血小板膜糖蛋白CD6 2P和CD6 3的检测方法及其在糖尿病患者中的表达

The Examination Method of Glycoprotein CD62P and CD63 on Platelet Membrane and Their Expression in Adults Patients with Diabetes Mellitus

血小板α 颗粒膜蛋白 (CD62P)和溶酶体膜蛋白 (CD63)是血小板活化标志物 ,但血小板膜蛋白极不稳定 ,易活化 ,造成实验结果不可靠 ,为此应采用统一标准的检测方法。为探讨该目的并了解糖尿病患者血小板的活化情况 ,实验中采用全血单克隆抗体直接免疫荧光标记法和流式细胞术 ,对正常人及 37例成人糖尿病患者进行了研究。正常人无固定组在标记后 30 ,60 ,90和 12 0分钟时 ,CD62P和CD63阳性率 (% )分别为 7.5 7± 2 .33,2 0 .5 0± 5 .70 ,2 8.70± 5 .67,36.5 2± 6.13及 0 .89± 0 .36,1.11± 0 .84 ,2 .35± 2 .0 2 ,5 .4 3± 3.66,其相应点的平均荧光强度各自为 1.5 7± 0 .13,1.88± 0 .0 8,2 .0 0±0 .0 9,2 .38± 0 .2 2及 3.91± 0 .11,4 .0 7± 0 .16,4 .38± 0 .14,4 .4 4± 0 .19,二项均随检测时间推移逐渐升高 ,其中阳性率增高更加显著。而正常人多聚甲醛固定组CD62P和CD63在相同各时间点的阳性率和平均荧光强度基本相同 ,未出现明显变化。此外 ,37例成人糖尿病患者CD62P和CD63的阳性率分别为 (14.11± 6.68) %及 (2 .71± 1.74 ) % ,比相对正常对照组显著升高 (P <0 .0 0 1,P <0 .0 5 )。由此可见 ,血小板CD62P和CD63非常敏感 ,在体外易受激活 ,采血后需立即标记 ,全部操作应在 30分钟内完成 ,

To study the optimal examination method of CD62P and CD63 and investigate platelet activation in patients with diabetes mellitus (DM), whole blood labeled directly with monoclonal antibodies CD62P and CD63 and flow cytometry were used to evaluate the positive percentages and the mean fluorescence intensity of CD62P and CD63. The specimens of peripheral blood obtained from 10 healthy adults were divided into two groups. In the unfixing group, the positive percentages of CD62P and CD63 at the periods of 30, 60, 90 and 120 minutes after staining were (7.57±2.33)%,(20.50±5.70)%,(28.70±5.67)% and (36.52±6.13)%, and, (0.89± 0.36)%,(1.11±0.84)%,(2.35± 2.02)% and (5.43±3.66)% respectively, their respective MFI were 1.57±0.13, 1.88±0.08, 2.00±0.09 and 2.38±0.22 and 3.91±0.11, 4.07±0.16, 4.38±0.14 and 4.44±0.19. However, in fixing group with 1% paraformaldehyde, the results had not any obvious change and almost were same. Besides it, the positive percentages of CD62P and CD63 in 37 adult patients with DM were (14.11±6.68)% and (2.71±1.74)%, significantly higher than that in the normal controls. It is concluded that the CD62P and CD63 on platelet membrane were very sensitive and would be easily activated in vitro, all manipulations that includes labeling with antibody, incubation and detection using flow cytometry should be finished within 30 minutes after samples collected. While fixing by using 1% paraformaldehyde can steady the labeling compounds and effectively prevent the artificial activation of platelet, and keep the stable results within two hours after the samples labeled. In adult patients with DM, the relationship between the cardiovascular complication of diabetes and platelet activation might be existed.