Usage of lactoferrin in prevention and treatment of tumor metastasis
A technology of lactoferrin and tumor metastasis, which is applied in the fields of medicine, biopharmaceuticals and tumor treatment, and can solve the problems of lack of tumor metastasis methods and drugs
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Embodiment 1
[0075] The preparation of embodiment 1 recombinant human lactoferrin
[0076] 1 Materials and methods
[0077] 1.1 Reagents and raw materials
[0078] buffer A (400mM NaCl, 20mM PB, pH7.5), buffer B (1000mM NaCl, 20mM PB, pH7.5); physiological saline.
[0079] The transgenic goat milk of recombinant human lactoferrin (purchased from Shanghai Transgenic Research Center) was determined to contain about 40-66 g of human lactoferrin and a small amount of about 0.05-0.12 g of goat lactoferrin per liter of goat milk.
[0080] 1.2 Method
[0081] 1.2.1 Degreasing:
[0082] Take 20 mL of transgenic goat milk highly expressing recombinant human lactoferrin, and degrease the goat milk as follows: centrifuge at 10,000 rpm for 30 min, and filter with 4 layers of gauze to obtain the filtrate.
[0083] 1.2.2 Cheese off:
[0084] Mix the filtrate with 20mL 800mM NaCl (40mM PB, pH7.5), adjust the pH to 4.6 with concentrated hydrochloric acid; after incubating at 40°C for 30min, centrifug...
Embodiment 2
[0097] Example 2 In vitro growth inhibition test and metastasis inhibition test of recombinant human lactoferrin on non-small cell lung cancer A549 cell line
[0098] 1 Materials and methods
[0099] 1.1 Reagents and materials
[0100] Transwell chamber (pore size 8 μm), DMEM basal medium, DMEM complete medium, DMEM medium (containing 1% fetal bovine serum), DMEM medium (containing 20% fetal bovine serum), trypsin, penicillin and streptomycin, 0.1 % crystal violet staining solution, MTT.
[0101] Non-small cell lung cancer A549 cell line.
[0102] 1.2 Method
[0103] 1.2.1 In vitro growth inhibition test of rhLF on non-small cell lung cancer A549 (MTT method)
[0104] Collect logarithmic phase A549 cells, adjust the concentration of cell suspension to 10 4 cells / mL, add 100 μL per well to make the cell density to be tested as 1000 wells. 5%CO 2 , incubate at 37°C until the cells adhere to the wall, add concentration gradient rhLF, 100 μL per well, the final drug conce...
Embodiment 3
[0114] Example 3 Recombinant human lactoferrin inhibits the growth of mouse melanoma lung metastases
[0115] In order to further study the activity of lactoferrin in inhibiting tumor metastasis in animals, in this example, the mouse melanoma B16F10 lung metastasis model was used to evaluate the activity of lactoferrin in inhibiting tumor metastasis, colonization and growth in vivo.
[0116] 1 Materials and methods
[0117] 1.1 Experimental animals
[0118] Animal source: C57BL / 6 mice, provided by the Experimental Animal Research Center of the Chinese Academy of Military Medical Sciences (Experimental Animal Production License: SCXK (Army))
[0119] Animal age: 6-8 weeks; animal weight: 18-22g; animal sex: male.
[0120] 1.2 Test drugs
[0121] 1) Human lactoferrin
[0122] Purified from transhuman lactoferrin gene goat milk, preparation method: prepare the required concentration with physiological saline before each administration. The purification method is the same as ...
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