| In recent years, with people of uterine cavity operation, medicine flow operation and increasing, lead to endometrial basal parts all or part of the defect, thinning, cause uterine wall adhesion, before and after uterine closure of uterine cavity volume decreases, and lead to patients caused by menstrual changes and reproductive abnormalities.Caused by intrauterine adhesions, therefore, has become one of common disease of department of gynaecology, and become an important cause of infertility, miscarriage, premature birth.Caused by intrauterine adhesions cure rate is low, after the separation surgery adhesions prone, seriously affecting the majority of women’s bodies and mental health.Caused by intrauterine adhesions separation of how to prevent the recurrence of adhesion, promote the growth of endometrium after treatment is a big problem in clinical work.Hysteroscopy(TCRA) is caused by intrauterine adhesions caused by intrauterine adhesions resection under the standard operation for treatment,Postoperative placed intrauterine device, drug therapy, artificial cycle caused by intrauterine placed balloon, the application of xanthan gum and antiblocking membrane method for preventing the adhesion have certaincurative effect.However, in patients with severe caused by intrauterine adhesions, intrauterine wound is larger, less normal endometrium tissues, are more likely to form another adhesion, the above method for such patients curative effect is poor.The study found that amniotic membrane transplantation to treat caused by intrauterine adhesions, can reduce the incidence of again caused by intrauterine adhesions and promote endometrial regeneration.Amniotic membrane has low antigenicity, promote epithelial, reduce inflammation, fibrous tissue proliferation inhibition,Amniotic membrane transplantation in ocular surface reconstruction and has applications in skin grafts, and have achieved satisfactory results.As a special kind of material of low immunogenicity has special structure and secretion function of fresh amniotic membrane contains a large number of bioactive substances, can promote cell proliferation differentiation of epithelial cells, reduce scar formation and prevention of adhesion, etc。Amniotic membrane has been widely development in the field of interdisciplinary surgery.In recent years, the amniotic membrane was gradually applied in ophthalmology and burns, etc。In the department of gynaecology, amniotic membrane can be used as a biological dressing applied in vaginal reconstruction, cervical form,Also has the experiment confirmed that the separation of fresh amniotic membrane transplantation into the IUA postoperative uterine cavity, can better reduce the incidence of uterine cavity adhesion again, have a good application prospect.Objective:This topic through the cultivation of endometrial cells on surface of the fresh amniotic membrane matrix, observe the cells grow in the amniotic membrane on the surface of the substrate, and USES the Real time PCR and ELISA method compared with amniotic membrane as the carrier of endometrial cells and endometrial cells in vitro culture of hepatocyte growth factor(HGF), matrix metalloproteinases(MMP- 9) and vascular endothelial factor(VEGF) expression level, preliminary exploration with human amniotic membrane as the carrier of human endometrial cells feasibility, to explore the transplantation of endometrial reconstruction to provide effective and rich source of material, and to discuss molecular mechanism of amniotic membrane transplantation to prevent caused by intrauterine adhesions accumulate experimental data.METHODS:1. By adopting the method of collagenase digestion and sieve filtration separation to cultivate human endometrial stromal cells in vitro, by morphological observation and immunohistochemical method appraisal for culture of cells;By adopting the method of trypsin digestion and mechanical shave their preparation to get rid of the amniotic membrane matrix scaffold amniotic epithelial cells. 2.To grow in vitro amplification represented the endometrial cells in removing amniotic epithelial cells on amniotic membrane matrix scaffolds as experimental group, without amniotic membrane carrier develop endometrial cells as blank control group.Observed under inverted microscope in the two groups in the form of cell growth, cell groups in terms of real-time fluorescent quantitative PCR method HGF, MMP- 9, the expression of VEGF m RNA level, ELISA method for determination of MMP- 9 groups of cells the quantity of protein expression. 3.The data from experiment to mean ±standard deviation said, m RNA and protein expression of relative quantity between the different groups of comparative adopt single factor analysis of variance, two more accord with normal distribution of quantitative data by using two independent sample t test, inspection level of alpha =0.05.RESULTS:1. The endometrial cells morphological characteristics and immunohistochemical results:Cultivate the stromal cells are polygonal, there are multiple small bumps on both ends of the cell, the cell arrangement of polarity, stick a wall.As the cell subculture, cells gradually elongated spindle, with fibroblasts. Cell growth cycle is commonly 7 to 10 days, 1 day for latent phase, 2-4 days into the logarithmic phase, 5 or 6 days later reached a plateau, shrinking fade in 7 days. Stromal cells specific antibody of Vimentin(Vimentin) staining positive, positive cell cytoplasm a tan, dyeing keratin antibody negative. 2. P2 generation endometrial cells inoculated in amniotic membrane carrier after 2 h, cell adhesion amniotic membrane matrix, a small balls, scattered distribution, refraction sex is good, but not yet attached. A small stick wall after 12 h, cells, gradually gathered in groups, into growth, and the proliferation of the petri dish endometrial cells form similar, but cells arranged owe rules, close connection between cells and cells, and cells elongated, traffic net-like to each other. L weeks or so can be observed clearly into the growth of cells. Amniotic membrane carrier in about 10 days started to shrivel and cell apoptosis. 3. The training of the amniotic membrane substrate surface endometrial cells, HGF m RNA content was 2.033 ± 0.922, MMP- 9 m RNA content was 1.356 ± 0.399, VEGF m RNA content was 0.976 ± 0.597; Control group m RNA HGF content was 1.299 ± 1.003, MMP- 9 content was 0.871 ± 0.529, VEGF content was 0.426 ± 0.193; Three groups of experimental factor m RNA expression are higher than the control group, the differences are obvious statistical significance(P < 0.05). 4. ELISA to detect cells that MMP- 9 protein expression supernatant fluid volume, amniotic membrane carrier group was(0.762±0.085)ng/ml, the control group was(0.089±0.008)ng/ml, amniotic membrane carrier group than control group in MMP-9 high quantity of protein expression, significant difference was statistically significant(P < 0.05)CONCLUSION:1. The endometrial cells on amniotic extracellular matrix scaffold can grow well, with amniotic membrane as the carrier to develop endometrial cells has certain feasibility. 2. The amniotic membrane carrier HGF can enhance endometrial cells, the expression of VEGF, MMP- 9. 3.Amniotic membrane transplantation to prevent TCRA caused by intrauterine adhesions provide new idea and method... |
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