【作者】 赵书君；

【导师】 丰有吉； 殷莲华； 周先荣； 郁茵华；

【作者基本信息】 复旦大学 ， 妇产科学， 2006， 博士

【摘要】 子宫内膜癌的发病年龄平均为63岁，其中5—14％的患者发病年龄小于40岁，多合并有未产、不育、月经失调、肥胖及多囊卵巢刺激症，保留生育功能成为这些年轻妇女的迫切需求。孕激素大量、长期应用为子宫内膜不典型增生及子宫内膜癌保守治疗的主要方法，目前子宫内膜癌的保守治疗对象严格限定于□型子宫内膜癌、临床ⅠA期、组织病理学Ⅰ级、雌、孕激素受体(estrogen receptor ER，progesterone receptor PR)阳性、有良好随访条件、无激素和抗癌药物应用禁忌症、有强烈生育要求的年轻患者。常用的孕激素有甲地孕酮及醋酸甲羟孕酮(medropxyprogesterone acetate MPA)。临床观察表明孕激素保守治疗子宫内膜样腺癌的成功率达到57％—75％，辅以助孕技术可获得一定的妊娠率，对年轻未孕子宫内膜癌患者具有很大的鼓舞性。但并非所有的患者均对孕激素反应良好，或开始反应良好到后来不反应，存在着30％左右治疗失败率。保守治疗失败相应延迟了手术根治的时间，可能会影响病人的预后。探讨孕激素治疗不敏感的机制对于临床保守治疗具有重大的指导意义。本研究利用分化好、雌孕激素受体阳性、对孕激素反应良好的子宫内膜癌Ishikawa细胞，以MPA为诱导剂，采用持续作用、梯度递增的方法诱导孕激素耐药的Ishikawa细胞亚系的建立，探讨雌、孕激素受体表达的改变及TGFa／EGF-EGFR信号通路活性的增强在孕激素耐药发生中的作用，为子宫内膜癌合理、安全、有效的保守治疗提供理论依据。本课题共分三部分：□子宫内膜癌孕激素耐药细胞的建立及其生物学特点；□雌、孕激素受体表达的改变及EGFR信号通路活性增强与孕激素耐药发生的关系；□外源性生长因子TGFa、EGF与子宫内膜癌孕激素耐药发生的关系。第一部分子宫内膜癌孕激素耐药细胞的建立及及其生物学特点目的建立子宫内膜癌孕激素耐药细胞，并研究其生物学特点。方法利用子宫内膜癌Ishikawa细胞，以MPA为诱导剂，采用浓度递增(1-10μM)、持续给药的方法诱导建立子宫内膜癌孕激素耐药细胞；应用四甲基偶氮唑蓝(MTT)法、流式细胞技术(FACS)及Brdu掺入法检测孕激素对母代Ishikawa细胞及孕激素耐药细胞增殖的影响；Transwell小室检测MPA对母代Ishikawa细胞及耐药细胞侵袭能力的影响；RT-PCR技术及Westem-blot技术检测MPA对母代Ishikawa细胞及耐药细胞CyclinD1表达的影响；RT-PCR技术及明胶酶谱技术检测MPA对母代Ishikawa细胞及耐药细胞MMP2、MMP9表达及活性的影响。结果(1)历时10个月MPA诱导后，获得了对MPA耐药的子宫内膜癌Ishikawa细胞亚系，暂且命名为孕激素耐药Ishikawa细胞，简称为耐药细胞。耐药细胞在含10μM MPA的完全培养基中的生长速率与母代Ishikawa细胞在不含MPA的完全培养基中的生长速率基本一致，细胞群体倍增时间分别为34.18±3.15h、35.14±2.68h，差异无统计学意义(t=0.331，P=0.762)。(2)MTT结果表明：MPA抑制母代Ishikawa细胞增殖，抑制效应具有浓度依赖性，不同作用浓度0.1、1、10μM的抑制率分别为20％、28％、41％；与母代Ishikawa细胞相反，相对高浓度的MPA(1.0、10μM)反而促进耐药细胞增殖，促增殖率分别为21％、46％。FACS结果表明：MPA可阻滞母代Ishikawa细胞于G1，阻滞效应具有时间依赖性，MPA作用24、48、72、96h细胞增殖指数分别下降14.2％、18.13％、26％、45％；与母代Ishikawa细胞相反，MPA作用72、96小时明显促进耐药细胞由G1期进入S期，增殖指数分别增加21.9％、20.85％。Brdu掺入实验进一步证实了MPA对母代Ishikawa细胞增殖的抑制作用及对耐药细胞增殖的促进作用。(3)耐药细胞的侵袭能力略高于母代Ishikawa细胞(P＞0.05)。MPA可抑制母代Ishikawa细胞的侵袭能力，MPA作用72h，细胞的侵袭能力下降了64％(t=6.107，P=0.026)；与母代Ishikawa细胞不同，MPA促进耐药细胞的侵袭能力，在MPA的作用下，耐药细胞的侵袭能力增加了26.3％(t=8.660，P=0.013)。(4)MPA抑制母代Ishikawa细胞CyclinD1、MMP2、MMP9的表达并降低MMP2活性，促进耐药细胞CyclinD1、MMP2、MMP9的表达并提高MMP2活性，与对照相比差异均有统计学意义(P＜0.05)。结论MPA的长期作用能够导致对孕激素抑制作用敏感的Ishikawa细胞对MPA耐药，一旦耐药发生，MPA可能通过增强CyclinD1、MMP2、MMP9表达及提高MMP2活性促进肿瘤细胞增殖、侵袭能力。提示用孕激素保守治疗子宫内膜癌是可行的，但要警惕长期用药孕激素耐药的发生。第二部分雌、孕激素受体表达的改变及EGFR信号通路活性增强与孕激素耐药发生的关系目的探讨雌、孕激素受体亚型表达的改变及内源性生长因子TGFa及其受体EGFR表达的提高及活性的增强与孕激素耐药发生的关系。方法采用免疫细胞化学、RT-PCR、Western-blot法检测耐药细胞及母代Ishikawa细胞ER、PR亚型的表达及EGFR的表达；RT-PCR检测两株细胞TGFa的表达及MPA对两株细胞TGFa表达的影响；Western-blot检测两株细胞EGFR-TK的表达情况；MTT及流式细胞术检测EGFR-TK抑制剂AG1478对两株细胞增殖的影响；RT-PCR、Western-blot检测AG1478对两株细胞CyclinD1表达的影响；RT-PCR检测AG1478对两株细胞MMP2、MMP9表达的影响。结果(1)与母代Ishikawa细胞相比，耐药细胞ERa、PRB的阳性表达率明显下降，ERβ的阳性表达率增加(均有P＜0.05) (2)耐药细胞TGFa、EGFR的表达水平高于母代Ishikawa细胞(均有P＜0.05)，且耐药细胞EGFR自身酪氨酸磷酸化的水平也高于母代Ishikawa细胞。(3)MPA下调母代Ishikawa细胞TGFa表达，上调耐药细胞TGFa表达。(4)MTT结果显示AG1478抑制耐药细胞的增生，抑制作用成浓度依赖性(F=18.148，P=0.00；各用药组与对照相比均有P＜0.05)，而较低浓度的AG1478对母代Ishikawa细胞没有明显的抑制作用，高浓度(10μM)的AG1478显示出抑制作用(P=0.009)。细胞周期分析结果表明AG1478可阻滞耐药细胞于G1期，AG1478作用24、48、72h抑制率分别为19.4％、11.2％、10.7％(均有P＜0.05)，而对母代Ishikawa细胞细胞周期分布没有明显影响，仅于作用48h显示出阻抑作用(t=5.074，P=0.037)，抑制率为9％。