| Objective1. To confirm the relationship between pregnancy/delivery and the immune function of T lymphocytes in TA2 mice by investigating T lymphocyte subsets in spleens and the concentration of serum IL-2, IL-4, IL-10 and IFN-γduring different stages of multiple pregnant and postpartum. To reveal the effect of T lymphocytes function on genesis of spontaneous breast cancer in TA2 mice by comparing the immune function of T lymphocyte in non-pregnant, pregnant and spontaneous cancer groups.2. To reveal the relationship between the level of serum estradiol(E2), progesterone(P) and T lymphocytes function in TA2 mice by correlating the changes in some parameters indicating quantity and function of T lymphocytes with the different level of E2 and P during different stages of pregnancy and puerperium. And to further confirm their relationship through the exogenous injection of E2 and P.3. To study the status of mammary epithelium before genesis of breast cancer in TA2 mice by observing the change of proliferation and apoptotic index of mammary epithelium during different stages of pregnancy and puerperium. To reveal the possible mechanism of genesis of spontaneous breast cancer in TA2 mice by detecting the expression of Bcl-2, Bax, caspase-3 and caspase-9 proteins in mitochondrial apoptosis pathway and estrogen receptor(ER) and proestogen receptor(PR) in mammary epithelium, and by comparing these index in different status of mammary epithelium during pre-/post-oncogenesis.Methods1. Sera and spleens were collected from pregnant or postpartum TA2 mice at different time points and also from cancer-bearing TA2 mice. We investigated the T lymphocyte subsets including CD3+, CD4+ and CD8+T using flow cytometry and calculated the CD4+/CD8+ ratio. The concentration of IL-2, IL-4, IL-10 and IFN-γin sera was analyzed by enzyme-linked immunosorbent assay (ELISA) and Th1/Th2 ratio was calculated.2. We correlated the level of E2/P with the changes of CD3+, CD4+ and CD8+T lymphocyte subsets and the concentration of IL-2, IL-4, IL-10 and IFN-γ. 3. Non-tumor adult TA2 mice after ovariectomy received exogenous injection of E2 and P. Their effects on T lymphocyte function of OVX mice were analyzed.4. We collected the mammary gland tissue from pregnant or postpartum TA2 mice at different time points and also from cancer-bearing TA2 mice. The expression of PCNA in mammary epithelial cells was measured by immunohistochemistry and proliferation index was calculated. Apoptosis of mammary epithelial cells was detected by TUNEL and apoptotic index was analyzed. The expression of proteins including ER, PR, Bcl-2, Bax, caspase-3 and caspase-9 was measured using immunohistochemistry and Western blotting. Their levels of mRNA were detected by Real-time PCR.Results1. The effect of pregnancy/delivery on immune function of T lymphocytes in TA2 mice(1) The effect of pregnancy/delivery on immune function of T lymphocytes:CD3+, CD4+, CD8+ and CD4+/CD8+ ratio were significantly different during different stages of pregnancy and puerperium (P<0.01). The level of CD3+, CD4+ T lymphocytes was the lowest on P10 (12.79±3.64,58.36±6.57, P<0.01). CD4+ /CD8+ ratio reached the minimum (1.81±0.14) and CD8+ T lymphocytes did the maximum (32.68±2.93) on P20. CD4+, CD8+ and CD4+/CD8+ ratio increased gradually. However, CD3+ still had a tendency to decrease and it recovered to the normal until 42 days after delivery. CD3+, CD4+ T lymphocytes on P10, P20 in different gravidities were significantly lower than those in 8MV (P<0.05). There was no difference between CD3+ lymphocytes on P10 and those in SBC (P>0.05). The level of CD4+lymphocytes on both P10 and P20 was close to that in SBC (P>0.05). And no difference was found between CD4+ lymphocytes on 3PP42,4PP42 and those in SBC (P>0.05). CD4+/CD8+ ratios on 1P20,2P10 and on different time during 3P,4P were lower than those in 8MV (P<0.01). Significant difference in the level of CD3+, CD4+, CD8+ T lymphocytes and CD4+/CD8+ ratio was showed between each time point in 4P and in 8MV (P<0.01), while it was not found between each time point in 4P and in SBC (P>0.05). Among the four pregnancy, CD3+, CD4+ and CD4+/CD8+ ratio in 4P were significantly lower than those in 1P and 2P (P<0.05).