2.1 Materials

Primary Antibodies in this study were purchased from Abcam (Cambridge, UK). The HRP-conjugated secondary antibody was acquired from Bioss Biotechnology (Beijing, China). The FASL siRNA (sc-29313) and ELISA kits for interleukin (IL)-10, IL-2, transforming growth factor (TGF)-β, and interferon (IFN)-γ were obtained from Santa Cruz Biotechnology (CA, USA). The DMEM medium and DMEM/F12 medium were both bought from Hyclone (UT, USA). The alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) assay kits were obtained from Sigma-Aldrich Co., Ltd. (MO, USA). The cell culture reagents were obtained from Gibco BRL Life Technologies (MD, USA). Other chemicals used in this research were of analytical grade.

2.2 Animals

Healthy adult male Lewis and Brown Norway (BN) rats (8–10 weeks, 210–250 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animals were housed at a constant temperature and humidity with free access to food and water. Animals were permitted to acclimate to the environment for 7 days before the experiments began.

2.3 Isolation and expansion of GMSCs and BMSCs

Ten healthy individuals who needed crown lengthening surgery or extraction of the third mandibular molar incisors, aged between 20 and 50 without any evidence of dental caries, were recruited in this study. The criteria included: (1) no gingival bleeding and swelling, no loss of attachment, and the depth of probing was less than 3 mm; (2) no smoking history; (3) no systemic diseases. The gingival tissues collected in oral surgery unit of Tianjin Medical University Stomatological Hospital.

GMSCs were isolated as following procedures. After washing with PBS, gingival tissues were digested at 37 °C for 40 min in sterile PBS containing 4 mg/mL dispase II and 3 mg/mL collagenase I (Sigma, USA). The cells were separated by centrifugation for 6 min at 1000 rpm, and then cultured with DMEM containing 15% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and cultured at 37 °C in a culture incubator with 5% CO₂. The cells were sub-cultured every 5 days.

BMSCs were isolated from femurs and bilateral fibula of 4 to 5 week old BN rats as previously described (Chen et al., 2018). The BMSCs were cultured in DMEM/F12 medium.

2.4 FASL knockdown in GMSCs

2 × 10⁵ GMSCs were seeded in a 12-well culture plate with 1 mL complete medium per well. 3 µL FASL siRNA were blended with 3 µL lipofectamine 2000 and then, this mixture were dissolved in 200 µL FBS free medium, standing for 20 min. To knockdown FASL, 10 µL siRNA solutions were added into every well to incubating GMSCs. Seven days after puromycin screening, the levels of FASL mRNA and protein were detected according to the methods as previously described (Saad, 2011) and the Specific primers used for genes were as follows: FASL forward, 5′-GAACTGGCAGAACTCCGTGAGT-3′; and reverse, 5′-CAGAGATCAGAGCGGTTCCATA-3′; β-actin forward, 5′-CTGGGACGACATGGAGAAAA-3′; and reverse, 5′-AAGGAAGGCTGGAAGAGTGC-3′. The empty vector of siRNA had no influence on the expression of FASL.

2.5 Establishment of rat liver transplantation model

In this experiment, the liver organs for transplantation in male BN rats were obtained from male Lewis rats. Based on the “Two-Cuff Technique” proposed by Kamada and Calne (Kamada et al., 1979), we established an rat orthotopic liver transplantation model without hepatic rearterialization. After the donor liver was successfully implanted, BN rats were randomly divided into four groups: normal saline (NS) group, FASL-/-GMSCs group, BMSCs group, and GMSCs group. In NS group, each recipient was injected with 1 mL physiological saline through dorsal vein of penis. As FASL-/-GMSCs group, the recipients were injected with 1 mL physiological saline containing 5 × 10⁶ FASL-/-GMSCs via vena dorsalis penis. Similarly, rats in BMSCs and GMSCs groups were respectively injected with an equal volume and cell number of BMSCs and GMSCs.

