| Introduction and ObjectiveEndometriosis is a disease caused by the survival endometrium outside of the uterine cavity, which can invade any part of the whole body, predominantly in the pelvic cavity. The incidence rate of endometriosis is increasing in China with unknown mechanism but it is more in the productive women who marry and delivery late than those who are multiparity. Although it is a benign disease, endometriosis has the abilities of invasion and metastasis likely as a behavior of malignant tumors, which can cause abdominal pain, dysmenorrheal and infertility. The pathogenesis of endometriosis remains unclear, and there are still many problems, such as without specific early diagnosis, without curable treatment, with bad results and so on.The basic pathologic change of endometriosis is that the endometriotic lesions present periodic bleeding, leading to the proliferation of fibrosis and the formation of cysts. In the development of endometriosis, typical endometrial structures are destroyed and uneasy to be found. Accordingthe endometrium implanting theory, shed retrograde endometrium implants at the different times with the menstrual period, which makes the molecular study of endometriosis difficult and results in different conclusions. So it is a kind of method to establish an experimental animal model as to resolve this problem. Immune deficiency mice, which have definite and stable genetic background, no immune rejective reaction, repeatability and could keep morphology and biology characteristics of implants, have been successfully applied in the study of endometriosis in the foreign countries, but there is no report in our country.Endometriosis is a disease associated with genetics and immune. Specific target gene of endometriosis is the key way to resolve the problem of early diagnosis, while no research has discovered the specific target. Peritoneal adhesion of shed endometrial tissue, neoangiogenesis and invasive growth are essential steps in the pathogenesis of endometriosis. Associated factors, such as ICAMs, VEGF, MMPs, play an important role in the genesis and development of endometriosis, while they all lack of specificity as in the malignant tumors. EG-VEGF is an angiogenic mitogen selective for endocrine gland endothelium, which functional resembles and complements VEGF in its ability to induce formation of neoangiogenesis, but structurally dissimilar with VEGF. However, there is no report in the study of endometriosis. But its tissue-specific character has attracted many researches attentions.Aromatase is a rate-limiting enzyme to estrogen biosynthesis, which not only affects the circulating estrogen biosynthesis by endocrine mechanism but also affects local estrogen biosynthesis by autocrine and paracrine mechanism. Endometriosis is a estrogen-dependent disease, which may be also modulated by local estrogen proved by clinical observation, such as relapse after curative operation, development on the menopausal women. So the mechanism of aromatase on the endometriosis attracts extensive attentions.In the present study, we investigated the establishment of nude mice endometriosis model and its biological behavior study. Based on the key role of aromatase on the estrogen biosynthesis, the specific expression of aromatase was investigated on eutopic and ectopic endometrium, aimed to provide a new idea to the early diagnosis of endometriosis.MethodsEndometrium of the late secretory phase was obtained from 24 patients with endometriosis and 24 normal pre-menopausal woman undergoing endometrial biopsy or hysterectomy. Nude mice were implanted with the late secretory endometrium into pelvic and abdominal cavity with estrogen support, and implanted with greater omentum as control at the same time. Nude mice were randomly killed at 5d, 15d and 30d to observe the growth of endometriotic lesions. 30 cases ofendometriotic cyst wall (15 I ~ II phase, 15 III—IV phase) and 15 cases of normal ovarian tissues were obtained at the time of surgery. The histological changes were observed by light microscope and transmission electron microscope. The expressions of EG-VEGF at the nude mice endometriotic lesions, human eutopic endometrium, endometriotic cyst wall and normal ovarian tissues were detected by RT-PCR method and the relationship between them were analysed. The expressions of aromatase, VEGF and MMP-9 at the nude mice endometriotic lesions and human eutopic endometrium were detected by RT-PCR method. The expressive positions and degrees of VEGF and MMP-9 mRNA at the nude mice