(2) The effect of pregnancy/delivery on cytokines:Concentration of Serum IL-2, IL-4, IL-10, IFN-γand Th1/Th2 ratio were significantly different between different time during pregnancy and puerperium in TA2 mice (P<0.05). IL-2, IL-4, IFN-γand Th1/Th2 ratio decreased during pregnancy while IL-10 went up, especially in Day10 and Day20 with a significance (P<0.05). IL-4, IL-10 and Th1/Th2 ratio recovered gradually after delivery. IL-2 reached the normal progestation level one day after delivery, and then went down gradually. It recovered to the normal 42 days after delivery. IFN-γcontinued to decrease after delivery and did not reached the normal 42 days after delivery although it started to increase. IL-2, IFN-γon 2P10,3P10, 3P20 and on different time during 4P were significantly lower than those in 8MV (P <0.05). IL-4 only in 4P10,4P20 was lower than that in 8MV (P<0.05). IL-10 on 1P10,1P20 and 2P20 was significantly higher than that in 8MV. Th1/Th2 ratios on P10 in different gravities and on different time point in 4P were lower than those in 8MV (P<0.05). There was almost no difference in the level of Th1/Th2 ratios between each time point in 4P and in SBC (P>0.05) while Th1/Th2 ratios in 4P were lower than those in 1P～3P.2. Effect of estradiol and progesterone on T lymphocyte function in TA2 mice(1) the effect of pregnancy/delivery on the level of E2 and P in TA2 mice:Significant difference was found between different time during pregnancy and puerperium in TA2 mice (P<0.01). Concentration of Serum E2and P increased gradually with the time of pregnancy, with the highest level on P20 (E2:184.48±72.94, P:78.32±34.17, P<0.01). After delivery, E2 was reduced gradually to the normal level. P decreased quickly just after delivery, then went up and down for a period of time, and finally recovered to the normal on PP21 (P>0.05). Concentration of Serum E2and P on 4P20 showed the highest level on 4P20 during multiple pregnancy, dramatically higher than that of 1P and 2P (P<0.01).(2) Correlation analysis:Positive correlations between E2/P and IL-10 were observed (P<0.01). Negative correlations between E2 and CD3+, CD4+, CD4+/CD8+, IL-2, IL-4, and IFN-γwere showed (P<0.05). Similarly, there were negative correlations between P and CD3+, CD4+, CD4+/CD8+ and IL-2 (P<0.01). No correlations were showed between P and IL-4, IFN-γ(P>0.05).(3) Treatment with exogenous injection of E2 and P:Each index in combination group showed a significant difference comparing with the control (P<0.01). CD3+T lymphocytes in single E2 group were much lower than those of control (P<0.05) and CD4+/CD8+ ratio and IL-2,IL-4 were the same (P<0.01). Only CD3+ T lymphocytes and IL-2 in single P group were dramatically lower than the control (P <0.05) while other index in this group were not different from the control. E2 in combination with P had the strongest effect. And the higher concentration of them resulted in a marked effect than the lower.3. Proliferation and apoptosis of mammary epithelial cells in different gravidities Significant difference in proliferation index (PI) and apoptosis index (AI) was showed on different time during pregnancy and puerperium in TA2 mice (P<0.01). AI increased gradually with the time of pregnancy, with the highest level on P20 (47.23±15.89, P<0.01). After delivery, AI did not decreased immediately with a still high level on PP1 and PP7, and then started to go down gradually until after PP21. AI remained at a low level during pregnancy, which increased after delivery and reached the highest level on PP1 (59.48±11.37, P<0.01). It started to go down after that. PI and AI on 4P20 were the highest during multiple pregnancy (86.20±7.09, 74.80±10.99), much higher than those in 1P and 2P (P<0.01).4. Expression of ERa and PR in mammary epithelium in TA2 mice(1) Immunohistochemistry results showed that significant difference was found in the Expression of ERαand PR in mammary epithelium between different stages of pregnancy and puerperium (P<0.01). ERa and PR in mammary epithelium during pregnancy had much higher expression than those in nonpregnant mice, which reached the maximum on P20 (ERa:84.60±9.40, PR:2.52±0.81, P<0.01). Their expressions started to decrease gradually after delivery and were very close to the level of nonpregnant mice on P42. ERαand PR on different time points in 4P were significantly higher expressed than those in 1P and 2P (P<0.01 or P<0.05).