整合素β1对人滋养层细胞粘附的影响
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摘要
在人胎盘发育过程中,滋养层细胞对子宫内膜血管的入侵至关重要。滋养层细胞的整个入侵过程是先进入毛细血管、再迁移至子宫螺旋动脉并在此过程中对子宫动脉血管系统进行重构。这一迁移过程提示,滋养层细胞的迁移方向是与血液的流动方向相反的,即滋养层细胞是逆血流而上的。为了弄清滋养层细胞在妊娠过程中的生理作用以及入侵子宫脉管系统的机理,学术界已做了大量卓有成效的工作,但是关于滋养层细胞在血管系统中逆流迁移机制的研究却极其有限。因此,为了深入了解和认识体内滋养层细胞逆血流迁移行为和调节机制,本文从细胞粘附分子的角度分析了滋养层细胞β1整合素的表达与其迁移行为的相关性。本论文的具体研究工作和结果、结论如下:
首先,本文建立了一种简单稳定的人体滋养层细胞原代培养方法。结果表明:采用胰酶+DNA酶辅助消化的方法可显著提高原代培养滋养层细胞的产量;同时,通过Percoll非连续梯度离心以及相应的差速贴壁和机械刮除等纯化步骤后得到的滋养层细胞纯度相对较高,可达95 %以上,且细胞形态单一,生长稳定,细胞活力强。这说明本文采用的方法适合滋养层细胞的分离、纯化和体外培养。
其次,本文还采用微管吸吮技术对滋养层细胞的粘附力学特性进行了研究。结果表明:滋养层细胞的粘附力随I型鼠尾胶原浓度的增加而呈显著的线性增加趋势,表现出明显的浓度依赖关系。当与内皮细胞接触时,滋养层细胞表现出较高的粘附力,表明与内皮细胞的接触可显著增强滋养层细胞的粘附力学行为。此外,本文还从粘附分子的角度对滋养层细胞与胶原和内皮细胞的粘附力学特征进行了分析,结果提示在滋养层细胞的粘附过程中,β1整合素也起着重要的调节作用。
最后,本文研究了体内绒毛组织和体外培养的滋养层细胞整合素表达的特性。结果表明:β1整合素在迁移显型滋养层细胞上高度表达。在剪切应力作用下,滋养层细胞β1整合素的表达随剪切应力作用的时间的延续而显著变化,剪切应力增强了滋养层细胞β1整合素的表达。当滋养层细胞与内皮细胞共培养时,β1整合素的表达较单独培养时显著增强。这些结果提示,β1整合素在滋养层细胞对内皮细胞的粘附和迁移过程中有着重要的关系。
本文的研究结果可进一步揭示人滋养层细胞生长、侵袭、迁移过程与滋养层细胞力学特性的相关关系,为全面认识人滋养层细胞入侵子宫脉管系统的机理提供定量的参考依据。
During the development of human placenta, invasion and migration of trophoblast cells (TCs) within the uterine vasculature is an essential process for the remodeling of the uterine vasculature. In vivo, the processes of cell migration are that TCs firstly invade into the capillary vessels, then migrate within uterine vasculature, and eventually reside within the spiral arteries. This indicates that the direction of TC migration within the uterine vasculature is against the blood stream. In order to understand the physiological roles of TCs in the pregnancy process and the mechanism of TC invasion into the uterine vasculature, many progress were made. However, little has been known about the hemodynamic mechanism of TC migration against the blood stream. Therefore, the correlation between integrinβ1 and the migratory behaviors of TCs was analyzed on the adhesion molecule level in order to understand the mechanism of TC invasion against the blood stream. The main works and results、conclusions in this paper are as follow:
First, a simple and stable protocol for human trophoblast cell culture was established. The results revealed that the population of isolated human TCs were significantly augmented using the digesting method of trypsin and DNase at 37℃. The purification of TCs was carried out by serial progress, and cytokeratin positive of TCs was greater than 95%. Configuration of the TCs isolated and cultured were singularity and the growth was stable with stronger viability.
Then, using a micropipette aspiration technique, the adhesive mechanical properties of TCs were measured. The results revealed that the adhesion forces of TCs to type I rat collagen increased with increasing concentration of collagen I. When contacted with HUVECs, the adhesion force of TCs was significantly enhanced. Moreover, from the point of view of adhesion molecules, the adhesive mechanical properties of TCs to collagen I was compared with those of TCs to HUVECs. The results suggested that trophoblast integrinβ1 played an important role in the adhesion of TCs.
Finally, integrinβ1 expression of TCs on the tissue sections and the isolated TCs were analyzed. The results demonstrated that migratory TCs was accompanied by higher integrinβ1 expression when compared to the non-migratory TCs. Shear stress up-regulated the integrinβ1 expression of TCs, and expression of integrinβ1 of TCs significantly changed with time. TCs co-cultured with endothelial cells increased the expression of integrinβ1 when compared with the TCs cultured alone, suggesting again that trophoblast integrinβ1 played a role in the adhesion and migration of TCs.
