兔三维子宫培养系统的优化及胚胎植入的研究
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摘要
人类对早期胚胎发育,尤其是在体内子宫上的发育过程知之甚少,而其体内的变化受激素、神经、体液以及内分泌的影响,鉴于子宫在胚胎发育以及人类生殖研究中具有无法替代的重要作用与功能,越来越多的国内外研究人员意识到体外人造子宫具有重要的科学研究价值和临床应用前景,建造组织工程化人造子宫已成为组织工程研究领域的热点之一。
本研究通过对兔子宫内膜细胞和平滑肌细胞的分离和培养方法的探讨,结果在体外成功的分离和纯化了三种细胞;以这三种细胞为种子细胞将其接种在鼠尾胶原和Matrigel的混合胶内,在体外构建具有三层细胞结构(从下到上依次为平滑肌层,基质层和上皮层)的三维子宫片层;体外优化三维培养系统,改变培养条件,如改变雌激素和孕激素的浓度或者在培养液中添加一些生长因子,体外诱导腺管结构和血管结构,使其更类似于在体结构;以三维子宫组织为模型,在雌激素和孕激素的作用下,诱导基质层细胞发生蜕膜化,模拟在体的接受态子宫。发育到囊胚期的小鼠胚胎与三维子宫组织共培养,观察胚胎在子宫片层上的发育情况,以期对胚胎早期发育事件有更深入的了解,尤其是希望能够部分模拟胚胎在体内子宫早期发育阶段的生理过程,主要结果如下:
1.本研究采用筛网法和差速离心法分离子宫内膜细胞,比较2种分离方法,结果差速离心法比较简单快捷,而且污染少,获得的细胞量大。再通过差速贴壁法纯化2种细胞,结果能够获得较纯的子宫内膜基质细胞和上皮细胞。
2.通过免疫组织化学染色法鉴定细胞的类型,结果发现小鼠抗人的CK18染色,上皮细胞的胞质呈棕色阳性;小鼠抗人的Vimentin染色,基质细胞胞质呈棕色阳性;小鼠抗人的α-actin免疫组织化学染色,平滑肌细胞的胞质呈棕色阳性。免疫荧光染色鉴定细胞的纯度,结果发现上皮细胞的纯度为97%,基质细胞的纯度为98%,平滑肌细胞的纯度为98%。
3.以体外培养3代以内的兔子宫内膜细胞和平滑肌细胞为种子细胞,以液态I型鼠尾胶原和Matrigel为支架材料,在静态拉力存在的条件下,体外构建三维子宫组织。培养14d左右的子宫组织取出后固定,H.E染色和免疫组织化学染色观察结构。结果构建的子宫组织具有明显的三层细胞结构,细胞在静态拉力的作用下具有一定的方向性,上皮细胞表现为一定的极性,呈柱状分布。通过扫描电镜和透射电镜观察子宫组织的结构,结果细胞结构完整,细胞表面出现微绒毛,细胞间存在紧密连接。
4.以原代分离的兔子宫内膜细胞和平滑肌细胞为种子细胞,将上皮细胞和基质细胞混合接种在鼠尾胶原和Matrigel内。在此基础上优化培养条件,促进子宫内膜细胞中混有的血管内皮细胞和腺上皮细胞形成一定的形态结构。首次构建出类似于在体子宫的形态和功能的子宫组织,体外构建的子宫组织出现类似于在体的腺管结构和血管结构,而且还观察到组织表面出现大的弯曲结构,弯曲处聚集大量的细。CK7和Ⅷ因子分别进行免疫组织化学染色,结果发现构成腺管和血管的细胞分别呈阳性,说明原代分离的子宫内膜细胞中混有血管内皮细胞,而且细胞具有一定的移动性和方向性。这些结构的形成为人造子宫的实现带来了希望。
5.通过改变雌激素和孕激素的比例,诱导三维子宫处于容受状态,即“植入窗口期”,H.E染色观察,结果基质细胞发生蜕膜化,基质层细胞变大,变为多核细胞。囊胚与三维子宫组织共培养,结果发现,20h胚胎黏附到三维子宫组织上,32h后胚胎侵入到三维子宫组织中,胚胎周围出现凹陷,可能胚胎在体外构建的三维子宫组织上成功的植入。
People learn a little of the development of early embryo, especially the course of development in uterus, while hormone ,nerve, body fluid and endocrine influence the change in vivo. Respecting uterus have an unable to be substituted important contribution and function in the development of embryos and the study of human generation, more and more researchers aware that artificial uterine have important value of science and perspective in the clinical application, establishing tissue engineering artificial uterine has become one of the hot spot in the research sphere of tissue engineering.
