COX-2抑制剂干预对体外培养的EMs间质细胞增殖的影响
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摘要
目的
(1)观察COX-2抑制剂对体外培养的内异症间质细胞增殖的影响。(2)探讨COX-2抑制剂对体外培养的内异症细胞干预的机制。(3)探讨COX-2抑制剂在治疗子宫内膜异位症中的应用价值。
方法
实验标本选取12例异位内膜标本取自山东省立医院妇产科2006年3月至2007年1月因卵巢型内异症行手术的患者,均经病理证实为EMs。正常对照组:25例取自同期因各种原因行诊断性刮宫或宫腔镜检查后经病理学诊断为正常的增生期或分泌期的子宫内膜标本。无菌收集实验组患者异位囊肿增厚的部分囊内壁及对照组内膜组织进行,一部分用10%多聚甲醛固定,用于组织学研究,一部分分离间质细胞培养,用于Nimesulide与Celecoxib对异位内膜细胞增殖影响的研究。步骤如下:1.取10%多聚甲醛中固定的异位内膜及正常内膜组织,脱水、包埋、切片,烤干。石蜡切片行HE染色,并行免疫组化检测COX-2水平。2.分离并培养内膜间质细胞,传代并进入对数生长期后分别行Celecoxib及Nimesulide干预,MTT法检测不同浓度及时间下抑制率,绘制细胞生长曲线。3.EIA法检测COX-2抑制剂干预后PGE_2水平。4.流氏细胞术检测COX-2抑制剂干预后凋亡率及细胞周期分布。
结果
1.免疫组化检测COX-2水平
异位内膜组COX-2水平显著高于对照组(P＜0.05)。
2.MTT检测
选择性COX-2抑制剂Celecoxib及Nimesulide均能抑制体外培养的异位内膜间质细胞的增殖,且呈时间浓度剂量依赖关系。
3.PGE_2水平检测
选择性COX-2抑制剂Celecoxib及Nimesulide能抑制体外培养的子宫内膜异位症间质细胞产生的PGE_2,且呈时间浓度剂量依赖。
4.凋亡检测
COX-2抑制剂干预后的细胞其凋亡率明显增加,与对照组相比有统计学意义,且呈时间浓度剂量依赖关系。
5.细胞周期检测
流氏细胞术检测结果显示,COX-2干预后的细胞,其位于G_0/G_1期比率明显增加,与对照组相比有统计学意义,且呈时间浓度剂量依赖。
结论
1.COX-2在内异症发生及发展中起重要作用
2.COX-2抑制剂可以明显抑制体外培养的内异症间质细胞,并呈时间浓度计量依赖性
3.COX-2抑制剂通过COX-2依赖及非COX-2依赖途径抑制间质细胞的增殖:
(1)COX-2抑制剂通过抑制COX-2的表达,减少PGE_2的产生,并可能进而下调芳香化酶、E_2及VEGF等的表达,发挥对异位内膜的抑制作用。
(2)COX-2抑制剂通过明显上调异位内膜间质细胞凋亡,并使细胞在G_0/G_1期停滞,发挥对内异症间质细胞增殖的抑制作用;
Objectives 1.To explore the influence of COX-2 inhibitors on the endometriotic stromal cells cultured in vitro;2.To explore the mechanism of inhibitory influence of COX-2 inhibitors on the endometriotic stromal cells;3.To evaluate the practical value of COX-2 inhibitor in treating endometriosis.
Methods Endometriotic tissue was picked from 12 patients of EMs from March 2006 to January 2007 who underwent surgery in our department,and all of them were confirmed endometriosis by histology examination.We used 25 normal dendometrium picked from patients who underwent diagnostic curettage and laparoscopy.All the endometrium were divided into two groups.one group was fixed by immersion in 10%buffered formalin for 6-24h,embedded in paraffin and sectioned at 6 mm,and mounted on silane-coated slides.Expression of COX-2 were examined immumohistochemically.Endometriotic stromal cells were isolated and cultured in the absence and in the presence of serum and with or without celecoxib or nimesulide.PGE_2 and apoptosis rates were detected by EIA and flowcytometry.
Main Results
1.Expression of COX-2 measurement by immunohistochemistry
The expression of COX-2 in EMs group was significantly higher than that of the control group(P＜0.05).
2.MTT detection
Both selective COX-2 inhibitors,celecoxib and nimesulide,could inhibit the proliferation of ectopic stromal cells cultured in vitro in a time and dose dependent mode.
