内异症患者血浆及腹腔液中MIF含量的检测及意义
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摘要
目的
子宫内膜异位症(endometriosis，EM)，是生育年龄妇女的常见病，发病率高达10％，但其发病机制不清。目前多数学者认为，血管生成在EM的发病中是一个关键现象。腹腔镜手术已证实，在典型的EM病灶内或病灶周围有大量的新生毛细血管包绕。
巨噬细胞移动抑制因子(macrophage migration inhibitory factor，MIF)最早被描述为能够抑制巨噬细胞游走的T淋巴细胞的一种产物。现在已知它有很多功能，包括炎症和免疫反应、细胞增殖、血管生成及肿瘤形成。最新研究提示，MIF可能在与EM相关的血管生成中起作用。在EM患者的在位内膜、异位内膜和腹腔液(peritoneal fluid，PF)中均可检测到MIF。
MIF在EM患者血浆中的检测，目前在国内外未见报道。本研究用酶联免疫吸附法(enzyme linked immune sorbent assay，ELISA)第一次同时测定了EM患者血浆和PF中MIF的水平，并分析MIF水平与疾病的分期和月经周期之间的关系，探讨MIF在EM发病过程中的作用。
方法
1研究对象：
EM患者29例，分别取血浆标本和PF标本29例，EM分期根据美国生育学会提出的修正子宫内膜异位症分期法确定，其中早期(Ⅰ-Ⅱ期)病例12例，进展期(Ⅲ-Ⅳ期)病例17例；根据病人月经周期史进行月经周期分期(增殖期或分泌期)。对照组为行输卵管吻合术或卵巢冠囊肿或卵巢浆液性囊腺瘤患者10例，分别取血浆标本和PF标本10例，且经腹腔镜检查或开腹检查证实无EM及炎症。
2标本的收集和处理：
术前采空腹静脉血2ml，置入抗凝管；在腹腔镜或开腹手术时抽吸PF2ml。收集到的血浆和PF立即于4℃下以3000r／min离心10分钟，取上清夜，分装，在-80℃冰箱中待测。
3检测方法:
按照人MIF定量EUSA试剂盒所定程序检测血浆及PF中MIF水平。
4统计分析:
两样本均数的比较采用非配对.t检验，相关分析采用Pearson检验。
结果
1实验组与对照组患者血浆中MIF水平检测结果
实验组患者血浆中MIF水平与对照组比较，有显著差异(P<0.01)。
实验组早期患者血浆中MIF水平与对照组比较，有显著差异(P<0.01)。
实验组进展期患者血浆中MIF水平与对照组比较，有显著差异(P<0.05)。
实验组早期病例与进展期病例患者血浆中MIF水平比较，无显著差异(P>
0 .05)。
2实验组与对照组患者PF中MIF水平检测结果
实验组患者PF中MIF水平与对照组比较，有显著差异(P<0.01)。
实验组早期患者PF中MIF水平与对照组比较有显著差异(P<0.01)，实
验组进展期患者PF中MIF水平与对照组比较，有显著差异(P<0.01)。
实验组早期病例与进展期病例患者PF中MIF水平比较，有显著差异(P<
0 .01)。
3实验组患者血浆中MIF水平与PF中MIF水平的比较及关系
实验组患者PF中MIF水平与血浆中MIF水平比较，有显著差异(P<
0.01)，且二者呈正相关(r二0.823，P<0.01)。
4实验组患者血浆与PF中MIF水平及月经周期的关系
实验组患者血浆MIF水平在增殖期与分泌期比较，有显著差异(P<0.
