跨膜型TNF-α正向与反向信号在活化诱导的淋巴细胞凋亡中的作用及机制

发布时间：2018-03-11 01:10

  本文选题：活化诱导的细胞凋亡(AICD)　切入点：反同信号(reverse　出处：《华中科技大学》2006年博士论文　论文类型：学位论文

【摘要】： TNF-α(tumor necrosis factor，TNF-α)是由激活的单核／巨噬细胞，淋巴细胞等分泌的具有多种生物学活性的细胞因子，它能直接介导肿瘤细胞的凋亡和坏死，参与机体局部及全身炎症反应，介导免疫细胞的增殖分化，发挥免疫调节作用。在体内以两种形式存在：即26kD的跨膜型TNF-α(membrane associated TNF-α，mTNF-α)和17kD的分泌型TNF-α(secreted TNF-α，sTNF-α)，后者由mTNF-α酶解而来。两型TNF-α通过与两型TNF受体(TNFR1与TNFR2)结合而发挥广泛的生物学作用。 成熟T细胞的AICD是机体清除免疫应答中过量的效应T细胞，调节免疫反应，维持免疫稳态和建立外周免疫耐受的重要机制。目前，已证实FasL／Fas是介导T细胞AICD重要的、但非唯一的死亡分子。关于TNF-α在AICD的作用主要限制于对sTNF-α的研究，mTNF-α正向信号在AICD中的作用则未见报道。 近来研究发现，mTNF-α除了作为配体可与TNFR结合，向靶细胞传递正向信号而发挥生物学效应外，还可同时作为受体向效应细胞传递反向信号(reverse signaling)。有学者报道Infliximab可以诱发Crohn's病患者肠相关淋巴组织固有层(lamina propria)T细胞凋亡及外周血活化的T细胞凋亡。提示mTNF-α的反向信号可以参与AICD。 本课题以PHA活化白血病CD4~+淋巴细胞系Jurkat建立体外的AICD模型，研究和比较mTNF-α和sTNF-α对T细胞AICD的影响，以期阐明两型TNF-α正向信号及mTNF-α的反向信号在AICD中的生物学特征及生物学作用，从而深入探讨AICD的分子机制，为临床治疗自身免疫病提供新的线索。一、AICD实验模型的建立 1．PHA诱导Jurkat细胞AICD的剂量依赖性 体外以不同剂量的PHA刺激Jurkat细胞24h，借助Annexin／PI法观察AICD。结果显示，PHA可诱导Jurkat细胞发生凋亡，且呈剂量依赖性，即凋亡率随PHA的浓度的递增而升高，当PHA为1μg／ml时其凋亡率已达50％，5μg／ml为63％，20μg／ml则超过80％。 2．PHA诱导Jurkat细胞AICD的时间动力学 选取5μg／ml的PHA，观察其诱导Jurkat细胞AICD的时间动力学。凋亡在2h时已达22％，6h已升高至56％，随着PHA刺激时间的延长，其凋亡率缓慢上升，24h时凋亡率为63％，而72h时则达82％。选取PHA既可有效诱导AICD，凋亡值又适中的剂量(5μg／ml)和作用时间点(24h)作为诱导体外AICD模型的条件。 3．TNF-α参与PHA诱导Jurkat细胞的AICD 用本实验室制备的对mTNF-α无反向信号作用的抗TNF-α单克隆抗体中和内源性TNF-α，可部分抑制PHA诱导的AICD(抑制率14.01％，p＜0.05)，提示TNF-α参与PHA诱导Jurkat细胞的AICD。 二、mTNF-α和sTNF-α正向信号在AICD中的作用 1．mTNF-α正向信号在AICD中的作用 1．1 AICD中Jurkat细胞表达内源性mTNF-α 实验证实未刺激的Jurkat表达少量mTNF-α(4.5％)，PHA活化的T细胞表达mTNF-α明显增加，并随着刺激时间的延长而逐渐增高。24h时mTNF-α的表达率为45.8％。 1．2 mTNF-α对未活化Jurkat的杀伤作用 用1％多聚甲醛固定PHA活化24h的Jurkat细胞，以不同的效靶比与未活化的Jurkat细胞共同孵育24h，借助WST-1法检测活化T细胞的杀伤活性。结果显示活化T细胞对未活化细胞具有明显的杀伤作用，且随效靶比的升高而增强，5：1时杀伤率为71％。应用兔抗人TNF多克隆抗体(TNFpAb)及鼠抗人sTNF-α单克隆抗体(TNFmAb)，可部分阻断其杀伤作用(抑制率分别为53.86％和43.37％，p＜0.01)，提示活化T细胞的杀伤作用部分是由mTNF-α介导的。 1．3 mTNF-α对活化Jurkat的杀伤作用 用多聚赖氨酸使将PHA活化24h的Jurkat细胞贴壁，以效靶比5∶1加入PHA活化2h的Jurkat细胞(作为靶细胞)共同孵育48h。结果显示，活化Jurkat细胞对活化Jurkat细胞有明显致凋亡作用(44.37％，p＜0.01)，且抗TNF pAb和mAb可以部分阻断AICD(p＜0.01)。提示mTNF-α可能是AICD的效应分子之一。 2．sTNF-α正向信号在AICD中的作用 2．1活化Jurkat细胞分泌内源性sTNF-α 收集PHA作用24小时的Jurkat细胞培养上清，以ELISA法未能检出sTNF-α的分泌。 