| Part? The extraction and function of exogenous mitochondriaObjective: Studies have found that extracted mitochondria from cells or tissue can keep its original activity and function, and maintain its original integrity. Also some studies have shown that mitochondria separated from the cell lines of the rodents could be injected into homologed oocytes, or one-cell embryos. Here,Plasmid that encoded a fusion of Discosoma sp. Red fluorescent protein and the mitochondrial targeting sequence from subunit VIII of human cytochrome C oxidase(p Ds Red2-Mito) was transfected into Chinese Hamster Ovary(CHO) cells, from which mitochondria extracted with specific markers were used to observe their selectivity to enter the primary cultured cells in CNS.Method: Using the recombinant plasmid p Ds Red2-Mito to transfect and monoclone CHO cells, to build the cell linesexpressing mitochondria with red fluorescence stably(Mitochondria-CHO, Mito-CHO). Fluorescence detected the expression of mitochondria in the Mito-CHO cells. In addition, extract mitochondria from tissues, then liver mitochondria were marked by Mito Tracker? Deep Red FM. JC-1(fluorescent probe to detect mitochondrial membrane potential) kit was used to analyse mitochondrial membrane potential changes after extracted from Mito-CHO. Fluorescence observed the specificity that exogenous mitochondria from different sources entered the primary cultured cells in CNS. Layer scanning with laser confocal microscope was conducted further to investigate wether mitochondria entered rat cortical astrocytes.Results: Fluorescence revealed that Mito-CHO cell lines could fully express red fluorescence labeled mitochondria; and Mito Tracker? Deep Red FM could be used to mark the mice liver mitochondria. And we found that mitochondrial membrane potential after extracted(Mito: 106.00 ± 13.70 %, n = 3, p > 0.05 vs control) had no difference from CHO cells(Con: 100 ± 12.64 %, n = 3), which was just to say that the mitochondrial membrane potential after extracted was normal. Then we found that mitochondria extracted from Mito-CHO cells and mice liver both could enter the rat cortical astrocytes. While mitochondria extracted from Mito-CHO cells could selectively enter cortical astrocytes and microglia, but not enter cortical and hippocampal neurons. Layer scanning showed that exogenous mitochondria were intracellular rather than extracellular or on the cell membrane after entering cells.Conclusion: Mitochondrial functionafter extracted from Mito-CHO cells had no change. And exogenous mitochondria could selectively enter cortical astrocytes and microglia, but not enter cortical and hippocampal neurons.Part ? The effects of exogenous mitochondria on rat cortical astrocytesObjective: Mitochondria are involved in astrocyte ATP synthetic, ion buffer, as well as glutamic acid oxidation. Also psychiatry disorders such as depression, bipolar disorder, autism and schizophrenia were closely related to mitochondrial morphology variation and function injury. Here we observed the effects of exogenous mitochondria on the activity and function of astrocytes.Methods: Fluorescence observed the proper concentration and time of mitochondria extracted from Mito-CHO cells into rat cortical astrocytes. 3-(4,5-dimethyl-2-thiazolyl)-2,5-Diphenyl-2-H-tetrazolium bromide(MTT) method was used to detect the proper incubation conditions for H2O2 to injury astrocytes. JC-1 kit was employed to analyze the changes of overall mitochondrial membrane potential after exogenous mitochondria into normal and H2O2 injuried astrocytes. Also MTT method was used to detect the effects of H2O2 on the livability of injuried astrocytes. Td T-mediated d UTP Nick-End Labeling(TUNEL) method was used to analyze the effects of exogenous mitochondria on the apoptosis of normal and H2O2 injuried astrocytes. Cell migration experiment was used to detect the protective effects of exogenous mitochondria on scratch astrocytes.Results: Fluorescence to observe the concentration and time dependence of exogenous mitochondria into astrocytes found that 24 ?g / 1×106 cells should incubate for 24 h. We chose that 400 ?M(Con: 100 ± 13.40 %; 400 ?M: 48.95 ± 5.40 %, n = 5, p < 0.01 vs control) H2O2 treated astrocytes for 1 h(Con: 100 ± 21.92 %; 1 h: 38.97 ± 13.12 %, n = 7, p < 0.05 vs control) by MTT method. Then we found that exogenous mitochondria had no effects on overall mitochondrial membrane potential of normal(Con: 100 ± 6.16 %; Con+Mito: 92.35 ± 2.56 %, n = 3, p > 0.05 vs control) and H2O2 treated(H2O2: 78.02 ± 7.36%, n = 3, p < 0.01 vs control; H2O2+Mito: 78.89 ± 13.65 %, n = 3, p > 0.05 vs H2O2) astrocytes after exogenous mitochondria into cells by JC-1 method. Also MTT method observed that exogenous mitochondria had no effects on the activity of astrocytes(Con: 100 ± 20.35%, Con+Mito: 95.66 ± 28.35%, n = 5-6, p > 0.05 vs control; H2O2: 90.07 ± 18.76 %, n = 6, p < 0.05 vs control; H2O2+Mito: 83.94 ± 15.51 %, n = 6, p > 0.05 vs H2O2); the parallel operation that exogenous mitochondria incubated for 24 h, resulted in cell activity of H2O2 group down a bit, perhaps because that cell activity in 24 h reversed slightly. And TUNEL method observed that exogenous mitochondria had no impact on the apoptosis of normal astrocytes(Con: 100 ± 33.56 %; Con+Mito: 107.78 ± 15.40 %, n = 3, p > 0.05 vs control), but could obviously decreased the apoptosis of H2O2 injured astrocytes(H2O2: 1316.4. ± 115.47 %, n = 3, p < 0.01 vs control; H2O2+Mito: 390.48 ± 128.84 %, n = 3, p < 0.01 vs H2O2). Then we observed that exogenous mitochondria could protect astrocytes from scrath. After scratch 24 h, the number of astrocytes migration(Con: 100 ± 8.73 %; Mito: 157.56 ± 13.89%, n = 3, p < 0.01 vs control) was more than control.Conclusion: After 24 ?g mitochondria with 1×106 cells incubated for 24 h; most of mitochondria could enter astrocytes. Exogenous mitochondria had no effects on overall mitochondrial membrane potential of normal and H2O2 injuried astrocytes after exogenous mitochondria into cells; Also exogenous mitochondria had no impact on the apoptosis of normal astrocytes, but could obviously decreased the apoptosis of of H2O2 injuried astrocytes; As well as that exogenous mitochondria could protect astrocytes from scrath. |
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