【作者】 王璇；

【导师】 孙保存； 张诗武；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2010， 博士

【摘要】 研究目的1.通过观察TA2小鼠不同次数妊娠期间及分娩后不同时间脾细胞中T淋巴细胞亚群以及IL-2、IL-4、IL-10、IFN-γ四种细胞因子的变化规律,揭示妊娠及分娩与TA2小鼠T细胞免疫功能的关系。通过比较经产、未产及自发瘤组TA2小鼠机体T细胞免疫状况,初步探讨T细胞免疫功能与TA2小鼠自发性乳腺癌的发生机制。2.通过将TA2小鼠不同次数妊娠期间及分娩后不同时间T细胞免疫状况与其机体雌二醇(E2)、孕酮(P)分泌水平进行相关性分析,初步揭示E2、P与T细胞免疫功能的关系,并采用外源性注射雌激素与孕激素的实验验证其间关系。3.通过观察TA2小鼠不同次数妊娠期间及分娩后不同时间乳腺上皮细胞增殖指数以及凋亡指数的变化,初步推断TA2小鼠发瘤前乳腺上皮细胞的状态；同时,通过检测线粒体凋亡通路中,Bcl-2、Bax、Caspase-3和Caspase-9蛋白表达,以及乳腺上皮细胞中雌激素受体(ER)和孕激素受体(PR)的表达,对比发瘤前及发瘤后的状态,探讨TA2小鼠自发性乳腺癌发生的可能机制。研究方法1.收集TA2小鼠不同次数妊娠期间及分娩后不同时间以及自发瘤小鼠脾细胞和血清,采用流式细胞术检测脾细胞CD3+、CD4+、CD8+T淋巴细胞亚群,计算CD4+/CD8+T细胞比值。利用ELISA方法检测血清中IL-2、IL-4、IL-10、IFN-γ四种细胞因子水平,并计算Th1/Th2。2.采用相关性分析,分析E2、P与CD3+、CD4+、CD8+T淋巴细胞亚群以及IL-2、IL-4、IL-10、IFN-γ四种细胞因子之间的相关性。3.对成年TA2雌鼠行卵巢切除术(OVX),外源性给与雌激素、孕激素干预,观察E2和/或P对OVX小鼠T细胞免疫功能的影响。4.收集TA2小鼠不同次数妊娠期间及分娩后不同时间以及自发瘤小鼠乳腺组织,采用免疫组化方法检测乳腺上皮细胞中PCNA的表达,计算增殖指数；采用TUNEL方法检测乳腺上皮细胞中的细胞凋亡,计算凋亡指数；采用免疫组化、Real-time PCR和Western blotting方法检测组织中ER、PR、Bcl-2、Bax、Caspase-3和Caspase-9蛋白表达及mRNA相对表达水平。结果1.妊娠及分娩对TA2小鼠T细胞免疫功能的影响(1)对T淋巴细胞亚群的影响：TA2小鼠妊娠及分娩后不同时间内CD3+、CD4+、CD8+和CD4+/CD8+差别具有统计学意义(P<0.01)。其中CD3+、CD4+T淋巴细胞在P10下降至最低(12.79±3.64,58.36±6.57,P<0.01),在P20,CD4+/CD8+降至最低(1.81±0.14),CD8+T升至最高(32.68±2.93)。分娩后,CD4+、CD8+和CD4+/CD8+逐渐恢复,但CD3+T仍有下降趋势,在分娩后42天时才恢复正常。多次妊娠不同时段与8月龄处女鼠(8MV)和自发乳腺癌(SBC)小鼠比较,CD3+、CD4+T在各次妊娠P10、P20均显著低于8MV(P<0.05),且P10CD3+T与SBC无差别(P>0.05),P10、P20 CD4+T均接近SBC (P>0.05),CD4+T在3PP42、4PP42时亦与SBC无差别(P>0.05)。CD4+/CD8+在1P20、2P10和3P、4P各时段均低于8MV (P<0.01)。CD3+、CD4+、CD8+T和CD4+/CD8+在4P各时段均低于或高于8MV(P<0.01),而与SBC无差别(P>0.05)。