2．2 PHA诱导Jurkat细胞表达TACE 进一步观察TNF转换酶(TACE)的表达是否受到抑制，流式细胞术结果显示PHA可以明显刺激Jurkat细胞表达TACE(表达率44.39％)。推测TACE可能导致大量可溶性TNFR的产生，从而影响sTNF-α的免疫学检测。 2．3外源性sTNF-α在AICD中的作用 为探索sTNF-α对T细胞是否具有致凋亡作用，用外源性sTNF-α(50U／ml)作用Jurkat细胞24h，结果显示sTNF-α可杀伤未活化(凋亡率23.81％，p＜0.01)和活化Jurkat细胞(凋亡率78.13％，p＜0.01)。 三、mTNF-α反向信号在AICD中的作用 1．mTNF-α反向信号促进PHA诱导的AICD 1．1抗TNF-α多克隆抗体可促进PHA诱导的AICD 用抗TNF-α多克隆抗体激活mTNF-α反向信号，结果显示，，其不能诱导未活化Jurkat细胞发生凋亡，但可明显提高活化Jurkat细胞的凋亡率，增强率为20.11％(p＜0.01)。提示mTNF-α反向信号可增强AICD效应。 1．2 sTNFR1也可促进AICD 为进一步确认mTNF-α反向信号促进AICD的效应，我们用可溶性TNFRI(sTNFR1 20μg／ml)刺激mTNF-α反向信号，结果sTNFR1不能诱导未活化的Jurkat细胞凋亡，但可明显提高活化Jurkat细胞的凋亡率(凋亡率86.82％)，且呈剂量依赖性(p＜0.01)。提示mTNF-α反向信号确可促进AICD。 2．mTNF-α反向信号增强AICD的机制 2．1 mTNF-α反向信号促进PHA诱导的Fas和FasL的表达 实验证实未经活化的Jurkat细胞低表达Fas／FasL；PHA可诱导Jurkat细胞表达Fas(33.28％)和FasL(34.98％)。用抗TNF-α多克隆抗体激活mTNF-α反向信号，可明显促进PHA诱导的Fas(52.19％)和FasL(47.74％)的表达(p＜0.01)，其增强率分别为56.82％，36.47％。 2．2 mTNF-α反向信号促进PHA诱导的mTNF-α表达 实验证实未活化Jurkat细胞低表达mTNF-α，PHA诱导其表达增加(45.51％)，而mTNF-α反向信号则可促进PHA诱导的mTNF-α表达率增至61.15％，与PHA刺激组相比，增强率为34.36％(p＜0.01)。 2．3 mTNF-α反向信号促凋亡中TNFR1和TNFR2的变化 实验证实未活化Jurkat细胞低表达TNFR1和TNFR2，PHA主要诱导Jurkat细胞表达TNFR1(45.29％)，而TNFR2表达则轻度上升(仅6.54％)，提示TNF-α在PHA诱导的AICD中主要通过含有死亡结构域的TNFR1介导凋亡。mTNF-α反向信号可明显上调TNFR1(59.32％％)和TNFR2的表达(55.66％，p＜0.01)。 结论：上述结果提示①mTNF-α可能为AICD的效应分子之一，其正向和反向信号均参与AICD。②PHA主要诱导Jurkat细胞表达TNFR1，提示TNF-α主要通过含有死亡结构域的TNFR1介导凋亡。③mTNF-α反向信号促进PHA诱导AICD的主要分子机制：上调Fas和FasL、mTNF-α、TNFR1和TNFR2的表达。
[Abstract]:TNF- (tumor necrosis factor, alpha TNF- alpha) by activated monocytes / macrophages, lymphocytes and cytokine secretion has various biological activities, it can directly mediated apoptosis and necrosis of tumor cells, are involved in local and systemic inflammation, proliferation and differentiation is mediated by immune cells, play a role in immune regulation. In the body in two forms: 26kD transmembrane TNF- (membrane alpha associated alpha TNF- alpha, mTNF-) and 17kD TNF- (secreted TNF- secreted alpha sTNF- alpha, alpha mTNF- alpha), the latter by enzymolysis. Type two and type two receptor alpha TNF- by TNF (TNFR1 and TNFR2 combined) play a biological role widely.
