| Mitochondrial transplantation is a new technology developed in recent years.Healthy mitochondria can be injected directly into the damaged site or its upstream blood vessels to play a therapeutic role.It has been studied in many animal models of various diseases,such as myocardial ischemia,cerebral stroke,liver and lung injury,and even has been successfully used in the treatment of childhood heart disease.In view of the fast onset action,mitochondrial transplantation can quickly improve tissue function within a few minutes after injection,it is hard to be explained by the whole mitochondria transplant to the damaged sites within such short period of time,for it has to take a related much longer time(such as an hour or more)because mitochondria should pass through blood vessel walls,tissues,intercellular spaces,and cell membranes to reach and enter cells for function.Therefore,it is questioned that how does mitochondrial transplantation onset action so fast.Does it really because the whole mitochondria been transplanted into the damaged cells?This is what we want to clarify in this thesis,and thus propose that some mitochondrial component rather than the whole mitochondria may function to the mitochondrial transplantation.To test this hypothesis,we established a much simple UV-irradiated cell model instead of the more complex animal models,and applied cell biological and molecular biological methods to explore the mechanism of mitochondrial transplantation.Materials and methods: HeLa cells were irradiated with UVC,which is commonly used in laboratory for sterilization,so the UVC irradiation-damaged HeLa cell model was established.Healthy mitochondria isolated from HeLa cells without irradiation were then co-incubated with the irradiated cells for mitochondrial transplantation.(1)Cell morphology changes before and after mitochondrial transplantation were observed by microscope.(2)Clone formation rate assay was used to evaluate the effect of mitochondrial transplantation on cell clone formation ability.(3)SRB(Sulforhodamine B)method was used to study the effects of different concentrations of mitochondria for mitochondrial transplantation on cell proliferation.(4)Fluorescent probes Hoechst 33342 and PI double staining method were used to observe and evaluate cell viability using a research-level inverted fluorescence microscope,and cell survival rate was calculated with Image J software to simplify the evaluation method of mitochondrial transplantation effect.(5)Flow cytometry assay was used to detect mitochondrial membrane potential,apoptosis and cell cycle.(6)Western blotting assay was used to detect the protein expression of apoptosis signaling pathway.(7)Different treatments,including heat inactivation,ultrasonic crushing and repeated freeze-thaw,were evaluated to explore the effective mitochondrial components which may play a protective role in the process of mitochondrial transplantation.(8)Structural integrity of mitochondria was evaluated through detecting malate dehydrogenase(MDH)release or using transmission electron microscopy.Results:(1)Mitochondrial transplantation significantly improved the morphology of UVC irradiation damaged HeLa cells.(2)All the results of clone formation rate,SRB,Hoechst 33342 and PI double staining strongly proved that mitochondrial transplantation exhibited a significant protective effect on UVC irradiation damage.(3)SRB results also identified that a 6.25 μg/m L is the optimal mitochondria concentration for mitochondrial transplantation under the experimental system established in this study.(4)Mitochondrial transplantation improved the mitochondrial membrane potential,cell cycle and apoptosis of UVC irradiated cells.(5)Mitochondrial transplantation decreased the expression and levels of NF-κB,p-p65 and activated caspase3 proteins through the NF-κB signaling pathway and caspases-dependent pathway.(6)No anti-UVC irradiation effect could be observed when mitochondria were treated with thermal inactivation and ultrasonic crushing treatments,while repeated freezing and thawing treatment still retained some effect.(7)Denied this hypothesis that mitochondrial component may function to the mitochondrial transplantation,and excluded the function of mtDNA,protein,lipids,ATP and other small molecules.(8)The structural integrity of mitochondria is a key factor in the anti-UVC irradiation effect of mitochondrial transplantation.Conclusion: Mitochondrial transplantation has anti-UVC irradiation effect,and the effect of mitochondrial transplantation is not determined by some active components of mitochondria,but the structural integrity is the key factor to determine the effect of mitochondrial transplantation.Our findings answered the question,"Is mitochondrial transplantation really a mitochondrial transplant?" And more works need to be done to illustrate how do the whole integrated mitochondria can help cells to repair the UVC irradiation damages. |
