Effect Of Lentivius-mediated TLR2 Gene Silencing On Rat Corneal Transplantation Rejection

BackgroundCorneal blindness is one of the major causes of blind eye diseases, ranking only second to cataract. It severely affects the life quality as well as physical and mental health of patients. Most of the patients could accept cornea transplantation as treatment. A majority of patients accepted the surgery could survive from the cornea diseas. However, as the transplantation operation with the highest success rate, postoperative immune rejection is still the most crucial factor affecting the therapy effect. Cornea transplantation is a complicated immune process participated by multiple kinds of immune cells and molecules. Even though the mechanism of immune rejection is becoming much clearer than before, and there have been lots of medical interventions to prevent immune rejection after transplantation, immune rejection is still unpreventable. Therefore, the mechanism of corneal transplantation rejection still needs more exploration.There have been various kinds of comprehensive researches to study cornea transplantation rejection. Cornea transplantation have been developed to be the maturest transplantation surgery, the clinical practice have begun 100 years before. Because the cornea owns the capability of immune privilege, the success rate of cornea transplantation is the highest amongst all the transplantation surgeries. The definition of immune privilege is listed below:The probability and rate of development of cornea transplantation rejection is relatively lower than other organ transplantation under the similar pre-transplantation conditions. Up to now, cornea transplantation rejection is till the primary risk factor of seriously affecting the outcome of cornea transplantation surgery. The major mechanism of transplantation immune rejection according to the researches is listed below:There are plenty of Langerhans cells carrying HLA-DR on the edge of cornea, the presence of those cells may induce the early phase of transplantation rejection, and contribute to the development of the rejection. MHC and minor antigen molecules on the cornea participate in the cornea transplantation rejection. The foreign antigens were intaken and processed by the APCs, and were presented by MHC-II on those cells, forming costimulatory molecules with TCRs expressed on the surface of T cells, inducing the T cells to recognize alien antigens. The acute cornea transplantation immune rejection is the type IV hypersensitivity mainly activated by CD4+T cells. The release of a series of immune inflammatory cells would lead to irreversible serious damage and harm to the corneal graft, finally causes the failure of the transplantation surgery.In the present time, there have been various kinds of preventive and treating methods for the precaution and treatment of transplantation rejection. The common ways we have used in the clinical practice are listed below:1, etiological treatment, anti-transplantation rejection therapy; 2,prevent and control the post-surgery inflammation; 3, symptomatic therapy, improve the nutrition support. Among all the treatment methods, treating with drugs is the most useful and common way. The most commonly-used drugs are:corticosteroid, interleukin-1 receptor antagonist, FK506, antineoplastic drug etc. However, the application of those drugs still have significant side effects. Long-term application of corticosteroid could cause serious inflammation due to the weakened immune system; The side effects of CsA are less serious than corticosteroid, but it could raise the morbidity risks of getting lymphoma and other malignant tumors, especially the skin cancer, interleukin-1 receptor antagonist could reduce the chemotaxis of APCs through the inhibition of inflammation factors, but the long-term application could cause dose-dependent symptoms; FK506 belongs to fungus metalolite, the mechanism of it is similar to CsA, but due to the high cost of making this drug, the clinical application is still limited. Therefore, to further clarify the immune mechanisms of cornea transplantation, seek an absolutely new target spot of this disease and develop a wholely new drug with fewer side effects, is still the essential task for the clinical and scientific researchers.TLRs (Toll like receptors) is a family of membrane bound proteins which could recognize PAMPS (pathogen associated molecular patterns) and DAMPs (Damage associated molecular patterns). They play an important role in the immune system. Up to now, scientists have found 13 kinds of TLRs. TLR2 (Toll like receptor 2) is the protein which expresses on the widest kinds of cells and tissues. What’s more, TLR2 could recognize the most kinds of pathogen microbes with their products. TLR2 could recognize and combined with exogenous ligands such as lipoprotein, peptidoglycan and endogenous ligands such as hot shock protein 70 and hyanuronic acid. TLRs could activate the downstream signals via at least two pathways including MyD88 independent pathway and MyD88 dependent pathway. TLR2 mainly initiates the signaling pathway through MyD88 dependent pathway and participate in the transplantation immune rejection process. Large numbers of experimental studies have shown that in the transplantation rejected organs such as lung, liver and kidney, TLR2 and MyD88 expressed much more than that in the normal specimens. Scholars have found that the application of steroid, CSA, antibodies and specific retardant could block TLR2/MyD88 signaling pathway and significantly prolong the survival of transplants. In addition, inflammatory cell infiltration in TLR2-/-mice is milder than that in the normal mice, with a prolongation of survival. Our previous studies have also shown that TLR2 and MyD88 express extremely higher, while the application of steroid and TLR2 monoclonal antibody could significantly down regulate the expression of TLR2 and MyD88, inhibiting the occurrence and development of cornea transplantation rejection and prolonging the survival of the transplantation grafts. Based on the study results, we could postulate that TLR2/MyD88 signaling pathway participate in the process of cornea transplantation immune rejection process, and plays an essential role in the corneal transplantation rejection.Gene interference is a updated and universal tool of gene therapy by inhibiting and shutting down the gene expression to treat diseases. RNAi (RNA interference) is a common way of gene interference. The siRNA (small interference RNA) was transmitted into the cells, leading to the specific degradation