3.Detection of PGE_2 level
Both selective COX-2 inhibitors,celecoxib and nimesulide,could decrease the level of PGE_2,which were produced by ectopic stromal cells cultured in vitro in a time and dose dependent mode。
4.Assessment of apoptosis by flow cytometry
The rate of apoptosis was significantly higher than that of control group after incubated with celecoxib or nimesulide in a time and dose dependent mode(P＜0.05)。
5.Analysis of cell cycle by flow cytometry
The effect of cox-2 inhibitors on the cell cycle of the ectopic stromal cells was determined by flowcytometry.Ectopic stromal cells cultured for 24 hours in the presence of celecoxib or nimesulide showed an accumulation of these cells in the G_0/G_1 phase of cell cycle.Similar results were obtained in all repeated experiments.
Conclusions
1.COX-2 involves in the development of endometriosis.
2.COX-2 inhibitors could sigficantly inhibit the proliferation of ectopic stromal cells cultured in vitro in a time and dose dependent mode(P＜0.05).
3.COX-2 inhibtors could inhibit the proliferation of ectopic stromal cells via the mechanisms dependent on COX-2 and independent on COX-2.
(1) COX-2 inhibitors down-regulated the level of PGE_2,exhibiting the inhibitory influence to ectopic endometrium..
(2) COX-2 inhibitors inhibited endometriotic stromal cells via inducing the apoptosis rate of ectopic stromal cells and G0/G1 arrest.
(1)观察COX-2抑制剂对体外培养的内异症间质细胞增殖的影响。(2)探讨COX-2抑制剂对体外培养的内异症细胞干预的机制。(3)探讨COX-2抑制剂在治疗子宫内膜异位症中的应用价值。
方法
实验标本选取12例异位内膜标本取自山东省立医院妇产科2006年3月至2007年1月因卵巢型内异症行手术的患者,均经病理证实为EMs。正常对照组:25例取自同期因各种原因行诊断性刮宫或宫腔镜检查后经病理学诊断为正常的增生期或分泌期的子宫内膜标本。无菌收集实验组患者异位囊肿增厚的部分囊内壁及对照组内膜组织进行,一部分用10%多聚甲醛固定,用于组织学研究,一部分分离间质细胞培养,用于Nimesulide与Celecoxib对异位内膜细胞增殖影响的研究。步骤如下:1.取10%多聚甲醛中固定的异位内膜及正常内膜组织,脱水、包埋、切片,烤干。石蜡切片行HE染色,并行免疫组化检测COX-2水平。2.分离并培养内膜间质细胞,传代并进入对数生长期后分别行Celecoxib及Nimesulide干预,MTT法检测不同浓度及时间下抑制率,绘制细胞生长曲线。3.EIA法检测COX-2抑制剂干预后PGE_2水平。4.流氏细胞术检测COX-2抑制剂干预后凋亡率及细胞周期分布。
结果
1.免疫组化检测COX-2水平
异位内膜组COX-2水平显著高于对照组(P＜0.05)。
2.MTT检测
选择性COX-2抑制剂Celecoxib及Nimesulide均能抑制体外培养的异位内膜间质细胞的增殖,且呈时间浓度剂量依赖关系。
3.PGE_2水平检测
选择性COX-2抑制剂Celecoxib及Nimesulide能抑制体外培养的子宫内膜异位症间质细胞产生的PGE_2,且呈时间浓度剂量依赖。
4.凋亡检测
COX-2抑制剂干预后的细胞其凋亡率明显增加,与对照组相比有统计学意义,且呈时间浓度剂量依赖关系。
5.细胞周期检测
流氏细胞术检测结果显示,COX-2干预后的细胞,其位于G_0/G_1期比率明显增加,与对照组相比有统计学意义,且呈时间浓度剂量依赖。
结论
1.COX-2在内异症发生及发展中起重要作用
2.COX-2抑制剂可以明显抑制体外培养的内异症间质细胞,并呈时间浓度计量依赖性
3.COX-2抑制剂通过COX-2依赖及非COX-2依赖途径抑制间质细胞的增殖:
(1)COX-2抑制剂通过抑制COX-2的表达,减少PGE_2的产生,并可能进而下调芳香化酶、E_2及VEGF等的表达,发挥对异位内膜的抑制作用。
(2)COX-2抑制剂通过明显上调异位内膜间质细胞凋亡,并使细胞在G_0/G_1期停滞,发挥对内异症间质细胞增殖的抑制作用;
Objectives 1.To explore the influence of COX-2 inhibitors on the endometriotic stromal cells cultured in vitro;2.To explore the mechanism of inhibitory influence of COX-2 inhibitors on the endometriotic stromal cells;3.To evaluate the practical value of COX-2 inhibitor in treating endometriosis.
