Male Wistar rats (n=60) aged 11 weeks and weighing 250–300 g were obtained from the Academy of Military Medical Science [Beijing, China; certificate no. SCXK (JUN) 2007–004]. These animals were maintained in a standard animal laboratory with free activity and free access to water and rodent chow. They were maintained in a temperature-controlled environment at 22–24°C with a 12-h light-dark cycle. The rats were fasted for 12 h prior to surgery, and were provided with free access to 10% glucose water following surgery. All the surgical procedures were performed under sterile conditions, and all experiments were performed according to the National Institutes of Health Guide for Care and Use of Laboratory Animals (13) and were approved by the ethics committee of Tianjin First Central Hospital, Tianjin Medical University (Tianjin, China).

For the introduction of 70% PH, the 60 rats were anesthetized with isoflurane inhalation (Lunan Pharmaceutical Co., Ltd., Shandong, China) via an isoflurane vaporizer (Matrx VMR; Midmark corporation, Dayton, OH, USA), and 70% of the liver of each rat, comprising the left lateral and median lobes, was excised, using the technique described by Saito et al (14). Suturing of the peritoneum and skin were performed independently. The remaining 30% of the liver started to grow for 7 days, following which R-PH was performed, in which 40 of 60 rats were anesthetized and the right lateral lobe was ligated and excised.

To obtain adequate cells and avoid the requirement for a long duration following establishment of the PH model, the autologous ADSCs were isolated and expanded 2 weeks prior to surgery. Adipose tissue cells were isolated from all 60 rats using a described previously method (15). Briefly, the rats were anesthetized via inhalational isoflurane. The hemi-inguinal fat pads were carefully excised and minced into pieces of ~1 mm³. The adipose tissue was digested in collagenase type I solution (Sigma-Aldrich, St. Louis, MO, USA) for 60 min at 37°C with constant agitation (100 rpm). The stromal cells were separated from the floating adipocytes by centrifugation at 200 g for 5 min at room temperature. The cells released were then resuspended in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and then sieved through 70 µm mesh (BD Biosciences, Franklin Lakes, NJ, USA). The resulting ADSCs were cultivated in DMEM/F12 medium containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). Following in vitro culture for 14 days at 37°C, 5% CO₂ and 95% humidity, a sufficient number of ADSCs were obtained for the autologous transplantation. The ADSCs (3×10⁶) from each experimental rat were cryopreserved in liquid nitrogen (Air Products and Chemicals (Tianjin) Co., Ltd., Tianjin, China) with cell name marked on tube prior to injection.

The expression levels of cell surface antigen were evaluated using flow cytometry. When cultures reach >80% confluency at 37°C, 5% CO₂ and 95% humidity, adherent cells were removed from the tissue culture polystyrene flasks via trypsinization (Invitrogen; Thermo Fisher Scientific, Inc.) and washed twice with DMEM/F12. All cells were incubated with fluorescein isothiocyanate-conjugated mouse anti-rat monoclonal antibodies against rat CD45 (cat. no. 554877), CD73 (cat. no. 551123), CD90 (cat. no. 554894) all obtained from BD Biosciences), CD34 (cat. no. sc-7324; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and CD105 (cat. no. ab11414; Abcam, Cambridge, MA, USA) for 40 min at room temperature at a dilution of 1:50. Following antibody incubation, data were acquired using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest 6.0 software (BD Biosciences).

The differentiation of the cells into osteogenic and adipogenic lineages, and subsequent detection were performed using established methodologies (15). Briefly, the ADSCs were seeded in medium at 2×10⁴ cells/cm² in six-well tissue culture plates. When the cells reached 100% confluency, DMEM/F12 was subsequently replaced with osteogenic inducer medium containing 100 nmol/l dexamethasone (Sigma-Aldrich), 10 mmol/l β-sodium glycerophosphate (Sigma-Aldrich) and 50 µg/ml vitamin C (Sigma-Aldrich), or adipogenic inducer medium containing 1 µmol/l dexamethasone, 0.5 mmol/l 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 5 mg/l insulin (Sigma-Aldrich) and 100 µmol/l indomethacin (Sigma-Aldrich), in DMEM/F12. Cells were maintained at 37°C in a 5% CO₂ incubator and the medium was changed every 3 days. Following a 14 day induction period, the cells were assayed for mineral content by Von Kossa staining (Shanghai Genmed Gene Pharmaceutical Technology Co., Ltd., Shanghai, China) and for lipid accumulation using Oil Red O staining (Sigma-Aldrich).

