Ocular Surface Reconstruction Surgery Early Donor Stem Cell Survival Status And Influencing Factors Of The Research

Purpose:To investigate the survival state of the donor cells after ex vivo cultured limbal epithelial transplantation and analyze the possible influencing factors.Methods:Rabbit limbal stem cell deficiency mode was induced by alkali burn, and ex vivo cultured limbal epithelial transplantation was performed at stable stage after alkali burn. Clinical manifestations were observed by slit lamp microscope at immediate postopereative, and1day,3day,7day,14day,21day,28day postoperative. HE stain was performed at preoperation and each time point, and the expression of p63, Ki-67, CD68in donor and host tissue were detected by immunohistochemistry. RT-PCR was used to detect the expression of ANp63α、 Ki-67、 ABCG2、 C/EBPA、 CK3and inflammatory factors IL-lβ、IL-6、 IL-4、 IL-10、 IL-13、 TNF-α、 MCP-1、TGF-β、 VEGF、CD4、CD8at each time point.Results:In moderate group, the corneal epithelium is integral and without neovascularization within1month. But in severe group, corneal epithelial defected repeatedly and neovascularization was occurred at14days postoperation. HE stain and immunohistochemistry shows that p63and Ki-67were expressed within28days in moderate group, but gradually decreased. P63could be detected within7days in severe group, and Ki-67was rarely detected. A large number of CD68+macrophage infiltrated in preoperative corneal strom in both group. Neutrophils infiltrated in amniotic was detected at1day and3days postoperation and significantly reduced at7days postoperation. Macrophage consistently elevated in severe group, but gradually reduced14days later in moderate group. RT-PCR results show that the expression of ANp63a in donor cells was reduced about40%immediately postopereative, and significantly reduced at3days postopereative. The expression of ABCG2could be detected but gradually reduced within28days. In moderate group, the expression of Ki-67, C/EBPA, CK3were significantly reduced or even disappear at14days postoperative. But in severe group, the expression of Ki-67, C/EBP△, CK3were significantly reduced at3days postoperative, and at7days postoperative we can only detect very a low expression level or even disappear of these factors. Most inflammatory factors appear two peaks postoperative. The first peak appear immediately postopereative, was early inflammation mediated by macrophages. The expression of CD4was significantly increased at7days postopereative, and the second peak appears at this time. So the possible reason of this peak was immune rejection induced by CD4+cells. The expression of host△Np63a, ABCG2, CK3was increased compared with the preoperative in moderate group, but in severe group, it does not change or even decreased.Conclusions:The donor cells could be exist within1month after ex vivo cultured limbal epithelial transplantation, but its proliferation, differentiation, and self-renewal capacity could significantly reduce or loss within14days under the hit of mechanical injury during the surgery, early inflammatory response postoperative and immune rejection. Macrophages may play an important role in reduce the early inflammatory and immune rejection. Functional recovery of the host stem cells maybe an important factor to maintain a long-term stable ocular surface.