CAPN5在子宫内膜异位症中的表达意义及丹参酮ⅡA对其的干预作用
摘要
研究背景
子宫内膜异位症(endometriosis, Ems,简称内异症),是现代妇女的常见病,指有生长能力的子宫内膜组织即腺体与间质在宫腔外种植生长,引起临床症状的一种疾病。育龄期妇女中有10-15%的发病率,80%的患者伴有盆腔慢性疼痛,50%合并不孕,30-50%伴有痛经,严重影响了患者生活质量,给患者从心理到身体带来了极大的痛苦。子宫内膜异位症近年来发病呈上升趋势,其病变部位广泛,病理形态多样性,目前为止发病机制并不清楚。
有研究指出,子宫内膜异位症的发生是由于在位内膜基因的异常表达,导致细胞异常生物学行为所致,内膜细胞凋亡的异常调控,也可能是内异症发病的原因之一。细胞凋亡是指生理状态下,细胞的程序性死亡。细胞凋亡的抑制会导致细胞增殖旺盛,活力加强。CAPN5作为半胱氨基酸蛋白酶家族的一员,是一种钙依赖蛋白酶,可以参加多种细胞活动,包括细胞的分化和凋亡。HOXA10是CAPN5的上游基因,可以通过与辅助因子结合启动对下游靶基因的转录,调控CAPN5的表达水平。二者在子宫内膜腺体细胞与基质细胞均有表达,可持续整个月经周期,对内膜细胞凋亡有调控作用。内异症虽然是良性疾病,却具有侵袭、远处转移等恶性生物学行为,目前的治疗对策有限,且复发率高。Tan Ⅱ A是丹参的一种主要提取成分,有抗肿瘤作用,目前认为是通过杀伤肿瘤细胞,阻碍细胞分化与增殖、诱导细胞凋亡等途径实现的。
建立良好的模型,对细胞生长形态的观察、凋亡指标的检测、探讨TanⅡA的干预机制有重要意义,是进一步揭示内异症发病机制及研究中药对内异症治疗策略的基础。
研究目的
1.建立子宫内膜腺体细胞、基质细胞体外共培养的内异症模型。
2.应用该模型观察对比内异症患者(内异症组)与非内异症患者(对照组)在位内膜细胞的生长情况、形态学特征、免疫鉴定。
3.观察内异症组内膜细胞中CAPN5、HOXA10的表达水平与对照组内膜细胞有何差异,二者是否有一致性,探讨其与内异症发生的关系。
4.检测Tan Ⅱ A干预内异症组内膜细胞后,CAPN5、HOXA10表达水平的变化,及二者是否有一致性。
5.观察Tan Ⅱ A是否可以抑制内异症组内膜细胞生长,对细胞凋亡率有何影响,探讨中药对子宫内膜异位症治疗的机制与意义
方法
1.取20例子宫内膜异位症患者在位子宫内膜(内异症组)和13例非子宫内膜异位症患者子宫内膜(对照组)组织,消化分离,原代混合培养子宫内膜腺上皮细胞与基质细胞。
2.HE染色观察内异症组与对照组细胞形态学特征
3.SP免疫细胞化学鉴定法鉴定细胞。
4.RT-PCR法检测CAPN5mRNA、HOXA10mRNA在两组细胞中的表达差异；
5.Western blot法检测CAPN5蛋白在两组中的表达差异。
6.应用AnnexinV-PE/7-AAD凋亡试剂盒、流式细胞仪检测两组细胞凋亡率的差异。
7.以不同浓度Tan Ⅱ A作用内异症组细胞,用MTT法测算细胞抑制率。
8.RT-PCR法检测Tan Ⅱ A干预后内异症组细胞CAPN5mRNA. HOXA10mRNA表达水平的变化情况。
9.Western blot法检测Tan Ⅱ A干预后内异症组细胞CAPN5蛋白表达水平的变化。
10.应用Annexin V-PE/7-AAD凋亡试剂盒、流式细胞仪检测TanⅡA干预后内异症组细胞凋亡率的变化。
结果
1.内异症组成功16例,对照组成功10例。内膜腺上皮细胞与基质细胞共培养,细胞混合个自贴壁,生长良好,免疫细胞化学染色鉴定CK19、vimentin阳性。
2.内异症组与对照组腺上皮细胞及基质细胞的形态学特征相似,但内异症组细胞生长铺满速度快。
3. RT-PCR结果显示,16例内异症患者在位内膜细胞HOXA10mRNA的相对表达水平(0.486±0.070)明显低于10例对照组内膜细胞HOXAlOmRNA的相对表达水平(1.091±0.105),两组比较有统计学意义(P<0.05)。
