Transcriptome Profiling in a Porcine Vascular Composite Allotransplantation Model
Transcriptome Profiling in a Porcine Vascular Composite Allotransplantation Model
We employed multiple MHC-mismatched Swiss Landrace pigs as both donors and recipients in our VCA procedures. These operations were conducted using the heterotopic porcine hindlimb VCA model, previously established and comprehensively documented. In summary, we procured osteo-myo-cutaneous limb allografts, including lymph nodes and skin paddles, from the donor pigs and transplanted them into a subcutaneous pocket created on the dorsolateral abdominal wall of the recipient pigs. Vessel anastomosis was performed between the graft and the recipient. The study encompasses six groups: Groups 1, 2, and 3 consist of skin tissue, while Groups 4, 5, and 6 comprise muscle tissue. Groups 1 and 4 represent the healthy control group, composed of three healthy donor pigs that underwent no prior manipulation, while others were rejection tissues. Groups 2 and 5 form the TGMS-TAC rejection group, where three recipients received TGMS-TAC. TGMS-TAC, as previously described (with a Tacrolimus concentration of 7 mg/mL), was injected into the specific graft at a dose of 140 mg per kg on post-operative day (POD) 0, 30, and 60, with 0.5 ml depots of TGMS-TAC subcutaneously injected into the skin graft. Groups 3 and 6 belong to the complete rejection group, featuring three recipients: one received oral administration of tacrolimus daily for the initial 14 days post-transplantation, while the other two recipients received a short course of systemic tacrolimus for 14 days, followed by intra-graft injections of 1.25 mL/kg-graft Rapa-loaded in situ forming implant (ISFI) loaded with 5 mg of rapamycin per 0.31 mL at multiple sites on POD 15. The animals were closely monitored until the graft skin displayed extensive epidermolysis, desquamation, and necrosis, serving as the endpoint criterion (corresponding to grade III-IV rejection) or until POD 90. Samples from the graft's skin and muscle were collected for analysis at the endpoint. We analyzed the mRNA transcriptome of 18 VCA samples, including skin and muscle from VCA transplant recipient pigs. The total RNA was extracted according to the instruction manual of the TRlzol Reagent (Life technologies, California, USA). Tissue samples (50-100 mg) were homogenized in 1 ml of TRI reagent and incubated for 5 minutes at 15-30 °C. We added 0.2 ml of chloroform and shook the mixture for 15 seconds. After an additional 2-3 minutes of incubation, we centrifuged it at 12,000 * g for 15 minutes. The RNA was precipitated by mixing the aqueous phase with isopropanol, followed by a 10-minute incubation at 15-30 °C. After a 10-minute centrifugation at 4 °C and 12,000 * g, we removed the supernatant, washed the RNA with 75% ethanol, and air-dried it for 5-10 minutes. The purified RNA was dissolved in RNase-free water, incubated at 55-60 °C for 10 minutes, and stored at -70 °C. We measured RNA concentration, purity, and integrity using the NanoDrop 2000 and the Agilent Bioanalyzer 2100 system. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology (Shanghai) Co., Ltd.) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads.First strand cDNA was synthesized and second strand cDNA synthesis was subsequently performed. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.