The study on isolation and culture of rabbit endometrial cells and smooth muscle cells, the results showed that three kinds of cells were successful isolated and purified in vitro; these three kinds of cells acted as seed cells, which were seeded in the mixture of collagen and Matrigel, three dimensional uterine lamellar were built in vitro, which composed of three layers of cells (smooth muscle cell, stromal cell and epithelial cell), through changing the condition of culture , for example: the concentration of estrogen and progestin or added to some growth factors in the culture medium, glandular tube and blood vascular were induced in vitro, three dimensional endometrium was similar to normal structure; Under the effect of estrogen and progestin, three dimensional uterine tissue acted as a model, stromal cells generated decidualization, which imitated normal receptive uterus. Mouse blastocysts and three dimensional uterine tissue coculture, which was used to observe embryos development on the uterine slice, in order to even more learn the early development event of embryo, which is more feasible for deeply learned on early event of embryos development, especially wished that the model could imitate the normal physiological course of embryo early development in the uterine. The mainly results of the research as following:
1. The research adopted the way of filtration through copper screen and centrifuge at different speed to isolate endometrial cells, comparing two kinds of ways, the conclusion indicated that the way of centrifuge at different speed was more simple and shortcut, and cells were less contaminated, the number of acquired cells was a great quantity. Two kinds of cells were purified through differential attachment technique, which could acquire more purify endometrial stromal cells and epithelial cells.
2. Immunohistochemisty identified the type of cells, mouse antihuman cytokeratin 18 stained, epithelial cells positive, cells show brown, mouse antihuman vimentin stained, stromal cells positive, cells show brown, mouse antihumanα-actin stained, smooth muscle cells positive, cells show brown. The purity of cells was identified by immunofluorescence, the purity of epithelial cells was 97%, the purity of stromal cells was 98%, and the purity of smooth muscle cells was 98%.
3. Endometrial cells and smooth muscle cells ahead of 3 generation acted as the seed cells, liquidⅠtype collagen and Matrigel acted as scaffold, under the condition of static state tensile force, uterine tissue were established in vitro. Cultured uterine tissue were taken out of the flask and fixed on 14th days, the structure were observed through H.E staining and immunohistochemistry staining. Conclusion indicated that established uterine tissue emerged evident three layers of structure, the growth of cells showed directionality under condition of static state tensile force, epithelial cells showed some polarity and column aspect. Through scan electron microscope and transmission electron microscope observed cells structure of uterine tissue, the conclusion indicated that the cellular structure integrity, the surface of epithelial cells emerge some microvilli, between cells and cells had tight junction.
4. Primary endometrial cells and smooth muscle cells of rabbit acted as seed cells, the mixture of epithelial cells and stromal cells seeded in liquidⅠtype collagen and Matrigel, in this base, the condition of culture was optimized, which precipitated vascular epithelial cells and glandular epithelial cells to emerge shape and construction. First time established uterine tissue was similar to normal uterus in shape and function, which emerged glandular tube and blood vessel, and the surface of tissue emerged big crook, which aggregated some cells. Immunohistochemisty stained respectively with CK7 andⅧ, the conclusion indicated that cells of glandular tube and blood vessel were positive respectively, cells had property of migration and directivity. The emergence of these structures brings hope for the implementation of artificial uterus.
5. Through changing the concentration of hormone, three dimensional uterus was induced to being receptive state, that is to say“the window of implantation”, H.E staining observed the uterine structure; the stromal cells took place decidualization, cells turned to be dilatants and multinuclear. Blastula and three dimensional uterine tissue cocultured, the result indicated that embryos stick to the three dimensional uterine tissue after being cultured 20h, embryos invaded into three dimensional uterine tissue after being cultured 32h, around of embryos appeared umbilication, embryo implanted three dimensional uterine tissue in vitro.
