Research On The Expression Of Costimulatory Molecules MRNA In Aging And Tumour

Objective: To construct the recombinant plasmid standards of CD28,CTLA-4,CD86,CD40L,ICOS,ICOSL cDNA,in addition,to detect the costimulatory molecules mentioned above gene expression by the Taqman Probe techonology.To explore the role of costimulatory molecules in aging and tumour by detecting the expression of the costimulatory molecules mRNA in different ages and cancerous people with the method of Taqman probe real-time fluorescent quantitative polymerase chain reaction(FQ-PCR) technology.Methods:Part one:First,the cDNA was synthesized by reverse tanscripting with Random 9 mers as the pimer and the total RNA from the gastricadenocarcinoma tissues as the template.Second,the target sequences of CD28,CTLA-4,CD86,CD40L,ICOS,ICOSL gene were amplifiedu using the cDNA synthesized at the first step as the template.Then the PBS-TⅡ-CD28/CTLA-4/CD86/CD40L/ICOS/ICOSL/β-ACTIN recom -binant plasmids were constucted.Standard curves were made by Taqman Probe fluerescene PCR techonology after gene sequencing.Part two: Collected 165(age:20-88 years old) peripheral blood samples of healthy people.The samples were divided into 6 groups according to different age stages. Explored the relationship between the expression of costimulatory molecules mRNA and aging by detecting the costimulatory molecules mRNA of the samples .Part three: Detected the costimulatory molecules CD28,CTLA-4,ICOS,ICOSL mRNA expression of the peripheral blood samples of esophageal carcinoma and lung cancer patients.And detected ICOS,ICOSL mRNA expression in gastricadenocarcinomas and their adjacent tissues.Results:Part one: The composition of the RNA extracted from the tissues was complete and the sequences of the recombinant plasmid was consistent with the target fragments.The original The diluted concentration of the original plamids(CTLA-4,CD86:E12-E4 copies/mL,CD28,CD40L,ICOS,ICOSL:E12-E3copies/mL) had good linear relationship(R2=1) afer amplifying by Taqman FQ-PCR.Part two: (1)The difference between male and female from 51 to 70 years old age in the expression of CD28,CTLA-4,CD86,CD40L,ICOS,ICOSL mRNA had no statistical significance.(2)The level of CD28,ICOS mRNA expression was decreasing with aging,but the CTLA-4 mRNA expressin was increasing.(3)Results showed that there was no correlation between the expression of CD86,CD40L,ICOSL mRNA and age(P>0.05).Part three: (1)The expression of CD28 and ICOS mRNA of esophageal carcinoma and lung cancer patients decreased comparing with the same age group of healthy people.(2)In the 42 gastric carcinomas tissues, the relative mean expression of ICOS mRNA was lower than the adjacent tissues.(3)There was no statistical significance in the expression of CD28,CD40L,ICOS,ICOSL mRNA among different clinical stage,tumor type,differentiation degree and lymph node metastasis groups.Conclusion:1.The method which can quantitatively detect the concentration of CD28,CTLA-4,CD86,CD40L,ICOS,ICOSL gene by Taqman Probe fluerescene PCR techonology has been successfully constructed.2.The chang of CD28,CTLA-4,ICOS mRNA expression accompanying with aging may be realated to the occurrence and development of aging.3.The expression level of the positive costimulatory molecules CD28 and ICOS mRNA in peripheral blood or tissues of cancerous people was sharply reduced compared with healthy people ,that may promote the immune evasion.4. The declining expression level of CTLA-4 mRNA in the peripheral blood of patients may suggest that the immune systems were seriously damaged.