of mRNA and inhibit the expression of the gene. At the present time, LV (Lentiviral vector) construction based siRNA has been widely used in the ophthalmic experimental field. Studies have shown that LV mediated target gene to transfect the cornea could reach long-term, effective and stable expression without significant toxicity, and it has become a potential vector in the gene therapy of corneal diseases in the future. In the following experiment, we have constructed LV-TLR2-siRNA-EGFP(enhanced green fluorescent protein), and perform the functional identification and titer determination. We performed the subsequent experiments:(1) Transfect the primary cultured cornea epithelial and stromal cells with LV-TLR2-siRNA-EGFP, analysis the affection by TLR2 expression (2) Applicate LV-TLR2-siRNA-EGFP to the rats operated with cornea transplantation, observe the corneal buttons under the slip-lamp microscope and evaluate the rejection index. Detect the expression of TLR2 and the downstream factor MyD88 on the mRNA, molecular level. Our experiments explored the feasibility of blocking TLR2 to prevent the rejection, and provide experimental proofs to the clinical treatment.Part I Experimental research of lentivirus mediated TLR2 gene interference on the corneal epithelial and stromal cellsObjectiveEvaluate the effectiveness and safety of LV-TLR2-siRNA-EGFP transfecting the corneal epithelial and stromal cells.MethodThe rat eyes were exteriorized under aseptic condition in the Horizontal Flow Clean Bench, the rat cornea tissues were cut off from the eye balls, the corneal epithelial layer and the stromal layer were detached, washed 2-3 times with aseptic PBS. lml parenzyme was added into the culture medium, digested 30min. Put into the DMEM culture medium, and place in the incubator (37±,5% CO2). Epithelial cells and stromal cells were separately divided into 3 groups. The transfected group was transfected with LV-TLR2-siRNA-EGFP; the negative control group was transfected with LV-EGFP, and the blank group was added saline. The transfected group was transfected with MOI 10,50,100,200, optimize the MOI after observing the highest EGFP expression under the fluorescent microscope. Observe the morphological changes of the transfected cells, detect whether the cells swell, pathologically changed. Detect the proliferation of cells after Oh,72h and 96h after incubating with CCK-8 under the microplate reader under 490nm. Epithelial cells and stromal cells were digested with parezyme and made into cell suspension, the fluorescence expression were detected by flow cytometery to evaluate the transfection efficiency. Operate the q-RT-PCR according to the illustration of DBI corporation and detect the TLR2 mRNA in different groups. The amplification condition:94℃ 2min, 4℃ 30s,58℃ 30s,72℃ 20s,40 circulations, using ABI 9700 PCR instrument to perform the assays.ResultsWhen transfected under MOI 200 the transfected cells showed the strongest fluorescent expression. The transfected cells showed no significant change compared with the blank group. CCK-8 results showed that IOD presented no significance among all the groups. The transfection efficiency were 77.600%±1.100% in the epithelial cells and 76.300%±1.387% in the stromal cells. RT-PCR showed the TLR2 mRNA was significantly down regulated in the transfect groups compared with the control groups.ConclusionLV-TLR2-siRNA-EGFP could effectively and stably transfect the cultured corneal epithelial and stromal cells in vitro, and showed no significant toxicity on the cells.Part Ⅱ Experimental study of inhibition of rat cornea transplantation rejection by Lentivirus mediated encoding TLR2 gene transfectionObjectiveExplore the feasibility of using LV-TLR2-siRNA-EGFP to inhibit postoperative rejection in the penetrating keratoplasty(PKP) rats.MethodFive groups were established:Normal coreas(N), allograft without any treatment(A), allograft treated with LV-EGFP(B), allograft treated with LV-TLR2-siRNA-EGFP(L), isograft group(I). Group A, B and L accepted allograft penetrating keratoplasty. Group L accepted 20μL LV-TLR2-siRNA-EGFP injection into the conjunctival sac after surgery. Group A and B were given the same volume of saline and LV-EGFP. The degree of edema, opacity and neovascularization were observed under the slip-lamp microscope every other day. Evaluate and grade the condition of the corneal grafts according to the Holland criteria. Mark down the survival time of rat corneal grafts for the subsequent survival analysis. Excise the eyes on the 9th after surgery for paraffin embedding, observe each layer under the microscope after HE and IHC staining. In addition, corneal tissues were detached to perform the q-RT-PCR to detect the TLR2 and MyD88 mRNA expression.Results1.The edema, opacity and neovascularization of corneal grafts of different groups aggravated gradually with time after operation. On the 9th day after surgery, group A and B showed severe edema and thickened stroma, the neovascular grew to the grim of the cornea button. At the same time, group L and I showed less edema, opacity and neovascular compared with group A and B.2.HE results showed:the normal rat cornea tissue comprised 5 layers, different layers arranged regularly, the stromal layer showed no edema and inflammatory cell infiltration. On the 9th postoperative day, the stromal layer became thickened and the structure showed irregularly in group A and B, with a serious inflammatory cell infiltration. The group L and I showed no significant thickened stroma, and few inflammatory cell infiltration.3.IHC results:The IHE results showed that TLR2 and MyD88 expressed in all the grafts, group A and B were significantly higher than group L and Ⅰ.4. On the 9th and 14th postoperative day, RT-PCR showed that TLR2 and MyD88 mRNA were detected in all groups. The relative expression in group A(15.350±1.562; 4.113±0.572) and B (13.848±1.475; 3.646±0.227)were significantly higher than that in group L(8.825±1.333; 2.416±0.479). On the 14th day group Ⅰ(5.153±0.256; 2.504 ±0.057) and group L(5.269±0.251; 2.628±0.146) were still lower than that in group A(12.104±0.430; 3.581±0.107) and B(10.567±0.277; 3.192±0.193). TLR2 and MyD88 showed a consistent variation tendency.5. The median survival time of group A is 9d and B 10d, which were significantly shorter than that in group L (more than 50% of the rats survived when it was 30d),p<0.001.ConclusionLV-TLR2-siRNA-EGFP could effectively prolong the survival time of rat allografts via down regulating TLR2/MyD88 signaling pathway, which plays a crucial role in the transplantation rejection process.