Preparation method and application of humanized skin fibroblast intracellular effective components

[0054] Example 1 Preparation method of intracellular active ingredients of human skin fibroblasts

[0055] The present embodiment provides a method for preparing an intracellular active ingredient in human skin fibroblasts, comprising the following steps:

[0056] (1) The obtained human abdominal full-thickness skin was placed in a PBS buffer containing 100 U / mL penicillin and 100 g / mL streptomycin for 40 times of repeated rinsing to remove subcutaneous fat and excess tissue;

[0057] (2) Cut the above skin into 2mm 3 A small piece, attached to the culture bottle on the dermis side, 2.5 mL of 2 mg / mL type II collagenase, placed at 37°C, 5% CO 2 Digest in cell incubator for 13 hours;

[0058] (3) Take out the digested skin, carefully take out the epidermal tissue, rinse the remaining part with PBS buffer containing 100U / mL penicillin and 100g / mL streptomycin repeatedly 8 times, and cut into pieces thoroughly;

[0059] (4) Then add 4 mL of 2 mg / mL type II collagenase

[0064] Example 2 The preparation method of the intracellular active ingredients of human skin fibroblasts

[0065] The present embodiment provides a method for preparing an intracellular active ingredient in human skin fibroblasts, comprising the following steps:

[0066] (1) The obtained human abdominal full-thickness skin was placed in a PBS buffer containing 100 U / mL penicillin and 100 g / mL streptomycin for 30 times of repeated rinsing to remove subcutaneous fat and excess tissue;

[0067] (2) Cut the above skin into 1mm 3 A small piece, attached to the culture bottle on the dermis side, 3 mL of 2 mg / mL type II collagenase, placed at 37°C, 5% CO 2 Digest in cell incubator for 12 hours;

[0068] (3) Take out the digested skin, carefully take out the epidermal tissue, and rinse the remaining part with PBS buffer containing 100U / mL penicillin and 100g / mL streptomycin for 5 times, and cut it into pieces;

[0069] (4) Then add 5mL of 2mg / mL type II collagenase, put i

[0074] Example 3 Preparation method of intracellular active ingredients of human skin fibroblasts

[0075] The present embodiment provides a method for preparing an intracellular active ingredient in human skin fibroblasts, comprising the following steps:

[0076] (1) The obtained human abdominal full-thickness skin was placed in a PBS buffer containing 100 U / mL penicillin and 100 g / mL streptomycin for 50 times of repeated rinsing to remove subcutaneous fat and excess tissue;

[0077] (2) Cut the above skin into 3mm 3 Small piece, dermal side attached to the culture bottle, 2mg / mL type II collagenase 2mL, placed at 37°C, 5% CO 2 Digest in cell incubator for 15 hours;

[0078] (3) Take out the digested skin, carefully take out the epidermal tissue, and wash the remaining part in PBS buffer containing 100U / mL penicillin and 100g / mL streptomycin repeatedly for 10 times, and cut into pieces thoroughly;

[0079] (4) Then add 3 mL of 2 mg / mL type II collagenase, place at