2.6 Tissue and specimen collection

To collect tissues, five recipients were randomly selected from every group. Other transplanted BN rats were maintained to acquire the information of their survival time. Serum was collected through the inferior vena cava, stored at −20 °C for usage, and the liver graft tissue was harvested on the postoperative days 3, 5, 7 and 14.

2.7 Survival of rats

The survival time of the rats was recorded for analysis and Kaplan-Meier survival curve was drawn. Log-rank test was used for statistical analysis.

2.8 Hematoxylin and eosin (H & E) staining and liver function test

The liver graft tissue was fixed in formalin for 24 h and H & E staining was performed to confirm histopathological features. The levels of peripheral serum ALT, AST and TBIL which are indicative of liver function in each group were detected by an automatic biochemical analyzer.

2.9 Enzyme-linked immunosorbent assay (ELISA)

The immunological response in rat liver transplantation model after GMSCs or BMSCs treatment was monitored by measuring the levels of IL-10, IL-2, TGF-β, and IFN-γ in peripheral serum, according to the ELISA kit manufacturer’s protocol.

2.10 Flow cytometry assay

Regulatory T cells (Treg) and T‐helper type 17 (Th17) cells were detected by flow cytometry. For analysis of Treg cells, the whole peripheral blood samples were first stained with PerCP-CD3, FITC-CD4 and APC-Cy7-CD25 for cell surface markers. Then cells were incubated with FoxP3 for intracellular staining. For Th17 detection, cells were stimulated with PMA and ionomycin, and then incubated with anti-CD4 and stained with PE-anti-IL17. The staining process of BMSCs and GMSCs were similar as above cells with antibodies of PE-CD29, -CD45, -RTIA; FITC-CD34, -CD90, -RT1B for BMSCs and FITC-OCT4, -SSEA4, −146 for GMSCs. All labeled cells were analyzed using BD FACSAria III (BD Biosciences, US).

2.11 Statistical analysis

GraphPad Prism 5.0 software and SPSS17.0 statistical software were applied for data analysis. All data was represented by mean ± standard deviation (SD). The student's t test was used for data comparison between two groups, and one-way ANOVA was used for data comparison between three or more groups. Survival analysis was drawn by Kaplan-Meier survival curve, and the survival rate was tested by Log-rank method. Differences were considered statistically significant when p ≤ 0.05.

2.12 Ethics

All animal procedures conformed to the Council of Europe Convention Directive (2010/63/EU) for the protection of vertebrate animals used for scientific purposes. All individuals have been signed the informed consent form. All experiments were approved by the ethical committee of Tianjin First Central Hospital.

3.1 Identification of BMSCs and GMSCs

As shown in Fig. 1, with passage and exchange, the BMSCs were purified, and the morphology became uniform. Most of them were slender spindle cells, and there were also flat-shaped cells with fissures (Fig. 1A). The cell surface MSC markers, CD29 and CD90 and the histocompatibility class I marker RT1A, were positively expressed, while hemacyte antigen, CD34 and CD45 and the histocompatibility class II marker RT1B, were negatively expressed (Fig. 1B). The primary GMSCs were arranged in a fusiform shape after being attached for 6 h (Fig. 1C). The differentiation capacity markers, OCT-4 and SSEA-4 and MSC markers, CD-146 and CD90, were detected with positive rate of 88.7%, 17.3%, 98.6%, and 99.9% (Fig. 1D). This result suggested that the cells obtained by the isolation and culture in this experiment were BMSCs and GMSC with higher purity.

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Fig. 1

Morphological and molecular characterization of BMSCs and GMSCs. The morphological observation of BMSCs (A) and GMSCs (C) (magnification, 100×) and surface marker molecular detection of BMSCs (B) and GMSCs (D) by flow cytometry. 1, 2, 3 respectively represented the primary generation, the Second-generation and the third-generation of BMSCs.

