Experimental Study Of The Role Of Brain Derived Neurotrophic Factor And Transforming Growth Factor-β₂ In Form Deprivation Myopia

Myopia is commonly and frequently encountered disease in ophthalmology, especially high myopia, which is also called pathological myopia or degeneration myopia. It makes a lot of difficulties to people's daily life, because of its syndrome and corrected visual acuity is not ideal. There is still no satisfactory explanation to myopia pathogenesis. The foundation of form deprivation myopia(FDM)model help people to deeply understand how high myopia occur and develop. Form deprivation modulate the development of local sclera by changing the level of various neurotransmitter and growth factors in the retina. These neurotransmitter and growth factors work by means of cooperate parallel and across.BDNF is a member of neurotrophins. Studies showed that monocular deprivation decreased BDNF mRNA level in the visual cortex contralateral to the deprived eye and BDNF level of ganglion cell of the deprived eye. TGF-P is a multi-function super family of growth factor, TGF-β₂ is major in the biological function of TGF-p in the eyes. It has been reported that monocular deprivation can reduce the level of mRNA and protein of TGF-P in retina-retinal pigment epithelium-choroid of the deprived eye. It is essential in the regulation of the dynamic balance of Matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of metalloproteinases(TIMP-2)and remodeling of ECM of sclera .The pathological change of sclera is the important mechanism of myopia. It has been reported that in FDM the level of MMP-2 of sclera is increase but the levelof TIMP-2 is decrease.Animal experiments have testified that lots of medicines are effective in the inhibition of myopia, but great breakthrough has not been made in the clinical application. The role of growth factor in diseases of ophthalmology FDM has been understood gradually, but it is difficult to be absorbed orally, can not get across the blood retinal barrier, is only effective in a short term, and can induce syndrome by intravitreal administration frequently. So people want to combine the growth factor to the gene therapy, the construction of eukaryotic expression vector of growth factor and transfection has been applied in experimental study in ophthalmology, but it has never been reported in the studies of myopia.By using the establishment of a form deprivation myopia model in guinea pigs, the aim of this experiment is to detect the expression of BDNF and TGF-β2 protein in retina by immunohistochemistry, and to detect the expression of BDNF and TGF-β2 mRNA in retina choroid and sclera by reversetranscription-polymerase chain reaction(RT-PCR), furthermore to detect the expression of MMP-2 and TIMP-2 mRNA and the level of its protein in sclera by RT-PCR and Western blotting. Also, by constructing the eukaryotic expression vector of BDNF and TGF-β2 and by using liposome to transfect the vitreous of guinea pig, the green fluorescene protein (GFP) expression was observed with fluorescene microscopy, the expression of BDNF and TGF-β2 mRNA was detected by RT-PCR . we investigated the mechanism of FDM. All of these has provided a new idea to the research of the therapy of myopia. The first part: Experimental study of the expression of BDNF in form deprivation myopia Methods1. Establishment of a form deprivation myopia model: Twenty guinea pigs at age of 3 days were selected. Ten guinea pigs were used for immunohistochemistry,the other ten guinea pigs were used for RT-PCR. All the right eyes underwent form deprivation with translucent goggles and the left eyes were used as control. 