01)。实验组患者PF中MIF水平在增殖期与分泌期比较，有显著差异(P<
0 .01)。
结论
1本研究第一次证实了MIF在EM患者血浆中明显升高。EM患者血
浆和PF中MIF水平在早期、进展期均较对照组明显升高，提示MIF水平在
EM疾病过程中持续增高，说明MIF参与疾病发生的整个过程。
2本研究发现EM患者PF中MIF水平在疾病早期比进展期明显增高，
提示MIF主要参与EM的早期发生。已有研究证实早期EM病变血管化
程度较显著，因此我们推测MIF在疾病早期可能通过血管生成作用参与发
病。
3 EM患者PF中MIF水平明显高于血浆中MIF水平，且二者呈正相
关，提示MIF主要在盆腹腔局部环境起作用，再扩大炎性反应，然后进人血
循环，使其血浆水平升高。
4 EM患者血浆及PF中MIF水平在增殖期比分泌期升高明显，原因可
能是在月经周期的增殖期腹腔巨噬细胞浓度较高及子宫内膜有高度的血管
生成，提示MIF主要在增殖期直接或间接地通过血管生成参与EM的发病。
Objective
Endometriosis ( EM) is a common gynecological disease affecting nearly 10% of reproductive - aged women. The cause of EM remains obscure. Recently, most investigators believe that angiogenesis is a crucial phenomenon in the development of endometriosis. Neovascularization is observed within and sur-rouding active endometrotic lesions by laparoscopy.
Macrophage migration inhibitory factor( MIF) was originally described as a product of activated T - lymphocytes that inhibited the random migration of cultured macrophages. It is now well known for a variety of functions, including inflammatory and immune responses, cell proliferation, angiogensis, and tumor progression. Recent findings suggest that MIF may play an important role in endometriosis - associated angiogenesis . Increased concentration of MIF was found in eutopic endometrium , ectopic endometrium and PF of women with EM .
To date,there was no reports regarding the presence of MIF in plasma of women with EM . The present study was undertaken to evaluate the levels of MIF in plasma and PF of women with EM by enzyme linked immune sorbent assay (ELISA) , to examine the correlation between MIF levels and the stage of the disease and the phase of menstrual cycle, and to determin the role of MIF in promoting the disease.
Methods
1. Subjects.
Plasma and PF samples were obtained from 29 women with EM in this stud-
y. The stage of EM was determined according to the revised classification of The American Fertility Society (12 with stage I and II ,17 with stage III and IV). Moreover, the menstrual cycle phase(proliferative or secretory) was determined according to the patients'cycle history. Control subjects (n = 10) were women undergoing surgery for tubal reanastomosis or patients with parovarian cyst or ovarian serous cystadenoma, who have no visible evidence of endometriosis and inflammation at laparoscopy or laparolomy. Plasma specimens (n = 10) and peritoneal fluid (n = 10) were collected from them, respectively.
2. Collection and processing of samples
Venous blood was drawn form women with and without EM before undergoing surgery , then put into tube containing anticoagulin; Peritoneal fluids were obtained at the time of laproscopy or laparotomy before any surgical intervention. Immediately after collection, the samples were centrifuged at 3000 rpm for 10 min at 4C ,and the supernatants were aliquoted and sored at -86C until analyzed
3. Measured procedure.
The concentration of MIF in plasma and PF were measured by the Sandwich ELISA, following the established procedure of test kit.
4. Stastical analysis
Unpaired t - test was used for the comparision of means , and Pearson ?test for correlation analysis.
Results
1. Concentration of MIF in plasma
MIF Concentrations of plasma were markedly higher in EM patients as compared with controls ( P < 0. 01). The significant increase was seen either in EM stages I - n ( P < 0. 01) or M - IV ( P < 0. 05 ). No significantly differences were shown between the case in stages I - II and stages III-IV(p>0.05).
2. Concentration of MIF in peritoneal fluid
The mean Concentration of MIF in PF was significantly higher in EM patients as compared with controls ( P <0. 01). The most significant increase was
seen in EM stages I - II. In stages III - IV, MIF concen trations slightly decreased as compared with the case in stages I -II(P<0. 01), but remained significant higer than those in the control group ( P <0. 01).