Methods Endometriotic tissue was picked from 12 patients of EMs from March 2006 to January 2007 who underwent surgery in our department,and all of them were confirmed endometriosis by histology examination.We used 25 normal dendometrium picked from patients who underwent diagnostic curettage and laparoscopy.All the endometrium were divided into two groups.one group was fixed by immersion in 10%buffered formalin for 6-24h,embedded in paraffin and sectioned at 6 mm,and mounted on silane-coated slides.Expression of COX-2 were examined immumohistochemically.Endometriotic stromal cells were isolated and cultured in the absence and in the presence of serum and with or without celecoxib or nimesulide.PGE_2 and apoptosis rates were detected by EIA and flowcytometry.
Main Results
1.Expression of COX-2 measurement by immunohistochemistry
The expression of COX-2 in EMs group was significantly higher than that of the control group(P＜0.05).
2.MTT detection
Both selective COX-2 inhibitors,celecoxib and nimesulide,could inhibit the proliferation of ectopic stromal cells cultured in vitro in a time and dose dependent mode.
3.Detection of PGE_2 level
Both selective COX-2 inhibitors,celecoxib and nimesulide,could decrease the level of PGE_2,which were produced by ectopic stromal cells cultured in vitro in a time and dose dependent mode。
4.Assessment of apoptosis by flow cytometry
The rate of apoptosis was significantly higher than that of control group after incubated with celecoxib or nimesulide in a time and dose dependent mode(P＜0.05)。
5.Analysis of cell cycle by flow cytometry
The effect of cox-2 inhibitors on the cell cycle of the ectopic stromal cells was determined by flowcytometry.Ectopic stromal cells cultured for 24 hours in the presence of celecoxib or nimesulide showed an accumulation of these cells in the G_0/G_1 phase of cell cycle.Similar results were obtained in all repeated experiments.
Conclusions
1.COX-2 involves in the development of endometriosis.
2.COX-2 inhibitors could sigficantly inhibit the proliferation of ectopic stromal cells cultured in vitro in a time and dose dependent mode(P＜0.05).
3.COX-2 inhibtors could inhibit the proliferation of ectopic stromal cells via the mechanisms dependent on COX-2 and independent on COX-2.
(1) COX-2 inhibitors down-regulated the level of PGE_2,exhibiting the inhibitory influence to ectopic endometrium..
(2) COX-2 inhibitors inhibited endometriotic stromal cells via inducing the apoptosis rate of ectopic stromal cells and G0/G1 arrest.
引文
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1 Attar E,Bulun SE.Aromatase and other steroidogenic genes in endometriosis:translational aspects[J].Hum Reprod Update,2006,12(1):49-56.
2.MacDonald PC,Rombaut RP,Siiteri PK.Plasma precursors of estrogen,Ⅰ:Extent of conversion of plasma -androstenedione to estrone in normal males and non-pregnant normal,castrate and adrenalectomized females[J].J Clin Endocrinol Metab 1967;27:1103-1111.
3.MacDonald PC,Edman CD,Hemsell DL,et al.Effect of obesity on conversion of plasma androstenedione to estrone in postmenopausal women with and without endometrial cancer[J].Am J Obstet Gynecol 1978;130:448-455.
4 Bulun SE,Price TM,Mahendroo MS,et al.A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription[J].J Clin Endocrinol Metab 1993;77:1622-1628.
5 Yue W,Wang JP,Hamilton CJ,et al.In situ aromatization enhances breast tumor estradiol levels and cellular proliferation[J].Cancer Res 1998;58:927-932.
6.Bulun SE,Simpson ER,Word RA.Expression of the CYP19 Gene and its product aromatase cytochrome P450 in human leiomyoma tissues and cells in culture[J].J Clin EndocrinolMetab 1994;78:736-743.
7.Noble LS,Takayama K,Zeitoun KM,et al.Prostaglandin E2 stimulates aromatase expression in endometriosis-derivedstromal cells[J].J Clin Endocrinol Metab 1997;82:600-606.
8 Brueggemeier RW,Quinn AL,Parrett ML et al.Correlation of aromatase and cyclooxygenase gene expression in human breast cancer specimens[J].Cancer Lett 1999;140:27-35.
9 Michael MD,Michael LF,Simpson ER.A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450(CYP19) gene[J].Mol Cell Endocrinol 1997;134:147-156
10 陈必良,樊杨,蔡国青,等.COX-2基因在子宫内膜异位症组织的表达及作用机制探讨[J].实用妇产科杂志,2005;21(3):154-156.