细胞侵袭实验结果表明AG1478抑制母代Ishikawa细胞及耐药细胞的侵袭能力，抑制效率分别为42.4％±4.1％及58.1％±6.9％(t=6.083，P=0.026；t=38.105，P=0.001)，对耐药细胞侵袭能力的抑制效率要高于母代Ishikawa细胞(t=4.614，P=0.044)。(5)耐药细胞及母代Ishikawa细胞经AG1478作用48h后，CyclinD1、MMP2、MMP9三种基因的表达均下降，在母代IshikaWa细胞中的表达分别下降为原水平的0.767、0.336及0.348倍，在耐药细胞中的表达分别下降为原水平的0.45、0.022、0.148倍。与母代Ishikawa细胞相比，AG1478引起的耐药细胞CyclinD1、MMP2、MMP9表达的下降程度均高于母代Ishikawa细胞(均有P＜0.05)。结论(1)ERa表达的下降，ERβ表达的升高、PRB表达的下降及TGFa-EGFR信号通路活性增强可能参与了子宫内膜癌孕激素耐药的发生。(2)EGFR-TK抑制剂有望成为孕激素耐药子宫内膜癌的一种新型有效的治疗方法。第三部分外源性生长因子TGFa、EGF与子宫内膜癌孕激素耐药发生的关系目的探讨外源性生长因子TGFa／EGF对子宫内膜癌Ishikawa细胞孕激素作用的影响。方法采用MTT法、划痕实验检测TGFa／EGF、MPA及TGFa／EGF联合MPA对母代Ishikawa细胞增殖、迁移运动能力的影响：Western-blot检测TGFa／EGF、MPA及TGFa／EGF联合MPA对母代Ishikawa细胞CyclinD1、E-cadherin蛋白表达的影响；RT-PCR及Western-blot技术检测TGFa／EGF及MPA对母代Ishikawa细胞PRB表达的影响；Western-blot技术检测TGFa／EGF刺激MAPK磷酸化的情况及MPA预作用48h对TGFa／EGF刺激MAPK磷酸化强度的影响。结果(1)TGFa／EGF促进母代Ishikawa细胞的增殖、迁移运动能力，MPA抑制母代Ishikawa细胞的增殖及迁移运动能力，与对照相比差异均有统计学意义(P＜0.05)，TGFa／EGF联合MPA促进母代Ishikawa细胞的增殖、迁移运动能力，与单用TGFa、EGF组差异无统计学意义(P＞0.05)。(2)TGFa、EGF促进细胞周期蛋白CyclinD1的表达，下调E-cadherin的表达，MPA与TGFa、EGF的作用相反，降低CyclinD1的表达，促进E-cadherin的表达，TGFa／EGF联合MPA与TGFa、EGF单独作用的结果相一致，引起CyclinD1表达的上调及E-cadherin表达的下调。(3)TGFa、EGF、MPA均下调PRB的表达，下调作用具有剂量依赖性，小剂量的生长因子TGFa(1ng／ml)、EGF(1ng／ml)、MPA(0.1μM)即能引起PRB表达的下降，随着剂量的增加，下调作用愈明显。TGFa、EGF作用12h，PRB表达下降，24h达到最低点；MPA作用6h，PRB表达开始下降，24h达到最低点，与对照相比差异均有统计学意义(P＜0.05)，之后表达又逐渐恢复，但不能恢复至原来水平。(4)TGFa、EGF能激活MAPK的活性分子P-ERK的表达，MPA预作用于母代Ishikawa细胞48h，此时再加入生长因子TGFa／EGF作用5min、15min、30min，P-ERK的表达强度要高于无血清预处理组(不含MPA)。结论(1)外源性生长因子TGFa／EGF促进子宫内膜癌Ishikawa细胞的增殖、迁移运动能力，拮抗MPA的抑制作用，可能通过上调CyclinD1的表达及下调E-cadherin的表达而发挥作用。(2)MPA抑制子宫内膜癌细胞的增殖及侵袭能力，下调CyclinD1的表达及上调E-cadherin的表达，却不能拮抗生长因子的促进作用及其引起的CyclinD1表达的升高及E-cadherin表达的下降。(3)生长因子TGFa／EGF与MPA都能够下调Ishikawa细胞PRB的表达，降低了细胞对MPA的反应，且MPA的预作用增强了生长因子TGFa／EGF刺激MAPK磷酸化的强度。上述结果提示：MPA的长期作用能够导致对孕激素抑制作用敏感的Ishikawa细胞对MPA耐受，雌孕激素受体亚型表达的失衡及内外源性TGFa／EGF-EGFR信号通路活性的增强可能为子宫内膜癌孕激素耐药发生的机制之一。一旦耐药发生，MPA可能通过增强CyclinD1、MMP2、MMP9表达及提高MMP2活性促进子宫内膜癌细胞的增殖、侵袭能力。在耐药发生过程中，子宫内膜癌由激素依赖型转为生长因子依赖型。EGFR-TK抑制剂有望成为孕激素耐药的子宫内膜癌新型有效的治疗药物。更多还原

【Abstract】 Endometrial carcinoma is often diagonised at the average age of sixty-thre years old. 5%-14% of endometrial adenocarcinoma occurs in women (<40 years of age), who very often still desire pregnancy. Progestin, especially medroxyprogesterone acetate (MPA) have been used as conservative treatment in atypical hyperplasia and endometrial adenocarcinoma for a long time.The response rate varies from 57%-75%. It has been reported that conservative treatment followed by IVF enable such patients to obtain a successful pregnancy. These relatively good response rates are encouraging with respect to the possibility of conservative treatment by progestins in young patients who desire for pregnancy, but over a third of patients with endometrial adenocarcinoma will display resistance to endocrine therapies at the time of presentation and most cancer patients that initially respond to progestin treatment would develop resistance later, resulting