The rats were divided into the following three groups: PH (n=20); R-PH (n=20), subjected to a R-PH and treated with saline by portal vein injection; and R-PH/ADSC group (n=20), subjected to R-PH and treated with autologous ADSCs (2×10⁶ cells/rat) by portal vein injection. Subsequent to these procedures, five animals in each group were sacrificed using anesthesia, as described above, at 24, 72 and 168 h following hepatectomy, respectively. Blood samples (2 ml) were collected by puncturing the vena cava, and the residual liver lobes were then rapidly excised and weighed. The livers were fixed in formalin (Tianjin Kemiou Chemical Reagent Co., Ltd., Tianjin, China) overnight, prior to processing and embedding in paraffin wax (Tianjin Kemiou Chemical Reagent Co., Ltd.). Sections (5-µm thick) were deparaffinized and fixed. The sections were stained with hematoxylin and eosin (H&E; Sigma-Aldrich) and observed using a Nikon Ni-U fluorescence microscope (Nikon Corporation, Tokyo, Japan) Additional samples were stored in liquid nitrogen.

For each time point, the total body weight of each of the fasted rats were weighed prior to sacrifice. The regenerating ratio of the liver following hepatectomy was calculated as the liver wet weight to body weight ratio (LBR), rather than the weight of the remnant lobes alone. The 2 ml blood samples were centrifuged at 4,000 × g for 10 min at room temperature prior to serum collection. The serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were measured using an automatic biochemical analyzer (Hitachi 7600; Hitachi, Ltd., Tokyo, Japan) 24, 72, and 168 h postoperatively in the R-PH/ADSC, R-PH and PH groups.

The expression level of PCNA, determined by immunohistochemistry, correlates with the degree of cell proliferation (16). Briefly, following fixation with formalin and paraffin embedding, the liver tissue sections were incubated with rabbit anti-rat polyclonal antibody against PCNA (cat. no. GTX100539; 1:500 dilution; Genetex Inc. Irvine, CA, USA) at 4°C overnight, and subsequently with a 3′,3-diaminobenzidine kit (Beyotime Institute of Biotechnology, Haimen, China). The proliferation index of the PCNA-stained cells was measured by counting the number of positive nuclei of hepatocytes under Ni-U fluorescence microscope, with data expressed as the percentage of PCNA-stained hepatocytes of the total number of hepatocytes.

The mRNA levels of HGF in liver tissue were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Total RNA was extracted from the frozen remnant lobe samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and then subjected to RT using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed using an ABI 7500 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using 1 µl cDNA template and 1X SYBR-Green I (Takara Bio, Inc., Tokyo, Japan) in a 25 µl reaction mixture (Takara Bio, Inc.; 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, 200 µM dNTP mix, 0.2 µM of each primer and 1 unit of Taq DNA polymerase). Primer Premier V5.0 software was used to design the primers, according to HGF gene sequences (GenBank; www.ncbi.nlm.nih.gov/genbank). Primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA). The primer sequences were as follows: HGF, sense 5′-ACA GCTTTTTGCCTTCGAGCTA-3′ and anti-sense 5′-CATCAAAGCCCTTGTCGGGATA-3′; β-actin, sense, 5′-ATATCGCTGCGCTCGTCGTC-3′ and anti-sense 5′-TCTTGCTCTGGGCCTCGTC-3′. The conditions for each qPCR reaction were as follows: 30 sec at 95°C, followed by 40 cycles of denaturation for 5 sec at 95°C, annealing for 30 sec at 58°C and extension for 30 sec at 72°C. The level of expression was calculated using the 2^−ΔCq method, in which ΔCq was calculated as Cq of target molecule − Cq of β-actin (17).

