人子宫内膜干细胞体外分离、培养及鉴定

发布时间：2018-05-13 05:33

本文选题：子宫内膜干细胞 + 分离　；参考：《郑州大学》2011年硕士论文

【摘要】：不孕症是妇科常见病之一,其中,女性因素占40-55%,在引起女性不孕的众多原因中9.2%为宫腔粘连导致。宫腔粘连的发生主要与宫腔手术创伤和宫腔感染有关,是宫腔手术后的远期主要并发症。患者大多有过多次人工流产史,尤其是人工流产术中过度刮宫,或术后继发宫腔感染,破坏了子宫内膜,严重时即可形成宫腔粘连,导致闭经和继发不孕。宫腔镜手术分离配合雌孕激素人工周期治疗是目前主要的治疗方案。但治疗效果并不太满意,子宫内膜的发育并不能很好的改善。子宫内膜是一个高度再生组织,在整个妇女的生育期要经历生长、分化和脱落400多次的循环。研究显示子宫内膜中含有子宫内膜干细胞。并将其定位于子宫内膜和子宫肌层交界部位。子宫内膜干细胞作为一种成体干细胞,已被证实具有高增殖潜力和多向分化的功能。子宫内膜干细胞在子宫内膜周期性增生和脱落中起到了关键作用。鉴于子宫内膜干细胞与子宫内膜生长的密切关系。我们从体外分离培养子宫内膜干细胞,然后移植到子宫内膜治疗子宫内膜发育不良性疾病,可能为因子宫内膜原因而导致的不孕症提供一种新的治疗方法。本实验主要探讨从切除的子宫中分离、培养子宫内膜干细胞,并对常用的三种细胞分离方法进行比较,旨在找出一种简便、实用、高效的体外分离、培养子宫内膜干细胞的方法,为进一步的研究提供基础的参考数据。目的 1.探索从切除的子宫中分离培养子宫内膜干细胞。 2.通过对三种不同分离方法的比较,从中优选出一种简便、实用、高效的体外分离、培养人子宫内膜干细胞的方法。材料与方法 1材料选取2010年3月～2010年6月在郑州大学第三附属医院妇科因子宫肌瘤、子宫脱垂、原位宫颈癌等而进行子宫切除手术的住院治疗的患者,年龄在31～49岁。要求术前3个月未使用过任何激素。详细记录病人的信息,如年龄,手术切除子宫的原因等。 2方法 2.1标本的处理子宫全切术后,无菌剃取子宫内膜组织并包括5 mm的子宫肌层。将收集的子宫内膜标本放入无菌的青、链霉素溶液中,2h内送入实验室。用无Ca2+Mg2+的缓冲液(PBS)进行冲洗,直到液体清亮。移入无菌的培养皿中,用眼科剪剪至1 mm3大小,呈糊状。 2.2组织分离和培养采用三种不同的分离方法：胰蛋白酶法、胶原酶Ⅰ法和混合法(胰蛋白酶法+胶原酶Ⅰ法)进行细胞分离和提取,并对三种不同的分离方法进行比较。将分离的细胞悬液用台盼兰染色,计数活细胞。按照2×105个/ml的细胞密度分别接种于25 cm2的培养瓶中,DMEM/F12培养液中包含10%胎牛血清,青、链霉素溶液,37℃,5%CO2培养箱孵育48 h后换液一次。弃去未贴壁细胞,以后每2～3天换液一次,并观察细胞生长情况。每种方法实验重复10次,取第2代生长到第5天的细胞,用台盼兰染色,倒置显微镜下计数活细胞数,每高倍镜下计数三次,取其平均数。以比较那种分离方法得到的活细胞数量多。 2.3细胞的鉴定方法用造血干细胞标志物CD90和间质干细胞标志物CD146单克隆抗体,采用免疫细胞化学法和RT-PCR法,对培养的子宫内膜干细胞进行鉴定。 2.4绘制细胞生长曲线图取对数生长期的细胞,接种于24孔板中,置培养箱培养；每24 h计数一次,每次计三孔,取其平均数；连续计数7天,将所得资料以天数为横坐标,细胞数为纵坐标绘制曲线,绘制细胞生长曲线。 2.5统计分析实验所得数据用SPSS16.0软件包进行统计学分析,各组数值变量参数以均数±标准差(x±s)表示,组间均数比较采用单因素方差分析(one-way ANOVA),两两比较采用LSD(least significant differerence)检验,方差分析前对各组数据进行正态性检验(Shapiro-Wilk Test)和方差齐性检验(Levene's Test),以a=0.05为检验水准。结果 1.子宫内膜干细胞形态观察培养的子宫内膜干细胞形态呈梭形,具有成纤维细胞形态,细胞贴壁平铺生长,细胞排列无极性。 2.子宫内膜干细胞的鉴定经干细胞标志物CD90和CD146单抗免疫细胞化学染色为阳性；经RT-PCR鉴定子宫内膜干细胞CD90和CD146的DNA分别在150bp和500bp高表达,而对照组不表达。证明培养的细胞具有干细胞特性。 3.三种分离方法培养的子宫内膜干细胞生长周期的比较所得单细胞悬液经培养后,第2代细胞经细胞计数法所得生长曲线图比较,无明显差异(F=0.291,P0.05)。均为近似“S”形,具有相同的细胞生长周期。 4.三种分离方法的比较对三组间年龄比较,差异均无统计学意义(P0.05)。三种分离方法获得的活细胞比较,胶原酶Ⅰ法获得的活细胞数(0.721±0.004)明显多于胰蛋白酶法(0.672±0.002)和混合法获得的的细胞数(0.671+0.001),差异有统计学意义(F=103.882,P0.01)。结论 1.从切除的子宫中成功获得子宫内膜干细胞。 2.胶原酶Ⅰ法分离子宫内膜干细胞是一种实用、高效、稳定的体外子宫内膜干细胞分离培养方法。
[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.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【参考文献】