| Proliferative vitreous retinopathy was proposed in 1983 by the American association of retinal experts. It is used to describe the disease of the anterior and/or posterior surface proliferative membrane of the vitreous and retina after the rhegmatogenous retinal detachment. The contracting membrane can lead to the retina traction, detachment and fixation. It is a common intractable blinding eye disease. According to reports 5% ~ 10% RRD can be secondary PVR, and in recurrent retinal detachment(retinal detachment,RD), the incidence of PVR is increased to 75%.In recent years, with the research on PVR pathological mechanism gradual deeply, a growing number of studies have found that the epidermal growth factor(epidermal growth factor, EGF) plays an important role on RPE cell migration, proliferation. It is one of the key factors to promote RPE cells migrate of PVR process. At present, the main method of clinical treatment of PVR is vitreous retinal surgery, but the surgical treatment effect is not ideal, the recurrence rate of postoperative PVR is higher. Therefore, with deeper understanding of a variety of cells and growth factors in the process of PVR pathogenic mechanism, the method of prevention and treatment of PVR according to the different stages of development of PVR and related factors to use different drugs is becoming hot trend. Drug researches are mainly concentrated in the field of western medicines which including metabolic drugs, corticosteroids, vitamin and its derivatives, extracellular matrix synthesis inhibitors and cell signal transduction inhibitors. Although the research of prevention the PVR in western medicine has been many years, there is no drug can be successfully applied in clinical because of larger intraocular drug toxicity, single pharmacological effect, expensive price and many other limitations. At present, the western medicine research is difficult to break through, the traditional Chinese medicine prevention and treatment of PVR research has become a new breakthrough direction. Traditional Chinese medicines have many advantages such as the rich medicine source, extensive effects, smaller side effects, and so on.Curcumin, a kind of traditional Chinese medicine monomer composition, is abstracted from rhizoma of Curcuma. It has many pharmacological effects including anti-proliferative, anti-inflammatory, anti-microbial and some others and many advantages including safety, low toxicity, wide medicine sources, low price. The pharmacological activity can meet the conditions of drugs for prevention and treatment of PVR. Previous researches have shown that curcumin could inhibit retinal pigment epithelial cells proliferation. Whether the curcumin play an important role to the epidermal growth factors in the formation of PVR, the experiment research the role and the related mechanism of the medicine to PVR by cells research in vitro and vitreous injection of curcumin experiment in animal vivo respectively and provide the basis for prevention of PVR. Part one The expression of EGF in RPE cells and the function to RPE cells cultured in vitroObjective: To research the expression and the function of EGF in rabbit RPE cells cultured in vitro.Methods: Extract and culture the chinchilla rabbit RPE cells, continously passage to the third generation and identify cells. Selecting the third generation of RPE cells in good condition to do experiment. Culture the RPE cells on the culture plate which have covered glass in it, then do the EGF immunohistochemical staining. The EGF groups can be divided into 3, 6, 9 and 12 ng/m L 4 groups and 1 blank control group(10%FBS. DMEM). Each group set 6 holes and a total of 3 culture plates were inoculated, randomly selecting 1 culture plates after dosing 24 h, 48 h and 72 h to detect the EGF influences to the RPE proliferation of the different concentrations at the different time by MTT.Results: The RPE cells cultured in the early proliferate actively. The nucleus is transparent. The cytoplasm contains abundant melanin granules. The expression of keratin is strongly positive by immunohistochemical staining. The expression of the EGF is in the cytoplasm of the rabbit RPE cells presenting claybank. At the same time, with the different concentration of EGF, the absorbance of RPE cells(OD value)is increasing with the increased of the concentration of EGF, and there are significant differences compared with the control group(P <0.05). With the concentration of EGF greater than or equal 9 ng/ml, there is no statistical significance between the two groups which have the adjacent concentration(P>0.05). With the same concentration of EGF, the absorbance(OD value) of RPE cells is increasing with the increased of the time, and comparing with the adjacent groups is no significan difference(P <0.05).Conclusions: The RPE cells cultured in vitro can be used for the vitro experiments; The expression of EGF is in the cytoplasm of the rabbit RPE cells; The regulation of EGF to the rabbit RPE cells proliferation in vitro has certain dose effect relationship, time effect relationship. The growth promoting effect of EGF on RPE cells is saturated when the concentration of EGF is more than 9 ng/ml; The research results indicate that the optimal concentration of EGF for promoting the proliferation of rabbit RPE cells in vitro is 9ng/ m1. Part two The inhibitory effect of curcumin on the expression of EGF of the RPE cells cultured in vitroObjective: To study the effect of curcumin on the expression of EGF of the RPE cells cultured in vitro. Detecting the influence of curcumin to the expression of EGF in the rabbit RPE cells by the method of immunohistochemistry, looking for the best concentration of curcumin in inhibiting the expression of EGF; To explore the mechanism of curcumin inhibiting EGF by the methods of PT-PCR, Western blot to test the expression of EGFm RNA and protein in RPE cells.Methods: Selecting the third generation of RPE cells in good condition to do experiment. Making the cells growing on the glass slide. The 4 groups were divided into blank control group(10%FBS.DMEM) and 10, 