摘要
随着稀土应用的日益广泛,特别是在我国稀土已大量应用于农业和饲养业,稀土正在越来越多地进入环境,进入食物链,因而对其生理、毒理、药理学的研究引起了广泛关注。同时,其生物效应的研究一直是个热点。稀土化合物具有多种生物学活性和药物活性,但其确切的作用机制尚未完全弄清楚,已限制了稀土合理安全的应用。离子通道在非兴奋性细胞的分化、生长发育中具有重要作用,然而,对稀土影响非可兴奋细胞增殖与离子通道的关系的研究却很少。因此探讨离子通道与细胞增殖分化的关系及其作用机制具有重要意义。本文利用全细胞膜片钳技术研究了稀土镧和镱对培养的大鼠成纤维NIH3T3细胞和大鼠成骨MC3T3细胞膜外向钾通道电流和动力学过程的作用、利用细胞生物学方法研究了镧和镱对培养的大鼠成纤维NIH3T3细胞和大鼠成骨MC3T3细胞增殖、凋亡和细胞周期的影响,试图揭示稀土影响钾通道在细胞增殖过程中的作用及其分子机制。主要研究结果如下：
MTT法和流式细胞仪检测结果表明：(1)La3+可显著促进成纤维NIH3T3细胞的生长,且具有一定的时间和浓度依赖性特征。100州的La3+作用72小时后增殖率可达到46.7%。(2)与此同时,Ca2+也可时间和浓度依赖性地促进NIH3T3细胞增殖,并且La3+和Ca2+的促增殖作用具有协同效应。这些说明镧离子对NIH3T3细胞的促增殖作用与钙离子相关,钙离子对细胞增殖是必不可少的。(3)La3+同样促进成骨MC3T3细胞的增殖,具有一定的时间和浓度依赖性。50μM的La3+作用72小时后对细胞的增殖率可达到38.7%。(4)与La3+相反,Yb3+抑制成纤维NIH3T3细胞的生长,且具有一定的时间和浓度依赖性特征。1mM的Yb3+作用NIH3T3细胞72小时后抑制率可达到57.2%。(5)而Yb3+对成骨MC3T3细胞的生长基本上没有太大的影响。此外,(6)在所选用的浓度和作用时间下,稀土La3+和Yb3+对NIH3T3细胞和MC3T3细胞凋亡没有明显的影响。
流式细胞仪检测细胞周期结果表明：(1)不同浓度的La3+作用NIH3T3细胞72h后,与对照组相比,La3+使Gl期细胞数量明显减少,S期细胞数明显上升,并且具有一定的时间和浓度依赖性。100μM的La3+使S期细胞数从6.6%上升到26.1%。说明La3+通过促进G1／S细胞进程而促进细胞增殖。(2)不同浓度的Ca2+；不同浓度的La3+和5mM的Ca2+的细胞周期结果与对照组相比,Ca2+使G1期细胞数量有一定的减少,S期细胞数有一定的上升,但不是很明显；La3+和Ca2+使G1期细胞数量有更明显的减少,S期细胞数有更明显的上升。结果提示镧和钙具有协同效应,1000M的La3+和5mM的Ca2+使S期细胞数从26.1%上升到30.6%。(3)不同浓度的La3+作用MC3T3细胞72h后,稀土La3+对MC3T3细胞周期没有明显的影响,这说明La3+促NIH3T3细胞和MC3T3细胞增殖的作用机制不完全一致,可能是由于不同类型的细胞差异造成的。(4)不同浓度的Yb3+作用NIH3T3细胞72h后,对NIH3T3细胞周期的影响不显著,使G2/M细胞的百分数略微有所增加,结合其对NIH3T3细胞增殖的作用表明,Yb3+可能使细胞阻滞于G2/M期,从而抑制细胞的生长。(5)不同浓度的Yb3+对其细胞周期的影响与对照组相比没有差异性。
全细胞膜片钳研究结果Ⅰ：(1)NIH3T3细胞外向钾通道电流具有内钙依赖性特征,其半数增大浓度EC50为252 nmol/L。但与兴奋性细胞相比较,其电流密度要小得多。这可能是非兴奋性NIH3T3细胞上钾通道数目少或者是钾通道开放几率小的缘故所造成的。(2)镧和镱这两种稀土离子均浓度依赖性地抑制大鼠成纤维NIH3T3细胞外向钾通道电流,EC50值分别为1.58和10.63μmol/L,因为EC50值越小即表示抑制作用越强,所以稀土离子镧对大鼠成纤维NIH3T3细胞外向钾通道电流的抑制作用比镱离子的要强。(3)通过镧和镱这两种稀土离子对钾电流抑制作用的动力学分析,我们发现镧离子使钾电流的半数激活电压向正电位方向移动16mV,使钾电流的半数失活电压向负电位方向移动9.48mV。相反,镱离子使外向钾电流的激活曲线向负电位方向移动11.41mV,而对失活曲线基本上没有影响。激活曲线右移使得激活过程推迟,电流减小；失活曲线左移使得失活过程提前,电流也减小,这是镧离子抑制作用强于镱离子的一个重要原因。
全细胞膜片钳研究结果Ⅱ：(1)MC3T3细胞外向钾通道电流具有内钙依赖性特征,其半数增大浓度EC50为39.8nmol/L。(2)镧离子浓度依赖性地抑制MC3T3细胞钾通道电流,其半抑制浓度EC50值为8.23μmol/L。(3)通过镧离子对钾电流抑制作用的动力学分析,我们发现LaCl3使钾电流的半数激活电压向正电位方向移动21mV,使半数失活电压向负电位方向移动9.66mV。(4)镱离子对钾电流及其动力学过程都没有明显的影响。这些结果说明,稀土镧和镱对大鼠成骨MC3T3细胞外向钾通道电流及其动力学的作用不相同。
总之,论文采用了MTT, FACS流式细胞仪研究了两种稀土离子La3+、Yb3+对NIH3T3和MC3T3细胞增殖、细胞周期及凋亡的作用；采用全细胞膜片钳技术,首次研究了稀土离子La3+、Yb3+对NIH3T3和MC3T3细胞外向钾电流大小及激活和失活动力学的影响,从通道水平阐明了钾通道在细胞生长中的重要作用。通过本文的研究进一步揭示了稀土的生物效应及其分子机制,对深入了解稀土生物效应的作用机制及其潜在的药用价值具有重要意义。