3. The comparison and correlation between MIF level of plasma and that of PF
Significant differences ( P < 0. 01) and posiitive correlation ( r = 0. 823, P <0. 01) were shown between MIF level in plasma and PF of women with EM.
4. Correlation between MIF levels and the phase of the menstrual cycle. The increased concentration of MIF in PF ( P < 0. 01) and plasma ( P <
0. 01 ) of women with EM was markedly more significant in the proliferative phase than in the secretory.
Conclusion
1. In the present sudy, we first find that concentration of MIF in plasm
子宫内膜异位症(endometriosis，EM)，是生育年龄妇女的常见病，发病率高达10％，但其发病机制不清。目前多数学者认为，血管生成在EM的发病中是一个关键现象。腹腔镜手术已证实，在典型的EM病灶内或病灶周围有大量的新生毛细血管包绕。
巨噬细胞移动抑制因子(macrophage migration inhibitory factor，MIF)最早被描述为能够抑制巨噬细胞游走的T淋巴细胞的一种产物。现在已知它有很多功能，包括炎症和免疫反应、细胞增殖、血管生成及肿瘤形成。最新研究提示，MIF可能在与EM相关的血管生成中起作用。在EM患者的在位内膜、异位内膜和腹腔液(peritoneal fluid，PF)中均可检测到MIF。
MIF在EM患者血浆中的检测，目前在国内外未见报道。本研究用酶联免疫吸附法(enzyme linked immune sorbent assay，ELISA)第一次同时测定了EM患者血浆和PF中MIF的水平，并分析MIF水平与疾病的分期和月经周期之间的关系，探讨MIF在EM发病过程中的作用。
方法
1研究对象：
EM患者29例，分别取血浆标本和PF标本29例，EM分期根据美国生育学会提出的修正子宫内膜异位症分期法确定，其中早期(Ⅰ-Ⅱ期)病例12例，进展期(Ⅲ-Ⅳ期)病例17例；根据病人月经周期史进行月经周期分期(增殖期或分泌期)。对照组为行输卵管吻合术或卵巢冠囊肿或卵巢浆液性囊腺瘤患者10例，分别取血浆标本和PF标本10例，且经腹腔镜检查或开腹检查证实无EM及炎症。
2标本的收集和处理：
术前采空腹静脉血2ml，置入抗凝管；在腹腔镜或开腹手术时抽吸PF2ml。收集到的血浆和PF立即于4℃下以3000r／min离心10分钟，取上清夜，分装，在-80℃冰箱中待测。
3检测方法:
按照人MIF定量EUSA试剂盒所定程序检测血浆及PF中MIF水平。
4统计分析:
两样本均数的比较采用非配对.t检验，相关分析采用Pearson检验。
结果
1实验组与对照组患者血浆中MIF水平检测结果
实验组患者血浆中MIF水平与对照组比较，有显著差异(P<0.01)。
实验组早期患者血浆中MIF水平与对照组比较，有显著差异(P<0.01)。
实验组进展期患者血浆中MIF水平与对照组比较，有显著差异(P<0.05)。
实验组早期病例与进展期病例患者血浆中MIF水平比较，无显著差异(P>
0 .05)。
2实验组与对照组患者PF中MIF水平检测结果
实验组患者PF中MIF水平与对照组比较，有显著差异(P<0.01)。
实验组早期患者PF中MIF水平与对照组比较有显著差异(P<0.01)，实
验组进展期患者PF中MIF水平与对照组比较，有显著差异(P<0.01)。
实验组早期病例与进展期病例患者PF中MIF水平比较，有显著差异(P<
0 .01)。
3实验组患者血浆中MIF水平与PF中MIF水平的比较及关系
实验组患者PF中MIF水平与血浆中MIF水平比较，有显著差异(P<
0.01)，且二者呈正相关(r二0.823，P<0.01)。
4实验组患者血浆与PF中MIF水平及月经周期的关系
实验组患者血浆MIF水平在增殖期与分泌期比较，有显著差异(P<0.