4. RT-PCR及Western blot(?)结果显示,内异症组在位内膜细胞CAPN5mRNA (0.237±0.051)和蛋白(0.221±0.049)的相对表达水平低于对照组内膜细胞CAPN5mRNA.454±0.083)和蛋白(0.512±0.120)的相对表达水平,统计学比较有意义(P<0.05)。
5.流式细胞仪测定细胞凋亡率结果显示：内异症组细胞凋亡率为6.869±2.050%,对照组细胞凋亡率为14.327±3.265%,前者明显低于后者,有统计学意义(P<0.05)。
6. Tan Ⅱ A可以抑制内异症患者在位内膜细胞的生长,并随着Tan Ⅱ A浓度的升高,作用时间的延长,抑制率增加,呈时间-剂量依赖性。
7. RT-PCR检测显示,Tan Ⅱ A能够促进内异症患者在位内膜细胞HOXA10mRNA的表达水平,并随Tan Ⅱ A浓度的增加分别为未加药组、1.0mg/L、2.0mg/L、4.0mg/L,HOXA10mRNA相对表达值也相应上升分别为0.486±0.070、0.658±0.071、0.956±0.131、1.130±0.109,呈剂量依赖性组间比较有统计学意义(P<0.05)。
8. RT-PCR及Western blot结果显示,TanⅡA能够促进内异症患者在位内膜细胞CAPN5及蛋白的表达水平,并随TanⅡA浓度的增加分别为未加药组、1.0mg/L、2.0mg/L、4.0mg/L,CAPN5mRNA和蛋白的相对表达水平也一致性上升分别为0.237±0.051、0.314±0.063、0.471±0.058、0.521±0.052；0.221±0.049、0.331±0.057、0.492±0.067、0.540±0.057,呈剂量依赖性,组间比较有统计学意义(P<0.05)。
9.流式细胞仪测定未加药组、1.0mg/L、2.0mg/L、4.0mg/L浓度TanⅡA作用后,细胞凋亡率结果显示,细胞凋亡率上升分别为6.869±2.050%、9.572±3.353%、21.602±6.501%、33.110±7.494%,组间比较有统计学意义(P<0.05)。
结论
1.成功建立了稳定的,子宫内膜腺上皮细胞与基质细胞的体外共培养模型。能够在细胞水平对内异症组与对照组在位内膜细胞的生长情况及形态特征进行实时观察记录。
2.内异症组在位内膜细胞中CAPN5mRNA、HOXAlOmRNA及CAPN5蛋白的表达水平均低于对照组内膜细胞。CAPN5与HOXA10有一致性。
3.与对照组比较内异症组细胞的凋亡率低,提示CAPN5的异常低水平表达可能与内异症的发病机制相关。
4. Tan Ⅱ A可以上调子宫内膜异位症患者在位内膜细胞CAPN5mRNA、 HOXA10mRNA及CAPN5蛋白的表达水平,呈剂量依赖性。CAPN5与HOXA10表达的变化有一致性
5. Tan Ⅱ A可以抑制子宫内膜异位症患者在位内膜细胞的生长,提高其凋亡率,提示Tan Ⅱ A可以诱导其细胞的凋亡,为中药治疗子宫内膜异位症提供新的靶点及依据。
Background
Endometriosis is a common disease of fertile women, defined by the presence of ectopic endometrial glands and stroma, affecting approximately10-15%of fertile women,80%with pelvic pain,30-50%with algomenorrhea and50%with infertility. The incidence of endometriosis is raising recently, the disease performance diversity,so far the pathogenesis of it is unclear.