in tumor progression. Moreover, the conservative treatment failure might delay radical treatment and alter the prognosis. Progestin therapy is limited by the development of resistance in the tumor. The cellular mechanisms of primary and acquired resistance to progestin are poorly understood. In order to investigate the molecular mechanisms we have developed a subcell line resistant to the growth inhibitory effects of progestins from Ishikawa (human endometrial adenocarcinoma cell line) by stepwise selection in increasing concentrations of the synthetic progestin: MPA and investigate the relationship between progestin resistance and the imbalance of ER and PR subtype and the relationship between progestin resistance and the intrinsic/ extrinsic upregulation of the signaling of TGFa/EGF-EGFR.These experiments were divided into 3 parts, as following: Establishment of endometrial carcinoma cell resistant to medroxyprogesterone and its biological characteristic; The imbalance of ER and PR subtype and the intrinsic upregulation of the signaling of TGFa-EGFR might contribute to progestin resistance; D The extrinsic of TGFa/EGF might contribute to progestin resistance.Section Establishment of endometrium carcinoma cell resistant to medroxyprogesterone and its biological characteristicObjective To develop a subcell line of Ishikawa cells resistant to the growth-inhibitory actions of medroxyprogesterone acetate (MPA) and investigate its biological characteristic.Methods Progestin-resistant cell line was developed from Ishikawa human endometrial adenocarcinoma cells by stepwise selection in increasing concentrations of the synthetic progestin, MPA. MTT, FACS and Brdu were used to detect the effect of MPA on the proliferation of the original Ishikawa cell line and the progestin-resistant subcell line. Matrigel invasion assay was used to detect the effect of MPA on the invasive capability of the two cell lines. RT-PCR and Western-blot were used to detect the expression of CyclinD1 under the treatment of MPA over different time. RT-PCR was used to detect the expression of MMP1, MMP9 under the treatment of MPA over different time. Zymographic analysis was used to detect the activity of MMP2, MMP9 secreted from the two cell lines over MPA treatment. Results (1) The progestin-resistant cell line was established from human Ishikawa cell (endometrial cancer cell line) by stepwise selection in continual and increasing concentrations of MPA (1-10μM) over ten months. The doubling time of the progestin-resistant cells (34.18±3.15 hours) grown routinely in the medium containing 10μM MPA was not significantly different from the doubling time of the original Ishikawa cell line (35.14±2.68 hours) grown in the absence of MPA (t=-0.331, P=0.762). (2) MPA inhibited the original Ishikawa cell growth, the inhibitory action was concentration dependent (F=29.525, P=0.000), low concentration of MPA (0.01, 0.1μM) had no effect on the growth of the