| Purpose:This study explored t the effects of curcumin on vascular astrocytoma U87MG cells in mimicry from cellular and molecular level?vasculogenic,mimicry,VM?the phenomenon of influence,and the mechanism of curcumin on the development of U87MG cell line VM by interfering with the signal transduction pathway VE-cadherin/EphA2/PI3K/MMP-2,based on cell biology,molecular biology techniques,through in organization of pathology and cell experiments.Our purpose is to provide the basis for molecular biology experiments in the future,to find effective target of astrocytoma and traditional Chinese medicine,and the theory of the pathogenesis of management of astrocytoma and treatment of new ideas and methods.Material and method:1.The pathology department of the General Hospital of Shenyang military region from 2014 to 2015 the diagnosis of astrocytoma specimens,90cases of complete clinical data,including grade II tumors,diffuse astrocytoma in 30 cases;III grade tumor,aplastic astrocytoma in 30 cases,grade IV tumors,30 cases of thrombocytoma its'contiguous as in normal tissue,the tissues were fixed and sliced,and CD31 and PAS,GFAP and PAS were used to observe the pathological changes and the VM phenomenon in two ways.2.The expression of VE-Cadherin and EphA2 protein in astrocytoma were detected by immunohistochemistry and Western Blotting methods.U87MG and SHG44 cells were cultured in vitro to observe the formation of VM lumen like structure.3.U87MG and SHG44were cultured in vitro and the growth curves of two kinds of cells were measured;The SHG44and U87MG cells were divided into low dose curcumin group(culture medium containing curcumin at concentration of 7.2?mol·L-1),dose group(curcumin in culture medium containing curcumin at concentration of 14.4?mol·L-1),high dose group(curcumin medium containing curcumin at concentration of 28.8?mol·L-1)and blank control serum group,and blank group set without cell,cultured for 12 h,24 h,48 h and 72 h,cells were collected to MTT colorimetric assay and determination the inhibitory effect of different doses of curcumin on the proliferation of SHG44 and U87MG cells,and screened the optimal concentration and time effect of curcumin.4.U87MG cells were divided into 4 groups,group A was not drug in the blank control group,B group of low dose curcumin group,curcumin culture a final concentration of 7.2?g·mL-1 system,group C dose group of curcumin,curcumin cultured a final concentration of 14.4?g·mL-1system,D group Jiang Huang in the high dose group,medium curcumin concentration as 28.8?g·mL-1 system,the invasion of U87MG cells was determined by Transwell method.Observed inhibition of curcumin on astrocytoma U87MG vasculogenic mimicry VM under the inverted microscope of three-dimensional cell culture medium.5.Cultured SHG44 cells and U87MG cells,which can be divided into SHG44control group,U87MG control group,U87MG curcumin group and U87MG inhibitor group,after 48h,immunocytochemistry,Western Blotting and real-time PCR methods were used to detect the protein and mRNA expression of vascular rendothelial-cadherin?VE-cadherin?,epithelial cell kinase?EphA2?,phosphatidylcholine?PI3K?,matrix metalloproteinase-2?MMP-2?.In this study,SPSS 17.0 statistical software was used to analyze and analyze the experimental data.The experimental data were repeated more than three times.The measured data were expressed as"meanħstandard deviation".The data of each group were normal distribution and the variance was homogeneous.Analysis of variance,analysis of whether there is statistical significance;two comparison,the use of SNK method for analysis;do not meet the parameters of the test,the use of multiple doses of data for rank sum test.P<0.05,the statistical significance.Results:1.The blocks of tissue sections witn Double staining of CD31 and PAS we can seen,vascular endothelial cells lining vascular classic in most of the tumor tissue,by CD31 staining of endothelial cells and PAS positive stained red basement membrane,CD31 staining negative Of the cells and PAS dyed red basement membrane composed of laryngeal structure,which contains red blood cells,which is the tumor of the mimicry blood vessels?VM blood vessels?,the typical mimic blood vessels composed by the GFAP-positive tumor cells and PAS-positive basement membrane.The VM positive rate of WHOII grade tumor tissue was3.33%,The VM positive rate of WHO III grade tumor tissue was 13.33%,The VM positive rate of WHOIV grade tumor tissue was 26.67%.The difference was statistically significant?p<0.05?,the higher the level of astrocytoma,the more obvious the phenomenon of angiogenesis mimicry.2.The expression of VE-Cadherin and EphA2 protein in WHOIII and IV tumor tissues were significantly higher than those in normal tissues by immunohistochemistry and Western Blotting methods.?p<0.05?,the higher grade of astrocytoma,the expression of VE-Cadherin and EphA2 