15, 20 ug / m L curcumin; Each group sets 6 holes and a total of 3 culture plates are inoculated. Rrandomly selecting 1 culture plate after dosing 24 h, 48 h, 72h; then doing the experiment of immunohistochemistry. To observe the expression of EGF in RPE cells. Respectively testing the m RNA expression and protein expression of the EGF of RPE cells of blank control group(containing 10% FBS. 0.5 ‰ DMSO DMEM), curcumin(15 ug/m L), EGF(ng/m L) 0.5 ‰ DMSO, EGF(9ng/m L) + curcumin(15 ug/m L) after 24, 48, 72 h by the method of RT-PCR and Western blot.Results:1 The inhibitory effect of different intensity curcumin on the expression of EGF in RPE cells(Immunocytochemistry staining): Time-dependence:the inhibitory effect of curcumin on the expression of EGF in RPE cells is increased with the prolonging of time, and there are significant differences between different time points(P<0.05). The inhibitory effect of curcumin on the expression of EGF in RPE cells is time-dependent. Dose dependence: the inhibition intensity of curcumin on expression of EGF in RPE cells at each time point is increased with the increasing drug concentrations, in addition to the difference between the curcumin 15ug/ml and 20ug/ml group have no statistical significance(P> 0.05), the differences between the rest groups have statistically significant(P< 0.05).2 Effect of curcumin on the transcription of EGFm RNA in RPE cells: Curcumin of 15 ug/m L role for 24 h, 48 h and 72 h, the EGF m RNA expression in cells gradually decreased with the extension of time, there are statistically significant difference compared with the control group(P<0.05); among the different time points, the adjacent time groups have statistically significant differences(P<0.05). The expression of EGF m RNA in the RPE cells with the effect of curcumin on EGF, there are significant differences compared with the control groups at various time points(P<0. 05), among the different time points, comparing the adjacent time groups, the differences are statistically significant(P<0. 05).3 Effect of curcumin on the expression of EGF protein in RPE cells: Curcumin of 15 ug/m L role for 24 h, 48 h and 72 h, the expression of EGF protein in cells is decreased with the time extension, there are statistically significant differences compared with the control group(P<0.05), among the different time points, the adjacent time groups have statistically significant differences(P<0.05). The expression of EGF protein in the RPE cells with the effect of curcumin on EGF, there are significant differences compared with the control groups at various time points(P<0.05), among the different time points, comparing the adjacent time groups, the differences are statistically significant(P<0. 05).Conclusions: The best concentration of Curcumin inhibiting the expression of EGF is 15ug/ml. Curcumin significantly inhibited the expression of m RNA and protein of EGF in rabbit RPE cells in vitro. Part three Effects of curcumin on EGF in rabbit eyes with PVRObjective: To explore the effects of curcumin on EGF in rabbit eyes with PVR and the function of preventing and treating PVR.Methods: Select 30 chinchilla rabbit whose vitreous body are extracted 0.2ml before injection. One eye is included in control group randomly, 30 eyes in all, the vitreous cavity of the eyes is injected with 0.1m L(2×106) well-grown RPE cells of the 3rd generation and 0.1ml normal saline which containning 0.5‰DMSO; the other one is included in study group, 30 eyes in all, the vitreous cavity of the eyes is injected with 0.1m L(2×106) well-grown RPE cells of the 3rd generation and 0.1ml curcumin with mass concentration of 1 mg/m L. At 3, 7, 14, 21 and 28 days after injection, to observe the situation of anterior segment and anterior vitreous with the slit lamp microscope, the situation of eyeground with indirect ophthalmoscope, the situation of vitreous opacity and the retinal detachment with fundus photochromy and B ultrasonography; after examination, select 6 Chinchilla with 12 eyes(6 eyes from the control group,6 eyes from the study group) at each time point, extracting vitreous body and detecting the content of EGF in vitreous body fluid by ELISA kit.Results:1 Anterior chamber effection: The third day after the vitreous cavity injection, a small amount of floating matter was observed in both the control group and the study group, they all subside on the seventh day.2 Vitreous and Retina: On the third day, the vitreous of the two groups are cloudy, the study group relative lighter; on the seventh day, some proliferation membrane form in the vitreous, the proliferation membrane is more and thick in the control group and is limited and thin in the study group; on the fourteenth day, the incidence of retinal detachment in control group is(11/18) 61%, the vitreous proliferation membrane of the study group is thin and the incidence of retinal detachment in study group is(2/18) 11%; On the twenty-first day, the incidence of retinal detachment in the control group was(8/12) 67%, with new blood vessels growing on the proliferation membrane, the study group retinal detachment rate is(2/12) 16%, a small-ranged retinal detachment; on the twenty-eighth day, the retinal detachment of the control group gradually increased, with blood vessel increasedly growing on the proliferation membrane, the retinal detachment rate is(5/6) 83%, meanwhile, the incidence rate of retinal detachment in the study group is(1/6) 16%. The difference of retinal detachment rate between the study group and the control group is statistical significant(P<0.05).3 The detection of EGFcontaining in vitreous body(ELISA result): The content of EGF in vitreous body fluid of the control group is higher than the study group. At each time point, the difference between the study group and the control group is statistical significant(P<0.05).Conclusions: Curcumin can effectively inhibit the level of EGF in the formation of PVR induced by RPE cells in the experimental study of rabbit eyes, and then inhibit the occurrence and development of PVR. |
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