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3.2 GMSCs attenuate allograft rejection and prolong recipient survival

In order to explore the effect and mechanism of GMSCs on liver transplantation rejection, we successufully constructed FASL-/-GMSCs. As shown in Fig. 2 A and B, the expression of FASL was sharply decreased in FASL-/-GMSCs, which indcated the successful establishment of FASL-/-GMSCs. In rat liver transplantation model, most rats in NS and FASL-/-GMSCs groups, died within 21 days. When administrated with BMSCs and GMSC, the rats generally had better conditions with surviving for more than one month. The median survival time of rats in NS, FASL-/-GMSCs, BMSCs, and GMSCs groups were 15, 16, 30 and 50 days, respectively (Fig. 2C). The results suggested that GMSCs infusion had a beneficial effect on liver allograft survival.

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Fig. 2

FASL protein (A) and mRNA (B) expression in GMSCs and the survival curve analysis of rats in each group (C). The Ratios of FasL mRNA were expressed as mean ± SD (n = 3). * p < 0.05, compared with control group.

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AST, ALT and TBIL that are closely associated with injured liver cells, are important indicators for liver function evaluation. The ALT and AST levels of each group began to increase immediately after the operation. They greatly decreased on the 3rd day, then increased, and subsequently began to decrease after the 5th day (Fig. 3A). After surgery, the TBIL levels in each group increased gradiently from the 3th day to 14th day. There was no significant difference between the FASL-/-GMSCs group and NS group for the levels of ALT, AST, and TBIL at each time point. However, the BMSCs group gave a statistical difference when copmared with FASL-/-GMSCs group and NS group (p < 0.05). At the same time, the levels of AST, ALT and TBIL in GMSCs group were lower than those in BMSCs group (p < 0.05).

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Fig. 3

(A) The levels of ALT, AST and TBIL in each group from 3 to 14 days after surgery. The values of ALT, AST and TBIL were expressed as mean ± SD (n = 5). ap < 0.05 vs. Normal group; bp < 0.05 vs. FasL-/-GMSCs group; cp < 0.05 vs. BMSCs group. (B) Histologic examination (H & E staining) of liver grafts. In normal group, hepatic cells were arranged neatly and the structure of hepatocytes was intact; In model group, much inflammatory cell infiltration and complete disruption of structure of hepatic lobules; In FASL-/-GMSC group, the similar to model group, disturbance of structure of hepatic plate and inflammatory cell infiltration; In BMSCs and GMSCs groups, structural integrity of liver cells.

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Histologic examination of liver allografts from recipients with nomal saline and FASL-/-GMSCs administration revealed severe acute rejection on 14 day. However, acute rejection of allografts was reduced in transplanted rats infused with BMSCs and GMSCs (Fig. 3B).

3.3 GMSCs regulated the peripheral serum cytokine and Th17/Treg levels

The levels of IL-10 and TGF-β in the peripheral blood of the GMSCs group were significantly increased, while IL-2 and IFN-γ levels were significantly reduced, compared with NS group (p < 0.05) (Fig. 4). The serum levels of immune regulatory Treg in the GMSCs group were also significantly improved, while inflammatory Th17 were decreased (p < 0.05) (Fig. 5A and B). The ratio of Th17/Treg reached to the highest level on the 5th day in each groups and this ratio was much higher in FASL-/-GMSCs group than that in other groups (Fig. 5C), suggesting that severe inflammation ocurred in FASL-/-GMSCs group.

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Fig. 4

Peripheral serum cytokine levels of IL-10, IL-2, TGF-β and IFN-γ in each group, determined by specific ELISA kits. The values were expressed as mean ± SD (n = 5). ap < 0.05 vs. Normal group; bp < 0.05 vs. FasL-/-GMSCs group; cp < 0.05 vs. BMSCs group.

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Fig. 5

Treg and Th17 levels in peripheral serum from each group, detected by flow cytometry. (A) Th17 cells were decreased with days after surgery. (B) Treg cells in normal and FASL-/-GMSCs groups were significantly lower than that in BMSCs and GMSCs groups. (C) The balances of Th17/Treg were calculated according to the detection data. The values were expressed as mean ± SD (n = 5). ap < 0.05 vs. Normal group; bp < 0.05 vs. FasL-/-GMSCs group; cp < 0.05 vs. BMSCs group.

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