progestin-resistant cells, but relatively higher concentration (1,10μM) of MPA significantly increased the proliferation of progestin-resistant cells(F=36.20, P=0.00). The inhibitory effects of MPA on the original Ishikawa cells and the stimulatory effects on the progesterone-resistant cells were further confirmed by the cell cycle analysis. The results of cell cycle analysis showed that MPA caused time-dependent inhibition of cell cycle of the original Ishikawa cells, MPA produced a 14.2% fall in the proliferation index on day 1 and a 45% reduction by day 4; reversely, MPA exert no effect on the cell cycle of the progestin-resistant cells on dayl and day 2, by day 3 and day 4, MPA produced an increase in the proliferation index 21.9% and 20.85% respectively. These results were further confirmed by Brdu experiment. (3) Theinvasiveness capability of the progestin-resistant cells were higher than that of the original Ishikawa cells, but the difference was not stastically significant (P>0.05). MPA inhibit the invasiveness capability of the original Ishikawa cells dramatically (t=6.107, P - 0.026) and promote the invasiveness capability of the progestin-resistant cells (t=8.660, P=0.013). (4) Consistent with the phenomena, treatment with MPA on the original Ishikawa cells reduced the expression of CyclinD1, MMP2, MMP9 and the activity of MMP2, and reversely, treatment with MPA on the progestin-resistant cell increased the expression of CyclinD1, MMP2, MMP9 and the activity of MMP2.Conclusions These results suggested that prolonged treatment with MPA on Ishikawa cell could give rise to the resistant effect to MPA, when the resistance was acquired, treatment with MPA could enhance the cancer cell proliferation, invasiveness and metastasis, which might be due to up-regulating CyclinD1, MMP2 and MMP9 expression and the activity of MMP2.Section D The imbalance of ER and PR subtype and the intrinsic upregulation of the signaling of EGFR contribute to progestin resistanceObjective To investigate the relationship between progestin resistance and the imbalance of ER and PR subtype and the relationship between progestin resistance and the intrinsic upregulation of the signaling of TGFa-EGFR. Methods Immunocytochemistry, RT-PCR and western-blot were used to detect the expression of ERa, ERp, PR, PRB and EGFR in the two cell lines. RT-PCR was used to detect the expression of TGFa mRNA in the two cell lines and TGFa expression under the treatment of MPA over different time. Western-blot was used to detect the expression of EGFR-TK in the two cell lines. MTT and FACS were used to detect the effects of EGFR-TK inhibitor AG1478 on the proliferation of the two cell lines.Matrigel invasion assay was used to detect the effects of MPA on the invasive capability of the two cell lines. RT-PCR and western-blot were used to detect the expression of CyclinD1 over AG1478 treatment and RT-PCR was used to detect the expression of MMP2, MMP9 over