| Melanoma is a malignant tumor with strong metastatic ability and extremely high lethal rate.Ultraviolet rays are the most important exogenous carcinogen in the formation of melanoma.Recent studies have shown that the development of tumors is not an independent event,and the interaction between tumors and interstitial cells in the tumor microenviro nment plays an important role in the development of tumors.Exosomes are nano-sized vesicles actively secreted by cells,with a diameter of about 50-150 nm,and have a lipid bilayer membrane structure,which can be released from different cell types and contain complete m RNA,mi RNA,protein,lipid Qualitative molecules.Tumor exosomes can mediate the transfer of these information molecules from tumor cells to normal interstitial cells and play a variety of cancer-promoting functions.Therefore,tumor exosomes are considered to be an intercellular transmission medium for carcinogenic information and can mediate tumors.The regulatory effect of cells on the tumor microenviro nment.Fibroblasts are the main components of the tumor microenviro nment.A large number of studies have shown that fibroblasts in the tumor microenviro nment undergo matrix remodeling by secreting MMP2 and MMP9,which is conducive to tumor progression,but how tumor cells achieve this effect has not been reasonably explained.There are also reports that high-metastatic melanoma cells can transfer their invasion potential to low-metastatic melanoma or bone marrow progenitor cells through exosomes.Therefore,we propose a scientific hypothesis: exosomes secreted by melanoma cells may be used as an important information transmission medium to transfer the invasion potential of melanoma cells to MEF cells,and promote the expression of MMP2 and MMP9 in MEF cells and the change of invasion ability.This topic revolves around this speculation and carried out two parts of research: the first part is to extract and identify exosomes produced by B16F10 cells;the second part is to observe the uptake of exosomes by MEF through fluorescence confocal,and through Western blot,immune cells Chemistry,q RT-PCR and Transwell experiments were used to detect the expression of MMP2 and MMP9 protein and m RNA in MEF cells,as well as changes in MEF invasion ability.Objective 1.Use ultracentrifugation to prepare exosomes secreted by mouse melanoma B16F10 cells,and identify their structural characteristics,particle size distribution and marker proteins.2.Through fluorescence confocal observation,deter mine whether B16F10 exosomes can be taken up by MEF cells;use B1F10 exosomes to treat MEF cells,and use Western blot,immunocytochemistry,q RT-PCR methods to verify the exosomes to MEF cells MMP2,MMP9 The effect of expression.Finally,through the Transwell test to detect whether exosomes can cause changes in the invasion ability of MEF cellsMethods 1.Use ultracentrifugation to extract the exosomes in the melanoma cell B16F10 medium,observe the morphological structure of the exosomes obtained by electron microscope negative staining,use a particle size analyzer to detect the size distribution of the melanoma exosomes,and pass Western The expression of TSG101 and TYRP2 were detected by blotting.2.Extract mouse embryonic fibroblasts(MEF)from pregnant mouse embryos,incubate MEF cells with melanoma exosomes labeled with PKH26 fluorescent dye,and observe the uptake of exosomes by MEF cells using fluorescence confocal,and the flow cytometer was used to detect the uptake rate of exosomes by MEF cells.Subsequently,Western blot was used to detect different concentrations(0.31 μg/m L,0.62 μg/m L,1.25 μg/m L,2.50 μg/m L,5.00 μg/m L)and different treatment times(0 h,12 h,24 h,36 h,48 h)After co-cultivation of exosomes with MEF cells,the expressions of MMP2 and MMP9 in MEF cells changed to deter mine an optimal exosome concentration.Finally,immunocytochemistry verified the expression changes of MMP2 and MMP9 in MEF under the optimal exosome concentration;q RT-PCR detected the m RNA expression of MMP2 and MMP9 in MEF under the optimal exosome concentration;The Transwell experiment detected the change of MEF cell invasion ability under the optimal exosome treatment conditions.Result 1.Electron microscopy results show that the exosomes secreted by melanoma cells have a round membranous vesicle structure,with a three-dimensional sense and uniform size,and the outside is a clearly visible bilayer lipid molecular membrane,which generally shows a saucer shape,and such vesicles conform to the typical morphological characteristics of exosomes.2.By observing the uptake process of exosomes,it was found that the cytoplasm of MEF cells began to show red fluorescence after melanoma cell treatment for 12 h,and began to gather around the nucleus after 24 h.At 48 h,there was obvious red fluorescence around the nucleus.Flow cytometry showed that the positive rate of MEF cell uptake after B16F10 exosome treatment gradually increased with time,and reached the highest value at 48 h.Western blot results showed that under relatively low exosome concentrations(0.31 μg/m L,0.62 μg/m L),the expression of MMP2 increased significantly,and then gradually decreased with the increase of exosome concentration.The expression level of MMP9 gradually increased with the increase of exosome concentration until the exosome concentration reached 2.5 μg/m L,and then the expression decreased.The results of 0.62 μg/m L exosome treatment of MEF cells at 0 h,12 h,24 h,36 h,and 48 h showed that after 48 h of exosome treatment,the expression levels of MMP2 and MMP9 in MEF cells were significantly different.Immunocytochemistry verified this conclusion under the best conditions(0.62 μg/m L,48 h).The results of q RT-PCR showed that after 48 h of MEF cells treated with 0.62 μg/m L exosomes,MMP9 m RNA expression was 1.44 ± 0.14 times that of the control group,and MMP2 m RNA expression was 1.32 ± 0.10 times the control group.Transwell test found that the invasion ability of MEF cells in the exosome-treated group(104.7 ±3.8)was significantly higher than that in the control group(69.4 ± 1.3).Conclusions This study is successfully extracted and identified exosomes produced by melanoma B16F10 cells,and proved that B16F10 exosomes can be taken up by MEF cells,resulting in significantly enhanced expression of MMP2 and MMP9.The optimal condition for B16F10 exosomes to treat MEF is 0.62 μg/m L co-culture for 48 h.In addition,according to this optimal treatment condition,we have fully verified the experimental results from the three aspects of protein,m RNA expression and MEF invasion ability.This study proves that melanoma exosomes can promote the expression of MMP2 and MMP9 in MEF cells.From the perspective of exosome-mediated cell-to-cell communication,verifying that melanoma has an invasive communication method through exosomes to normal cells provides a certain research basis for seeking ways to control tumor invasion and metastasis. |
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