| Background and Objective:Myasthenia gravis(MG) is an auto-antibody mediated neuromuscular disorder. The auto-antibodies are directed against the nicotinic acetylcholine receptor(AChR) at the muscular endplate,thereby impairing the transmission of nerve impulse to the muscle,essential for its contraction.As auto-antibodies are the main culprit for the development of disease,it has been designated as a B cell mediated disorder.However, several studies highlight T cell pathogenesis in the exacerbation of the disease.Hence, it could be designated as a T cell dependent auto-antibody mediated disease.At the same time,70%-80%MG patients have abnormal thymus(including the thymus hyperplasia and thymoma),and thymectomy is an effective means of treatment of MG, It has become a relevant consensus,tha MG has some correlation with abnormal thymus.The antigen specificity T cell's activation needs two different signals.The first signal from antigen peptide - MHC the molecular composition compound and TCR the interaction;The second signal from the antigen-presenting cells(APC) on the surface expression of costimulatory molecules delivered to T cell。Over the last decade studies have shown that co-stimulatory signals promot of T cell growth, differentiation and cytokine production,as well as provide survival signals to prevent the occurrence of apoptosis of T cells,it is also critical to the induction and maintenance of the incompetence of T cells(anergy).More importantly,due to the control of co-stimulatory signals can be enhanced or termination of a means of immune responses,which have become an increasing concern.Recently,new ways of co-stimulatory B7-H1/B7-DC--PD-1 play an important regulated role to the effects of activated T cells.One,B7-H1 which is bind to T cells with different specific receptor is able to regulate the activation land differentiation of T cell,and is the new research hot spot.On the one hand,it may promotes the person T cell multiplication under the anti-CD3 immune body or the heterogenic antigen stimulation with the corresponding ligand PD-1 union and promotes IL-10 to secrete (10).Another situation is the opposite,such as B7-H1 to stimulate T cells,can inhibit the TCR-mediated cell proliferation and cytokine IL-2,IL-4,IL-10 and IFN-γproduction,Led to the cessation of the cell cycle.And it in the cell reply displays the negative regulation in this kind of T to affect.Recently,we have to use immunohistochemistry on the thymus frozen section B7-H1 expression was found to detect.It expresses the obvious reduction in the MG patient thymus.This had reflected the MG patient immunologic function is at the abnormal state,and prompts the B7-H1 member to be possible with the PD-1 interaction, participates in the MG morbidity through the transmission different signal.According to the above,this research use molecular biology and fluorescence activation cell sorting technique(fiow cytometry,FCM) and so on experiment methods,Excises the thymus take the MG patient surgery to organize the T lymphocyte as the object of study,examines B7-H1 /B7-DC--PD-1 altogether to stimulate the signal passage member the expression situation,discusses this circuit and MG has the development relevance,and studies it to the MG patient cellular immunity regulative function.MethodsFrom clinical to choose 21 cases of MG patients and 10 cases of congenital heart disease in patients with excision of the thymus,carries on the following experiment separately:1.The production of frozen sections,immunohistochemistry testing thymus tissue molecules B7-H1 expression.2.Thymus extract total RNA,RT-PCR amplification were B7-H1,B7-DC,PD-1 and internalβ-actin gene,1.2%agarose gel electrophoresis,Gelatin image analysis software determines B7-H1,B7-DC,PD-1 and the materials for internal referenceβ-actin gene electrophoresis banding grey level using Quantity the One4.6.3,and calculates B7-H1,B7-DC,the PD-1 gene banding grey level and corresponding specimenβ-actin gene banding grey level ratio,it takes this specimen goal gent mRNA the relative expression quantity.Using statistics software SPSS11.0,compared with goal gone in the MG patient and normal comparison thymus organization's expression situation.3.The mechanical separation method separation thymocite uses the indirect method to examine its superficial B7-H1 member the expression.Results1.Immunohistochemical results showed MG group and the normal group in B7-H1 protein expression between a statistically significant difference(χ~2=4.19,p＜0.05), B7-H1 protein expression in MG group was significantly reduced.2.Independent sample T-test demonstrated that the B7-H1 mRNA expression level in the MG group(n=21) is 0.46±0.21 with statistic significance(p＜0.05) when compared with the control group(n=11,0.144±0.144).There's no statistic significance (p＞0.05) in B7-DCmRNA expression between two the foemer being 0.710±0.080 and the latter 0.647±0.058.3.Independent sample T-test demonstrated that there's no statistic significance (p＞0.05) between B7-H1mRNA and B7-DCmRNA expression level in the MG group.4.Flows the type cell meter test result to indicate that the MG group and the control group thymocite surface expresses the B7-H1 member,expresses the masculine gender rate with the normal control group(n=11,33.48±2.05) to compare, accompanies the thymoma MG group to express(n=2,40.09±1.98) the remarkable markup,the thymus atrophy group(n=5,32.17±1.79) then the change is not big,but the proliferation MG group expresses(n=14,21.22±126) reduces obviously.Conclusions1.Abnormal expression of co-stimulatory molecules B7-H1/B7-DC--PD-1 exists.MG patients with hyperplasia revealed a marked reduction in B7-H1 and rise in B7-H1mRNA,which indicated that after this co-stimulatory pathway in the circuit member expression process to have the duplication and regulates.2.B7-H1/B7-DC--PD-1 was found in various thymus tissues,which showed the existence regulation of activated T lymphocytes. |
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