本研究通过对兔子宫内膜细胞和平滑肌细胞的分离和培养方法的探讨,结果在体外成功的分离和纯化了三种细胞;以这三种细胞为种子细胞将其接种在鼠尾胶原和Matrigel的混合胶内,在体外构建具有三层细胞结构(从下到上依次为平滑肌层,基质层和上皮层)的三维子宫片层;体外优化三维培养系统,改变培养条件,如改变雌激素和孕激素的浓度或者在培养液中添加一些生长因子,体外诱导腺管结构和血管结构,使其更类似于在体结构;以三维子宫组织为模型,在雌激素和孕激素的作用下,诱导基质层细胞发生蜕膜化,模拟在体的接受态子宫。发育到囊胚期的小鼠胚胎与三维子宫组织共培养,观察胚胎在子宫片层上的发育情况,以期对胚胎早期发育事件有更深入的了解,尤其是希望能够部分模拟胚胎在体内子宫早期发育阶段的生理过程,主要结果如下:
1.本研究采用筛网法和差速离心法分离子宫内膜细胞,比较2种分离方法,结果差速离心法比较简单快捷,而且污染少,获得的细胞量大。再通过差速贴壁法纯化2种细胞,结果能够获得较纯的子宫内膜基质细胞和上皮细胞。
2.通过免疫组织化学染色法鉴定细胞的类型,结果发现小鼠抗人的CK18染色,上皮细胞的胞质呈棕色阳性;小鼠抗人的Vimentin染色,基质细胞胞质呈棕色阳性;小鼠抗人的α-actin免疫组织化学染色,平滑肌细胞的胞质呈棕色阳性。免疫荧光染色鉴定细胞的纯度,结果发现上皮细胞的纯度为97%,基质细胞的纯度为98%,平滑肌细胞的纯度为98%。
3.以体外培养3代以内的兔子宫内膜细胞和平滑肌细胞为种子细胞,以液态I型鼠尾胶原和Matrigel为支架材料,在静态拉力存在的条件下,体外构建三维子宫组织。培养14d左右的子宫组织取出后固定,H.E染色和免疫组织化学染色观察结构。结果构建的子宫组织具有明显的三层细胞结构,细胞在静态拉力的作用下具有一定的方向性,上皮细胞表现为一定的极性,呈柱状分布。通过扫描电镜和透射电镜观察子宫组织的结构,结果细胞结构完整,细胞表面出现微绒毛,细胞间存在紧密连接。
4.以原代分离的兔子宫内膜细胞和平滑肌细胞为种子细胞,将上皮细胞和基质细胞混合接种在鼠尾胶原和Matrigel内。在此基础上优化培养条件,促进子宫内膜细胞中混有的血管内皮细胞和腺上皮细胞形成一定的形态结构。首次构建出类似于在体子宫的形态和功能的子宫组织,体外构建的子宫组织出现类似于在体的腺管结构和血管结构,而且还观察到组织表面出现大的弯曲结构,弯曲处聚集大量的细。CK7和Ⅷ因子分别进行免疫组织化学染色,结果发现构成腺管和血管的细胞分别呈阳性,说明原代分离的子宫内膜细胞中混有血管内皮细胞,而且细胞具有一定的移动性和方向性。这些结构的形成为人造子宫的实现带来了希望。
5.通过改变雌激素和孕激素的比例,诱导三维子宫处于容受状态,即“植入窗口期”,H.E染色观察,结果基质细胞发生蜕膜化,基质层细胞变大,变为多核细胞。囊胚与三维子宫组织共培养,结果发现,20h胚胎黏附到三维子宫组织上,32h后胚胎侵入到三维子宫组织中,胚胎周围出现凹陷,可能胚胎在体外构建的三维子宫组织上成功的植入。
People learn a little of the development of early embryo, especially the course of development in uterus, while hormone ,nerve, body fluid and endocrine influence the change in vivo. Respecting uterus have an unable to be substituted important contribution and function in the development of embryos and the study of human generation, more and more researchers aware that artificial uterine have important value of science and perspective in the clinical application, establishing tissue engineering artificial uterine has become one of the hot spot in the research sphere of tissue engineering.