8 weeks later ,the goggles were removed respectively.2. Determination of the refractive state and the axial length: Before occludingand after occluding for 4 weeks and 8 weeks , the refractive state and axial length were determined with retinoscopy and A-scan ultrasonography respectively.3. Hematoxylin-eosin staining and morphology: After the refractive state and axial length were determined ,the eyes were extracted after 2 month , Routine hematoxylin-eosin staining were done, and morphological changes in form deprived guinea pigs retina and sclera was detected under light microscope.4. The expression of BDNF protein in retina were detected by immunohistochemistry.5. The expression of BDNF mRNA were detected by RT-PCR in retina choroid and sclera.Results1. Comparation of refraction state between the occluded eyes and control eyes: Monocular occluding for 8 weeks could lead to myopia, the refraction showed significant difference between the occluded eyes(-3.40±1.33D) and the control ones(2.95±0.98D) (P<0.01). The longer the eyes were occluded, the more severe the myopia was. After monocular occluding for 8 weeks ,the change was -7.35±1.16 D in the occluded eyes, -0.90±0.70 D in the control eyes before occlussion and after occlussion, the occluded eyes became myopia(-7.3 5± 1.16 D Relatively.2. Comparation of axial length between the deprived eyes and control eyes: Monocular occluding for 8 weeks could lead to myopia, the axial length was longer, There was significant differences between the occluded eyes(8.13±0.18mm) and the control ones(7.33±0.20mm) (P<0.01). The longer the eyes were occluded, the more longer the axial length was.3.Morphology change in retina and sclera: hematoxylin-eosin staining showed the posterior retina of the occluded eyes were thinner than that of the control eyes, The posterior sclera of the occluded eyes were thin and rarely.4.1mmunohistochemistry: BDNF positive cells were mainly distributed in the inner nuclear layer and ganglion cell layer of the retina, immunohistochemistry staining shows that BDNF positive expression was detected in the cytoplasm,displaying as yellow or pale brown pellet. Through the image analysis, theexpression of BDNF protein in retina in the occluded eyes was decrease, there were significant difference compared with that in the control eyes(P<0.01).5. RT-PCR: The expression of BDNF mRNA in retina and choroid of the occluded eyes decrease, there were significant difference compared with that in the control eyes(P<0.01), the expression of BDNF mRNA in sclera of the deprived eyes were not detected.The second part: Experimental study of the expression of BDNF in form deprivation myopia Methods1. Establishment of a formdeprivation myopia model: refers to the first part.2. determination of the refractive state and the axial lengthrrefers to the first part.3. The expression of TGF-2 protein in retina was detected by immunohistochemistry.4. The expression of TGF-02 mRNA was detected by RT-PCR in retina ^ choroid and sclera.Results1. Comparation of refraction state and axial length between the occluded eyes and control eyes: refers to the first part.2. immunohistochemistry: TGF-02 positive cells were mainly distributed in the outer segments of photoreceotors cell of the retina, immunohistochemistry staining shows that TGF-P2 positive expression display as pale brown .Through the image analysis, the expression of TGF-P2 protein in retina of the occluded eyes decrease .There were significant difference compared with that in the control eyes(PO.Ol).3. RT-PCR: The expression of TGF^mRNA in retina ^ choroid and sclera of the occluded eyes decrease, there were significant difference compared with that in the control eyes(P<0.01).The third part: Experimental study of the expression of MMP-2 and TIMP-2 in the sclera of form deprivation myopiaMethods1. Establishment of a form deprivation myopia model: refers to the first part.2. Determination of the refractive state and the axial length:refers to the first part.3. The expression of MMP-2 and TIMP-2 mRNA in sclera were detected by RT-PCR.4. The expression of MMP-2 and TIMP-2 protein in sclera were detected by Western blotting .Results1. Comparation of refraction state and axial length between the occluded eyes and control eyes: refers to the first part.2. RT-PCR: The expression of MMP-2 mRNA in sclera of the occluded eyes increases greatly, There were significant difference compared with that in the control eyes(P<0.01), The expression of TIMP-2 mRNA in sclera of the occluded eyes decrease,There were significant difference compared with that in the control eyesCPO.Ol).3. Western blotting: The expression of MMP-2 protein in sclera of the occluded eyes increases ,There were significant difference compared with that in the