Accumulated evidences suggest that abnormal expression of some genes in eutopic endometrium with endometriosis, such as the abnormal expression of apoptosis, is believed to contribute to the establishment and maintenance of the disease. Apoptosis is a type of cell death in which the cell uses specialized cellular machinery to kill itself. Cell suicide mechanism that enables metazoans to control cell number and eliminates cells that threaten the animal's survival. An increase in cell proliferation without an equivalent change in apoptosis has been described.Calpain5is a member of the cytoplasmic cysteine protease family, a group of Ca++regulated enzymes. CAPNs in general, and in particular CAPN5, have been implicated in multiple cellular processes including cellular differentiation and apoptosis. CAPN5is expressed in human endometrium and regulated by HOXA10. The function of HOXA10in the uterus is well defined, HOXA10is a transcription factor that regulates a battery of target genes. The molecular mechanism by which HOXA10directs endometrial receptivity and decidualization includes the regulation of CAPN5in human endometrial cells. Both CAPN5and HOXA10are expressed in endometrial glandular epithelial cells and stromal cells throughout the menstrual cycle. This in turn likely regulates apoptosis of the endometrium. Endometriosis is prone to progression and invasion, exhibiting some characteristics of malignant tumor behavior, such as the invasion, distant metastasis, recurrence etc, so far the treatment measures for it is limited. The main components extracted from Salvia miltiorrhiza, a common herb, is Tan Ⅱ A. The antitumor function of Tan Ⅱ A dues to decrease in cell proliferation and regulate apoptosis.
In order to research the pathogenesis of endometriosis and discuss the treatment with herb, a good model must be established, so that we can observe the differentiation and morphography of endometrial cells, to determine expression of CAPN5and HOXA10, and to discuss the effect of Tan Ⅱ A.
Objective
1.To establish endometriosis endometrial glandular epithelial cells and stromal cells in vitro co-culture model.
2.Using the model, to observe and compare the growth and morphography of endometrial cells of endometriosis and controls.
3.To determine and compare expression of CAPN5and HOXA10in each group.
4.To determine and compare expression of CAPN5and HOXA10in endometriosis group after the effect of Tan Ⅱ A.
5.To observe the growth and apoptosis of endometrial cells of endometriosis after the effect of Tan Ⅱ A.
Methods
1.Eutopic endometrium were obtained from20women with endometriosis as a group,13eutopic endometrium were obtained from women without endometriosis as controls. Isolated and culture primary glandular epithelial cells and stromal cells.
2.To observe and compare the growth and morphogram of endometrial cells of endometriosis and controls with HE staining.
3.Identification of endometrial cell by immunocytochemistry.
4.RT-PCR was performed to determine expression of CAPN5and HOXA10 in each group.
5.Western blot was performed to determine expression of CAPN5in each group.
6.Annexin V-PE/7-AAD kit was performed to determine the apoptosis of each group.
7.Estimate the apoptosis of endometrial cells of endometriosis with MTT cell proliferation assay after the effect of Tan II A.
8.RT-PCR was performed to determine expression of Calpain5and HOXA10in endometrial cells of endometriosis after the effect of Tan II A.
9.Western blot was performed to determine expression of CAPN5in endometrial cells of endometriosis after the effect of Tan II A.
10.Annexin V-PE/7-AAD kit was performed to determine the apoptosis of endometrial cells after the effect of Tan II A.
Results
1.Both endometrial glandular epithelial cells and stromal cells grew well. the immunocytochemisty with antibodies against CK19and vimentin were positive.
2.The endometrial cells obtained from controls share a similar morphogram with the eutopic endometrial cells of women with endometriosis, but grew slower.
3.The result was confirmed using RT-PCR showed a decrease in HOXA10mRNA in endometriosis compared with endometrium obtained from controls (P<0.05).