The study on isolation and culture of rabbit endometrial cells and smooth muscle cells, the results showed that three kinds of cells were successful isolated and purified in vitro; these three kinds of cells acted as seed cells, which were seeded in the mixture of collagen and Matrigel, three dimensional uterine lamellar were built in vitro, which composed of three layers of cells (smooth muscle cell, stromal cell and epithelial cell), through changing the condition of culture , for example: the concentration of estrogen and progestin or added to some growth factors in the culture medium, glandular tube and blood vascular were induced in vitro, three dimensional endometrium was similar to normal structure; Under the effect of estrogen and progestin, three dimensional uterine tissue acted as a model, stromal cells generated decidualization, which imitated normal receptive uterus. Mouse blastocysts and three dimensional uterine tissue coculture, which was used to observe embryos development on the uterine slice, in order to even more learn the early development event of embryo, which is more feasible for deeply learned on early event of embryos development, especially wished that the model could imitate the normal physiological course of embryo early development in the uterine. The mainly results of the research as following:
1. The research adopted the way of filtration through copper screen and centrifuge at different speed to isolate endometrial cells, comparing two kinds of ways, the conclusion indicated that the way of centrifuge at different speed was more simple and shortcut, and cells were less contaminated, the number of acquired cells was a great quantity. Two kinds of cells were purified through differential attachment technique, which could acquire more purify endometrial stromal cells and epithelial cells.
2. Immunohistochemisty identified the type of cells, mouse antihuman cytokeratin 18 stained, epithelial cells positive, cells show brown, mouse antihuman vimentin stained, stromal cells positive, cells show brown, mouse antihumanα-actin stained, smooth muscle cells positive, cells show brown. The purity of cells was identified by immunofluorescence, the purity of epithelial cells was 97%, the purity of stromal cells was 98%, and the purity of smooth muscle cells was 98%.
3. Endometrial cells and smooth muscle cells ahead of 3 generation acted as the seed cells, liquidⅠtype collagen and Matrigel acted as scaffold, under the condition of static state tensile force, uterine tissue were established in vitro. Cultured uterine tissue were taken out of the flask and fixed on 14th days, the structure were observed through H.E staining and immunohistochemistry staining. Conclusion indicated that established uterine tissue emerged evident three layers of structure, the growth of cells showed directionality under condition of static state tensile force, epithelial cells showed some polarity and column aspect. Through scan electron microscope and transmission electron microscope observed cells structure of uterine tissue, the conclusion indicated that the cellular structure integrity, the surface of epithelial cells emerge some microvilli, between cells and cells had tight junction.
4. Primary endometrial cells and smooth muscle cells of rabbit acted as seed cells, the mixture of epithelial cells and stromal cells seeded in liquidⅠtype collagen and Matrigel, in this base, the condition of culture was optimized, which precipitated vascular epithelial cells and glandular epithelial cells to emerge shape and construction. First time established uterine tissue was similar to normal uterus in shape and function, which emerged glandular tube and blood vessel, and the surface of tissue emerged big crook, which aggregated some cells. Immunohistochemisty stained respectively with CK7 andⅧ, the conclusion indicated that cells of glandular tube and blood vessel were positive respectively, cells had property of migration and directivity. The emergence of these structures brings hope for the implementation of artificial uterus.
5. Through changing the concentration of hormone, three dimensional uterus was induced to being receptive state, that is to say“the window of implantation”, H.E staining observed the uterine structure; the stromal cells took place decidualization, cells turned to be dilatants and multinuclear. Blastula and three dimensional uterine tissue cocultured, the result indicated that embryos stick to the three dimensional uterine tissue after being cultured 20h, embryos invaded into three dimensional uterine tissue after being cultured 32h, around of embryos appeared umbilication, embryo implanted three dimensional uterine tissue in vitro.
引文
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