control eyesCPO.Ol), The expression of TIMP-2 protein in sclera of the occluded eyes decrease, there were significant difference compared with that in the control eyesCPO.Ol).The fourth part: Construction of eukaryotic expression vector and transfection of brain derived neurotrophic factor and transforming growth factor-p^ gene Methods1. Construction of the clone vector of BDNF and TGF-P2 gene: The total RNA was extracted from retina of guinea pig. RT-PCR were used to obtain the BDNF and TGF-p2 gene .The BDNF and TGF-p2 gene were cloned into pGEM-TEasy to amplify. Select and identify the recombinant.2. Construction of the eukaryotic expression vector of BDNF and TGF-f$2 and analyse the sequence of BDNF and TGF-fc: BDNF and TGF-P2 gene were cloned into eukaryotic expression vector pEGFP-C3 and pEGFP-N3 respectively. Theeukaryotic expression vector of pEGFP-C3-BDNF and pEGFP-N3-TGF-P2 were constructed and the sequence of BDNF and TGF-P2 were analysed.3.Gene transfection of The eukaryotic expression vector of pEGFP-C3-BDNF and pEGFP-N3-TGF-P2 : eighty-eight guinea pigs at age of 3 days were selected. Eight guinea pigs were used for observation for green fluorescene protein (GFP) , other eighty guinea pigs were divided into BDNF and TGF-P2 group and used for RT-PCR. The right eyes were used as transfected eyes, the left eyes were used as control eyes. pEGFP-C3-BDNF > pEGFP-N3-TGF-p2 and Liposome were mixed to transfect the vitreous of guinea pig with 1 u 1 respectively. The eyes were extracted after the eyes were transfected for 3 days, K 2> 4 weeks.The green fluorescene protein (GFP) expression was observed with fluorescene microscopy, The expression of BDNF and TGF-p2 mRNA was detected by RT-PCR . Results1. Construction of the clone vector of BDNF and TGF-P2 gene: The gene of BDNF and TGF-p2 were obtained and amplified by RT-PCR to Construct pGEM-TEasy- BDNF and pGEM-TEasy- TGF-P2, The recombinant was identified.2. Construction of the eukaryotic expression vector of BDNF and TGF-P2 and analyse the sequence of BDNF and TGF-P2: BDNF and TGF-p2 gene were cloned into eukaryotic expression vector pEGFP-C3 and pEGFP-N3 respectively, The eukaryotic expression vector of BDNF and TGF-P2 were constructed . The sequence analysis resultstestified that they were identical.3. Gene transfection of the eukaryotic expression vector of pEGFP-C3-BDNF and pEGFP-N3-TGF-P2 : In the third and 1 week , GFP was observed in part and all retina in BDNF group and TGF-P2 group, After 2 week , GFP was reduced gradually. In the fourth week .A small number of GFP was observed, GFP was not observed in choroid and sclera . The expression of BDNF and TGF-P2 mRNA in retina of the experimental eyes increases, There were significant difference compared with that in the control eyesCPO.Ol).Conclusions1. The form deprivation myopia of guinea pigs model were established byoccluding with translucent goggles for 8 weeks.2. The expression of BDNF protein in the ganglion cell layer and inner nuclear layer of the retina of the occluded eyes decrease . The expression of BDNF mRNA in the retina and choroid of the occluded eyes decrease . The expression of BDNF mRNA in sclera of the deprived eyes were not detected by Monocular occluding.These results suggested that BDNF participated in the development of FDM.3. The expression of TGF-P2 protein in retina of the occluded eyes decrease .The expression of TGF-P2 mRNA in retina ^ choroid and sclera of the occluded eyes were decrease by monocular occluding. These results suggested that TGF-P2 participated in the development of FDM .4. The expression of MMP-2 mRNA and protein in sclera of the occluded eyes decrease by monocular occluding. These results suggested that imbalance between MMP-2 and TIMP-2 participated in ECM remodeling process to develop FDM.5. The eukaryotic expression vector of pEGFP-C3-BDNF and pEGFP-N3-TGF-P2 were constructed successfully.6. Liposome-mediated the eukaryotic expression vector of pEGFP-C3-BDNF and pEGFP-N3-TGF-P2 transfect the vitreous of guinea pig, Liposome is able to deliver BDNF and TGF-p2to retinal with efficacy , expression of BDNF and TGF-p2 increase. All of these has provided a new idea to the research of the therapy of myopia.