With the widely used of rare earth elements, especially in agriculture and stock breeding in china, they are more and more entered into entironment and food cycle. So the researches of their physiology, toxicology and pharmacology are extensively interested. Researches on their biological effects have been a hot spot. Lanthanide compounds display a variety of biochemical and physiological effects, but the exact mechanism is not clear. it is limit to reasonably and safely use them. Ion channels play an important role in the processes of cell differentiation and upgrowth in non-excitable cells. So it is significant to study the relationship and it's mechanism between ion channel and cell proliferation. Effects of two lanthanide ions of La3+and Yb3+ on potassium currents and it's kinetics in non-excitable NIH3T3 cells and MC3T3 cells were investigated using the whole-cell voltage-clamp technique, and influences of La3+and Yb3+ on cell proliferation, cell apoptosis and cell cycles were studied using cell biological methods. We aim at posting some evidence to reveal the function and it's molecular mechanism of the effect on potassium channel by rare earth elements. The results show as below:
Cell proliferation and cell apoptosis in NIH3T3 cells and MC3T3 cells were mensurated by MTT assay and by flow cytometer. (1) La3+observably increased cell proliferation of NIH3T3 cells in a dose-and time-dependent manner, with a 46.7% increase in 100μM La3+treated cells for 72 h. (2) At the same time, Ca2+can also promote proliferation of NIH3T3 cells. However, it is lower than that of La3+'s. Furthermore, there have synergistic effects between La3+and Ca2+on increasing proliferation. All these results showed that exposure to La3+was able to increase NIH3T3 cell growth which was correlated with Ca2+and Ca2+is absolutely necessarily in cell proliferation. (3) La3+also increased cell proliferation of MC3T3 cells in a dose-and time-dependent manner, with a 38.7% increase in 50μM La3+ treated cells for 72 h. (4) Oppositely, Yb3+ inhibited observably cell proliferation of NIH3T3 cells in a dose-and time-dependent manner, with a 57.2% increase in 1mM Yb3+treated cells for 72h. (5) Yb3+had no obvious effect on cell proliferation of NIH3T3 cells. (6) In addition, under research condition of time and dose, La3+and Yb3+had no obvious effects on apoptosis of NIH3T3 cells.
