| Objective:(1)To investigate the localization and possible roles of fusome/spectral protein-prolyl 4-hydroxylase subunit ?(P4hb),adductin a(Addl)and haemoprotein ?(Sptbn2)in cell differentiation using the tissues of Eriocheir sinensis and mice at different developmental stages of testis and hepatocellular carcinoma.Methods:(1)The testis of young mice,adult mice and adult Eriocheir sinensis were used as materials.Real-time quantitative PCR(RT-qPCR)and Western blot(WB)techniques were used to analyze the expression of target gene and target protein at tissue level.Immunofluorescence localization(IF)technique was used to further analyze the expression distribution of target protein in different cells and different parts of cells.The target genes in cells with high expression of target proteins were inhibited by siRNA transfection,and the expression levels of cyclin gene Anln and kinesin-like protein gene Kifll were analyzed by RT-qPCR at messenger RNA(mRNA)level.(2)Mice were used as experimental animals.Hepatocellular carcinoma(HCC)tissue grew for 10 days after inoculation of mouse hepatocellular carcinoma(H22)under the axilla,HCC tissue,normal liver cells at 20 days were used as materials.The expression and localization of target gene and target protein were analyzed by RT-qPCR,WB and IF.Results:(1)P4hb,Addl and Sptbn2 mRNA and protein were found in the crabs testis and can be positioned to the crabs in the testis seminoma cells,the first stage of spermatocyte,spermatid and sperm cells in the second stage of the cytoplasm and cell membrane,mesenchymal cells and extracellular matrix,and the nucleus of a few specific areas,P4hb and Addl can locate in the sperm and the third phase of pure cell nuclei.The content of P4hb in all kinds of cells is in descending order:spermatogonia,spermatocyte,the first stage of spermatocyte,sperm cell,the third stage of spermatocyte and the second stage of spermatocyte.There was no significant difference in fluorescence intensity between sperm cells and sperm at the first stage,between sperm cells and sperm at the second stage,and between sperm cells and sperm at the third stage(n=3,P>0.05),and the difference of fluorescence intensity of P4hb in any other two kinds of cells was statistically significant(n=3,P<0.05).The content of Addl in all kinds of cells is in descending order:spermatogonia,first-stage sperm cells,spermatocytes,second-stage sperm cells,third-stage sperm cells,and sperm cells.There was no significant difference in the fluorescence intensity of Addl between spermatocytes and first-stage sperm cells,second-stage sperm cells and third-stage sperm cells,and second-stage sperm cells and spermatozoa(n=3,P>0.05),and the difference of Addl fluorescence intensity in any other two kinds of cells was statistically significant(n=3,P<0.05).The fluorescence intensity of Sptbn2 in all kinds of cells was from strong to weak:spermatogonia,first-stage sperm cells,second-stage sperm cells and spermatocytes,and almost no expression was found in third-stage sperm cells and sperm cells.There was no significant difference in fluorescence intensity between spermatogonocytes and first-stage sperm cells,spermatocytes and second-stage sperm cells,and sperm and third-stage sperm cells(n=3,P>0.05),there were statistically significant differences in the fluorescence intensity of Sptbn2 in any other two types of cells(n=3,P<0.05);(2)The mRNA and protein of P4hb,Add1 and Sptbn2 were all present in the testis of mice,but the content in the testis of young mice was higher than that of adult mice(n=3,P<0.05).If can be located in the cytoplasm,cell membrane,intercellular stroma,extracellular matrix,and a few specific regions of the nucleus of spermatogonia,primary spermatocyte,secondary spermatocyte,spermatocyte,etc.In addition,P4hb is located in the neck of the sperm tail.The content of P4hb in all kinds of cells is in descending order:primary spermatocytes,spermatogonia,secondary spermatocytes,sperm cells and sperm cells.There was no significant difference in P4hb fluorescence intensity between spermatogonial cells and spermatogonial cells,spermatogonial cells and sperm,primary spermatogonial cells and sperm,secondary spermatogonial cells and sperm(n=3,P>0.05),and the difference of fluorescence intensity of P4hb in any other two kinds of cells was statistically significant(n=3,P<0.05).Addl can be located in the nucleus,cytoplasm,cell membrane and the whole tail of sperm.The content of Addl in various kinds of cells with fluorescence intensity is in descending order:spermatogonia,spermatocyte,secondary spermatocyte,sperm and primary spermatocyte.There was no significant difference in fluorescence intensity between primary spermatocytes and sperm cells,between primary spermatocytes and sperm,and between secondary spermatocytes and sperm(n=3,P>0.05),and the difference of Addl fluorescence intensity in any other two kinds of cells was statistically significant(n=3,P<0.05).The content of Sptbn2 in all kinds of cells was in descending order:spermatogonia,spermatocyte,spermatocyte,primary spermatocyte and secondary spermatocyte.There was no significant difference in fluorescence intensity between spermatogonia and spermatogonia,spermatogonia and spermatogonia,primary spermatogonia and secondary spermatogonia,secondary spermatogonia and spermatogonia,spermatogonia and spermatogonia(n=3,P>0.05),and there were statistically significant differences in the fluorescence intensity of Sptbn2 in the other two types of cells(n=3,P<0.05).We randomly selected P4hb from the above three molecules for interference experiments.After adding small interfering RNA(siRNA)of P4hb into the culture plate of mouse spermatogonial cells,the mRNA expression levels of P4hb,Anln and Kifll in mouse spermatogonial cells were significantly lower than those in mouse spermatogonial cells without adding P4hb siRNA.(3)In the tumor tissues formed at different time periods and normal mouse liver tissues,the mRNA and protein expression levels of Addl in the tumor tissues at 10d,20d and liver tissues showed a downward trend,but the expression levels in the tumor tissues at 10d were higher than those in the liver tissues with statistical significance(n=3,P<0.05),and there was no significant difference in mRNA and protein expression levels between the other two groups(n=3,P<0.05),and Addl is mainly located in the cytoplasm,cell membrane and cell matrix of HCC cells.There was no significant difference in the expression levels of P4hb and Sptbn2 in tumor tissues and liver tissues at different time periods(n=3,P<0.05).Conclusions:(1)P4hb,Addl and Sptbn2 may participate in the construction of cytoskeleton through the biological processes of intercellular adhesion,integrin and motor protein formation in the testis of crab and mouse,and play an important role in the differentiation of spermatogonia;(2)During the occurrence of HCC in mice,Addl may participate in the construction of cytoskeleton by participating in the formation of actin,motor protein bundle and motor protein filament,and play an important role in liver cell dedifferentiation and canceration. |
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