4.The results were confirmed using RT-PCR and western blot, that both also showed a similar decrease in CAPN5mRNA and protein, respectively, in endometriosis compared with endometrium obtained from controls (P<0.05).
5.The result of apoptosis assay using Annexin V-PE/7-AAD kit showed a lower rate of eutopic endometrium in endometriosis compared with endometrium obtained from controls (P<0.05).
6.MTT confirmed Tan Ⅱ A up-regulated the restriction of the eutopic endometrium cells of women with endometriosis in a time-dose manner(P<0.05).
7.RT-PCR confirmed that, in the eutopic endometrium of women with endometriosis,there were significant increases in HOXA10mRNA expression after the effect of Tan Ⅱ A compared with non-dose controls (P<0.05).
8.The results were confirmed using the RT-PCR, and western blot, that both showed a similar increases in CAPN5mRNA and protein after the effect of Tan Ⅱ A, respectively, in the eutopic endometrium of women with endometriosis compared with non-dose controls (P<0.05).
9.The result of apoptosis assay using Annexin V-PE/7-AAD kit confirmed that Tan Ⅱ A up-regulated the apoptosis of the eutopic endometrium cells of women with endometriosis compared with non-dose controls(P<0.05).
Conclusion
1.Successfully established endometriosis in vitro cell co-culture model. Use this model we can get real time observation of the growth and morphography of endometrial cells from endometriosis and controls.
2.In the eutopic endometrium of women with endometriosis, there were significant decreases in CAPN5mRNA and protein expression, as well as HOXA10mRNA, compared with fertile controls.
3.Decreased apoptosis in the eutopic endometrium of women with endometriosis compared with endometrium obtained from controls implicate that, aberrant expression of CAPN5is associated with endometriosis.
4.TanⅡA could higher the expression of CAPN5mRNA and protein, as well as HOXA10mRNA in a dose-manner.
5.TanⅡA is associated with restricted proliferation and increased cell death of the eutopic endometrium of women with endometriosis, which can provide a theory of treating endometriosis with Chinese herb.
子宫内膜异位症(endometriosis, Ems,简称内异症),是现代妇女的常见病,指有生长能力的子宫内膜组织即腺体与间质在宫腔外种植生长,引起临床症状的一种疾病。育龄期妇女中有10-15%的发病率,80%的患者伴有盆腔慢性疼痛,50%合并不孕,30-50%伴有痛经,严重影响了患者生活质量,给患者从心理到身体带来了极大的痛苦。子宫内膜异位症近年来发病呈上升趋势,其病变部位广泛,病理形态多样性,目前为止发病机制并不清楚。
