1.1 The main reagent
The main reagent included 0.25% trypsin-0.53mmol EDTA (Invitrogen Company, USA), DMEM/F-12 (Invitrogen Company, USA), Rabbit Anti-FGFR-3 (Santa-cruz Company, USA), Fetal Bovine Serum ( Gibco Company, USA), DAB chromogenic kit (Fuzhou Maixin Biotechnology Co., Ltd.), rabbit anti-mouse collagen type II monoclonal antibody (Fuzhou Maixin Biotechnology Co., Ltd.), endogenous peroxidase blocker ( Fuzhou Maixin Biotechnology Co., Ltd.), animal non-immune serum (Fuzhou Maixin Biotechnology Co., Ltd.), Astragalus injection (10ml/bottle, each equivalent to 20g of original medicinal material, Zhengda Qingchunbao Pharmaceutical Co., Ltd.).
1.2 Experimental animals
① Healthy newborn 24h SD rats, clean grade, not limited to males and females. ②Healthy 4-week-old SD rats, clean grade, not limited to males and females, come from the same closed group (provided by the Animal Center of Zhejiang University of Traditional Chinese Medicine).
2.1 Cultivation of PSCs
The method of co-cultivation of epiphyseal growth plate LaCroix ring and quiescent zone Precartilaginous stem cells: Newborn 24h SD rats were selected and sacrificed by cervical dislocation. After disinfection, the knee joints were separated under a microscope (×10 times) under sterile conditions. Surrounding muscle tissue, exposing the distal femur and proximal tibia, cut out the epiphyseal growth plate LaCroix ring and resting area (Figure 1), cut to about 1mm cube, digested with 0.25% trypsin-0.53mmol EDTA for 20 minutes, 10% fetal bovine serum Digestion of the DMEM/F-12 medium was terminated for 2 minutes, filtered by a 40-mesh sieve, centrifuged for 5 minutes (1500 r/min), and then resuspended in DMEM/F-12 medium containing 10% fetal calf serum to culture in saturated humidity. Medium was changed every other day.
2.2 Identification of PSCs
Select the third-generation cells to be placed in DMEM/F-12 culture medium containing 10% fetal bovine serum to make a cell suspension, and inoculate them in the culture wells of a 6-well plate pre-installed with a cover glass and culture in saturated humidity. The cells covered approximately 60%, the culture medium was aspirated 6-well culture plates, were washed twice with PBS, followed by FGFR-3 immunohistochemistry, the specific operational steps performed according to the kit.
2.3 Astragaloside IV Intervenes PSCs
Set up the experimental group and the control group, select the 3rd generation cell to make a cell suspension and inoculate it on a 24-well culture plate, 1*104 cells per well, add 100ug/ml astragaloside IV to the experimental group, 3ml/d, Add culture medium to the control group, 3ml/d. After every 24 hours, 3 wells were taken from the control group and the experimental group, and the cells were digested with 0.25% trypsin for relevant tests.
2.4 MTT cell viability detection
Control group and experimental group as described above were prepared from 2d cells, 4d cells and 6d cells. Use ELX-800UV enzyme label detector to adjust the zero hole to zero, and Select light wave of 490nm to measure the absorbance of each hole (OD).
2.5 Monoclonal antibody staining of collagen type Ⅱ
Selects the third-generation cells into cell suspension, culture wells were seeded in 6 well plates previously cover glass placed in a humidity saturated culture, cells were grown until about 80%, 4% paraformaldehyde 4℃ under overnight.At room temperature Triton-100 + 0.5% BSA (bovine fetal protein) for 30min. The rabbit anti-mouse type II collagen monoclonal antibody was cultured overnight at 4°C. FITC-labeled secondary antibody was incubated for 30 min at room temperature. Observe under a laser confocal microscope.
2.6 Astragaloside IV Intervention in SD Rats
A total of 48 4-week-old clean SD rats with similar body weight were randomly divided into 2 groups with 24 rats in each group. Experimental group: intraperitoneal injection of Astragalus injection, the amount of which was calculated at 8.0 g per kilogram of the rat, 10ml per kilogram, once a day. Control group: Inject the same amount of normal saline, and do related inspections with the materials taken in the 3rd and 5th weeks after feeding.
2.7 Measurement of tibia length
The animals were killed by cervical dislocation.The muscle tissues around the tibia and the upper and lower articular surfaces were cleaned, and the tibia was exposed and placed flat. Choose a vernier caliper (with an accuracy of 0.02mm) to measure the length of the tibia, so that the vernier caliper clamps the two ends of the tibia. Do not press too tightly to avoid bending the tibia and thus affecting the experimental data.