Human Endometrial Stem Cells In Vitro Separation Cultivate And Identification

Infertility is one of the common gynecologic diseases, according to the domestic statistics,40-55 percent for women causes of infertility.Uterine adhesion of 9.2 percent of women causes. The occurrence of uterine adhesion main with uterine surgical trauma and uterine infection of uterine surgery is concerned, the forward main complications is uterine adhesion. There have been a number of abortions, especially caused by excessive GuaGong painles, or postoperative uterine infection, destroy the endometrial casuse uterine adhesion, cause can be formed secondary amenorrhoea and infertility. Surgical separation and female hysteroscopic treatment artificially cycle is the main treatment options. But the treatment effect is not quite satisfied, the development of the endometrium isn't a good improvementEndometrial is a highly tissue regeneration, in the whole women's growth period to undergo growth, differentiation and loss of 400 times cycle. Now research shows that endometrial contains endometrial stem cells. And its orientation in endometrial and myometrium border area. Endometrial stem cells as a kind of adult stem cells, has been confirmed with high proliferation potential regenerative powers and functions. Endometrial cells on endometrial cyclical hyperplasia and fall off to play a key role. Endometrial growth and endometrial stem cells have close. We separated from in vitro training endometrial stem cells, and then transplanted into the uterus to treat endometrial development for maladaptive disease, because of endometrial infertility caused provide a new treatment.This experiment mainly discussed in the womb from resection isolation, culture, and endometrial stem cells to commonly used three kinds of cell separation methods compare, in order to find out a simple, practical and efficient in vitro separation, cultivate people endometrial stem cell method for further studies on the reference data provide the basis.Purpose1. In vitro isolated and cultured endometrial stem cells from the resection of uterine.2. Using three different digestion methods for processing of samples from which to explore a simple, practical and efficient in vitro were isolated and cultured human endometrial stem cell method.Materials and methods1 MaterialsSelect Mar-Sep 2010 in zhengzhou university article affiliated hospital gynecological hospitalization of patients,the age of 31～49 years, uterine fibroids, uterine prolapse, in situ cervical cancer, etc. for hysterectomy patients. Asked 3 months before surgery did not use any hormones. Detailed record of patients information, such as age, reason for surgical removal of the uterus and so on.2 Methods2.1 Treatment of samplesAfter the womb cutting,sterile shave endometrial tissue including 5mm myometrium. The samples will be collected into penicillin and streptomycin,2 hours into the lab. With no Ca 2+Mg 2+buffer (PBS) to wash, until the liquid brighter. Into a sterile Petri dish, cut with scissors to 1mm3 size paste.2.2 Tissue digestion and cultureDigestion using three different methods:Method trypsin, collagenase I law and mixing (trypsin method, collagenase I method) for cell separation and extraction and separation of three different methods were compared.The isolated cell suspension with trypan blue staining of living cells. By 2×105/ ml cell density were seeded in 25 cm2 of the culture flask, DMEM/F12 medium containing 10% fetal bovine serum, penicillin and streptomycin,,37℃,5% CO2 incubator incubated 24～48 h (24 h after the medium was changed liquid). Adherent cells are not discarded, after medium was changed once every 2～3 days and observe the cell growth.Each method experimental repeats 10 times, take the second generation of growth to 5 days of cells with Taiwan dyeing,under fiberscopes, each cell count, at high magnification counting three times, taking the averages. Compare that to get cells digestion methods number.2.3 IdentificationUsing hematopoietic stem cell markers CD90 and interstitial stem cells CD 146 monoclonal antibodies, take immune cells chemical method and RT-PCR method of cultivation, the endometrial cells evaluations.2.4 Drawing cell growth curveTake logarithmic growth cells, vaccination in 24 orifice plate, the setting of incubator training; Once every 24 h count, and each plan three holes, take its average; Continuous count 7 days, the data to days, cell count for the abscissa denotes drawn for y-coordinate can get cell curve, the growth curve.2.5 Statistical analysisDatas are analyzed by SPSS16.0 software packet, data are shown as mean±SEM. It's comparison in groups deploy one-way mean square analysis (ANOVA), and comparison between each pair groups by least significant differerence analysis. Take Shapiro-Wilk Test and Levene's Test before variance analysis about group data.Use a=0.05 as inspection level. Results1. Endometrial stem cell morphology observationCultivate endometrial cells form a spindle, have fibroblasts form, cells, cells grow on the tile arranged no polarity.2. The identification of endometrial stem cellsCD90 and CD 146 by stem cell markers single anti-rejection cell chemistry dyeing as positive; After RT-PCR appraisal endometrial cells CD90 and CD 146 DNA in 150bp and 500bp respectively, and the control group not high expression expression. Proof the cultured cells have stem cell characteristics.3. Comparison cell growth cycle of three methods of separation of endometrial cellsBy train from single-celled levitation liquid, the second generation cells after by the cell count method income growth curve comparison, and there were no obvious difference (F=0.291, P>0.05).Are approximate"S"shape, have the same cell growth cycle.4. The three separation method comparisonThree methods of separation of living cells obtained by counting by compare collagenase I method acquired living cells (0.721±0.004) number is the more than trypsin method getting cell count (0.672±0.002) and hybrid method of cell counting acquired (0.671±0.001), the difference among the groups were statistically significant (F= 103.882, P<0.05).Conclusions1. From resection uterine successful get endometrial stem cells.2. By collagenase I separation endometrial cells is a practical, high efficiency, stable in vitro endometrial cells cultivation method.