Cloning And Screening Of Retina Damage Related Genes In Experimental Chronic Ocular Hypertension Rabbit

It has long been considered that intraocular hypertension is the major risk factor for glaucomatous optic neuropathy; retina and optic nerve are target organs of glaucoma damage. Pathological mechanism of glaucomatous optic neuropathy is progressive death of retina ganglion cells (RGCs) and optic nerve fiber loss, which leads to irreversible optic function damage. Lots of studies have shown that RGCs apoptosis is the death way of glaucomatous RGCs, and related genes and factors participate in glaucomatous occurrence and development. The purpose of this study is to search and find these unknown genes, so the mechanism of glaucomatous retina and optic nerve damage can be deeply understood on molecular level, which can provide significant basis for glaucomatous treatment and prevention. Suppression subtractive hybridization (SSH) is a kind of experimental techniques among gene clone strategies. By comparing the expressing difference between two groups of mRNA, we can get genes which express in one group but not or very little in another by SSH. So the technique is mainly used to search for unknown genes.Methods: In this study, a chronically elevated intraocular pressure (IOP) in rabbits was set for 5 weeks, then differential expressing genes were screened from the retina of experimental eyes but not in control by SSH and differential screening, so differential expressed sequence tags (ESTs) can be got. Then the ESTs were used to construct a subtractive library for advanced screening. Lastly we got retinal damage-related genes in rabbits of chronic intraocular hypertension. After these procedures, we analyzed related genes' function by comparision and verification so as to find molecular basis of retinal damage. This study included two experiments as below.1. Established rabbit model with chronic elevation of IOP and confirmed from their morphological changesChronically elevated IOP in rabbits was produced by injecting methylcellulose into anterior chamber once a week for 5 weeks, while injecting Linger solution of equal volume into anterior chamber as the control. A direct tonometer was used to measure IOP once a week to gurantee IOP value among 25-35mmHg. After 5 weeks, the retina and optic nervesamples were harvested for morphological observation to judge whether the model was successful.2. Construction of subtractive cDNA library and preliminary screening (1) Total RNA was extracted from retina of experimental and control eyes;(2) mRNA was purified from total RNA;(3) SSH; (4) Dot blot hybridization to screen related ESTs;(5) Construction of subtractive library with PMD18-T vector;(6) Selective sequencing and analyzing;(7) Cellular location of related ESTs through in situ hybridization (ISH). Results: IOP was determined using homemade direct tonometer, and IOP of experimental eyes maintained consistently among 25-35mmHg for 5 weeks. In experimental group, there were less RGCs and optic nerve fiber, thinner retina nerve fibre layer and vacuolation in retina nerve layer and inner reticular layer under light microscope. With electron microscope, in experimental retina there were RGCs karyopyknosis, nucleolus edge collection and some karyolysis; mitochondrion swelling; endoplasmic reticulum dilatation; karyopyknosis and perinuclear space amplification in inner nuclear layer; some chromatin concentration and nuclear deformation in outer nuclear layer. These accorded with pathological changes of chronic glaucoma.Identification of total RNA by electrophoresis and spectrophotometer showed no pollution and degradation. The electrophoresis of mRNA showed obvious smear, and identification of SSH efficiency proved former procedures were successful.After construction of subtractive library and screening, we found twenty-eight positive clones in forward subtraction and ninety positive clones in reverse subtraction. In following dot blot hybridization, eleven clones in forward subtraction and thirty clones in reverse subtraction were screened. Nineteen positive clones were...