4次妊娠中,4P CD3+、CD4+和CD4+/CD8+明显低于1P、2P(P<0.05)。(2)对细胞因子的影响：TA2小鼠妊娠及分娩后不同时间内IL-2、IL-4、IL-10、IFN-γ和Th1/Th2差别具有统计学意义(P<0.05)。IL-2、IL-4、IFN-γ和Th1/Th2在妊娠期间有所下降,IL-10有所上升,以10天和20天显著(P<0.05)。分娩后,IL-4、IL-10和Th1/Th2逐渐恢复,IL-2于分娩后1天恢复至正常未妊娠水平,但随后又逐渐下降,在分娩后42天时才恢复正常,IFN-γ在分娩后仍有下降,在分娩后42天虽有所恢复但未达正常。IL-2和IFN-γ在2P10、3P10、3P20、4P各时段均显著低于8MV(P<0.05),IL-4仅在4P10和4P20低于8MV(P<0.05),IL-10在1P10、1P20、2P20显著高于8MV, Th1/Th2在各妊娠次数P10以及4P各个时段均低于8MV (P<0.05)。4P各时段几乎均与SBC无差别(P>0.05),且4P各时段指标均不同程度低于1P～3P。2.雌激素与孕激素对TA2小鼠T细胞免疫功能的影响(1)妊娠及分娩对TA2小鼠E2和P的影响：TA2小鼠妊娠及分娩后不同时间内E2和P差别均具有统计学意义(P<0.01)。随着妊娠天数的增加,血清E2和P的浓度逐渐升高,以P20最为明显(E2：184.48±72.94,P：78.32±34.17,P<0.01)。分娩后,E2逐渐下降恢复正常,P立即下降后又有所波动,最后于PP21恢复正常(P>0.05)。多次妊娠间,以4P20血清E2和P浓度最高,且明显高于1P、2P(P<0.01)。(2)相关性分析结果显示：E2、P与CD8+T和IL-10呈正相关(P<0.01),而E2与CD3+、CD4+、CD4+/CD8+、IL-2、IL-4、IFN-γ呈负相关(P<0.05)；P与CD3+、CD4+、CD4+/CD8+、IL-2呈负相关(P<0.01)。P与IL-4、IFN-γ之间相关关系不明显(P>0.05)。(3)外源性给与雌激素、孕激素干预结果显示：与对照组比较,E2+P联合作用组各指标均与对照有差别,且具有统计学意义(P<0.01)；E2单独作用组CD3+T淋巴细胞、CD4+/CD8+和IL-2、IL-4均明显低于对照(P<0.05或P<O.01)；P单独作用组仅CD3+T淋巴细胞和IL-2明显低于对照(P<0.05),其它均与对照无差别。其中,E2+P作用最为明显,且高浓度作用大于低浓度作用。3.妊娠及分娩对TA2小鼠乳腺上皮细胞增殖和凋亡的影响TA2小鼠妊娠及分娩后不同时间内增殖指数(PI)和凋亡指数(AI)差别均具有统计学意义(P<0.01)。随着妊娠天数的增加,AI逐渐增加,以P20最为明显(47.23±15.89,P<0.01)。分娩后,AI并未立即下降,在PP1和PP7仍然持续较高水平,PP21以后逐渐下降。AI在妊娠期间较低,分娩后有所升高,以PP1最高(59.48±11.37,P<0.01),随后有所下降。多次妊娠间,以4P20 PI和AI最高(86.20±7.09,74.80±10.99),且明显高于1P、2P(P<0.01)。4.ERα和PR在TA2小鼠乳腺上皮细胞中的表达(1)免疫组化染色结果显示：妊娠和分娩不同阶段乳腺上皮细胞ERα和PR表达差异具有统计学意义(P<0.01)。妊娠后乳腺上皮细胞中ERα和PR表达明显高于未妊娠小鼠,到妊娠20天达到最大(ERa:84.60±9.40, PR:2.52±0.81, P<0.01)。