AG1478 treatment in the two cell lines. Results (1) Compared with original Ishikawa cells, ERa, PRB expression were downregulated and the expression of ERP was upregulated in progestin-resistant cells, the differences were statistically significant (P<0.05). (2) The expression of TGFa and EGFR were higher in progestin-resistant cells than that in the originalIshikawa cells (P<0.05), and the expression of EGFR-TK was higher in progestin-resistant cells than that in the original Ishikawa cells. (3) MPA downregulated the expression of TGFa in original Ishikawa cells, but upregulated the expression of TGFa in progestin-resistant cells, the differences were both statistically significant (P<0.05). (4) MTT results showed that AG1478 inhibited progestin-resistant cells proliferation, the inhibitory effect was dose-dependent (F=18.148,P=0.00; compared with vehicle control, P<0.05), but the inhibitory effects on Ishikawa cells were significant only when AG1478 amounted to a higher level(10μM).The results of cell cycle analysis showed that AG1478 caused inhibition of cell cycle of progestin-resistant cells, the inhibitory rates were 19.4%, 11.2%, 10.7% respectively over 24,48, 72h treatment; but AG1478 produced little fall in the proliferation index on day 1 and 9% reduction on day 2 in the progestin-resistant cells. (5)AG1478 inhibited the invasiveness capability of original Ishikawa cells and the progestin-resistant cells, the inhibitory effects were 42.4% and 58.1% respectively, the inhibitory effect on progestin-resistance cells was higher than the inhibitory effect on original Ishikawa cells(P<0.05). (6) AG1478 downregulated the expression of CyclinD1, MMP2, MMP9 in the two cell lines, the inhibitory effects (55%,97.8%,85.2%) on progestin-resistance cells were higher than the inhibitory effects on Ishikawa cells(23.3%,66.4%,65.2%). Conclusion The downregulation of ERa and PRB, the upregulation of ERβ and the highly activated TGF-EGFR signaling might contribute to progestin resistance in endometrial carcinoma. The inhibitory chemicals AG1478 of EGFR-TK might benefit the conservative treatment of progestin-resistant endometrial carcinoma.Section The extrinsic TGFa and EGF might contribute to progestinresistance in endometrial carcinomaObjective To investigate the relationship between extrinsic growth factors and progestin resistance in endometrial carcinoma.Methods MTT was used to detect the effect of extrinsic growth factors (TGFa/EGF), MPA and TGFa/EGF combined with MPA on the proliferation of Ishikawa cells. Wound healing assays was used to detect the effects of TGFa/EGF, MPA and TGFa/EGF combined with MPA on the metastatic capability of Ishikawa cells. Western-blot was applied to detect the expression of CyclinD1, E-cadherin in Ishikawa cells under the treatment of TGFa/EGF, MPA and TGFa/EGF combined with MPA over different time. RT-PCR and Western - blot were used to investigate the expression of PRB under the treatment