01)。实验组患者PF中MIF水平在增殖期与分泌期比较，有显著差异(P<
0 .01)。
结论
1本研究第一次证实了MIF在EM患者血浆中明显升高。EM患者血
浆和PF中MIF水平在早期、进展期均较对照组明显升高，提示MIF水平在
EM疾病过程中持续增高，说明MIF参与疾病发生的整个过程。
2本研究发现EM患者PF中MIF水平在疾病早期比进展期明显增高，
提示MIF主要参与EM的早期发生。已有研究证实早期EM病变血管化
程度较显著，因此我们推测MIF在疾病早期可能通过血管生成作用参与发
病。
3 EM患者PF中MIF水平明显高于血浆中MIF水平，且二者呈正相
关，提示MIF主要在盆腹腔局部环境起作用，再扩大炎性反应，然后进人血
循环，使其血浆水平升高。
4 EM患者血浆及PF中MIF水平在增殖期比分泌期升高明显，原因可
能是在月经周期的增殖期腹腔巨噬细胞浓度较高及子宫内膜有高度的血管
生成，提示MIF主要在增殖期直接或间接地通过血管生成参与EM的发病。
Objective
Endometriosis ( EM) is a common gynecological disease affecting nearly 10% of reproductive - aged women. The cause of EM remains obscure. Recently, most investigators believe that angiogenesis is a crucial phenomenon in the development of endometriosis. Neovascularization is observed within and sur-rouding active endometrotic lesions by laparoscopy.
Macrophage migration inhibitory factor( MIF) was originally described as a product of activated T - lymphocytes that inhibited the random migration of cultured macrophages. It is now well known for a variety of functions, including inflammatory and immune responses, cell proliferation, angiogensis, and tumor progression. Recent findings suggest that MIF may play an important role in endometriosis - associated angiogenesis . Increased concentration of MIF was found in eutopic endometrium , ectopic endometrium and PF of women with EM .
To date,there was no reports regarding the presence of MIF in plasma of women with EM . The present study was undertaken to evaluate the levels of MIF in plasma and PF of women with EM by enzyme linked immune sorbent assay (ELISA) , to examine the correlation between MIF levels and the stage of the disease and the phase of menstrual cycle, and to determin the role of MIF in promoting the disease.
Methods
1. Subjects.
Plasma and PF samples were obtained from 29 women with EM in this stud-
y. The stage of EM was determined according to the revised classification of The American Fertility Society (12 with stage I and II ,17 with stage III and IV). Moreover, the menstrual cycle phase(proliferative or secretory) was determined according to the patients'cycle history. Control subjects (n = 10) were women undergoing surgery for tubal reanastomosis or patients with parovarian cyst or ovarian serous cystadenoma, who have no visible evidence of endometriosis and inflammation at laparoscopy or laparolomy. Plasma specimens (n = 10) and peritoneal fluid (n = 10) were collected from them, respectively.
2. Collection and processing of samples
Venous blood was drawn form women with and without EM before undergoing surgery , then put into tube containing anticoagulin; Peritoneal fluids were obtained at the time of laproscopy or laparotomy before any surgical intervention. Immediately after collection, the samples were centrifuged at 3000 rpm for 10 min at 4C ,and the supernatants were aliquoted and sored at -86C until analyzed
3. Measured procedure.
The concentration of MIF in plasma and PF were measured by the Sandwich ELISA, following the established procedure of test kit.
4. Stastical analysis
Unpaired t - test was used for the comparision of means , and Pearson ?test for correlation analysis.
Results
1. Concentration of MIF in plasma
MIF Concentrations of plasma were markedly higher in EM patients as compared with controls ( P < 0. 01). The significant increase was seen either in EM stages I - n ( P < 0. 01) or M - IV ( P < 0. 05 ). No significantly differences were shown between the case in stages I - II and stages III-IV(p>0.05).