分娩后表达水平逐渐下降,到第42天,基本与未妊娠小鼠相近。多次妊娠间,4P小鼠各时段ERα和PR表达明显高于1P、2P(P<0.01或P<0.05)。(2) Real-time PCR结果显示：不同妊娠次数TA2小鼠ERαmRNA和PR mRNA表达之间差异具有统计学意义(P<0.01或P<0.05),且4P小鼠各时段ERαmRNA和PR mRNA表达均不同程度明显高于1P～3P(P<0.01或P<0.05)。5. Bcl-2、Bax、Caspase-3和Caspase-9在不同妊娠次数TA2小鼠乳腺上皮细胞中的表达(1)免疫组化染色结果显示：正常TA2小鼠乳腺上皮细胞中Bcl-2、Bax、Caspase-3和Caspase-9均呈低表达。妊娠期时均有所升高,其中以妊娠20天时最为明显(P<0.01或P<0.05)；在分娩后,Bcl-2、Bax、Caspase-3继续升高,到了分娩后21天,达到最大值(Bcl-2:8.04±0.83; Bax:9.92±0.77; Caspase-3: 9.60±0.40, P=0.000), Caspase-9变化不大。到分娩后42天基本恢复至正常。不同妊娠次数TA2小鼠Bcl-2、Bax、caspase-3和caspase-9表达之间差异具有统计学意义(P<0.01)。4P小鼠各时段Bcl-2和Bax表达明显高于1P～3P (P<0.01), Caspase-3和Caspase-9表达明显高于1P、2P(P<0.01或P<0.05)。(2)Real-time PCR结果显示：不同妊娠次数TA2小鼠四个基因表达差异具有统计学意义(P<0.01或P<0.05)。其中,4P小鼠各时段bcl-2, bax, caspase-3和caspase-9 mRNA表达明显高于1P～3P(P<0.01或P<0.05)。(3) Western blotting结果显示：基本与免疫组化结果一致。3P、4P小鼠各时段Bcl-2、Bax、Caspase-3和Caspase-9表达明显高于1P、2P(P<0.01或P<0.05),其中以妊娠20天最为显著。6. ER、PR、Bcl-2、Bax、Caspase-3和Caspase-9在正常乳腺、癌前乳腺及乳腺癌组织中的表达免疫组化染色,Real-time PCR以及Western blotting结果一致显示：与8MV比较,除PR低于8MV(P<0.01)外,SBC小鼠乳腺ERa, Bcl-2, Bax, Caspase-3和Caspase-9表达均明显高于8MV(P<0.01或P<0.05)；自发瘤组织中ERα, PR, Bcl-2, Bax, Caspase-3和Caspase-9蛋白表达均明显低于8MV(P<0.01),但bcl-2,,bax和caspase-3 mRNA表达均显著高于正常8MV小鼠(P<0.01), caspase-9 mRNA略高于8MV,但无统计学意义(P>0.05)。ERα和PR mRNA在肿瘤组织中表达均低于正常8MV小鼠(P<0.01),其中PR更为明显(P<0.01)。结论多次妊娠及分娩使TA2小鼠体内雌激素与孕激素持续处于高水平,如此,一方面使TA2小鼠机体长期处于细胞免疫被抑制的状态,而造成体液免疫机制被慢性激活；另一方面刺激乳腺上皮细胞增殖,并通过线粒体途径的特殊机制调节乳腺上皮细胞凋亡。雌激素与孕激素分别经其各自受体,破坏TA2小鼠Th1/Th2的平衡以及乳腺上皮细胞增殖和凋亡的平衡,以致TA2小鼠自发乳腺癌成为可能。更多还原

【Abstract】 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.更多还原