發(fā)布時間：2018-05-13 05:33

  本文選題：子宮內(nèi)膜干細(xì)胞 + 分離��； 參考：《鄭州大學(xué)》2011年碩士論文

【摘要】：不孕癥是婦科常見病之一,其中,女性因素占40-55%,在引起女性不孕的眾多原因中9.2%為宮腔粘連導(dǎo)致。宮腔粘連的發(fā)生主要與宮腔手術(shù)創(chuàng)傷和宮腔感染有關(guān),是宮腔手術(shù)后的遠(yuǎn)期主要并發(fā)癥。患者大多有過多次人工流產(chǎn)史,尤其是人工流產(chǎn)術(shù)中過度刮宮,或術(shù)后繼發(fā)宮腔感染,破壞了子宮內(nèi)膜,嚴(yán)重時即可形成宮腔粘連,導(dǎo)致閉經(jīng)和繼發(fā)不孕。宮腔鏡手術(shù)分離配合雌孕激素人工周期治療是目前主要的治療方案。但治療效果并不太滿意,子宮內(nèi)膜的發(fā)育并不能很好的改善。 子宮內(nèi)膜是一個高度再生組織,在整個婦女的生育期要經(jīng)歷生長、分化和脫落400多次的循環(huán)。研究顯示子宮內(nèi)膜中含有子宮內(nèi)膜干細(xì)胞。并將其定位于子宮內(nèi)膜和子宮肌層交界部位。子宮內(nèi)膜干細(xì)胞作為一種成體干細(xì)胞,已被證實具有高增殖潛力和多向分化的功能。子宮內(nèi)膜干細(xì)胞在子宮內(nèi)膜周期性增生和脫落中起到了關(guān)鍵作用。 鑒于子宮內(nèi)膜干細(xì)胞與子宮內(nèi)膜生長的密切關(guān)系。我們從體外分離培養(yǎng)子宮內(nèi)膜干細(xì)胞,然后移植到子宮內(nèi)膜治療子宮內(nèi)膜發(fā)育不良性疾病,可能為因子宮內(nèi)膜原因而導(dǎo)致的不孕癥提供一種新的治療方法。 本實驗主要探討從切除的子宮中分離、培養(yǎng)子宮內(nèi)膜干細(xì)胞,并對常用的三種細(xì)胞分離方法進行比較,旨在找出一種簡便、實用、高效的體外分離、培養(yǎng)子宮內(nèi)膜干細(xì)胞的方法,為進一步的研究提供基礎(chǔ)的參考數(shù)據(jù)。 目的 1.探索從切除的子宮中分離培養(yǎng)子宮內(nèi)膜干細(xì)胞。 2.通過對三種不同分離方法的比較,從中優(yōu)選出一種簡便、實用、高效的體外分離、培養(yǎng)人子宮內(nèi)膜干細(xì)胞的方法。 材料與方法 1材料 選取2010年3月～2010年6月在鄭州大學(xué)第三附屬醫(yī)院婦科因子宮肌瘤、子宮脫垂、原位宮頸癌等而進行子宮切除手術(shù)的住院治療的患者,年齡在31～49歲。要求術(shù)前3個月未使用過任何激素。詳細(xì)記錄病人的信息,如年齡,手術(shù)切除子宮的原因等。 2方法 2.1標(biāo)本的處理 子宮全切術(shù)后,無菌剃取子宮內(nèi)膜組織并包括5 mm的子宮肌層。將收集的子宮內(nèi)膜標(biāo)本放入無菌的青、鏈霉素溶液中,2h內(nèi)送入實驗室。用無Ca2+Mg2+的緩沖液(PBS)進行沖洗,直到液體清亮。移入無菌的培養(yǎng)皿中,用眼科剪剪至1 mm3大小,呈糊狀。 2.2組織分離和培養(yǎng) 采用三種不同的分離方法：胰蛋白酶法、膠原酶Ⅰ法和混合法(胰蛋白酶法+膠原酶Ⅰ法)進行細(xì)胞分離和提取,并對三種不同的分離方法進行比較。 將分離的細(xì)胞懸液用臺盼蘭染色,計數(shù)活細(xì)胞。按照2×105個/ml的細(xì)胞密度分別接種于25 cm2的培養(yǎng)瓶中,DMEM/F12培養(yǎng)液中包含10%胎牛血清,青、鏈霉素溶液,37℃,5%CO2培養(yǎng)箱孵育48 h后換液一次。棄去未貼壁細(xì)胞,以后每2～3天換液一次,并觀察細(xì)胞生長情況。 每種方法實驗重復(fù)10次,取第2代生長到第5天的細(xì)胞,用臺盼蘭染色,倒置顯微鏡下計數(shù)活細(xì)胞數(shù),每高倍鏡下計數(shù)三次,取其平均數(shù)。以比較那種分離方法得到的活細(xì)胞數(shù)量多。 2.3細(xì)胞的鑒定方法 用造血干細(xì)胞標(biāo)志物CD90和間質(zhì)干細(xì)胞標(biāo)志物CD146單克隆抗體,采用免疫細(xì)胞化學(xué)法和RT-PCR法,對培養(yǎng)的子宮內(nèi)膜干細(xì)胞進行鑒定。 2.4繪制細(xì)胞生長曲線圖 取對數(shù)生長期的細(xì)胞,接種于24孔板中,置培養(yǎng)箱培養(yǎng)；每24 h計數(shù)一次,每次計三孔,取其平均數(shù)；連續(xù)計數(shù)7天,將所得資料以天數(shù)為橫坐標(biāo),細(xì)胞數(shù)為縱坐標(biāo)繪制曲線,繪制細(xì)胞生長曲線。 