11 Rostom A,Dube C,Lewin G et al.Nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors for primary prevention of colorectal cancer:a systematic review prepared for the U.S.Preventive Services Task Force[J]Ann Intern Med.2007 Mar 6;146(5):376-389.
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1 Attar E,Bulun SE.Aromatase and other steroidogenic genes in endometriosis:translational aspects[J].Hum Reprod Update,2006,12(1):49-56.
2.MacDonald PC,Rombaut RP,Siiteri PK.Plasma precursors of estrogen,Ⅰ:Extent of conversion of plasma -androstenedione to estrone in normal males and non-pregnant normal,castrate and adrenalectomized females[J].J Clin Endocrinol Metab 1967;27:1103-1111.
3.MacDonald PC,Edman CD,Hemsell DL,et al.Effect of obesity on conversion of plasma androstenedione to estrone in postmenopausal women with and without endometrial cancer[J].Am J Obstet Gynecol 1978;130:448-455.
4 Bulun SE,Price TM,Mahendroo MS,et al.A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription[J].J Clin Endocrinol Metab 1993;77:1622-1628.
5 Yue W,Wang JP,Hamilton CJ,et al.In situ aromatization enhances breast tumor estradiol levels and cellular proliferation[J].Cancer Res 1998;58:927-932.
6.Bulun SE,Simpson ER,Word RA.Expression of the CYP19 Gene and its product aromatase cytochrome P450 in human leiomyoma tissues and cells in culture[J].J Clin EndocrinolMetab 1994;78:736-743.
7.Noble LS,Takayama K,Zeitoun KM,et al.Prostaglandin E2 stimulates aromatase expression in endometriosis-derivedstromal cells[J].J Clin Endocrinol Metab 1997;82:600-606.
8 Brueggemeier RW,Quinn AL,Parrett ML et al.Correlation of aromatase and cyclooxygenase gene expression in human breast cancer specimens[J].Cancer Lett 1999;140:27-35.
9 Michael MD,Michael LF,Simpson ER.A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450(CYP19) gene[J].Mol Cell Endocrinol 1997;134:147-156
10 陈必良,樊杨,蔡国青,等.COX-2基因在子宫内膜异位症组织的表达及作用机制探讨[J].实用妇产科杂志,2005;21(3):154-156.
11 Rostom A,Dube C,Lewin G et al.Nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors for primary prevention of colorectal cancer:a systematic review prepared for the U.S.Preventive Services Task Force[J]Ann Intern Med.2007 Mar 6;146(5):376-389.
12 Chishima F,Hayakaw S,sugita K,et al.Increased expression of cyclooxygenase -2 in local lesions of endometriosis patients[J].Am J Rep Immunology,2002,48:50-56.
13 Koks CA,Groothuis PG,Dunselman GA,et al.Adhesion of shed menstrual tissue in an in vitro model using amnion and peritoneum:a light and electron microscopic study[J].Hum Reprod.1999.14,816-822.
14 Harada M,Suganuma N,Furuhashi M,et al.Detection of apoptosis in human endometriotic tissues.Mol Hum Reprod.1996.2:307-315.
15 Nishida M,Nasu K,Ueda T,et al.Endometriotic cells are resistant to interferon-(?)-induced cell growth inhibition and apoptosis:a possible mechanism involved in the pathogenesis of endometriosis[J].Mol Hum Reprod 11:29-34.
16 Sheng H,Shao J,Morrow JD et al.Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells[J].Cancer Res 1998;58:362-366.
17 Laschke MW,Elitzsch A,Scheuer C et al.Selective cyclo-oxygenase-2 inhibition induces regression of autologous endometrial grafts by down-regulation of vascular endothelial growth factor-mediated angiogenesis and stimulation of caspase-3-dependent apoptosis.[J].Fertil Steril.2007 Jan;87(1):163-171.
18 张新艳,刘彦.子宫内膜异位症的血管生成和抗血管生成治疗[J].生殖与避孕.2007;27(2):133-136.
19 Donnez J,Smoes D,Gillerot S,et al.Vascular endothelial growth factor(VEGF)in endometriosis[J].Hum Reprod,1998,13(6) 1686-1690.
20 Hull ML,Charnock-Jones DS,Chan CL,et al.Antiangiogenic agents are effective inhibitors of endometriosis[J].J Clin Endocrinol Metab,2003,88:2889-2899.
21 Masferrer JL,Koki A,Seibert K.COX-2 inhibitors:A new class of antiangiogenic agents[J].Ann NY Acad Sci,1999,889:54-86.