2. Concentration of MIF in peritoneal fluid
The mean Concentration of MIF in PF was significantly higher in EM patients as compared with controls ( P <0. 01). The most significant increase was
seen in EM stages I - II. In stages III - IV, MIF concen trations slightly decreased as compared with the case in stages I -II(P<0. 01), but remained significant higer than those in the control group ( P <0. 01).
3. The comparison and correlation between MIF level of plasma and that of PF
Significant differences ( P < 0. 01) and posiitive correlation ( r = 0. 823, P <0. 01) were shown between MIF level in plasma and PF of women with EM.
4. Correlation between MIF levels and the phase of the menstrual cycle. The increased concentration of MIF in PF ( P < 0. 01) and plasma ( P <
0. 01 ) of women with EM was markedly more significant in the proliferative phase than in the secretory.
Conclusion
1. In the present sudy, we first find that concentration of MIF in plasm
引文
1. Strathy JH, Mollgaard CA, Coulam CB, et al. Endometriosis and infertility : a laparoscopic study of endometriosis among fertile and infertile women. Fertil steril 1982,38:667-672.
2. Sampson J. Peritoneal endometriosis due to menstrual dissemination of medometrial tissue into the peritoneal cavity. AM J obstet Gynecol 1927,14: 442 - 69.
3. Halme J, Hammorid MG, Hulka JF, et al. Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 1984, 64 : 151 - 154.
4. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995,1:27-31.
5. Eisermann J, Gast MJ, Pineda J, Odem RR, et al. Tumor necrosis factor in Peritoneal fluid of women undergoing laparosoopic surgery. Fertil steril 1988,50:573 - 579
6. Ryan IP,T seng Jf, Schriock ED, et al. Interleukin-8 concentrations are elevated in peritoneal fluid of women with endomeriosis. Fertil Steril 1995, 63:929 - 32.
7. Mclaren J. Vascular endothelial growth factor and endometriotic angiogenesis. Hum Reprod Update, 2000,6:45 - 55
8. Hull ML, Charnock-Jones DS, Chan CL, et al. Antiangiogennic agents are effective inhibitors of endometriosis. J Clin Endocrinol Metab 2003, 88 (6) : 2889 - 2899.
9．黄任和，许四宏．巨噬细胞移动抑制因子的研究进展．微生物学免疫进展99(4)：78-80
10. Kats R, Metz CN,Akoum A. MIF is markedly expressed in active and early-stage endometriotie lesions. J Clin Endoerinol Metab 2002; 87 (2) : 883-889.
11. Yang Y, Degranpre P, Rharfi A, et al. Identification of MIF as a potent endomethelial cell growth promoting agent released by ectopic human endometrial cells. J Clin Endocrinol Metab 2000,85(12):4721-4727:
12. American Fertility Society. Revised American Fertility Society classification of endonetriosis. Fertil Steril 1985,43:351-352.
13. Arcuri F, Ricci C, Letta F, et al. MIF in the human endometrium: expression and localization during the menstrual cycle and early pregnancy. Biol Reprod 2001,64 : 1200 - 1205.
14. Kats R, Collette T, Metz CN. Marked elevation of MIF in the peritoneal fluid of women with endometerosis. Fertil Steril 2002,78 (1) :69 -76.
15. Akoum A, Kong J, Metz C, et al. The macrophage is an important and previously unrecognized source of MCP-1 and MIF by peritoneal macrophages in women women with and without endometriosis. Fertil Steril 2002, 77 (5): 989 - 994.
16. Nisolle M, Casnas-Roux F, Anaf V, et al. Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil steril 1993,59:681-684.
17. Halme J, Becket S, Hammond MG, et al. Increased activation of pelvic macropha-es in infertile women with mild endometriosis. Am J Obstet Gynecol 1983,145:333-337
18. Haney AF, Jenkins S, Weinberg JB. The stimulus responsible for the peritoneal Fluid inflammation observed in infertile women with endometriosis. Fertil steril 1991,56:408 -413.