Mature T cells AICD scavenging effect of T cells is the excessive immune response, immune responses and maintenance of immune homeostasis and an important mechanism of peripheral immune tolerance. At present, it has been confirmed that FasL / Fas is mediated by T cell AICD, but not death only. The effect of AICD on TNF- alpha mainly limits in the research of sTNF- alpha, mTNF- alpha positive signal in AICD have not been reported.
A recent study found that, in addition to mTNF- alpha as ligands can bind to TNFR, transfer positive signal to the target cell and its biological effect, can also act as a receptor to deliver reverse signal to effector cells (reverse signaling). Some scholars have reported that Infliximab can induce Crohn's in patients with intestinal disease associated lymphoid tissue in lamina propria (lamina propria) T cell apoptosis the apoptosis of T cells and activated peripheral blood. Reverse signal mTNF- alpha can participate in AICD.
The activation of PHA leukemia CD4~+ cell lines AICD Jurkat model in vitro, study and compare the effects on mTNF- alpha and sTNF- alpha on T cell AICD, biological characteristics and biological effects of the reverse signal in order to clarify the type two positive signal and mTNF- alpha TNF- alpha in AICD, from which to study the molecular mechanism of AICD and provide a new clue for the treatment of autoimmune diseases. First, establish experimental models of AICD
1.PHA induced dose dependence of AICD in Jurkat cells
In vitro with different dosages of PHA stimulation of Jurkat cells with 24h, Annexin / PI method to observe the results of AICD. showed that PHA induced apoptosis of Jurkat cells in a dose-dependent manner. The apoptosis rate increased with increasing the concentration of PHA, when PHA is 1 g / ml, the apoptosis rate reached 50%, 5 g / ml 63%, 20 g / ml is more than 80%.
Time dynamics of AICD induced by 2.PHA in Jurkat cells
Select 5 g / ml PHA, observe the Jurkat cells induced by AICD time dynamics. Apoptosis has reached 22% in 2H, 6h has increased to 56%, with PHA stimulating time increasing, the apoptosis rate increased slowly, 24h apoptosis rate was 63%, while the 72h is selected as 82%. PHA can be effective AICD induced apoptosis, but the value of moderate dose (5 g / ml) and time point (24h) as an in vitro model of AICD induced conditions.
3.TNF- alpha participates in PHA induced AICD in Jurkat cells
Neutralization of endogenous TNF- alpha by anti TNF- alpha monoclonal antibody against mTNF- alpha without reverse signal effect can partially inhibit PHA induced AICD (inhibition rate 14.01%, P < 0.05), suggesting TNF- alpha is involved in PHA induced Jurkat cell AICD..
Two, the role of mTNF- - alpha and sTNF- - alpha forward signals in AICD
The role of 1.mTNF- alpha forward signal in AICD
Expression of endogenous mTNF- alpha in Jurkat cells in 1.1 AICD
The results showed that a small amount of mTNF- alpha (4.5%) was expressed in unstimulated Jurkat, and the expression of mTNF- alpha increased significantly in PHA activated T cells, and increased gradually with the prolongation of stimulation time. The expression rate of mTNF- was 45.8%. in.24h.
The killing effect of 1.2 mTNF- alpha on unactivated Jurkat
1% poly Jurkat PHA activated 24h cells fixed with formaldehyde, with different effect target ratio and non activated Jurkat cells were incubated with 24h, using WST-1 method to detect the cytotoxicity of activated T cells. The results showed that T cell activation has obvious killing effect on non activated cells, and enhanced with effect target ratio when 5:1 increases, the killing rate for the 71%. application of Rabbit anti human TNF polyclonal antibody (TNFpAb) and mouse anti human sTNF- alpha monoclonal antibody (TNFmAb), partially blocked the killing effect (inhibition rate were 53.86% and 43.37%, P < 0.01), which suggested that the killing of activated T cells by in part by the mTNF- alpha mediated.
