Medicinal composition for treating infarction
A pharmaceutical composition and composition technology, applied in drug combination, gene therapy, drug delivery, etc., can solve problems such as restenosis, and achieve the effect of improving life prognosis
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Embodiment 1
[0071] Example 1. Preparation of liposomes containing hsa-miR-145
[0072] Dissolve hsa-miR-145 (5 nM; obtained from Applied Biosystems) in 100 μl of RNase-free distilled water, add 400 μl of serum-reduced medium Opti-MEM (GIBCO), and shake with a Vortex mixer Mix for 30 seconds. Then, 10 µl of Lipofectamine RNAiMax (Invitrogen) was added to the resulting mixture, followed by shaking and mixing with a vortex mixer for 30 seconds. Next, the obtained mixture was left standing at room temperature for 10 minutes, and 300 µl of Opti-MEM was added thereto to make 800 µl. The hsa-miR-145-containing liposomes thus prepared were used in Example 2 below.
Embodiment 2
[0073] Embodiment 2. Application and therapeutic effect thereof in myocardial infarction
[0074] The liposome containing hsa-miR-145 prepared in Example 1 above was administered to the infarction site of a model animal with severe myocardial infarction, and the therapeutic effect of hsa-miR-145 on infarction was evaluated.
[0075] 1. Experimental method
[0076] A morbid model animal of severe myocardial infarction was prepared using Japanese white rabbits (male, about 2 kg in body weight) by coronary ischemia for 30 minutes.
[0077] The liposome containing hsa-miR-145 prepared in Example 1 was intravenously administered to this diseased model animal (n=5) at a ratio of 0.035 mg per 1 kg body weight, and was sacrificed under anesthesia on the 14th day after administration and removed. Heart (hsa-miR-145 administered group). In addition, as a control group, physiological saline was administered intravenously at a rate of 0.5 ml per 1 kg of body weight to morbid model a...
Embodiment 3
[0085] Example 3. Verification of the mechanism of action of hsa-miR-145
[0086] In Example 2 above, it was confirmed that hsa-miR-145 is effective in shrinking myocardial infarction lesions and preserving cardiac function. Therefore, the mechanism of action of this hsa-miR-145 was verified.
[0087] (1) Experimental method
[0088] (1-1) Preparation of samples
[0089] The hsa-miR-145-containing liposome prepared in Example 1 above was transfected into H9c2 cells, which are rat heart-derived cells. Specifically, the final concentration of hsa-miR-145 was adjusted to 20 nM or 40 nM, and H9c2 cells were cultured at 37° C. for 72 hours in the presence of the hsa-miR-145-containing liposomes.
[0090] After culturing, total protein was extracted from the resulting H9c2 cells. Specifically, using a cell scraper (cell scraper) (Sumitomo Bakelite, Japan), the cultured H9c2 cells were scraped from the culture dish and suspended in phosphate buffered saline (PBS(-), TAKARA, J...
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