| Objective:Autophagy is an evolutionarily conserved catabolic path-way from yeast to mammals for recycling intracellular.Components Microtubule associated protein 1 light chain 3-II(LC3-II)and autophagy degradation P62 protein play an important role in the process of autophagy.This experiment detected the expression of LC3-II and P62 protein and gene in newly diagnosed children with acute leukemia.By testing the expression level of autophagy in bone marrow cells of children with acute leukemia,we can provide the basis for explaining the pathogenesis children with acute leukemia from a new perspective,and provide new ideas for the treatment of children with acute leukemia.Methods:1 Immunohistochemical streptavidin Perosidase1.1 Cases and group From January 2014 to June 2016,50 children with Acute Leukemia,first diagnosed in the Second Hospital of Hebei Medical University were randomly chosed as experience group.the 50 children includ 30 children with Acute Lymphoblastic Leukemia(ALL),including the standard risk 13 cases,moderate14 cases and 3 cases of high risk.Another 20 children are Acute Myelocytic Leukemia(AML),including M1 1 cases,M2 5 cases,5 cases of M3,4 cases of M4,3 cases of M5 and M7 2 cases.All cases of AL includ Male 30 and female20.Aged 1 ~14 years.All patients were confirmed by clinical manifestations,cell morphology(Morphology,M),immunology(Immunology,I),cytogenetics(Cytogentics,C)and Molecular Biology(Molecular,M)(MICM).In addition,randomly select 20 children without malignant hematological diseases(including 14 cases with Immune Thrombocytopenia,6 cases of children with Nutritional Anemia),witch age and sex matched with the experimental group.1.2 Specimen collection and processing Extract 2ml bone marrow from the experimental group and the control group,naturally freezing in the glass tube.Then pour into glass bottles with4% paraformaldehyde,Paraffin embedded.The specimens were qualified which number of leukemic cells ranges from 50% to 95% of the total number of nucleated cells by HE staining.Immunohistochemistry was used to detect the expressions of LC3-? and P62 proteins.The slices were blinded to the researchers.LC3-? and P62 were determined by the method of semi quantitative classification of Fromowitz positive cells,Randomly select 5high-power fields(* 400),0r 10 high-power fields(* 400)if the slice with less cells.The ratio of positive cells to total cell number and the depth of staining were evaluated according to the sections.2 Reverse transcription-PCR(RT-PCR)Randomly select 20 children newly diagnosed with Acute Leukemia,Confirmed by the above method,including 12 cases of ALL patients,8 cases of children with AML,and 12 children without malignant hematological diseases,including 7 cases with Immune Thrombocytopenia,5 cases of children with Nutritional Anemia at the same time.RT-PCR was used to detect those children.After the extraction of RNA,reverse transcription and PCR,we can detect with UV lamp.3 Statistical Analysis In immunohistochemical data,SPSS21.0 statistical package is used to analyze all the datas,and statistical methods contain the Chi-square test and Spearman correlation ananlysis,and the difference is statistically significant when P<0.05.In RT-PCR experimental data,Graphpad prism 5.0 is used to analyze the two sample with t test.Results:1 Immunohistochemical streptavidin Perosidase:LC3-II protein and P62 protein positive staining was brown to sepia particles,LC3-II protein was located in the cytoplasm,and part of the nucleus,P62 in cytoplasm.1.1 The results showed that the rates of LC3-? positive expression in the experimental group and the control group were 66.00%(33/50)and15.00%(3/20),P<0.05.The difference was statistically significant.The rates of P62 were 76.00%(38/50)and 55.00%(11/20),P>0.05.The difference was no statistically significant.1.2 The rates of LC3-? positive expression in children with ALL and the control group were 63.33%(19/30)and 15.00%(3/20),P<0.05.The difference was statistically significant.The rates of P62 were 73.33%(22/30)and 55.00%(11/20),P>0.05.The difference was no statistically significant.1.3 The rates of LC3-? positive expression in children with AML and the control group were 70.00%(14/20)and 15.00%(3/20),P<0.05.The difference was statistically significant.The rates of P62 were 80.00%(16/20)and 55.00%(3/20),P>0.05.The difference was no statistically significant.1.4 The rates of LC3-? positive expression in children with ALL and AML were 63.33%(19/30)and 70.00%(14/20),P>0.05.The difference was no statistically significant.The rates of P62 were 73.33%(22/30)and 80.00%(16/20),P>0.05.The difference was no statistically significant.1.5 The rates of LC3-? positive expression in male AL,ALL and AML were 66.67%(20/30)?60.00%(12/20)and 80.00%(8/10),and the rates of LC3-? in female AL,ALL,and AML were 65.00%(13/20)?70.00%(7/10)?60.00%(6/10).There was no significant difference between male and female(P>0.05).The positive expression rates of P62 in male AL,ALL and AML were 80.00%(24/30)?75.00%(15/20)?90.00%(9/10),in female AL,ALL and AML were 70.00%(14/20)?70.00%(7/10)?70.00%(7/10).There was no significant difference between male and female(P>0.05).1.6 LC3-II in ALL children with type B and type T positive rates were66.67%(16/24)and 50.00%(3/6).The difference was not statistically significant(P>0.05).P62 in ALL children with type B and type T positive rates were 75.00%(18/24)and 66.67%(4/6).The difference was not statistically significant(P>0.05).1.7 Spearman correlation analysis showed that there was no significant correlation between the expression of LC3-II and P62 protein(RS =0.249,P=0.103,P>0.05)in children with AL.2 Reverse transcription-PCR The expression of LC3-II gene in the experience group and the control group showed a normal distribution,the mean and standard deviation was0.360 ± 0.047 in the experience group.In the control group,it was 1.107±0.136,two samples of t=5.169,P=0.000,P<0.05.The difference was statistically significant.The expression of P62 in two data showed a normal distribution.In the experience group,the mean and standard deviation was1.336 ±0.139.In the control group the mean and standard deviation was1.610±0.449,t=0.583,P=0.570,P>0.05,the difference was not statistically significant.Conclusions:1 The expression of autophagy related gene and protein LC3-? in acute leukemia were up-regulated suggesting that maybe the level of autophagy increased in acute leukemia cells in children.Maybe the enhancement of autophagy is related to the occurrence of children with AL.2 There was no significant difference in the expression level of LC3-?and P62 in AML and ALL.The positive rates of LC3-? and P62 in male and female children with AL and the positive rates of LC3-II and P62 in ALL children with type B and type T were not statistically significant,suggesting that maybe the type,sex and the immunophenotype of ALL children cannot influent the activity of autophagy.3 Spearman correlation analysis showed that there was no correlation between the expression of LC3-and P62 protein in AL children. |
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