Developmental Changes Of PTEN In Chicken Before And After Hatching And Effect Of Green Tea On Its Expression

PTEN(phosphatase and tensin homolog deleted on chromosome ten)is the first discovered tumor suppressor with protein and lipid phosphatase activity.With the deepening of research,it is found that PTEN,as a tumor suppressor,also plays an important role in the regulation of cell proliferation,differentiation,apoptosis and a series of pathophysiological processes.However,PTEN is less studied in poultry than in mammals.In this present study,the tissue distribution and developmental changes of PTEN during the development of chicken embryo and chick were studied by real-time quantitative PCR(Quantitative real-time PCR,q RT-PCR)and immunohistochemistry staining.The effect of green tea on PTEN gene expression in chicken was also investigated to provide ideas for the physiological and pathological research of PTEN in chicken development.1.Tissue difference of PTEN transcription.In this research,the heart,skin,pectoralis major,cecum,lung,jejunum,duodenum,ileum,hypothalamus,proventriculus,kidney,liver,bursa of Fabricius,cecum tonsil,thymus and spleen were collected in chicken embryos on the 18 th day of hatching(E18)and the 15 th day after hatching(D15),and the distribution of PTEN in different samples was studied by q RT-PCR.The results showed that the relative expression level of Pten m RNA in the tissues was shown as skeletal muscle and immune organs > viscera organ > digestive organs at E18.However,at D15,the PTEN m RNA expression in heart was the highest among all the tissues detected,and followed by liver,proventriculus and kidney,which were 89.99%,41.23%and 39.63% as much as that of heart(P < 0.05).PTEN m RNA expression in the other tissues were very low,which were only 1.2%-19.47% as much as that of heart(P < 0.05).2.Developmental changes of PTEN transcription.In this research,tissues of the bursa of Fabricius,spleen,thymus,pectoralis major,liver,duodenum,jejunum,ileum,and cecum were collected respectively in chicken embryos of E15,E18,E20 and D1,D5,D10,D15 after hatching.The developmental changes of PTEN expression in different tissues were explored by immunohistochemistry and q RT-PCR methods.The results showed that the expression of Pten m RNA in the chick’s bursa of Fabricius showed an upward trend from D1 to D5.In bursa,an increase of around 3.02-and 3.11-fold of PTEN m RNA expression was seen at E18,E20 compared to that of E15(P< 0.05).The level of PTEN m RNA expression at D5 increased abruptly and reached 7.4-fold as high as that of E15(P < 0.05),which was the highest during the whole experiment time.From D1 to D5,the relative expression level of Pten m RNA showed a downward trend in the spleen and thymus.The relative expression level of Pten m RNA in the thymus reached the highest level on D1,which was approximately3.79-fold that of E15(P < 0.05).Pten m RNA expression in the spleen did not change significantly in the early stage,but it decreased rapidly at D10 and increased suddenly at D15,which were 0.37-fold and1.75-fold of E15(P < 0.05).The relative expression level of Pten m RNA in skeletal muscle showed an upward trend,reaching the highest level at D15,which was approximately 5.1-fold that of E15(P < 0.05).The relative expression level of Pten m RNA in liver reached the highest level on D1,which was approximately 10.46-fold of E15(P < 0.05).In the intestine,the relative expression levels of Pten m RNA in the duodenum and ileum have the same trend.The relative level of Pten m RNA expression in the duodenum reached the highest level at D5,which was approximately 4.1-fold that of E15(P < 0.05).The expression of Pten m RNA in the ileum reaching the highest level at D1,which was approximately16.6-fold that of E15(P < 0.05).The relative level of Pten m RNA expression in the jejunum reached the highest level on D10,which was approximately 1.46-fold that of E15(P < 0.05).In the cecum,we can find that the relative level of Pten m RNA expression reaches the highest level at E18,which is approximately3.7-fold that of E15(P < 0.05).While PTEN-immunopositive cells(PTEN-ip cells)in bursa were mainly distributed in epithelial cells before hatching and in bursal follicles after hatching.In spleen,PTEN-ip cells were expressed mainly in periarterial lymphatic sheath.During the embryonic stage,PTEN-ip is expressed in both thymic cortex and medulla.After incubation,PTEN-ip is mainly distributed in the medulla.PTEN immunopositive cells in skeletal muscle are mainly expressed in the nucleus during the embryonic stage,and PTEN-ip can be clearly observed in the cytoplasm at the 20 th day of the embryonic stage.It can be observed that PTEN-ip is mainly distributed in the cytoplasm after the shell,and only a small part of the nucleus has positive expression.PTEN-ip in the liver is mainly expressed in the nucleus during the embryonic stage.PTEN-ip is not only expressed in the cytoplasm but also in the nucleus after hatching.3.Effect of green tea on PTEN transcription.In this research,1 m L of green tea water(10 g tea/100 m L water)was given to the experimental chickens on the 7th day(7d)after the chickens were raised.The bursa of Fabricius,spleen,duodenum and jejunum were collected at 7d,12 d,15d,18 d and 21 d after feeding(0d,5d,8d,11 d and 14 d after gavage of green tea water),the effect of green tea on the expression of PTEN in these tissues was studied by q RT-PCR.The result showed that,the relative expression level of Pten m RNA in the bursa of Fabricius of the blank group reached the highest level on the 18 th day(11d after feeding green tea water),which was approximately 4.3-fold that of 7d(0d after feeding green tea water)(P < 0.05).The blank group was significantly different from the experimental group(P < 0.05)on 12 d and 18d(5d and 11 d after feeding green tea water).On the 18 th day(11d after feeding the green tea water),the relative expression level of Pten m RNA in the blank group was approximately 2.03-fold that of the green tea group(P < 0.05).In the spleen of the blank group,the relative expression of Pten m RNA showed an uptrend integrally.The expression of Pten m RNA in the green tea group was significantly lower than that in the blank group at 12days(5d after feeding green tea water),which was approximately 60% of the blank group(P < 0.05).At 15days(8d after feeding green tea water),it was significantly higher than that of the blank group.The relative expression level of Pten m RNA in the green tea group was approximately 2.98-fold that of the blank group(P < 0.05).In the duodenum of the blank group,the relative expression level of Pten m RNA showed an overall downtrend,which reaching the lowest at 21d(14d after feeding green tea water),which was approximately 11.5% of that of 7d(0d after feeding green tea water)(P < 0.05).At 21d(14d after feeding green tea water),the relative expression level of Pten m RNA between the blank group and the experimental group was extremely significant,and the relative expression level of Pten m RNA in the green tea group was approximately 9.7-fold that of the blank group(P < 0.05).In the jejunum,the relative expression level of Pten m RNA in the blank group increased first and then decreased.The relative expression levels of Pten m RNA in the blank group and the experimental group were significantly different for 12 days(5d after feeding green tea water),and the relative expression levels of Pten m RNA in the blank group was approximately 3.2-fold that of the green tea group(P < 0.05).Conclusion: PTEN exists widely in chicken embryo and chick tissues,and its expression is different in time and space;The developmental changes of PTEN in different types of tissues are different,and the expression position and location of PTEN also change with different development periods;Green tea has different effects on immune organs and intestinal tissues.