protein in tumor tissue were higher,two were positively correlated.3.In the Matrigel gel culture for 24-48 h,the results of the inverted phase contrast microscope showed that the low-grade astrocytoma cell SHG44 could not form a vascular-like three-dimensional structure,and the highly malignant glioblastoma cell line U87MG cells could form similar to the structure of the vascular cavity,indicating that in vitro experiments,poorly differentiated tumor cells are more prone to angiogenic mimicry.4.The results of SHG44 and U87MG cell growth curves showed that the OD values of the two cells increased at the time of 12 h,24 h and 48 h,and reached the peak at 48 h.At 72 h,the OD value of the two cells decreased significantly,Suggesting that the growth capacity of72 h cells has been reduced,the death of cells increased.5.With the increase of the time of action,the inhibition rate gradually increased,reaching a peak at 48 h,with time-dose effect.The inhibitory rate of curcumin middle and high-dose curcumin group on U87MG cells at 48 h was significantly different from that of other groups?p<0.05?,When the inhibition rate was less than 10%,there was no obvious cytotoxicity on the cell lines.The cytotoxic dose was used as the optimal dose.Therefore,the choice of middle dose of curcumin for 48 h was the best dose and best time of action.The follow-up dose and duration of action in this experiment.6.The inhibitory effect of curcumin on the angiogenic mimicry of U87MG in astrocytomas was observed by inverted microscope,transwell method was used to determine the invasion of U87MG cells by curcumin,the two results are similar.With the increase of curcumin concentration,the inhibition of VM phenomenon and invasion of U87MG cells also increased.The protein and mRNA expression of VE-cadherin,EphA2,PI3K,MMP-2 were detected by immunocytochemistry,immunoblotting and real-time quantitative polymerase chain reaction?RT-PCR?.The results showed that the protein expression of VE-cadherin,EphA2 and MMP-2 in U87MG blank control group was significantly higher than that in SHG44 group?p<0.05?.Compared with U87MG blank control group,the protein expression of VE-cadherin,EphA2 and MMP-2 in the optimal dose group and U87MG inhibitor group was significantly lower than that in U87MG control group?p<0.05?,There was no significant difference in the expression of VE-cadherin,EphA2 and MMP-2 protein in the best dose group and U87MG inhibitor group?U87MG??P>0.05?;PI3K protein expression in U87MG blank control group was significantly higher than the SHG44 control group,the difference was statistically significant?P<0.05?;Compared with U87MG blank control group,the protein expression of PI3K in the optimal dosage group of U87MG curcumin was significantly lower,the difference was statistically significant?P<0.05?,There was no significant difference in the expression of PI3K protein in U87MG inhibitor group and U87MG blank group?P>0.05?,Compared with SHG44 control group,there was no significant difference in the expression of PI3K protein in the optimal dose group of U87MG curcumin?P>0.05?.The results of mRNA showed that the expression of VE-cadherin,EphA2 and MMP-2 in U87MG blank group was increased compared with SHG44 control group,the difference was statistically significant?P<0.05?,The expression in U87MG of optimal dose group and U87MG inhibitor group decreased significantly,compared with the U87MG blank group,the difference was statistically significant?P<0.05?,There was no significant difference in the expression of U87MG of optimal dose group and U87MG inhibitor group?P>0.05?;Compared with SHG44 control group,the mRNA expression of PI3K in U87MG blank group increased,the difference was statistically significant?P<0.05?,The PI3K mRNA expression of U87MG of optimal dose group was significantly lower than that in U87MG blank group,and the difference was statistically significant?P<0.05?,there was no significant difference in the mRNA expression of PI3K between U87MG inhibitor group and U87MG blank group?P>0.05?,compared with SHG44 control group,the mRNA expression of PI3K in U87MG of optimal dose group was not significantly different?P>0.05?.Conclusion:1.There are angiogenic mimicry?VM?in astrocytoma,the higher the WHO grade of astrocytoma,the higher the proportion of VM phenomenon;The proportion of VM phenomenon was not significantly correlated with gender,age,preoperative status,and so on.The presence of VM in the astrocytoma was related to the protein expression of VE-Cadherin and EphA2.2.Curcumin inhibited the growth of SHG44 and U87MG cells,and inhibits angiogenesis mimicry VM phenomenon.3.Curcumin inhibits angiogenesis mimicry VM phenomenon and invasion in highly malignant glioblast cell line U87MG cells,this was achieved by modulating the VE-cadherin/EphA2/PI3K/MMP-2 cell signaling pathway. |
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