有研究指出,子宫内膜异位症的发生是由于在位内膜基因的异常表达,导致细胞异常生物学行为所致,内膜细胞凋亡的异常调控,也可能是内异症发病的原因之一。细胞凋亡是指生理状态下,细胞的程序性死亡。细胞凋亡的抑制会导致细胞增殖旺盛,活力加强。CAPN5作为半胱氨基酸蛋白酶家族的一员,是一种钙依赖蛋白酶,可以参加多种细胞活动,包括细胞的分化和凋亡。HOXA10是CAPN5的上游基因,可以通过与辅助因子结合启动对下游靶基因的转录,调控CAPN5的表达水平。二者在子宫内膜腺体细胞与基质细胞均有表达,可持续整个月经周期,对内膜细胞凋亡有调控作用。内异症虽然是良性疾病,却具有侵袭、远处转移等恶性生物学行为,目前的治疗对策有限,且复发率高。Tan Ⅱ A是丹参的一种主要提取成分,有抗肿瘤作用,目前认为是通过杀伤肿瘤细胞,阻碍细胞分化与增殖、诱导细胞凋亡等途径实现的。
建立良好的模型,对细胞生长形态的观察、凋亡指标的检测、探讨TanⅡA的干预机制有重要意义,是进一步揭示内异症发病机制及研究中药对内异症治疗策略的基础。
研究目的
1.建立子宫内膜腺体细胞、基质细胞体外共培养的内异症模型。
2.应用该模型观察对比内异症患者(内异症组)与非内异症患者(对照组)在位内膜细胞的生长情况、形态学特征、免疫鉴定。
3.观察内异症组内膜细胞中CAPN5、HOXA10的表达水平与对照组内膜细胞有何差异,二者是否有一致性,探讨其与内异症发生的关系。
4.检测Tan Ⅱ A干预内异症组内膜细胞后,CAPN5、HOXA10表达水平的变化,及二者是否有一致性。
5.观察Tan Ⅱ A是否可以抑制内异症组内膜细胞生长,对细胞凋亡率有何影响,探讨中药对子宫内膜异位症治疗的机制与意义
方法
1.取20例子宫内膜异位症患者在位子宫内膜(内异症组)和13例非子宫内膜异位症患者子宫内膜(对照组)组织,消化分离,原代混合培养子宫内膜腺上皮细胞与基质细胞。
2.HE染色观察内异症组与对照组细胞形态学特征
3.SP免疫细胞化学鉴定法鉴定细胞。
4.RT-PCR法检测CAPN5mRNA、HOXA10mRNA在两组细胞中的表达差异；
5.Western blot法检测CAPN5蛋白在两组中的表达差异。
6.应用AnnexinV-PE/7-AAD凋亡试剂盒、流式细胞仪检测两组细胞凋亡率的差异。
7.以不同浓度Tan Ⅱ A作用内异症组细胞,用MTT法测算细胞抑制率。
8.RT-PCR法检测Tan Ⅱ A干预后内异症组细胞CAPN5mRNA. HOXA10mRNA表达水平的变化情况。
9.Western blot法检测Tan Ⅱ A干预后内异症组细胞CAPN5蛋白表达水平的变化。
10.应用Annexin V-PE/7-AAD凋亡试剂盒、流式细胞仪检测TanⅡA干预后内异症组细胞凋亡率的变化。
结果
1.内异症组成功16例,对照组成功10例。内膜腺上皮细胞与基质细胞共培养,细胞混合个自贴壁,生长良好,免疫细胞化学染色鉴定CK19、vimentin阳性。
2.内异症组与对照组腺上皮细胞及基质细胞的形态学特征相似,但内异症组细胞生长铺满速度快。
3. RT-PCR结果显示,16例内异症患者在位内膜细胞HOXA10mRNA的相对表达水平(0.486±0.070)明显低于10例对照组内膜细胞HOXAlOmRNA的相对表达水平(1.091±0.105),两组比较有统计学意义(P<0.05)。
4. RT-PCR及Western blot(?)结果显示,内异症组在位内膜细胞CAPN5mRNA (0.237±0.051)和蛋白(0.221±0.049)的相对表达水平低于对照组内膜细胞CAPN5mRNA.454±0.083)和蛋白(0.512±0.120)的相对表达水平,统计学比较有意义(P<0.05)。
5.流式细胞仪测定细胞凋亡率结果显示：内异症组细胞凋亡率为6.869±2.050%,对照组细胞凋亡率为14.327±3.265%,前者明显低于后者,有统计学意义(P<0.05)。
6. Tan Ⅱ A可以抑制内异症患者在位内膜细胞的生长,并随着Tan Ⅱ A浓度的升高,作用时间的延长,抑制率增加,呈时间-剂量依赖性。
7. RT-PCR检测显示,Tan Ⅱ A能够促进内异症患者在位内膜细胞HOXA10mRNA的表达水平,并随Tan Ⅱ A浓度的增加分别为未加药组、1.0mg/L、2.0mg/L、4.0mg/L,HOXA10mRNA相对表达值也相应上升分别为0.486±0.070、0.658±0.071、0.956±0.131、1.130±0.109,呈剂量依赖性组间比较有统计学意义(P<0.05)。
8. RT-PCR及Western blot结果显示,TanⅡA能够促进内异症患者在位内膜细胞CAPN5及蛋白的表达水平,并随TanⅡA浓度的增加分别为未加药组、1.0mg/L、2.0mg/L、4.0mg/L,CAPN5mRNA和蛋白的相对表达水平也一致性上升分别为0.237±0.051、0.314±0.063、0.471±0.058、0.521±0.052；0.221±0.049、0.331±0.057、0.492±0.067、0.540±0.057,呈剂量依赖性,组间比较有统计学意义(P<0.05)。
9.流式细胞仪测定未加药组、1.0mg/L、2.0mg/L、4.0mg/L浓度TanⅡA作用后,细胞凋亡率结果显示,细胞凋亡率上升分别为6.869±2.050%、9.572±3.353%、21.602±6.501%、33.110±7.494%,组间比较有统计学意义(P<0.05)。
结论
1.成功建立了稳定的,子宫内膜腺上皮细胞与基质细胞的体外共培养模型。能够在细胞水平对内异症组与对照组在位内膜细胞的生长情况及形态特征进行实时观察记录。
2.内异症组在位内膜细胞中CAPN5mRNA、HOXAlOmRNA及CAPN5蛋白的表达水平均低于对照组内膜细胞。CAPN5与HOXA10有一致性。
3.与对照组比较内异症组细胞的凋亡率低,提示CAPN5的异常低水平表达可能与内异症的发病机制相关。
4. Tan Ⅱ A可以上调子宫内膜异位症患者在位内膜细胞CAPN5mRNA、 HOXA10mRNA及CAPN5蛋白的表达水平,呈剂量依赖性。CAPN5与HOXA10表达的变化有一致性
5. Tan Ⅱ A可以抑制子宫内膜异位症患者在位内膜细胞的生长,提高其凋亡率,提示Tan Ⅱ A可以诱导其细胞的凋亡,为中药治疗子宫内膜异位症提供新的靶点及依据。