The experimental results in this paper may provide quantitative referenced gist in opening out relations between mechanical properties of TCs and its growth, invasion, migration; finding out the mechanism of all-sidely TC invasion into the uterine vasculature.
首先,本文建立了一种简单稳定的人体滋养层细胞原代培养方法。结果表明:采用胰酶+DNA酶辅助消化的方法可显著提高原代培养滋养层细胞的产量;同时,通过Percoll非连续梯度离心以及相应的差速贴壁和机械刮除等纯化步骤后得到的滋养层细胞纯度相对较高,可达95 %以上,且细胞形态单一,生长稳定,细胞活力强。这说明本文采用的方法适合滋养层细胞的分离、纯化和体外培养。
其次,本文还采用微管吸吮技术对滋养层细胞的粘附力学特性进行了研究。结果表明:滋养层细胞的粘附力随I型鼠尾胶原浓度的增加而呈显著的线性增加趋势,表现出明显的浓度依赖关系。当与内皮细胞接触时,滋养层细胞表现出较高的粘附力,表明与内皮细胞的接触可显著增强滋养层细胞的粘附力学行为。此外,本文还从粘附分子的角度对滋养层细胞与胶原和内皮细胞的粘附力学特征进行了分析,结果提示在滋养层细胞的粘附过程中,β1整合素也起着重要的调节作用。
最后,本文研究了体内绒毛组织和体外培养的滋养层细胞整合素表达的特性。结果表明:β1整合素在迁移显型滋养层细胞上高度表达。在剪切应力作用下,滋养层细胞β1整合素的表达随剪切应力作用的时间的延续而显著变化,剪切应力增强了滋养层细胞β1整合素的表达。当滋养层细胞与内皮细胞共培养时,β1整合素的表达较单独培养时显著增强。这些结果提示,β1整合素在滋养层细胞对内皮细胞的粘附和迁移过程中有着重要的关系。
本文的研究结果可进一步揭示人滋养层细胞生长、侵袭、迁移过程与滋养层细胞力学特性的相关关系,为全面认识人滋养层细胞入侵子宫脉管系统的机理提供定量的参考依据。
During the development of human placenta, invasion and migration of trophoblast cells (TCs) within the uterine vasculature is an essential process for the remodeling of the uterine vasculature. In vivo, the processes of cell migration are that TCs firstly invade into the capillary vessels, then migrate within uterine vasculature, and eventually reside within the spiral arteries. This indicates that the direction of TC migration within the uterine vasculature is against the blood stream. In order to understand the physiological roles of TCs in the pregnancy process and the mechanism of TC invasion into the uterine vasculature, many progress were made. However, little has been known about the hemodynamic mechanism of TC migration against the blood stream. Therefore, the correlation between integrinβ1 and the migratory behaviors of TCs was analyzed on the adhesion molecule level in order to understand the mechanism of TC invasion against the blood stream. The main works and results、conclusions in this paper are as follow:
First, a simple and stable protocol for human trophoblast cell culture was established. The results revealed that the population of isolated human TCs were significantly augmented using the digesting method of trypsin and DNase at 37℃. The purification of TCs was carried out by serial progress, and cytokeratin positive of TCs was greater than 95%. Configuration of the TCs isolated and cultured were singularity and the growth was stable with stronger viability.
Then, using a micropipette aspiration technique, the adhesive mechanical properties of TCs were measured. The results revealed that the adhesion forces of TCs to type I rat collagen increased with increasing concentration of collagen I. When contacted with HUVECs, the adhesion force of TCs was significantly enhanced. Moreover, from the point of view of adhesion molecules, the adhesive mechanical properties of TCs to collagen I was compared with those of TCs to HUVECs. The results suggested that trophoblast integrinβ1 played an important role in the adhesion of TCs.
Finally, integrinβ1 expression of TCs on the tissue sections and the isolated TCs were analyzed. The results demonstrated that migratory TCs was accompanied by higher integrinβ1 expression when compared to the non-migratory TCs. Shear stress up-regulated the integrinβ1 expression of TCs, and expression of integrinβ1 of TCs significantly changed with time. TCs co-cultured with endothelial cells increased the expression of integrinβ1 when compared with the TCs cultured alone, suggesting again that trophoblast integrinβ1 played a role in the adhesion and migration of TCs.
The experimental results in this paper may provide quantitative referenced gist in opening out relations between mechanical properties of TCs and its growth, invasion, migration; finding out the mechanism of all-sidely TC invasion into the uterine vasculature.
引文
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