Effect Of Exogenous Mitochondria On MPP~+-induced SH-SY5Y Cell Injury

Parkinson's disease?PD?is an age-related neurodegenerative disease,and often characterized by movement disorders in those of middle to old age,such as bradykinesia,resting tremor and postura instability.The lose of dopaminergic neurons is recognized as the pathophysiology of the disease.And the constant festure of PD are Lewy bodies.At present,the drugs for this disease only can improve symptoms,but not completely cured,including levodopa agents,MAO-B?B-type monoamine oxidase?inhibitors,DA agonists and nerve repair agents.Parkinson's disease is associated with environmental and genetic factors,but the precise mechanism that cause PD is unclear.In recent years,a large number of studies have shown that mitochondrial dysfunction is an important reason of Parkinson's disease.It is well known that mitochondria are important organelles in eukaryotic cells,and a number of cellular activity are related to it.What,s more,mitochondria poss its own unique genetic material,and the mitochondrial disfunction lead to many human diseases.However,mitochondria repair therapy offers a new way for the treatment of this disease.When the mitochondria with normal function are internalized by impaired receptor cells,the cell viability can be restored and the mitochondria function of cells are also repaired.Then we isolated and purificated the mitochondria from liver of healthy mice and then incubated with Parkinson's disease in vitro model,to study the effect of mitochondria on the model cell in Parkinson's disease.In this work,the mitochondria are isolated from the liver of healthy Kunming mice,and then co-incubation with the cells of PD model,as a result the cell viability and other biochemical indicators were measured to evaluate the therapy effect.The work is divided into three parts,the first part: the pDsRed-Mito and pECFP-Mito were used to transfection the liver hepatocellular cells?HepG2 cells?to mark the mitochondria,respectively.In order to obtain the stable expression cell line,the lentiviral transfection was used.Then the fluorescent tagged mitochondria of HepG2 cells were isolated,and incubation with SH-SY5 Y cells,the internalize process was observed under confocal microscope.The cell viability and growth curve were also measur ed after incubation for 24 h,48 h and 72 h.The second part: To isolate and purify the mitochondria from the liver of healthy kunming mice,and the mitochondrial particle size was determinated by dynamic light scattering,the electron transmission microscope was used to observated the internal structure of isolated mitochondrial.What,s more the isolated mitochondria were stained with James green B and MitoTrack Red CMXRos respectively,and the mitochondria morphology were observed under fluorescence microscope and oil mirror.The flow cytometry,fluorescence spectrophotometer and ultraviolet spectrophotometer were also used to detected the membrane potential and mitochondria swelling after treatment with JC-1?mitochondrial membrane potential detection probe?and different concentration of CaCl₂.The third part: The 1-methyl-4-phenyl-pyridineion?MPP+,the commonly agent to build the Parkinson's cell model?was used to damage SH-SY5 Y cells,and constructed the Parkinson's cell model in vitro.Afterwards,the mitochondria isolated from liver of Kunming mice were incubated with Parkinson's disease in vitro model cells,and alamar blue was used to detected the cell viability,TEM results showed that after treated with MPP+,the mitochondria in SH-SY5 Y cells were been swelling,cavitation and burst.When incubated with mitochondria for three days,the most mitochondria in SH-SY5 Y cells emerged normal form,the cell apoptosis and necrosis were decreased,and intracellular reactive oxygen species?ROS?levels decreased,adenosine triphosphate?ATP?content increased,and the mitochondrial membrane potential restored.The results showed that the exogenous mitochondria could be internalized SH-SY5 Y cells,and the mitochondria isolated from the mouse liver had certain activity,and had some therapeutic effect on PD in vitro model,which might provide a new novel therapeutic approach.