Mesenchymal stem cells (MSCs) have been applied in basic and clinical researches of organ transplantation.Our previous study showed that intravenous injection of MSCs prolonged corneal allograft survival in rat.However, the effect of local administration of MSCs on corneal allograft rejection remains unclear.

The aim of this study was to investigate the effect of subconjunctival injection of MSCs on corneal allograft rejection in rat model of keratoplasty.

MSCs were isolated and cultured from femur and tibia bone marrow of clean Wistar rats, and then the cells were identified by induced differentiation of osteoblast and adipocyte.The third generation of MSCs was used in subsequent experiment.Allogenic penetrating keratoplasty was performed with the bilateral corneas of 20 Wistar rats as donor grafts and the right eyes of 40 Lewis rats as recipients.PBS 0.1 ml containing 2×10⁶ MSCs and 0.1 ml PBS only was subconjunctivally injected immediately and postoperative 3 days respectively in randomized two groups, and another 6 normal Lewis rats served as the normal control group.Corneal rejection response was evaluated under the slit lamp after surgery based corneal opacity, edema and neovascularization, and the grafts were scored according to the criteria of Larkin.The corneal samples were extracted from 12 rats of the PBS control group and the MSCs group separately 10 days after surgery.The relative expressions of Th1 cytokines (interferon-γ [IFN γ] mRNA and interleukin-2 mRNA) and Th2 cytokines (IL-4 mRNA and IL-10 mRNA) were detected by real-time quantitative PCR.Protein levels of IL-4 and IL-10 proteins in the corneas were assayed by ELISA.All experimental protocols involving rats were approved by the laboratory animal care and use committee of the Tianjin Medical University and treated with the ARVO statement for the use of animals in ophthalmic and vision research.

The cells grew well with the orange stain for alizarin red in differentiated the osteoblasts and red stain for Oil red O in differentiated adipocytes.The survival time of corneal graft in the MSCs group was (11.8±1.6) days, it was significantly longer than (9.6±1.4) days in the PBS control group (P=0.004). The levels of IL-4 mRNA and IL-10 mRNA in the MSCs group were significantly higher than those in the PBS control group (both at P=0.00); while the levels of IFN-γ mRNA and IL-2 mRNA were not significantly different between the groups (both at P>0.05). The IL-10 protein contents were (22.74±7.06), (68.40±12.83) and (215.41±44.66)pg/ml in the normal control group, PBS control group and MSCs group, showing significant difference among the three groups (F=55.06, P=0.00) and a significant increase in the MSCs group compared with the PBS control group and the normal control group (both at P<0.05).

Subconjunctival injection of MSCs prolongs the survival time of cornea allograft in penetrating keratoplasty probably by modulating the balance between Th1 and Th2 cytokines, especially by up-regulating Th2 cytokines.

Mesenchymal stem cell transplantation; Corneal transplantation; Graft survival/immunology; T-lmphocytes; Interferon-gamma; Interleukin; Rats, Lewis