2.5統(tǒng)計分析 實驗所得數(shù)據(jù)用SPSS16.0軟件包進行統(tǒng)計學(xué)分析,各組數(shù)值變量參數(shù)以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,組間均數(shù)比較采用單因素方差分析(one-way ANOVA),兩兩比較采用LSD(least significant differerence)檢驗,方差分析前對各組數(shù)據(jù)進行正態(tài)性檢驗(Shapiro-Wilk Test)和方差齊性檢驗(Levene's Test),以a=0.05為檢驗水準(zhǔn)。 結(jié)果 1.子宮內(nèi)膜干細(xì)胞形態(tài)觀察 培養(yǎng)的子宮內(nèi)膜干細(xì)胞形態(tài)呈梭形,具有成纖維細(xì)胞形態(tài),細(xì)胞貼壁平鋪生長,細(xì)胞排列無極性。 2.子宮內(nèi)膜干細(xì)胞的鑒定 經(jīng)干細(xì)胞標(biāo)志物CD90和CD146單抗免疫細(xì)胞化學(xué)染色為陽性；經(jīng)RT-PCR鑒定子宮內(nèi)膜干細(xì)胞CD90和CD146的DNA分別在150bp和500bp高表達,而對照組不表達。證明培養(yǎng)的細(xì)胞具有干細(xì)胞特性。 3.三種分離方法培養(yǎng)的子宮內(nèi)膜干細(xì)胞生長周期的比較 所得單細(xì)胞懸液經(jīng)培養(yǎng)后,第2代細(xì)胞經(jīng)細(xì)胞計數(shù)法所得生長曲線圖比較,無明顯差異(F=0.291,P0.05)。均為近似“S”形,具有相同的細(xì)胞生長周期。 4.三種分離方法的比較 對三組間年齡比較,差異均無統(tǒng)計學(xué)意義(P0.05)。 三種分離方法獲得的活細(xì)胞比較,膠原酶Ⅰ法獲得的活細(xì)胞數(shù)(0.721±0.004)明顯多于胰蛋白酶法(0.672±0.002)和混合法獲得的的細(xì)胞數(shù)(0.671+0.001),差異有統(tǒng)計學(xué)意義(F=103.882,P0.01)。 結(jié)論 1.從切除的子宮中成功獲得子宮內(nèi)膜干細(xì)胞。 2.膠原酶Ⅰ法分離子宮內(nèi)膜干細(xì)胞是一種實用、高效、穩(wěn)定的體外子宮內(nèi)膜干細(xì)胞分離培養(yǎng)方法。
[Abstract]:Infertility is one of the common diseases of Gynecology, among which female factors account for 40-55%, and 9.2% of the causes of female infertility are intrauterine adhesions. The occurrence of intrauterine adhesions is mainly related to uterine cavity surgery trauma and uterine cavity infection. It is a long-term major complication after uterine cavity surgery. Most patients have a history of artificial abortion, especially artificial abortion. Excessive curettage in the operation, or secondary uterine cavity infection after operation, destroy the endometrium, which can form intrauterine adhesions and cause amenorrhea and secondary infertility. Hysteroscopic surgery is the main treatment for the treatment of estrogen and progesterone, but the treatment effect is not satisfactory, and the development of the endometrium can not be improved well.
The endometrium is a highly regenerative tissue that goes through 400 cycles of growth, differentiation, and abscission throughout the birth of a woman. The study shows that endometrium contains endometrium stem cells in the endometrium and is located at the junction of the endometrium and the myometrium. Endometrial stem cells play a key role in the proliferation and detachment of endometrium.