19. Fujimoto J, Sakahguchi H, Hirose R, et al. Angiogenesis in endometriosis and angiogenic factors. Gynecol Obstet Invest 1999,48 suppl 1:14-20.
20. Lebovic DI, Mueller MO, Taylor RN. Immunobilogy of endometriosis. Fertil steril 2001,75 : 1 - 10.
21. Maclaren J, Prentice A, Charnock-Jones DS, et al. vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian sterioids. J Clin Invest 1996,98:482-489.
22. Halme J, Becker S, Hammond MG, et al. Pelvic macrophaes in normal and infertile women: the role of patent tubes. Am J Obstet Gynecol 1982,142: 890 - 895.
23. Nap AN, Griffioen AW, Dunselman GA, et al. Antiangiogenesis therapy for endometriosis. J Clin Endocrinol Metab 2004,89 (3) : 1089 - 1095.
2. Sampson J. Peritoneal endometriosis due to menstrual dissemination of medometrial tissue into the peritoneal cavity. AM J obstet Gynecol 1927,14: 442 - 69.
3. Halme J, Hammorid MG, Hulka JF, et al. Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 1984, 64 : 151 - 154.
4. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995,1:27-31.
5. Eisermann J, Gast MJ, Pineda J, Odem RR, et al. Tumor necrosis factor in Peritoneal fluid of women undergoing laparosoopic surgery. Fertil steril 1988,50:573 - 579
6. Ryan IP,T seng Jf, Schriock ED, et al. Interleukin-8 concentrations are elevated in peritoneal fluid of women with endomeriosis. Fertil Steril 1995, 63:929 - 32.
7. Mclaren J. Vascular endothelial growth factor and endometriotic angiogenesis. Hum Reprod Update, 2000,6:45 - 55
8. Hull ML, Charnock-Jones DS, Chan CL, et al. Antiangiogennic agents are effective inhibitors of endometriosis. J Clin Endocrinol Metab 2003, 88 (6) : 2889 - 2899.
9．黄任和，许四宏．巨噬细胞移动抑制因子的研究进展．微生物学免疫进展99(4)：78-80
10. Kats R, Metz CN,Akoum A. MIF is markedly expressed in active and early-stage endometriotie lesions. J Clin Endoerinol Metab 2002; 87 (2) : 883-889.
11. Yang Y, Degranpre P, Rharfi A, et al. Identification of MIF as a potent endomethelial cell growth promoting agent released by ectopic human endometrial cells. J Clin Endocrinol Metab 2000,85(12):4721-4727:
12. American Fertility Society. Revised American Fertility Society classification of endonetriosis. Fertil Steril 1985,43:351-352.
13. Arcuri F, Ricci C, Letta F, et al. MIF in the human endometrium: expression and localization during the menstrual cycle and early pregnancy. Biol Reprod 2001,64 : 1200 - 1205.
14. Kats R, Collette T, Metz CN. Marked elevation of MIF in the peritoneal fluid of women with endometerosis. Fertil Steril 2002,78 (1) :69 -76.
15. Akoum A, Kong J, Metz C, et al. The macrophage is an important and previously unrecognized source of MCP-1 and MIF by peritoneal macrophages in women women with and without endometriosis. Fertil Steril 2002, 77 (5): 989 - 994.
16. Nisolle M, Casnas-Roux F, Anaf V, et al. Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil steril 1993,59:681-684.
17. Halme J, Becket S, Hammond MG, et al. Increased activation of pelvic macropha-es in infertile women with mild endometriosis. Am J Obstet Gynecol 1983,145:333-337
18. Haney AF, Jenkins S, Weinberg JB. The stimulus responsible for the peritoneal Fluid inflammation observed in infertile women with endometriosis. Fertil steril 1991,56:408 -413.
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