HIV感染可通过多种机制引起机体内T细胞凋亡，其中，病毒的编码蛋白可以根据其在靶细胞内的表达水平调节细胞凋亡。gp120蛋白作为HIV包膜蛋白的主要组成部分，既可以在细胞外与细胞表面的CD4分子结合导致靶细胞和旁观者细胞凋亡，又可以在细胞内上调Fas、FasL、TNF-α、TRAIL受体DR4和DR5的表达诱导细胞凋亡，还可以使细胞滞留在G2期导致胞内ROS升高促进凋亡。

研究显示，CD45分子在HIV-1 gp120蛋白介导的CD4^＋T细胞凋亡中发挥调节作用，在gp120蛋白的作用下，CD45分子通过调节FasL的表达参与细胞凋亡。作为Ⅰ型跨膜糖蛋白，CD45分子主要以CD45RABC、CD45RO、CD45RA、CD45RB和CD45RC等异构体形式表达于淋巴细胞，其胞外结构域糖基化形式的差异对CD45异构体活性和功能具有重要影响。目前，CD45异构体在HIV感染引起的细胞凋亡方面研究甚少，为加深对CD45异构体在细胞凋亡过程中作用的理解，本研究通过构建稳定表达CD45异构体的细胞系，比较了CD45异构体在调节gp120蛋白介导的CD4^＋T细胞凋亡过程中的差异，并进一步分析了其下游分子Lck的活性变化。

在本研究中，我们首先比较了CD45^＋的Jurkat细胞和CD45^－的J45.01细胞受到gp120诱导时凋亡率的变化。结果显示，Jurkat细胞可发生明显凋亡现象，J45.01细胞不发生凋亡，说明CD45分子在gp120介导的凋亡过程发挥了调节作用；随后，我们构建了5种稳定表达CD45异构体的细胞系，并比较了这5种异构体在gp120介导T细胞凋亡过程中的作用。结果发现，CD45RO可以明显调节细胞凋亡，其他异构体对细胞凋亡无调节作用；接下来，我们分析了CD45的糖基化形式对细胞凋亡的影响。O-聚糖存在于除CD45RO以外的异构体上，我们通过O-糖基化抑制剂去除这几种异构体细胞表面的O-糖基化。gp120蛋白诱导凋亡处理后，去除O-糖基化后的CD45RA-J45.01细胞表现出凋亡特性，其它异构体细胞未发生明显凋亡。说明O-聚糖对HIV-1 gp120蛋白介导的细胞凋亡有一定的抑制作用，但在各异构体中发挥的作用大小并不一致，这可能与CD45异构体的空间结构有关。

为了进一步说明CD45异构体在调节细胞凋亡中的功能差异，在HIV-1 gp120蛋白诱导各个细胞系后，我们分析了CD45异构体酪氨酸磷酸酶的活性变化。通过比较CD45对其底物Lck的活性抑制位点Tyr505的去磷酸化程度，发现CD45RO可使Lck Tyr505发生明显的去磷酸化，说明HIV-1 gp120蛋白处理细胞后，CD45RO的活性增强；Lck Tyr394是Lck的活化位点，该位点磷酸化程度增强代表Lck活性增强。通过比较该位点磷酸化程度变化，发现在CD45RO-J45.01细胞中Lck Tyr394磷酸化程度增强，说明在HIV-1 gp120蛋白处理细胞后CD45RO促进了Lck的活化；Lck的活化在胞内信号转导中具有重要作用，于是我们利用Lck的特异性抑制剂PP2处理CD45RO-J45.01细胞和Jurkat细胞，再经HIV-1 gp120蛋白诱导凋亡处理，发现PP2可抑制CD45RO-J45.01细胞和Jurkat细胞凋亡，说明Lck的活化参与HIV-1 gp120蛋白介导的T细胞凋亡。

