cells cultivated in vitro in RPMI - 1640 culture solution,we collected samples at different time points,stained with PI,rabbit -anti -mouse HSP70 antibody and goat -anti -rabbit IgG antibody marked by FITC respectively to observe change of cell cycles of Hcaf,effect of expression of HSP70 in Hcaf cell,and ultramicro - structure of Hcaf cells by scanning electron microscope.ResultsThe primary course of the experiment showed that via ultrasonication,centrifugation, chromatography and gradient elution with FPLC -system of Hcaf cells to collect samples of all peaks.The sample of C peak was proved 70KD proteins by SDS - PAGE and identified as HSP70 by Western- blot.The secondary course showed that the difference of inhibiting function of HSP70 in healthy tissues of 615 mouse to tumor tissues was not significant compared with control group;however,difference of inhibition of tumor between LiCl group and HSP70 of Hcaf cells group was significant,but the difference was not statistically significant;the group combining LiCl with HSP70 of Hcaf cells demonstrated the most significant function of inhibiting tumor.TNF-αslightly increased after injection of HSP70 in normal tissues,but it didn' t possess statistical significance compared with control group.The increase of TNF -αin LiCl group and HSP70 of Hcaf cells group was statistically significant(p＜0.05 );but the increase of TNF -αamong the group combining LiCl with HSP70 of Hcaf cells, single LiCl group and HSP70 of Hcaf cells group had no statistical significance. The tertiary course showed that "hypodiploid peak" of experimental cells gradually increased,DNA content in G_0/1 period gradually decreased and diplont peak decreased at 8hrs,24 hrs and 48hrs;DNA content in G₂M period increased and tetraplont increased;DNA content in S period also increased,but cells of control group grew well without "hypodiploid peak" in cell cycle,and after interaction with LiCl,Hcaf cells developed periodic block.When Hcaf cells in the tertiary course interacted with LiCl for 8 hours,the average fluorescence intensity of HSP70 marked by FITC increased,indicating that expression of HSP70 in experimental group enhanced compared with control group.The fluorescence in-tensity of control group showed no obvious increase compared with single use of secondary antibody.In LiCl group(60ug/ml),after interaction for 8 hours,the results of Hcaf cells under transmission electron microscope showed that volume of Hcaf cells became larger,vi in the margin;kytoplasm had little change,nucleus was large with nucleolus,heterochromatin accumulated into bolus scattered inside of nucleolus;margin of cells became bulged and blebbing;apoptotic bodies appeared in cells and cells possessed apoptosis.ConclusionsHeat shock protein 70 could be achieved following the procedures of ultrasonication, chromatography of ConA- sephrose,and DEAE collumn,gradient elution,identification of SDS - PAGE and western blot,protein of C peak collected from elution with FPLC - system by use of Hcaf cells.LiCl and HSPT0 derived of Hcaf cells could increase levels of TNF -αin peripheral blood of mouse with function of inhibiting tumor growth.Combining use of LiCl and HSP70 derived of Hcaf cells could increase levels of TNF -αin peripheral blood of 615 mouse with significant function of inhibiting tumor;HSP70 derived of healthy 615 mouse had no significant function of inhibiting tumor compared with control group.LiCl possessed function of inhibiting tumor in Hcaf tumor cells cultured in vitro;it also enhanced expression of HSP70 in Hcaf cells cultured in vitro.