Production of partial liver resection model in mice
The mice were anesthetized with 10 % chloral hydrate intraperitoneal injection. After abdominal disinfection, the central vertical incision of the lower abdomen of xiphoid process was taken, with a length of 1.5 cm-3 cm, and the epidermis, muscularis and peritoneum were cut successively to fully expose the liver. The middle lobe of the liver and the left lobe were ligated and removed, and the abdomen was closed with sutures successively. After sutures, the incision was disinfected again.
Isolation and culture of bone marrow mesenchymal stem cells
The femur of the mice was taken, and the muscles and fascia were covered with ice packs in a super clean table. Both ends of the femur were cut off with scissors. A syringe was inserted into the medullary cavity, and the bone marrow was washed out, cut and blown into single-cell suspension. Then, DMEM/F12 culture medium containing FBS was added, and conventional culture was performed in an incubator at 37 ℃ and 5 % CO2.
Lentivirus transfection of CCR2
The BMSCs growing at log stage in the third generation were selected. When the cells grew to 80 % fusion degree, the CCR2 overexpressed plasmid lentivirus (PGMLV-PA4-CCR2) was used to infect the BMSCs according to the virus infection ratio of 50:1. After infection for 6-8 h, fresh medium was replaced to continue culture. After 48 h, the stable expression strains were screened with puromycin medium with the final concentration of 0.1 mg/L.
Liver index
BMSCs were transferred into partial hepatectomy mice through caudal veins. The weight of mice and the weight of regenerated liver were measured 72, 120 and 168 h after partial hepatectomy, and the liver index was calculated according to the ratio of regenerated liver weight to body weight.
HE staining
Mouse liver tissue was fixed in 4 % paraformaldehyde, embedded in paraffin, and sectioned to 4 μm thick. Baked at 65 ℃ for 4.5 h; The sections were dyed with hematoxylin dyeing solution for 3-5 min, washed with tap water, differentiated with differentiation solution, washed with tap water, returned blue solution returned blue, and washed with running water; Sections were dehydrated in 85 % and 95 % gradient alcohol for 5 min, respectively, and then stained in eosin solution for 5 min. The slices were put into anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol ⅲ for 5 min, dimethyl I for 5min, and xylene II for 5min in sequence. The slices were transparent and sealed with neutral gum. Observe with a light microscope.
Hepatic function detection
The mice in each group were anesthetized by intraperitoneal injection of pentobarbital sodium at 72 h after partial hepatectomy, the abdominal cavity was fixed in supine position, and 1 mL blood was collected from orbit. The mice were left standing at room temperature for 30 min, centrifuged at 4000 r/min for 5 min, and 300 μ L serum was collected. The contents of alanine amiotransferase (ALT) and aspartate aminotransferase (AST) in serum were detected by automatic biochemical instrument.
Migration capability of the MSCs
MSCs was vaccinated in the upper chamber (2.0 × 105/ hole), while the lower chamber was fitted with recombinant 100 ng/ml CCL2 for 4 hours, wiped the MSCs, remaining on the upper surface and stained with 0.1 % crystal purple. MSCs migrated to the lower surface were counted microscopically.
Real-time PCR.
Extract 1 μg RNA from tissue , in the extraction tissue was prepared as described in the Invitrogen reverse recording kit Then the target gene was amplified and PCR tested on PRISM 7900 Sequence Detector by the operating guidelines of QuantiTect SYBR Green RT-PCR Kit (Qiagen), and the copy number of the target gene in the sample calculated the relative content of the target gene from the copy number of β-actin.
Western blot
The liver tissue was cut into pieces, lysate was added into ice to homogenate, supernatant was collected by centrifugation at 4 ℃, and protein concentration was measured by BCA method. SDS-PAGE electrophoresis was performed on appropriate protein samples at 80 V for 2 min and then at 120 V for 2 h. 300 mA for 2 h. The PVDF membrane was sealed in 5 % skimmed milk powder for 2 h and washed with TBST for 3 times, 10 min each time. Primary antibody (1:500 dilution) was incubated at 4 ℃ overnight and washed with TBST for 3 times, 10 min each time. The secondary antibody (1:2000 dilution) was incubated at room temperature for 1 h, washed 3 times with TBST, 10 min each time, and developed with ECL.
Statistical analysis
SPSS 25.0 statistical software was used to conduct t test on the data of the experimental group and the control group, and P<0.05 was considered statistically significant.