In view of the close relationship between endometrial stem cells and endometrium growth, we isolate and culture endometrium stem cells from in vitro, and then transplant them into the endometrium for the treatment of endometrial dysplasia, which may provide a new treatment for infertility caused by endometrial causes.
In this experiment, we mainly discuss the isolation of endometrium stem cells from the excised uterus, and compare the three commonly used methods of cell separation. The aim is to find a simple, practical, efficient method for isolation and culture of endometrium stem cells in vitro, and provide basic reference data for further research.
objective
1. explore the isolation and culture of endometrial stem cells from the removed uterus.
2. by comparing three different separation methods, a simple, practical and efficient method for isolation and culture of human endometrial stem cells was selected.
Materials and methods
1 material
From March 2010 to June 2010, patients who were hospitalized for hysterectomy for hysteromyoma, uterine prolapse, and in situ cervical cancer at the Third Affiliated Hospital of Zhengzhou University were 31~49 years of age. No hormones were used in the 3 months before operation. The information of the sick people, such as age, and the reason of the operation of the uterus, was recorded in detail. Wait.
2 method
Treatment of 2.1 specimens
After total hysterectomy, the endometrium was shaved and the endometrium was shaved with 5 mm. The collected endometrium specimens were put into the sterile green, Streptomycin Solution and 2H into the laboratory. The samples were flushed with a Ca2+Mg2+ free buffer (PBS) until the liquid was clear.
2.2 tissue separation and culture
Three different separation methods: trypsin method, collagenase I method and mixed method (trypsin and collagenase I) were used to separate and extract the cells, and the three different separation methods were compared.
The isolated cell suspension was stained with trypan blue to count living cells. The cell density of 2 x 105 /ml was inoculated in the culture bottle of 25 cm2 respectively. The DMEM/F12 culture medium contained 10% fetal bovine serum, green, streptomycin solution, 37 C, and 5%CO2 incubator incubated for 48 h. Cell growth.
Each method was repeated 10 times, and second generations of cells were grown to fifth days. The number of living cells was counted with trypan blue, the number of living cells was counted under the inverted microscope, and the average number of the cells was counted under each high magnification. The number of living cells obtained by the method of separation was compared.
Identification of 2.3 cells
A monoclonal antibody of hematopoietic stem cell marker CD90 and interstitial stem cell marker CD146 was used to identify the cultured endometrium stem cells by immunocytochemistry and RT-PCR.
2.4 plot the curve of cell growth
The cells from the logarithmic growth period were inoculated in 24 Kong Banzhong and incubated in the culture box. The cells were counted every 24 h and the average number was taken each time. The number of data was counted for 7 days, and the number of cells was plotted in the longitudinal coordinates, and the cell growth curve was drawn.
2.5 statistical analysis
The experimental data were statistically analyzed with SPSS16.0 software package, and the parameters of each group were expressed as x + s. The average number of groups was compared with single factor analysis of variance (one-way ANOVA). 22 compared with LSD (least significant differerence) test, and the normal test of each group was tested before the analysis of variance (Shapiro-W, Shapiro-W). Ilk Test) and homogeneity test of variance (Levene's Test), taking a=0.05 as the test standard.
Result
Observation on the morphology of 1. endometrium stem cells
The cultured endometrial stem cells were spindle shaped and had fibroblast morphology. The cells adhered to the wall and grew flat.
Identification of 2. endometrium stem cells
The immunocytochemical staining of CD90 and CD146 McAbs was positive by stem cell markers, and the DNA of CD90 and CD146 in endometrial stem cells was highly expressed in 150bp and 500bp by RT-PCR, while the control group was not expressed in the control group, which showed that the cultured cells had the characteristics of stem cells.
3. comparison of the growth cycle of endometrial stem cells cultured by three different methods
After the single cell suspension was cultured, the growth curves of the second generation cells were compared with the cell count method. There was no significant difference (F=0.291, P0.05). All of the cells were similar to "S", with the same cell growth cycle.
4. comparison of three separate methods
There was no significant difference in age between the three groups (P0.05).
Compared with the living cells obtained by the three methods, the number of living cells (0.721 + 0.004) obtained by collagenase I (0.721 + 0.004) was significantly more than that of trypsin method (0.672 + 0.002) and the number of cells (0.671+0.001) obtained by mixed method. The difference was statistically significant (F=103.882, P0.01).
conclusion
1. endometrial stem cells were successfully obtained from the removed uterus.
2. collagenase I isolation of endometrial stem cells is a practical, efficient and stable method for isolation and culture of endometrial stem cells in vitro.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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