Method for controlling cartilage quality by utilizing cartilage specific gene expression degree
A gene expression and specific technology, applied in the field of biomedicine, to achieve good cartilage tissue formation ability, improve the quality of cartilage culture, and the method is convenient and quick.
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Embodiment 1
[0014] Example 1 Culture of chondrocytes, CRTL1 gene expression level of synoviocytes
[0015] Cut the discarded cartilage tissue or synovial tissue from artificial joint surgery into small pieces of 5mm×5mm, wash with PBS solution (Gibco) for 3 times, add 0.25% trypsin / EDTA and 0.2% type II collagenase, 500mg cartilage Add 10mL 0.25% trypsin / EDTA, 10mL 0.2% type II collagenase, digest at 37 ℃ for 10-20 h, filter out impurities through a 50-mesh sieve, and centrifuge at 350g for 5 min, remove the supernatant, add Cell suspension was made in high glucose DMEM (Gibco) medium containing 10% fetal bovine serum. 5000 pieces / cm 2 The cell density was seeded at 75cm 2 In the shake flask, culture in a saturated humidity, 37°C, 8% CO2 incubator, and exchange the culture medium once every 2-3 days. When the cells grew to 80-90%, they were digested with 0.25% trypsin / EDTA, passaged at a ratio of 1:5, and the third passage cells were recovered for extraction of total RNA for real-time ...
Embodiment 2
[0028] Example 2 Cartilage-specific matrix generation ability of 3D cultured chondrocytes
[0029] In order to check whether the various cultured chondrocytes in Table 1 have cartilage damage repairing effect, the present invention utilizes 3D culture technology to imitate the in vivo environment of chondrocytes, and checks whether these cultured cells have the ability to form cartilage tissue.
[0030] (1) 3D chondrocyte culture is using sodium alginate (Alginate) technology, the specific method:
[0031] The chondrocytes after passage 3 were concentrated by centrifugation at 350g×5min, and the supernatant was removed. Add 2% sodium alginate solution to the cell pellet to prepare 2×10 6 cells / mL cell suspension. Move the cell suspension to a 10ml syringe and suck it up, install a 22G injection needle, drop the cell suspension into 100mM calcium chloride solution to make sodium alginate hydrogel balls, wash with normal saline for 3 times, transfer To a 6well plate of a 6-we...
Embodiment 3
[0043] Example 3 In vivo test
[0044] In order to confirm the effect of the passage number of cultured chondrocytes on the ability of cultured chondrocytes to form cartilage in vivo. Cultured chondrocytes CH3 at passages 3, 5, and 9 were transplanted into articular cartilage defects (5mm×2.5mm) in the knees of 20-week-old rabbits. Two months later, the transplanted cartilage tissues were taken out, fixed with 4% paraformaldehyde, and stained with safranin-0 cartilage.
[0045] Table 4 shows the CRTL1 gene expression level (CRTL1 Coppet number / GAPDH Coppet number) of cultured CH3 chondrocytes at different passages, and the results of Safranin O cartilage staining. Two months after transplanting the cultured cartilage of passage 3 to the articular cartilage defect of the rabbit knee, the cartilage defect was almost filled with cartilage tissue-specific matrix, showing good cartilage damage repair ability. Although the repairing effect of cultured cartilage of passage 5 on car...
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