Cell cycles in cultured NIH3T3 cells and MC3T3 cells were assessed by flow cytometer. (1) La3+observably decreased the percentage of cells in G1 phase and increased the percentage of cells in S phase of NIH3T3 cells in a dose-and time-dependent manner, with 6.6% increase to 26.1% in 100μM La3+ treated cells. These showed that La3+ promoted cell proliferation of NIH3T3 cells through promoted G1/S cell cycle progression. (2) At the same time, Ca2+also can decreased the percentage of cells in G1 phase and increased the percentage of cells in S phase of NIH3T3 cells. However, it is not observably. Furthermore, 100μM LaCl3 and 5mM CaCl2 increased the percentage of cells in S phase of NIH3T3 cells from 26.1% to 30.6%. These indicated that La3+and Ca2+have synergistic effect on increase in the percentage of cells in S phase. (3) La3+had no visible effect on cell cycle of MC3T3 cells with different concentration of La3+treated cells for 72h. These results showed that there was different mechanism of La3+promoted cell proliferation between NIH3T3 cells and MC3T3 cells induced by different cell style. (4) The effect of Yb3+on cell cycle of NIH3T3 cells is slender, with little increase in the percentage of cells in G2/M phase. These showed that Yb3+inhibited cell proliferation through promoted G2/M cell cycle progression. (5) Yb3+had no obvious effect on cell cycle of MC3T3 cells.
Outward potassium currents (Ik) in cultured NIH3T3 cells were recorded by whole cell patch-clamp technique. (1) The outward potassium currents of NIH3T3 cells were concentration-dependent of Ca2+ and the EC50 value of Ca2+was calculated to be 252 nmol/L. Contrast to excitable cells, the currents density are very small. These may be the reason of non-excitable cell few channel or little open rate. (2) La3+and Yb3+all inhibited the outward potassium current in a concentration-dependent manner and EC50 was 1.58 and 10.63μmol/L respectively. The inhibition effect of La3+was stronger than that of Yb3+ due to his small inhibition EC50. (3) The kinetics of blockade of La3+and Yb3+on IK indicated that the activation curves was shifted by La3+to positive potential for 16mV and the inactivation curves was shifted to negative potential for 9.48mV; contrarily, the activation curves was shifted by Yb3+to negative potential for 11.41mV and the inactivation curves no changed. The suspendment of activation and the advance of inactivation all decreased current intensity. This was the main reason of La3+inhibition stronger than that of Yb3+.
Outward potassium currents (IK) in cultured MC3T3 cells were recorded by whole cell patch-clamp technique. (1) The outward potassium currents of MC3T3 cells were concentration-dependent of Ca2+and the EC50 value of Ca2+was calculated to be 39.8 nmol/L. (2) La3+inhibited the outward potassium current in a concentration-dependent manner and EC50 was 8.23μmol/L. (3) The kinetics of blockade of La3+indicated that the activation curves was shifted by La3+to positive potential for 21mV and the inactivation curves was shifted to negative potential for 9.66mV. (4) The activation curves and the inactivation curves by Yb3+had no changed. These showed that it is a different mechanism between La3+and Yb3+on K+currents and its kinetics.
In summary, in this dissertation, it has been researched the effects of La3+ and Yb3+on cell proliferation, cell apoptosis and cell cycles of NIH3T3 cells and MC3T3 cells; it has been studied, for the first time, the effects of La3+ and Yb3+on potassium currents and its kinetics of activation and inactivation in NIH3T3 cells and MC3T3 cells by whole cell patch-clamp technique. It is demonstrated that potassium channel plays a crucial roles in cell growth from channel level. In conclusion, our experiments have revealed the rare earth element biology effect and their molecular mechanism further. These results are significant for the further understand biology effect of rare earth element and its potential used in medicine.
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