of TGFa/EGF and MPA over different concentration and different time. Western-blot was used to detect the expression of P-ERK under different condition that the cells were treated with or without MPA for 48 h and with or without growth factors for the indicated time. Results (1) TGFa/EGF and TGFa/EGF combined with MPA stimulated the proliferation and the motility of Ishikawa cells, contrary to TGFa/EGF, MPA inhibited the proliferation and the motility of Ishikawa cells, compared with control, the differences were statistically significant (P<0.05), the stimulatory effects of TGFa/EGF and TGFa/EGF combined with MPA were not significant(P>0.05). (2) TGFa/EGF and TGFa/EGF combined with MPA increased the expression of CyclinDl and decreased the expression of E-cadherin, MPA decreased the expression of CyclinDl and increased the expression of E-cadherin, these actions were significant when treatment for 24 h. (3) TGFa/EGF and MPA downregulated the expression of PRB, the effects were dose-dependent and these actions were significant when MPA treatment for 12h and TGFa/EGF treatment for 24 h, then PRB expression refreshed but could not attain to the primary level. (4) TGFa/EGF activated MAPK, the expression of P-ERK were higher under the condition that the cells were pretreated with MPA for 48h than the cells not pretreated with MPA with TGFa/EGF for the indicated time.Conclusion (1) TGFa/EGF stimulated Ishikawa cell proliferation, motility and antagonized the inhibitory effect of MPA which might be through upregulating the expression of CyclinDl and downregulating the expression of E-cadherin associated with proliferation and metastasis. (2) MPA inhibited Ishikawa cell proliferation,motility which might be through downregulating the expression of CyclinD1 and upregulating the expression of E-cadherin, but could not antagonized the stimulatory effect of TGFa/EGF on the proliferation and metastasis and the effect of TGFa/EGF on the expression of CyclinDl and E-cadherin. (3) TGFa/EGF downregulated the expression of PRB and attenuated the effect of MPA, and MPA potentiated the stimulatory action of TGFa/EGF on the tyrosine phosphorylation of MAPK. In a word, extrinsic TGFa/EGF accelerated progestin resistance in endometrial carcinoma,.In conclusion, the results of the experiment suggested that prolonged treatment with MPA on Ishikawa cell could give rise to the resistant effect to MPA, the downregulation of ERa, PRB, the upregulation of ERβ and the highly activated intrinsic/ extrinsic TGF-EGFR signaling might contribute to progestin resistance in endometrial carcinoma. When the resistance subtype was acquired, treatment with MPA could enhance the cancer cell proliferation, invasiveness and metastasis, the progestin-resistance cell line progressed from steroid-dependent to growth factors dependent. The inhibitory chemicals of EGFR-TK might benefit the conservative treatment of progestin-resistant endometrial carcinoma.更多还原