(2) Real-time PCR results showed that there was a significant difference in the expression of ERαmRNA and PR mRNA between in different gravidities in TA2 mice (P<0.01 or P<0.05). ERαmRNA and PR mRNA on different time points in 4P were significantly higher expressed than those in 1P～3P (P<0.01orP<0.05).5. Expression of Bcl-2, Bax, Caspase-3 and Caspase-9 in mammary epithelium in TA2 mice in different gravidities(1) Immunohistochemistry results showed that Bcl-2, Bax, Caspase-3 and Caspase-9 were lower expressed in mammary epithelium in nonpregnant TA2 mice. All of them increased during pregnancy, with the maximum on P20 (P<0.01 or P<0.05). Bcl-2, Bax and Caspase-3 went up continuously after delivery, which reached the maximum on P21 (Bcl-2:8.04±0.83, P=0.000; Bax:9.92±0.77, P=0.000; Caspase-3:9.60±0.40, P=0.000). Caspase-9 did not show much change. All these indexes recovered to the normal 42 days after delivery. Significant difference was showed in the expression of Bcl-2, Bax, Caspase-3 and Caspase-9 between in different gravidities in TA2 mice (P<0.01). The expression of Bcl-2 and Bax in 4P was obviously higher than that in 1～3P (P<0.01). Caspase-3 and Caspase-9 in 4P were also higher than those in 1P and 2P(P<0.01 or P<0.05).(2) Real-time PCR results showed that the expression of these four genes had a significant difference in different gravidities in TA2 mice (P<0.01 or P<0.05). The expression of bcl-2, bax, caspase-3 and caspase-9 mRNA on each time point in 4P were higher than those in 1P～3P (P<0.01 or P<0.05).(3) Western blotting results showed the same trend as the result of immunohistochemistry. Bcl-2, Bax, caspase-3 and caspase-9 on each time point in 3P and 4P had a higher expression than those in 1P and 2P (P<0.01 or P<0.05), with the most obvious data on P20.6. Expression of ER, PR, Bcl-2, Bax, Caspase-3 and Caspase-9 in normal control, precancerous lesion and mammary cancer tissue Data from immunohistochemistry, Real-time PCR and Western blotting had the same trend. ERa, Bcl-2, Bax, Caspase-3 and Caspase-9 in mammary epithelium in TA2 mice had a higher expression than those in 8MV mice before tumorigeness (P<0.01 or P<0.05) except the lower expression of PR (P<0.01). ERa, PR, Bcl-2, Bax, Caspase-3 and Caspase-9 proteins in spontaneous tumor tissue had a lower expression than those in 8MV mice. However, the levels of bcl-2, bax and caspase-3 mRNA in TA2 mice were significantly higher than those in normal 8MV mice (P<0.01). caspase-9 mRNA in TA2 mice was a little higher than that in 8MV mice with no significance (P>0.05). ERαand PR mRNA expression in tumor tissue was lower than those in 8MV mice (P<0.01), especially in the expression of PR mRNA (P<0.01).ConculsionRepeated pregnancy and delivery can increase the level of estrogen and progesterone, which results in the continuous inhibition of the cellular immunity and chronic activation of humoral immunity in TA2 mice. On the other hand, the proliferation of mammary epithelium was stimulated during the course of frequent pregnancy and delivery. The apoptosis of mammary epithelium is induced by a specific mechanism through mitochondrial apoptosis pathway. When estrogen and progesterone combine with their receptors respectively, they shows their effect of destroying the balance both of Th1/Th2 and of proliferation/apoptosis in mammary epithelium, which may contribute to the genesis of spontaneous breast cancer in TA2 mice.
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