Caspase-3 Mediated Cyclic Stretch Induced Apoptosis In Myoblast In Vitro

Myoblasts are the precursor cells of muscular cells and belong to the skeletal muscle maternal cell line with myofilament in the endochylema. The cell differentiation process is from mesenchymal to myoblast and then to matured muscle cell. Cardiac muscle tissues and smooth muscle tissues in matured individuals do not contain myobalsts while myoblasts in skeletal muscle tissues are in the form of muscle satellite cells. In physiological conditions, muscle satellite cells are located under muscle fiber basal membrane. When skeletal muscle is injured, they will be actived and propagate, differentiate into tissue-specific cells and finally into new muscle fibers. Then the regeneration process of injured fibers is fulfilled. Differentiation process of skeletal muscle precursor cells is as follows: muscle originated stem cells, silent muscle satellite cells, myoblasts, mucle cells. According to Blau's research, myoblasts from embryo can differentiate 60-70 times and myoblasts from matured individuals can differentiate 45 times. Both are enough for researches on cellular transplant and related tissue engineering.Apoptosis plays important roles in healthy living of multicell creatures as well as generation and differentiation, but researches on apoptosis, especially caused by cyclic stretch, were much less. In the process of embryonic development, apoptosis can eliminate the excessive cells that have already fulfilled their duties and guarantee a normal embryonic development. In the developed stage of organism, apoptosis can eliminate the aged and sick cells to keep the organism healthy. Up to now, though great progresses had been made, too much are still unknown. Elucidating the mechanism of apoptosis is essential not only to organism development, tumor control and immunomodulation, but also to curing stubborn disease in clinic.Caspase belongs to cysteine proteinase family and the family has 14 members. They are essential to apoptosis. Once activated, they can initiate protein degradation and cell apoptosis inreversibly. Their main function is to cut substrate at the site behind aspartate. Caspase-3 is one of the most important members of the family not only because its core position in apoptosis process, but also its extensive substrates and multifunction. They widely exist in many tissues and cells in form of proenzyme. Caspase-3 proenzyme is composed of one large-subunit and one small-subunit and only after being cut and activated can Caspase-3 perform its function. During the process of apoptosis conducted by Caspase, one of the most important substrates of Caspase-3 is PARP (Poly ADP-Ribose Polymerase) and PARP can be divided into several subgroups: PARP-1, PARP-2, PARP-3, Vault-PARP and Tankyrases (TANK-1, -2). Of all these subgroups, function of PARP-1 is the most important. The molecular weight of PARP-1 is 116kDa. When apoptosis happens, PARP-1 can be decomposed by caspase-3 and the 85kDa breakdown product is an early marker of apoptosis.In this experiment, we evaluated the role of Caspase-3 in myoblasts apoptosis induced by cyclic stretch. First we investigated the relationship between cyclic stretch and apoptosis. 10% or 20% surface elongation was forced on myoblasts with cyclic stretch for 12 or 24 hours. Then we detected the apoptotic extents of myoblasts immediately after stretch to determine how magnitudes and time of cyclic stretch acted on myoblasts. We used transmission electron microscope to observe the form of apoptotic cells and used flow cytometer to count the percentage of apoptotic cells of each team. Second, we investigated the effects of Caspase-3 in myoblasts apoptosis. We detected the activity of caspase-3 to see whether this process was conducted by it. PARP is one of the most important substrates of caspase-3. In this experiment we detected changes of PARP mRNA by means of RT-PCR and PARP protein by means of Western-blot. From these results we could determine the activity of caspase-3 indirectly. Caspase-3 inhibitor was also been used to see what effect it had in the apoptosis process. So, we divided this experiment into four parts.Part 1. Stretch protocol research on myoblasts in vitro: this part mainly focuses on investigating proper mode, magnitude and time for cell stretch and making preparations for the following experiment. According to pertinent literatures and requirements of this experiment, we selected cyclic stretch as stretch mode, 10% or 20% surface elongation as stretch magnitude, 12 or 24 hours as stretch time with non-stretch team as control.Part 2. Effect of cyclic stretch on myoblasts cultured in vitro. In this experiment we used transmission electron microscope to observe histological characters of apoptotic cells after stretch and used flow cytometer to count the apoptosis percentage of each team. Then performed statistical analysis. The results showed: cyclic stretch could induce cell apoptosis in a magnitudes and time dependent way.Part 3. Research on mechanisms of myoblasts apoptosis caused by cyclic stretch in vitro. PARP is one of the most important substrate of Caspase-3 in apoptotic process. In this experiment, we detected the changes of PARP mRNA and PARP protein to determine the effect of Caspase-3 in this apoptotic process. The results showed: apoptosis caused by cyclic stretch is conducted by Caspase-3 and this is a post-transcriptional regulation process.Part 4. Effect of Caspase-3 on myoblasts apoptosis caused by cyclic stretch in vitro. In this experiment, we added Caspase-3 specific inhibitor z-DEVD-fmk in each experimental team, then counted the apoptotic cells and detected the Caspase-3 activity. The results showed: Caspase-3 inhibitor could block this apoptotic process partly, not thoroughly.