The Study Of Intracellular Calcium Regulation In Rat Bone Marrow Mesenchymal Stem Cells After Treatment With Stimuli Related To Cell Proliferation

Objective: Bone marrow mesenchymal stem cell (MSC) is a kind of adult stem cells derived of the bone marrow stroma in mammal animals. Many evidences showed that MSCs are self-renewal potential to divide symmetrically or asymmetrically and potential to differentiate into multilineage cells. The mechanism that regulates MSC proliferation and differentiation is current focus and challenge in MSC studies. And as a highlight, the intracellular calcium signals are involved in many physiological and pathological processes indispensably. Nevertheless, how calcium signal participate in MSC proliferation is unclear. In this experiment, some physical and chemical stimuli were separately applied to promote or inhibit MSC proliferation. Meanwhile, in the proliferation regulation, how intracellular calcium signal responds to and involves is studied.Methods: Firstly, primary SD rat MSCs were separated, purified and cultivated, then the subcultured ones were identified. Secondly, with 0.1% fetal bovine serum starvation and nocodazole treatment, MSCs from passage 3 to 5 were synchronized within the G₁ phase during the cell cycle.Thirdly, the G₁ ones were treated separately with 10% fetal bovine serum (FBS) in short time and long time, then the relative concentrations of intracellular calcium were examined through fluorescence microscopy. In the same way, 15 ng/ml epidermal growth factor (EGF) was used to stimulate MSCs. To suppress the proliferation, 10μg/ml Mitomycin C was added for short time and discarded 2.5 hours later to confine cells to the condition of long time division suppression, accompanying with calcium measurement. Lastly, MSCs were designed to different shapes in a certain area with micropattern technique. The effects of shapes and sizes on MSC proliferation were tested and then intracellular calcium change was detected in this condition.Results: MSCs were sufficiently confined in G₁ phase. When MSCs were stimulated by chemical factors such as FBS and EGF which could encourage cells to divide, they showed an ability of quick proliferation and intracellular calcium concentration climbed up to the highest level, then dropped down, at last kept at a higher degree with obvious calcium fluctuations than untreated MSCs. After the treatment of inhibitory chemical drug such as mitomycin C, MSC division was inhibited and intracellular calcium concentration ascended transiently then declined immediately and maintained at a lower level compared to untreated ones and the level didn't show a strong oscillation. When MSCs encountered some physical factors, such as controlled in several types of geometrical shapes with an area of 900μm², their propagation was partly repressed. Contrasted with unpatterned MSCs, the concentration of intracellular calcium sustained at a lower degree and at a weak fluctuation. Especially for round shape cells, they hardly had any fluctuatation.