| BackgroundAs the area of the largest organ, skin undertaking important function to protect the internal structure and every systemic organ. When injured, the skin must consistently and rapidly repair itself in order to maintain this external defense system. This course initiates a cascade of events that culminates in scar formation.)。Scar is a symbol of the clinical cure. Scar only in such growths case, and misshapen body and a functional impairment, it is pathological. The scar formation that sparks a pathologic phenomenon calls that it is healing in the wound the process for the pathologic sex scar formation which is effected by local micro environment, the changing of the expression occurrence of the gene eventually effect the revolution of scar formation to return. Clinical scars include hypertrophic scar and keloid. Because there are clinical similarities between hypertrophic scars and keloids, the terms hypertrophic scar and keloid are often used interchangeably. However, there are many pathological and biochemical differences that suggest that these entities are distinct from one another. Keloids are those hypertrophic-appearing scars that continue to extend beyond the margins of the original wound and evolve over time without a quiescent or regressive phase. Keloids are considered by many to represent the product of genetically abnormal wound healing cells; others believe these scars to be a type of benign fibrous tumor. Keloid tissue infiltrates into surrounding normal and has a high recrudescence after the surgical. But hypertrophic scars is most likely local to grow, and didn't continue to extend beyond the margins of the original wound, which happened are all excessively repairing themselves.。There are a great deal of deposition primaries and radicle guilder composition deposite inside scars is the main pathology expression of abnormal scars.Despite decades of research and a vast literature devoted to the topic of wound healing, the pathophysiology of keloids remains incompletely understood. Sometimes even a slight injuring such as injecting can cause the development of keloids. The patients of keloids often have a long and instability course of disease and this abnormal scar likes to infringe in the special position of body and its multiplication always keep in high rate. All of these clinical symptoms result in for patients to sufferer serious mental state burden. Particularly to keloids, people try a lot of therapies to cure the special disease, however, there is no excellent therapy to relieve the pain of patients nomatter surgical excision or adjunctive therapies by now. Although the keloid is a kind of positive disease, its pathologic characteristics and clinical character brought sufferer physiology with mentally of pain and sufferings.Lacking of satisfied treatment way, also brought plastic surgeon the harassment on the operate.As a result, the previous period researcher proposes significant importance of the efficiency prevention of keloids, and now spread investigation. Our study group has proved that the genotype polymorphism could be found in codon 72 exon 4 of p53 gene which was picked up from the lymphocyte in peripheral blood. And the important one is the genotype polymorphism has the differentia between the normal human and keloid patients. This differentia was so important that it can afford the theoretic and experimental evidence for keloid predisposition detection in clinic. Also,the keloid detected reagent kit was manufactured on the base of the differentia for clinical application. It was that applied ASO and polymerase chain reaction and reverse dot blot to discriminate the codon-72 polymorphism of the tumor suppressor gene p53.This topic set applied the kit to get the human genome DNA from the blood sample and desire the primer and probe of p53 gene. The codon-72 polymorphism of the tumor suppressor gene p53 were detected by using polymerase chain reaction and reverse dot blot. Finally, the result can be showed through three different coloured firm and get conclusion.The codon-72 polymorphism of the tumor suppressor gene p53, the Proline-encoding allele and Proline/Proline genotype are the genetic predisposition to keloid of the Han race in Guang Dong, China. The keloid reagent kit can pick up the genetic predisposition to keloid among the human beings and will be a assistant way to forecast the disease.Objective1,According to previous period p53 gene reagent kit, to establish a new method of molecular beacon with real-time PCR for p53 gene SNP detection, operating simply and cutting low cost, shrinking short time, making of much easier than clinical generalization.2,Applied molecular beacon with real-time PCR for p53 gene SNP detection to examine the large-scale clinic sample, and visited the patients who were subjected, observed clinical sufferer scar formation revolution to return whether agree with genotype. In order to make foundation for anticipating the outbreak risk of abnormal scar. MethodsThe test is an random-control experiment, we made up two groups. One is case set, blood samples of keloid were taken from 28 patients which were approved getting keloid by at least two clinic workers. Another is normal-cure set, blood samples were taken from 260 female patients(after caesarea section