综上所述，本研究明确了CD45异构体在调节HIV-1 gp120蛋白介导的T细胞凋亡中存在的差异，揭示了这一凋亡过程主要是CD45RO通过活化Lck促使凋亡信号向胞内传导，进而对细胞凋亡起调节作用。本研究结果为HIV-1感染引起的旁观者细胞凋亡奠定了理论基础。

The depletion of CD4⁺ T lymphocytes is a central pathogenic featureduring HIV-1 infection, and the loss of CD4⁺T cells is associated with apoptosis, which represents the major mechanism of CD4⁺ T cell depletion. However, activation-induced cell death (AICD) played an important rolein the apoptosis of CD4⁺ T cells, and the molecular mechanism of AICD is remaining uncertain. The protein encoded by HIV, gp120, which was a part of Env can induced apoptosis by AICD.

CD45 is regulated the apoptosis by induced the expression of Fas ligation. CD45 is a type I glycoprotein, containing a single trans-membrane domain. CD45 is expressed in multiple isoforms, such as CD45RABC, CD45RO, CD45RA, CD45RB and CD45RC. The CD45 isoforms modified by N-glycans and O-glycans on extracellular domain are reported to be involved in CD45-mediated apoptosis. Therefore, we decided to explorethe roles of CD45 isoforms on apoptosis.

In our study, regulation of gp120-induced cell death was investigated in various transfectants with different CD45 isoforms. We detected the expression of CD45 isoforms on Jurkat cells and J45.01 cells. CD45RA, CD45RB, CD45RC, CD45RABC and CD45RO was expressed respectively on Jurkat but not J45.01 cells.We found that gp120 could induce the apoptosis of CD45⁺ Jurkat cells, but not CD45^- J45.01 cells, suggesting the key role of CD45 in regulation of gp120-induced death of Jurkat cells. Then we examined the effect of gp120 on J45.01 expressed with CD45 isoforms.CD45RO-J45.01 cells undergo a significant apoptosis, J45.01 expressed with other CD45 isoforms could not undergo apoptosis. CD45 is a heavily glycosylated protein, therefore, we next investigated roles played by the glycans in CD45 molecule in the regulation of gp120-induced death. J45.01 expressed with CD45RA, RB, RC and RABC were modified by O-glycans and N-glycans. J45.01 expressed with CD45RO only contained N-glycans.Since the difference of apoptosis among these cells implying the O-glycans may play some role during the apoptosis process, so we conferred that the O-glycans inhibited the apoptosis induced by gp120. Furthermore, our results showed that gp120-induced apoptosis of CD45RA-J45.01 cells and CD45RABC-J45.01 cells could be improved by BG，which can inhibit O-glycosylation of cells. Therefore, it can be suggested that O-glycans on CD45 inhibited apoptosis of Jurkat cells induced by gp120.

Lck has suggested that play a key role in the TCR proximal signals leading to death of activated T cells. As the substrate of CD45, dephosphorylation of Lck Tyr505 reflect the activation of CD45.In our study, we compared the dephosphorylation of Lck Tyr505 among J45.01 cells expressed with CD45 isoforms. The result showed that Lck was most susceptible to CD45RO. And the dephosphorylation of Lck Tyr505 were increased when CD45RA-J45.01 cells pretreated by GB, namely, the activation of CD45RA was enhanced. Our result demonstrated that the activation of CD45 was increased by gp120, and the CD45RO is more susceptible to be activated. Lck Tyr394 is an activation site. When the phosphorylation of Tyr 394 increased, the activation of Lck are enhanced. The phosphorylation level of Tyr 394 was enhanced in CD45RO-J45.01 cells and Jurkat cells treated with gp120. Then, the cells were pretreated with P22 (the specific inhibitor of Lck) and HBA.The apoptosis was inhibited by PP2 in CD45RO-J45.01 cells and Jurkat cells.

In summary, our results illustrate that Lck kinase phosphorylates Tyr394 with resultant T cell activation upon gp120-induced cell stimulation. This would be the basis of the depletion of memory T cells in HIV infection process.