The killing effect of 1.3 mTNF- alpha on activated Jurkat
With poly-L-lysine to PHA activated 24h Jurkat cells adherent to effect target ratio of 5 to 1 PHA activated 2H Jurkat cells (as target cells) were incubated with 48h. results showed that the activation of Jurkat cells was induced by apoptosis of activated Jurkat cells (44.37%, P < 0.01), and anti TNF pAb and mAb could partially block AICD (P < 0.01) suggest that mTNF- alpha may be one of the effector molecules of AICD.
The role of 2.sTNF- alpha forward signal in AICD
2.1 activated Jurkat cells secrete endogenous sTNF- alpha
The Jurkat cell culture supernatant was collected for 24 hours by PHA, and the secretion of sTNF- alpha was not detected by ELISA.
2.2 PHA induced expression of TACE in Jurkat cells
We further observed whether the expression of TNF converting enzyme (TACE) was inhibited. The results of flow cytometry showed that PHA could stimulate Jurkat cells to express TACE (44.39%). It is speculated that TACE may lead to the production of a large number of soluble TNFR, thereby affecting the immunological detection of sTNF- alpha.
The role of 2.3 exogenous sTNF- alpha in AICD
To explore whether sTNF- alpha can induce apoptosis in T cells, Jurkat 24h was treated by exogenous sTNF- alpha (50U / ml). Results showed that sTNF- alpha could kill unactivated (apoptosis rate 23.81%, P < 0.01) and activated Jurkat cells (apoptosis rate 78.13%, P < 0.01).
Three, the role of mTNF- alpha reverse signal in AICD
1.mTNF- alpha reverse signal promotes PHA induced AICD
1.1 anti TNF- alpha polyclonal antibody can promote PHA induced AICD
Activation of mTNF- alpha reverse signal by anti TNF- alpha polyclonal antibody showed that it could not induce apoptosis of unactivated Jurkat cells, but significantly increased the apoptotic rate of activated Jurkat cells. The enhancement rate was 20.11% (P < 0.01). It suggests that mTNF- alpha reverse signal can enhance AICD effect.
1.2 sTNFR1 can also promote AICD
To further confirm the effect of promoting AICD mTNF- alpha reverse signal, we use soluble TNFRI (sTNFR1 20 g / ml) stimulation of mTNF- alpha sTNFR1 can not reverse signal, the Jurkat cell apoptosis induced by non activated, but can significantly improve the rate of apoptosis of activated Jurkat cells (apoptosis rate 86.82%), in a dose-dependent manner (P < 0.01) suggest that mTNF- can promote AICD. alpha reverse signal
The mechanism of 2.mTNF- alpha reverse signal enhancement of AICD
2.1 mTNF- alpha reverse signal promotes the expression of PHA induced Fas and FasL
The experiment confirmed that without activation of Jurkat cells with low expression of Fas / FasL; PHA can induce Jurkat cells to express Fas (33.28%) and FasL (34.98%). By using anti TNF- polyclonal antibody activated mTNF- alpha alpha reverse signals, can obviously promote PHA induced by Fas (52.19%) and FasL (47.74%) expression (P < 0.01), the increase rates were 56.82%, 36.47%.
2.2 mTNF- alpha reverse signal promotes the expression of mTNF- alpha induced by PHA
The results showed that the expression of mTNF- alpha was low in the unactivated Jurkat cells, and the expression increased by PHA (45.51%), while the mTNF- alpha reverse signal could promote the expression of mTNF- induced by PHA to 61.15%, which was 34.36% compared with the PHA stimulation group (P < 0.01).
Changes of TNFR1 and TNFR2 in apoptosis induced by 2.3 mTNF- alpha reverse signal
The experiment have demonstrated that the activation of Jurkat cells with low expression of TNFR1 and TNFR2, PHA expression in Jurkat cells was induced by TNFR1 (45.29%), while the expression of TNFR2 was increased slightly (only 6.54%), suggesting that TNF- alpha in PHA induced AICD through TNFR1 mediated death domain containing apoptotic signal to anti.MTNF- alpha could significantly increase the TNFR1 (the expression of 59.32%%) and TNFR2 (55.66%, P < 0.01).
Conclusion: These results suggest that mTNF- may be one of the molecular effects of AICD alpha, the forward and reverse signals are involved in the AICD. PHA induced Jurkat TNFR1 expression, suggesting that TNF- alpha mainly mediated by TNFR1 containing death domain. The apoptotic mTNF- alpha reverse signal to promote the molecular mechanism of PHA induced upregulation of Fas AICD: and FasL, mTNF- alpha, the expression of TNFR1 and TNFR2.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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