in a week Age). Race and gender in two groups were similar, at the same time, two groups have no abnormal scar and tumor history. Course:two groups all applied the method of molecular beacon with real-time PCR to detect p53 gene polymorphism.We get the human genome DNA from the blood sample and two molecular beacon probes of CCC/CGC SNP of p53 72th codon were synthesized and labelled with fluorescences. CCC probe was labelled with Fam and CGC probe was labelled with Hex. CCC/CGC SNP was detected by molecular beacon real-time PCR from genomic DNA of blood cell. The SNP type results were compared with the results of reverse dot hybridization and conformed by DNA direct sequensing.. According to the result of gene types of p53 with molecular beacon with real-time PCR we have already known, draw up a following discretion standard:With fluorescence intensity the≥blank tube fluorescence intensity 2.000% for the masculine gender respond. The pure MBC probe(Fam green fluorescence) masculine gender judgment purely matches son for CCC/CCC; The pure MBG probe(HEX red fluorescence) masculine gender judgment purely matches son for CGC/CGC; The MBC probe(Fam green fluorescence) and MBG probe(HEX red fluorescence) double male judgment matches son for miscellaneous CCC/CGC. The codon-72 polymorphism of the tumor suppressor gene p53 were detected by using three kinds of fluorescence. Finally the rayleing distribution rayleigh discrepancy of statistical analysis three kinds of genotypes, combine clinical with visit a result, observe genotype with the on the sick list relation of clinical pathologic sex scar formation.ResultThe genotype of the 260 sample normal curing and 28 keloid patients just like this:Proline/ Proline genotype in keloid group has 12 cases(42.87% in total); Proline/Arginine genotype in keloid group has 10 cases(35.7% in total); Arginine/Arginine genotype in keloid group has 6 cases(21.43.%)in total. The result conformed with DNA direct sequensing. Then to detect the result by using polymerase chain reaction and reverse dot blot and the result was showed that: Proline/Proline genotype has 14 cases; Proline/Arginine genotype has 9 cases; Arginine/ Arginine genotype has 5 cases. The result of the correlation between polymerase chain reaction and reverse dot blot and DNA direct sequensing is 92.86% The result of the correlation between molecular beacon with real-time PCR and DNA direct sequensing is 100%, it is higher than the method of polymerase chain reaction and reverse dot blot. The sensitive of the method of molecular beacon with real-time PCR kit is about 42.86%,and the specificity of the kit is about 87.5%. The sensitive of the method of polymerase chain reaction and reverse dot blot kit is about 50%,and the specificity of the kit is about 83.33%. It was seen that, the sensitive of the method of molecular beacon with real-time PCR kit is lower than polymerase chain reaction and reverse dot blot, but the specificity of the kit is higher than it.The genotype of normal curing samples and keloid patients after operation just like this: Proline/Proline genotype in normal curing group has 46 cases(19.3% in total); Proline/ Arginine genotype in normal curing group has 117 cases(49.2% in total); Arginine/Arginine genotype in normal curing group has 75 cases(31.5.%)in total. Clinical visit 260 famale patient and there are 22 patients have hypertrophic scar. The genotype of them just like this:Proline/ Proline genotype is 9(40.9% in total), Proline/Arginine genotype is 8(36.36%), Arginine/ Arginine genotype is 5 (22.72%). The experiment result showed that:the gene p53 codon 72 polymorphism of famale who after operation detected by molecular beacon real-time PCR, the result is similar to objection we have known, Proline/Proline genotype are the genetic predisposition to abnormal scar.ConclusionThe codon-72 polymorphism of the tumor suppressor gene p53, the Proline-encoding allele and Proline/Proline genotype are the genetic predisposition to keloid of the Han race in Guang Dong, China. The method of molecular beacon with real-time PCR for p53 gene SNP detection update the keloid reagent kit, can pick up the genetic predisposition to keloid much convenient and faster among the human beings, and will be a assistant way to forecast the disease.Clinical significanceThe extension and application of the reagent kit made the abnormal scar predisposition prevention possibly. The method of molecular beacon with real-time PCR is highly suitable for SNPs of p53 gene fast detection in clinic. The keloid predisposition can be advised to avoid the dangerous action and sensitive things to reduce the risk of keloid if he has a positive result. Also, the clinic workers are easy to find out the keloid predisposition and give them the integrant therapy before the surgical operation. The keloid-testing reagent kit is long with fast checking, convenient handling and less cost. Only 1ml blood and little time can get the answer of the test. The kit can help the clinic workers and keloid predisposition human beings fight back the attacking of the keloid. In a word, it is a good kit which can be use perfectly in the clinic condition. Make up the for nothing of scar formation examination item to lack. |
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