The Study On Inhibition Of PVR With Lentivirus Vector Mediated HSP47 SiRNA

ObjectiveTo observe the expressions of HSP47, TGF-β2 and collagen-I both in the epiretinal proliferative membranes of PVR patients and that in the retina of established PVR animal model in pigmented rabbits;To investigate the effect of LV-HSP47-si RNA transfection on epithelial-mesenchymal transition of cultured ARPE-19 cells induced by TGF-β2;To investigate the inhibitory action of LV-HSP47-si RNA on the process of PVR model.Methods1. Epiretinal membranes were made into pathological specimens from 30 patients with PVR by vitrectomy,and internal limiting membranes were collected as contrast from 12 donor eyes for corneal transplantation. To observe the pathological features, collagen deposition, and expression of TGF-β2, HSP47 and collagen type I in the proliferative membranes by HE staining, Masson staining and immunohistochemistry staining.2. PVR animal models were established with 40 healthy adult rabbits, the right eyes were selected as experimental eyes and the left eyes were as controls,the experimental eyes were enucleated and made into pathologic specimens. To observe the pathological features, collagen deposition, and expression of TGF-β2, HSP47 and collagen type I in the proliferative membranes by HE staining, Masson staining and immunohistochemistry staining.3.Cultured ARPE-19 cells in logarithmic growth phase were divided into 5 groups randomly:⑴ The untreated group; ⑵The TGF-β2 stimulated group; ⑶The LV-HSP47-si RNA transfected group; ⑷the lentiviral empty vector transfected group;⑸the PBS group. The cells in group ⑵~⑸ were pretreated by TGF-β2 to induce epithelial mesenchymal transition,then the group ⑶⑷⑸were coincubated with 10μl LV-HSP47-si RNA, lentiviral vector and PBS for 48 h,respectively. To observe the transfection efficiency, cell viability,HSP47 gene knockdown effection,and the expression of HSP47, α-SMA, FN and Col-I,fluorescence microscopy,CCK8 Kit, RT-PCR and Western blot were used.4. 36 healthy adult rabbits established of PVR models were divided into 3 groups randomly,with 12 rabbits in each group: ⑴The LV-HSP47-si RNA group; ⑵The lentiviral empty vector group;⑶The PBS group; The right eyes were selected as the experimental eyes. At 7th day, all rabbits were transfected with 10μl LV-HSP47-si RNA, lentiviral vector and PBS by intravitreal injection,respectively. To observe the clinical characteristics at different time points, investigate the histopathological features at the 28 th day, and detect the expression of HSP47,α-SMA,FN and Col-I in retinal tissues by fluorescence quantitative PCR and Western blot.Results1. We found that there were fewer cell components, and most of which were RPE cells, they adhered in extracellular matrix rich in collagen; Some of the RPE cells changed into spindle fibroblasts and shaped larger; More collagen component were found in the extracellular matrix by Masson staining; Significantly elevated expression of TGF-β2, HSP47 and Col-I were found by immunohistochemical staining, and they were mainly expressed in the cytoplasm and interstitial.2. Rabbits model of PVR presented vitreous opacity, proliferative membranes, partial and infundibular retinal detachment gradually, and the success rate of the animal model were 100%.The histopathological features of the PVR model were: the inflammatory cells and fibroblasts accumulated in the retinal surface gradually,proliferative membranes appeared and contracted,retinal detachment,retinal folds,disordered structure of the retina and increased fibrous connective tissue appeared; The expression of HSP47,TGF-β2, and Col-I increased gradually and got the peak at the 28 th day by immunohistochemical staining; Transparent vitreous body, structural integrity of retina, a small amount of collagen were found in normal rabbits control, and in which the expression of HSP47,TGF-β2,and Col-I were weak positive, immunehistochemical staining.3. ⑴Cultivated ARPE-19 cells were stimulated by 5ng/ml TGF-β2, the expression of decreased which was the marker of the epithelial cells,and the expressions of α-SMA, FN and Col-I were significantly up-regulated which were mesenchymal markers in both m RNA and protein levels(P <0.05);⑵Cells LV-HSP47-si RNA transfective efficiency was the highest(>75%) when the concentration of polybrene in the cultivation medium was 5μg/ml and the MOI was 10. In the LV-HSP47-si RNA transfective group, the cell viability appeared no significant difference(P > 0.05),compared with the LV-vector group and the PBS group; In the LV-HSP47-si RNA transfective group,the expression of HSP47 m RNA was significantly down regulated compared with the LV-vector group and the PBS group(P<0.05), and the knockdown efficiency can reach 75%.⑶In the LV-HSP47-si RNA transfective group, the EMT process induced by TGF-β2 were significantly inhibited, including the upregulated expression of HSP47,α-SMA, FN and Col-I both in m RNA and protein levels, compared with the LV-vector group and the PBS group(P <0.05).4.⑴The fundus photograph and B ultrasound scan showed the conditions were similar in the LV-vector group and the PBS group at different time points,which including vitreous proliferation, partial and infundibular retinal detachment.In the LV-HSP47-si RNA group, vitreous proliferation formed and be stable with no significantly progression.⑵Based on the Fastenberg grade at 14 th day and 28 th day, the ratio of retinal detachment in the LV-HSP47-si RNA group were less than that in the LV-vector group or in the PBS group,significantly(χ2=7.598,6.143;P=0.016,0.027).⑶At 28 th day, the expression of HSP47,αSMA,FN and Col-I was measured by fluorescence quantitative PCR and Western blot, the results showed transfection of LV-HSP47-si RNA by intravitreal injection could significantly down regulate the expressions of HSP47,αSMA, FN and Col-I(P all<0.05),compared with the LV-vector group and PBS group.Conclusions1.Increased expression of HSP47 and TGF-β2 were observed in proliferative membrane and internal limiting membrane,and the location of expression were identical with that of Col-I.2.Transfection of LV-HSP47-si RNA into cultured ARPE-19 cells can inhibit the EMT(epithelial- mesenchymal transition) induced by TGF-β2.3.Transfection of LV-HSP47-si RNA in experimental rabbits by intravitreal injection can inhibit the development and progress of PVR significantly,and reduce the rate of contractional retinal detachment.