【作者】 刘万钱；

【导师】 邓小燕；

【作者基本信息】 重庆大学 ， 生物医学工程， 2008， 博士

【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,它是子宫的脉管系统进行重构的必须条件,而子宫脉管系统的重构对于确保胎儿胎盘系统得到充足的血液供应具有非常重要的意义。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。在体内,血流环境作为滋养层细胞生物学行为和生理功能的重要潜在调节因素对其迁移行为的调节具有举足轻重的意义;同时,环境因素对细胞分裂/增殖、分化、凋亡和迁移等行为的影响也是当前细胞生物学和生物力学中的一个研究热点。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,本文以平板流动腔为加载方式模拟子宫脉管系统血流环境,从血流动力学角度对两个问题进行了初步的探讨:1)滋养层细胞在血管中移动的方向性是由什么决定的?2)它们逆血流而上的动力或者说它们逆血流而上的方式是什么?并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,本文研究了流动剪切应力对滋养层细胞迁移行为的影响。结果表明:在无流动条件下,滋养层细胞表现出持续无方向性的位置变化,但并未发现在单一方向上的显著性迁移现象。在7.5、15和30dyne/cm2的剪切应力作用下,滋养层细胞的平均运动速度和X轴(剪切应力方向)上的绝对位移都随剪切应力水平的增加而呈显著的线性增加趋势。同时,滋养层细胞在X轴垂直方向上的位移却并未为零,特别是在6-12h这段时间内细胞运动趋于稳定,表现出了一定的自主性迁移行为。在剪切应力作用下,细胞膜流动性的改变和敏感离子信号通道的阻断可显著的下调滋养层细胞的迁移速度和在X轴垂直方向上的净位移。这些结果表明剪切应力调控了人体滋养层细胞的迁移行为。但与体内的情况相反的是,剪切应力作用下滋养层细胞的迁移方向并不是逆流而上却是顺流而下的。尔后,本文研究了剪切应力作用下与内皮细胞共培养对滋养层细胞迁移行为的影响。结果表明:在不同大小的剪切应力作用下,虽然仍未观察到滋养层细胞在逆流方向上的显著性迁移现象,但与滋养层细胞单独培养时明显不同的是,共培养后,滋养层细胞的平均迁移速度和X轴向上的绝对位移不再随剪切应力的变化而发生显著性变化,而是几乎保持一致。这些结果暗示,与内皮细胞共培养时,剪切应力作用下滋养层细胞抵抗流体冲击引起位移的抗逆性行为增强,细胞粘附性和在X轴上运动的稳定性增加。同时,在用抗β1型整合素抗体处理后,滋养层细胞在15dyne/cm2剪切应力作用下的平均迁移速度有所减少但在X轴上的绝对位移却有所增加;非特异性对照组的结果与正常对照组的差异不显著。这些结果表明β1整合素在滋养层细胞的迁移过程中起着重要的调节作用。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。本文的研究结果提示我们,滋养层细胞在体内的逆流迁移行为可能并不是一种细胞直接从子宫毛细血管移动到螺旋动脉的逆血流运动,而可能是一种定向分裂/增殖行为。即,在与血管内皮细胞接触时,滋养层细胞通过上调粘附分子的表达而拥有足够强的粘附力对抗血流冲击并稳定地粘附于血管内壁;尔后,细胞通过逆血流方向的定向分裂/增殖,达到逆血流迁移的目的。更多还原

【Abstract】 During the development of human placenta, trophoblast cells (TCs) invasion of, and migration within the uterine vasculature is an essential process for the remodeling of the uterine vasculature, which is important in assuring an adequate blood supply to the developing fetal-placental unit. In vivo, the courses 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, a great amount of studies have been carried out. However, little has been known about the hemodynamic mechanism of TC migration against the blood stream. Apparently, local blood flow environment must be of paramount importance and involved in regulating biological behaviors and functions of TCs in the migratory process of TCs within the uterine vasculature. The effects of environmental factors on cell division/proliferation, differentiation, apoptosis and motility have become one of the hottest topics in cell biology and biomechanics. Using a 2-D flow chamber technique to mimic the local blood flow environment of the uterine vasculature, the present thesis studied the migratory behaviors of human TCs from the hemodynamic point of view and tried to understand what decides the direction of TC motility and what is the power or mode of TC motility against the blood stream. Meanwhile, the correlation between integrinβ1 and the migratory behaviors of TCs was analyzed on the adhesion molecule level.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, the effect of the flow-induced shear stress on the motility of TCs cultured on collagen I was studied. The results demonstrated that TCs were highly dynamic with continuous non-directional or dominant shifting with little net displacement in the direction of flow (x-coordinate) under the static conditions (0 dyn/cm2). Average cell migration velocity and net displacement of TCs in the direction of flow increased almost linearly with increasing shear stress. It was also found that the displacement of TCs in the perpendicular direction of flow is not zero. Remarkably, the velocity ratio, Vx/Vy was slowly decreased and the motility of TCs was stable with the cells’own initiative during the 6-12h period. Changes of cell membrane fluidity and interdiction of flow-sensitive ion channels regulated the migration of TCs under different shear stresses was studied. These results demonstrated that shear stress regulated the motility of TCs, however the migration direction of TCs was not against the flow direction but generally in the direction of flow.Then, the migration of TCs co-cultured with HUVECs under different shear stresses was studied. The results revealed that the average velocity and the absolute displacement of TCs in the direction of flow remained essentially unchanged at all levels of shear stress. Again, no apparent counter-flow migration of TCs was observed. These results suggested that the interaction between TCs and endothelial cells up-regulated the adhesion of TCs, hence enhanced their resistance against the displacement by flow. Meanwhile, under the shear stress of 15dyne/cm2, when monoclonal mouse anti-human integrinβ1 was used to block the action of integrinβ1, the average migration velocity of TCs was significantly reduced and their absolute displacement (x-coordinate) was increased when compared with the control group. However, there was no significant difference between the nonspecific antibody group and the control group. These results suggested that integrinβ1 played an important role in regulating the migration of TCs co-cultured with endothelial cells.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.In conclusion, the present in vitro study demonstrated that by contact with endothelial cells, the adhesion force of human TCs might be regulated according to local flow environment to ensure a stable adhesion of TCs in uterus spiral arteries. So long as the adhesion force of the TCs can resist displacement by blood flow, there is a reason to believe that migratory trophoblasts may reach the spiral arteries not by the actual movement of the cells but by a mechanism of directional cell division/proliferation.更多还原