endometriotic lesions and human eutopic endometrium were detected by in-situ hybridization method. The expressions of steroid receptors and aromatase protein at the nude mice endometriotic lesions and human endometrium were detected by immunohistochemistry method.ResultsAll the transplants could be rediscovered in the pelvic and abdominal cavity of mice. Sites of implantation of the endometrial fragments were preferentially the peritoneum and the adipose fat surrounding bladder. On day 5, when the mice were killed, the endometriotic lesions fixedly adhered to the normal tissue of the mice with the formation of new bloodvessels. On day 15, the lesions became harder and tighter, and were fused with the tissues of mice. On day 30, the lesions atrophied and seemed smaller. The lesions showed more severe adhesions.Under the light microscope, glandular cells were cube or flattened. In late stage, fibrosis was more obvious and glandular cells were not in integrity. Degeneration, necrosis and infiltration of inflammatory cells could be seen in all lesions. Under the electron microscope, on day 5, the endometrial cells presented with shortened cilia and clear organell. On day 15, organell of glandular cell was not in its integrity with cell nuclear shrinking. On day 30, organell of glandular cell was disappeared, nuclear chromatin was aggregated but secretory granules still could be seen. There was no difference at morphology and histology of endometriotic lesions implanted by endometrium of patients with endometriosis or without endometriosis.There was no difference in the relative content of EG-VEGF, VEGF, MMP-9 and aromatase mRNA between the eutopic endometrium obtained from patients with endometriosis or without endometriosis (P>0.05), while the positive rate of aromatase in eutopic endometrium from patients with endometriosis was higher than that without endometriosis. Compared with eutopic endometrium, the relative contents of EG-VEGF, VEGF, MMP-9 and aromatase mRNA were increased in nude mice endometriotic lesions (P<0.05). EG-VEGF mRNA expressedhighest at 15d group (PO.05 vs 30d group); VEGF and MMP-9 mRNA in 15d group was higher than 5d and 30d groups (P<0.05); there was no difference of aromatase at 5d, 15d and 30d groups (P>0.05). The expressions of EG-VEGF/VEGF mRNA were increased in the endometriotic cysts of I ~ II phases than III-— IVphases (PO.05).The hybridization signals of EG-VEGF mRNA distributed at the plasma of glandular and stroma cells. The expressions of endometriotic lesions at 5d and 15d were mostly strong positive while at 30d were mostly weak positive. The positive rate was higher in 5d+15d group than 30d group and eutopic endometrium group (PO.05). For the endometriotic cysts, the positive rate of I -—' II phases, which mostly strong positive, were higher than III~ IVphases (PO.05), which mostly weak positive.The positive rate of steroid receptors in 30d group has the significant difference compared with 5d and 15d groups (p< 0.05). There was no difference between that of normal woman and the patient with endometriosis. The expressions of steroid receptors were both mostly weak. The positive rate of aromatase protein in the eutopic endometrium from patients with endometriosis (91.67%) were higher than that without endometriosis (70.83%) (PO.05). There was no difference of the positive rate of aromatase protein between the nude mice endometriotic lesions (91.67%) and eutopic endometrium obtained from the patients withendometriosis (P>0.05); there was significant difference of positive rate of aromatase protein between nude mice endometriotic lesions (95.83%) and eutopic endometrium obtained from patients without endometriosis (P<0.05).Conclusions1. The genesis and development of endometriotic lesions in nude mice EM model are similar with that of human EM, which provides an appropriate EM model for the clinical and basic studies of human EM.2. The expression of EG-VEGF, increasing in the early stage, is similar with VEGF in the ovarian and nude mice endometriotic lesions, which perhaps associated with angiogenesis of endometriotic lesions. EG-VEGF maybe play specific role in the genesis of endometriosis.3. The expression of aromatase is high in the nude mice endometriotic lesions, but has no relationship with degrees and stages. The expression of aromatase is higher in the eutopic endometrium of patients with endometriosis than without. Aromatase maybe play specific role in the local estrogen biosynthesis of endometriosis. |
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