Data are expressed as the mean ± standard deviation. Differences in parameters were analyzed using one-way analysis of variance. Statistical analyses were performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The ADSCs were verified by analyzing the expression surface markers and multipotent differentiation of the cells. At passage three, the cultured ADSCs exhibited a fibroblast-like morphology (Fig. 1). The data showed that the ADSCs were positive for CD90, CD105 and CD73, but were negative for CD34 and CD45 when analyzed using flow cytometric analyses (Fig. 2). In addition, the ADSCs at passage three exhibited potential for osteogenic and adipogenic differentiation following culture in osteogenic and adipogenic growth media. Positive ALP and oil red O staining confirmed the cells as osteogenic and adipogenic, respectively (Fig. 3).

The outcomes in the 60 rats subjected to PH were examined in the present study. The LBR at 24 and 72 h post-hepatectomy was significant decreased in the R-PH group, compared with that in the PH group (P<0.01; Fig. 4). However, the LBR in the R-PH rats that received ADSC transplantation, was significantly higher, compared with that in the R-PH rats 72 h postoperatively (P<0.05). Furthermore, the LBR increased steadily in the three groups, and no further differences among the groups were observed 168 h postoperatively (Fig. 4).

At ~24 h post-hepatectomy, the sections of the remnant lobes were stained with H&E and examined under a light microscope. The histopathological analysis revealed that no liver cell inflammation or necrosis was present in any of the samples. In the PH group, the arrangement of hepatocytes was deranged, with microvesicular fatty degeneration observed in the cytoplasm and mild dilatation of the sinusoids (Fig. 5A). The remnant lobe in the R-PH group exhibited prevalent vacuolization degeneration in the hepatocytes, and dilatation of the sinusoids was more marked, compared with that in the PH group (Fig. 5B). Similarly, in the R-PH/ADSC group, vacuolization and sinusoidal dilatation was similar, but to a lesser extent, compared with the R-PH group (Fig. 5C).

In the R-PH/ADSC group, the levels of ALT and TBIL were substantially lower 24 h following hepatectomy, compared with those in the R-PH group, whereas the levels of AST were significantly lower 24 and 72 h postoperatively, compared with those in the R-PH group. Although a significant protective effect was observed in the rats transplanted with ADSCs, the liver function remained significantly higher in the R-PH group, compared with that in the PH group 24 h postoperatively. At 72 h postoperatively, the serum levels of ALT and TBIL in the three groups were similar. Furthermore, the liver function gradually decreased to the basal level in the three groups at 168 h post-hepatectomy (Fig. 6).

To evaluate whether ADSC transplantation enhanced the proliferation of hepatocytes in remnant lobes, the expression levels of PCNA were assessed. Although the number of PCNA-positive hepatocytes was significantly reduced in the R-PH rats transplanted with ADSCs, compared with the PH rats (P<0.01; Fig. 7), the R-PH rats transplanted with ADSCs exhibited a significant increase in the number of PCNA-positive hepatocytes at 24 h postoperatively, compared with the R-PH rats, indicating that ADSCs exerted hepatoprotective effects by promoting liver regeneration in the early phase. No statistically significant differences in the PCNA-labeling index were observed among three groups at 72 and 168 h (P>0.05; Fig. 7).

The mRNA levels of HGF peaked within 24 h in the PH group and R-PH/ADSC group, and then gradually decreased after 72 h. However, the mRNA levels of HGF in the R-PH group decreased significantly after 24 h, and peaked at 72 h, compared with PH and R-PH/ADSC groups, indicating that hepatic regeneration following R-PH was inhibited. The postoperative course of the mRNA levels of HGF are shown in Fig. 8.