Background
Endometriosis is a common disease of fertile women, defined by the presence of ectopic endometrial glands and stroma, affecting approximately10-15%of fertile women,80%with pelvic pain,30-50%with algomenorrhea and50%with infertility. The incidence of endometriosis is raising recently, the disease performance diversity,so far the pathogenesis of it is unclear.
Accumulated evidences suggest that abnormal expression of some genes in eutopic endometrium with endometriosis, such as the abnormal expression of apoptosis, is believed to contribute to the establishment and maintenance of the disease. Apoptosis is a type of cell death in which the cell uses specialized cellular machinery to kill itself. Cell suicide mechanism that enables metazoans to control cell number and eliminates cells that threaten the animal's survival. An increase in cell proliferation without an equivalent change in apoptosis has been described.Calpain5is a member of the cytoplasmic cysteine protease family, a group of Ca++regulated enzymes. CAPNs in general, and in particular CAPN5, have been implicated in multiple cellular processes including cellular differentiation and apoptosis. CAPN5is expressed in human endometrium and regulated by HOXA10. The function of HOXA10in the uterus is well defined, HOXA10is a transcription factor that regulates a battery of target genes. The molecular mechanism by which HOXA10directs endometrial receptivity and decidualization includes the regulation of CAPN5in human endometrial cells. Both CAPN5and HOXA10are expressed in endometrial glandular epithelial cells and stromal cells throughout the menstrual cycle. This in turn likely regulates apoptosis of the endometrium. Endometriosis is prone to progression and invasion, exhibiting some characteristics of malignant tumor behavior, such as the invasion, distant metastasis, recurrence etc, so far the treatment measures for it is limited. The main components extracted from Salvia miltiorrhiza, a common herb, is Tan Ⅱ A. The antitumor function of Tan Ⅱ A dues to decrease in cell proliferation and regulate apoptosis.
In order to research the pathogenesis of endometriosis and discuss the treatment with herb, a good model must be established, so that we can observe the differentiation and morphography of endometrial cells, to determine expression of CAPN5and HOXA10, and to discuss the effect of Tan Ⅱ A.
Objective
1.To establish endometriosis endometrial glandular epithelial cells and stromal cells in vitro co-culture model.
