Analysis Of Vβ Gene Usage Of Cytolytic T-lymphocytes In Coxsackievirus B₃ Induced Myocarditis

Objective: To ascertain the colonality of .specific cytolytic T-lymphocytes in Coxsackievirus B3-induced myocarditis through analysing v β gene usage of the cytolytic T-lymphocytes which result in immune-mediated myocyte injury, and to explore the pathogenesis of viral myocarditis and provide the precondition for blocking the combination of CTL with myocyte, finally preventing and curing viral myocarditis with immune-based therapy.Methods: An experimental murine model of myocarditis has been developed using inbred Balb/c male mice and a myocarditic variant of Coxsackievirus B, type 3. Mice were killed on day 7 after virus inoculation, a time when T cells show significant differentiation .ACTLs, VCTLs, ACTLs are respectively isolated from inguinal and mesenteric lymph nodes of Balb/c mice suffered from viral myocarditis with immunoadsorption. T-lymphocytes prepared from inguinal and mesenteric lymph nodes of uninfected Balb/c mice served as control group. Cytotoxicity assay was performed with MTT method. RNA extraction and cDNA synthesis was performed with standard methods. According to reports, we synthesized 20 different v β -specific oligonucleotides as a 5'sense primers and a c β -specific oligonucleotide as 3'anti-sense primer. RT-PCR was performed with standard method. V P gene usage was analysed by electophoresis of PCR products on agarose gel.Results: The mean of OD value of ACTL> VCTL and MCTL in experimental group is 0.42> 0.43and0.43, respectively, the mean of OD value of T cell in control group is 0.82. the difference is significant between experimental group and control group(P<0.01). But the difference is not significant between experimental groups(P>0.01). the cytotoxicity of ACTL> VCTL and MCTL is 72%> 69%and71%, respectively, the cytotoxicity of T cell in control group is 19%. the difference is significant between experimental group and control group(P<0.01). But the difference is not significant between experimental groups(P>0.01). T cell in control group expresses all v P gene of 20 families. But the v P gene usage of CTLs in experimental groups is markedly restricted. ACTL expresses predominantly Vj86, V/38.K V/88.2> V/38.3, MCTL expresses predominantly V/35.K V/38.K V/38.2, V/38.3 and VCTL expresses predominantly V/37, V/38.1 > V/S8.2, V/38.3. Notably, all of ACTL> VCTL and MCTL express mutually V/38.1 > V/38.2 andV|38.3.Conclusions: Cytolytic T-lymphocytes play important roles in myocyte injury, the v P gene usage of CTLs which result in immune-mediated myocyte injury in Coxsackievirus B3- induced myocarditis is markedly restricted and these CTLs differentiate colonally stimulated by specific antigen. Our study shows that analysis of v P gene usage and the colonality of .specific cytolytic T-lymphocytes in Coxsackievirus B3 induced myocarditis would provide basic experimental data for clinic immune-based therapy for viral myocarditis.