Analysis Of Differential Display Genes And Protein Between Keloid And Normal Skin

Background/Objective:Keloid is a specific phenomenon in pathology for human being which often produces the malformation and dysfunction in clinic and is a tricky problem for aesthetic medicine and orthopedics. Keloid is much different from any other kind of scar tissue, it can overgrow the ambit of the original wound, invade neighboring tissues and appear as the proliferation of tumor. Furthermore, it has the trait of constantly non-degenerate, and relapse if cut purely by operation. Due to the inclarity of the pathology of keloid, there is still no way to cure it suitably .The only way to solve this problem is to find out the pathology of this illness.Doctors have been searching for the causes for a long time,making a lot of researches from every aspect of the pathology, such as the mechanics which appears as the tensity of the wound;the anoxia which causes by the keloid is in a state of lacking blood; Immunity reaction, collagen synthesis and dissolution imbalance, cell growth factor, cell apoptosis and heredity. All those theories tell the relationship between keloid and other factors, but still cannot explain the real cause of the sickness.How to catch the cause of keloid through analysis is the key to the door.There are about 100,000 genes out of 23 pairs of chromosome, and of them there is only 15% applied in individual cell. The selectivity of genes give rise to many living activities. Besides, the change of expressions caused by various factors, and single gene mutation and the synergy of multigenes can cause sicknesses by changing the normal activities of the organisms. And by comparing the difference between the expressions of sick organism and normal organism genes, we can find a lot of useful informations.We use the SSH to filtrate the keloid and normal skin tissues. It is byusing cross-fertilize to pick out the mutual sequence. And the SELDI can detect the different protein. All those genes and proteins of different expressions will provide a trace for finding the cause of the keloid.Methods:1. Obtain specimens of keloid and normal skin, these specimens diagnosed with criterion of clinic and pathology. When these specimens were cut out from body, store samples in liquid nitrogen.2. We use the Clontech PCR-Select cDNA Subtraction kit to do the test.3. Firstly extract total RNA from keloid tissue and normal skin tissue respectively, then isolate mRNA from the total RNA.4. Fist-Strand cDNA synthesis. With these mRNA as the template, synthesis cDNA of keloid and normal skin. The cDNA of keloid is the tester, and the cDNA of normal skin is the driver.5. Rsa I digestion. This step generates shorter, blunt-ended ds cDNA fragments which are optimal for subtraction and necessary for adaptor ligation. After digestion these cDNA fragments are about 500 bp.6. Prepare adaptor-ligated tester cDNA. The tester cDNA are divided to two groups: one group is ligated with Adaptor 1, and the second is ligated with Adaptor 2R.7. First hybridization and second hybridization. After two hybridization the remaining ss cDNA are dramatically enriched for differentially expressed sequences.8. PCR amplification. Differentially expressed cDNA are selectively amplified during this reactions. In the first amplification , only ds cDNA with different adaptor sequences on each end are exponentially amplified. In the second, nested PCR is used to further reduce background and to enrich for differentially expressed sequences.9. After SSH, the PCR mixture is ligated with T-Vector, then transfect DH5-α bacteria. Select the positive clones and analysis the insert gene fragments.10. Southern hybridization identificate the keloid differentially display genes.11. The positive clones of Southern hybridization are selected, and analysis these sequence. The results compare with GeneBank, and analysis these gene's structure and function.12. using the technology of SELDI( Surface enhanced laser desorption/ionization), analysis differential protein of between keloid and normal skin tissue.Results:1. After total RNA isolation, examine the RNA's integrity by electrophoresing samples on a formaldehyde denaturing agarose/EtBr gel. Total RNA typically exhibits two bright bands, which correspond to ribosoma 28S and 18S RNA at -4.5 and 1.9 kb, respectively, with a ratio of intensities of about 1.5-2.5:1.2. The mRNA of these samples be as template for synthesizing cDNA.After Rsa I digestion, electrophoresing samples show these fragments size are about 0.1-1.5 kb. This result suggests the digestion is complete.3. Analysis of ligation. The PCR product using one gene-specific primer (G3PDH3' Primer) and PCR Primer 1 is about the same intensity as the PCR produt amplified using two gene-specific primers (G3PDH3' and 5' Primers). This result shows ligation was less than 25% complete.4. After the first PCR reaction, the test samples show band spaning from 200 bp to 750 bp, no special band in it. The control sample bands span more range. In the second, nested PCR is used to futher reduce background and to enrich for differentially expressed sequences, there are secial bands in electrophoresing.5. After SSH, the PCR mixture is ligated with T-Vector, then transfect DU5-a bacteria. There are whrite clones in the culture medium. Select these whrite clones, and analysis the insert gene fragment size. The ideal clones have about 500 bp insert gene fragment.6. Select ideal clones, and perform Southern hybridization. We find 20 positive clones. Analysis these gene's structure and function.7. These 20 gene fragments are keloid differentially displaying gene compared with normal skin tissue. 11 gene clones have been known their function, but 9 clones we don't know their structure and function. In these known function gene include BTAFl and E4TF1 which have relationship with DNA transcription and translation; EPHB2 which have key roles in patterning and morphogenesis; PDGFR which play critical roles in mesenchymal cell migration and proliferation, and GAGE which has relationship with cancer.8. Using the technology of SELDI, we find several differential protein of between keloid and normal skin tissue.Conclusion:1. We successfully construct keloid differentially expressed gene library by Suppression Subtration Hybridization.2. Some these differentially expressed gene's function are matching the biologic behavior of keloid, so it is worth to research ulteriorly.3. All those genes and proteins of different expressions will provide a trace for finding the cause of the keloid.