2.Using the model, to observe and compare the growth and morphography of endometrial cells of endometriosis and controls.
3.To determine and compare expression of CAPN5and HOXA10in each group.
4.To determine and compare expression of CAPN5and HOXA10in endometriosis group after the effect of Tan Ⅱ A.
5.To observe the growth and apoptosis of endometrial cells of endometriosis after the effect of Tan Ⅱ A.
Methods
1.Eutopic endometrium were obtained from20women with endometriosis as a group,13eutopic endometrium were obtained from women without endometriosis as controls. Isolated and culture primary glandular epithelial cells and stromal cells.
2.To observe and compare the growth and morphogram of endometrial cells of endometriosis and controls with HE staining.
3.Identification of endometrial cell by immunocytochemistry.
4.RT-PCR was performed to determine expression of CAPN5and HOXA10 in each group.
5.Western blot was performed to determine expression of CAPN5in each group.
6.Annexin V-PE/7-AAD kit was performed to determine the apoptosis of each group.
7.Estimate the apoptosis of endometrial cells of endometriosis with MTT cell proliferation assay after the effect of Tan II A.
8.RT-PCR was performed to determine expression of Calpain5and HOXA10in endometrial cells of endometriosis after the effect of Tan II A.
9.Western blot was performed to determine expression of CAPN5in endometrial cells of endometriosis after the effect of Tan II A.
10.Annexin V-PE/7-AAD kit was performed to determine the apoptosis of endometrial cells after the effect of Tan II A.
Results
1.Both endometrial glandular epithelial cells and stromal cells grew well. the immunocytochemisty with antibodies against CK19and vimentin were positive.
2.The endometrial cells obtained from controls share a similar morphogram with the eutopic endometrial cells of women with endometriosis, but grew slower.
3.The result was confirmed using RT-PCR showed a decrease in HOXA10mRNA in endometriosis compared with endometrium obtained from controls (P<0.05).
4.The results were confirmed using RT-PCR and western blot, that both also showed a similar decrease in CAPN5mRNA and protein, respectively, in endometriosis compared with endometrium obtained from controls (P<0.05).
5.The result of apoptosis assay using Annexin V-PE/7-AAD kit showed a lower rate of eutopic endometrium in endometriosis compared with endometrium obtained from controls (P<0.05).
6.MTT confirmed Tan Ⅱ A up-regulated the restriction of the eutopic endometrium cells of women with endometriosis in a time-dose manner(P<0.05).
7.RT-PCR confirmed that, in the eutopic endometrium of women with endometriosis,there were significant increases in HOXA10mRNA expression after the effect of Tan Ⅱ A compared with non-dose controls (P<0.05).
8.The results were confirmed using the RT-PCR, and western blot, that both showed a similar increases in CAPN5mRNA and protein after the effect of Tan Ⅱ A, respectively, in the eutopic endometrium of women with endometriosis compared with non-dose controls (P<0.05).
9.The result of apoptosis assay using Annexin V-PE/7-AAD kit confirmed that Tan Ⅱ A up-regulated the apoptosis of the eutopic endometrium cells of women with endometriosis compared with non-dose controls(P<0.05).
Conclusion
1.Successfully established endometriosis in vitro cell co-culture model. Use this model we can get real time observation of the growth and morphography of endometrial cells from endometriosis and controls.
2.In the eutopic endometrium of women with endometriosis, there were significant decreases in CAPN5mRNA and protein expression, as well as HOXA10mRNA, compared with fertile controls.
3.Decreased apoptosis in the eutopic endometrium of women with endometriosis compared with endometrium obtained from controls implicate that, aberrant expression of CAPN5is associated with endometriosis.
4.TanⅡA could higher the expression of CAPN5mRNA and protein, as well as HOXA10mRNA in a dose-manner.
5.TanⅡA is associated with restricted proliferation and increased cell death of the eutopic endometrium of women with endometriosis, which can provide a theory of treating endometriosis with Chinese herb.
引文
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