Application of MSC (mesenchymal stem cell) exosomes in preparation of pharmaceutic preparation for treating PF (pulmonary fibrosis)
A technology of mesenchymal stem cells and pharmaceutical preparations, applied in the application field of mesenchymal stem cell exosomes in the treatment of pulmonary fibrosis, can solve the problems of non-expression and no detection of MSC-specific surface markers, and achieve simple methods and low preparation costs Inexpensive, the effect of improving the survival rate
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Embodiment 1
[0033] Isolation and identification of exosomes from mesenchymal stem cells and normal lung fibroblasts
[0034] 1 Isolation, culture and identification of mesenchymal stem cells and normal lung fibroblasts (NLF)
[0035] Under sterile conditions, cut the umbilical cord into small pieces with a length of about 1 cm, wash them thoroughly, remove the arteries and veins of the umbilical cord, cut the small pieces of the umbilical cord into small pieces, place them in a petri dish, use Wharton glue to directly adhere to the bottom of the dish, and add a small amount of 10wt% FBS and 5wt% green chain double antibody low-sugar DMEM culture solution, placed at 37 ° C, 5% CO 2 Cultivate in an incubator, and add culture medium after 4 hours. After 3 days, the culture medium was changed, and the medium was changed every 3 days. After the MSCs reached 70-80% fusion, they were digested with 0.25% trypsin, subcultured, and the P3 generation cells were taken for experiments.
[0036] Tak...
Embodiment 2
[0043] MSC exosomes can enter target cells and inhibit the expression of α-SMA and Fibronectin in abnormally activated fibroblasts
[0044] 1MSC exosomes can enter target cells (human lung fibroblasts)
[0045] The extracted MSC exosomes and normal lung fibroblast exosomes were labeled with CM-Dil from Invitrogen, and added to the adherent cultured normal human lung fibroblasts. After 24 hours, the exosomes entered with a fluorescence microscope were observed. In the case of cells, the results are as follows image 3 As shown, both red fluorescent-labeled MSC exosomes and lung fibroblast exosomes can successfully enter human lung fibroblasts.
[0046] 2MSC exosomes can inhibit the expression of α-SMA and Fibronectin in normal human lung fibroblasts activated by TGF-β1
[0047] Isolate and culture normal human lung fibroblasts, plant P3 generation cells in a 6-well plate at an appropriate density, and starve the cells for 12 hours after the cells have reached 70% confluence. ...
Embodiment 3
[0053] Intratracheal administration of MSC exosomes reduces mortality and lung lesions in BLM-induced pulmonary fibrosis
[0054] 1 Establishment of BLM-induced pulmonary fibrosis mouse model
[0055] Take 6-week-old SPF grade BALB / C mice and anesthetize them by intraperitoneal injection of pentobarbital sodium. Under the direct vision of the laryngoscope, use the airway of small animals to administer drugs, and insert the drug catheter into the trachea with a high-pressure gun. The dose of kg was injected into PBS, and the experimental control group and the two treatment groups were injected into BLM at a dose of 3 mg / kg. After the injection, the animal was upright and rotated to make the drug evenly distributed in the lungs.
[0056] 2 Intratracheal administration of MSC exosomes reduces survival in BLM-induced pulmonary fibrosis
[0057] On the second day after BLM administration, the MSC exosome treatment group was given 50ul of MSC exosomes at a concentration of 1ug / ml i...
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