本文選題：活化誘導(dǎo)的細(xì)胞凋亡(AICD)　切入點(diǎn)：反同信號(hào)(reverse　出處：《華中科技大學(xué)》2006年博士論文　論文類型：學(xué)位論文

【摘要】： TNF-α(tumor necrosis factor，TNF-α)是由激活的單核／巨噬細(xì)胞，淋巴細(xì)胞等分泌的具有多種生物學(xué)活性的細(xì)胞因子，它能直接介導(dǎo)腫瘤細(xì)胞的凋亡和壞死，參與機(jī)體局部及全身炎癥反應(yīng)，介導(dǎo)免疫細(xì)胞的增殖分化，發(fā)揮免疫調(diào)節(jié)作用。在體內(nèi)以兩種形式存在：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酶解而來(lái)。兩型TNF-α通過與兩型TNF受體(TNFR1與TNFR2)結(jié)合而發(fā)揮廣泛的生物學(xué)作用。 成熟T細(xì)胞的AICD是機(jī)體清除免疫應(yīng)答中過量的效應(yīng)T細(xì)胞，調(diào)節(jié)免疫反應(yīng)，維持免疫穩(wěn)態(tài)和建立外周免疫耐受的重要機(jī)制。目前，已證實(shí)FasL／Fas是介導(dǎo)T細(xì)胞AICD重要的、但非唯一的死亡分子。關(guān)于TNF-α在AICD的作用主要限制于對(duì)sTNF-α的研究，mTNF-α正向信號(hào)在AICD中的作用則未見報(bào)道。 近來(lái)研究發(fā)現(xiàn)，mTNF-α除了作為配體可與TNFR結(jié)合，向靶細(xì)胞傳遞正向信號(hào)而發(fā)揮生物學(xué)效應(yīng)外，還可同時(shí)作為受體向效應(yīng)細(xì)胞傳遞反向信號(hào)(reverse signaling)。有學(xué)者報(bào)道Infliximab可以誘發(fā)Crohn's病患者腸相關(guān)淋巴組織固有層(lamina propria)T細(xì)胞凋亡及外周血活化的T細(xì)胞凋亡。提示mTNF-α的反向信號(hào)可以參與AICD。 本課題以PHA活化白血病CD4~+淋巴細(xì)胞系Jurkat建立體外的AICD模型，研究和比較mTNF-α和sTNF-α對(duì)T細(xì)胞AICD的影響，以期闡明兩型TNF-α正向信號(hào)及mTNF-α的反向信號(hào)在AICD中的生物學(xué)特征及生物學(xué)作用，從而深入探討AICD的分子機(jī)制，為臨床治療自身免疫病提供新的線索。一、AICD實(shí)驗(yàn)?zāi)Ｐ偷慕?1．PHA誘導(dǎo)Jurkat細(xì)胞AICD的劑量依賴性 體外以不同劑量的PHA刺激Jurkat細(xì)胞24h，借助Annexin／PI法觀察AICD。結(jié)果顯示，PHA可誘導(dǎo)Jurkat細(xì)胞發(fā)生凋亡，且呈劑量依賴性，即凋亡率隨PHA的濃度的遞增而升高，當(dāng)PHA為1μg／ml時(shí)其凋亡率已達(dá)50％，5μg／ml為63％，20μg／ml則超過80％。 2．PHA誘導(dǎo)Jurkat細(xì)胞AICD的時(shí)間動(dòng)力學(xué) 選取5μg／ml的PHA，觀察其誘導(dǎo)Jurkat細(xì)胞AICD的時(shí)間動(dòng)力學(xué)。凋亡在2h時(shí)已達(dá)22％，6h已升高至56％，隨著PHA刺激時(shí)間的延長(zhǎng)，其凋亡率緩慢上升，24h時(shí)凋亡率為63％，而72h時(shí)則達(dá)82％。選取PHA既可有效誘導(dǎo)AICD，凋亡值又適中的劑量(5μg／ml)和作用時(shí)間點(diǎn)(24h)作為誘導(dǎo)體外AICD模型的條件。 3．TNF-α參與PHA誘導(dǎo)Jurkat細(xì)胞的AICD 用本實(shí)驗(yàn)室制備的對(duì)mTNF-α無(wú)反向信號(hào)作用的抗TNF-α單克隆抗體中和內(nèi)源性TNF-α，可部分抑制PHA誘導(dǎo)的AICD(抑制率14.01％，p＜0.05)，提示TNF-α參與PHA誘導(dǎo)Jurkat細(xì)胞的AICD。 二、mTNF-α和sTNF-α正向信號(hào)在AICD中的作用 1．mTNF-α正向信號(hào)在AICD中的作用 1．1 AICD中Jurkat細(xì)胞表達(dá)內(nèi)源性mTNF-α 實(shí)驗(yàn)證實(shí)未刺激的Jurkat表達(dá)少量mTNF-α(4.5％)，PHA活化的T細(xì)胞表達(dá)mTNF-α明顯增加，并隨著刺激時(shí)間的延長(zhǎng)而逐漸增高。24h時(shí)mTNF-α的表達(dá)率為45.8％。 1．2 mTNF-α對(duì)未活化Jurkat的殺傷作用 用1％多聚甲醛固定PHA活化24h的Jurkat細(xì)胞，以不同的效靶比與未活化的Jurkat細(xì)胞共同孵育24h，借助WST-1法檢測(cè)活化T細(xì)胞的殺傷活性。結(jié)果顯示活化T細(xì)胞對(duì)未活化細(xì)胞具有明顯的殺傷作用，且隨效靶比的升高而增強(qiáng)，5：1時(shí)殺傷率為71％。應(yīng)用兔抗人TNF多克隆抗體(TNFpAb)及鼠抗人sTNF-α單克隆抗體(TNFmAb)，可部分阻斷其殺傷作用(抑制率分別為53.86％和43.37％，p＜0.01)，提示活化T細(xì)胞的殺傷作用部分是由mTNF-α介導(dǎo)的。 1．3 mTNF-α對(duì)活化Jurkat的殺傷作用 用多聚賴氨酸使將PHA活化24h的Jurkat細(xì)胞貼壁，以效靶比5∶1加入PHA活化2h的Jurkat細(xì)胞(作為靶細(xì)胞)共同孵育48h。結(jié)果顯示，活化Jurkat細(xì)胞對(duì)活化Jurkat細(xì)胞有明顯致凋亡作用(44.37％，p＜0.01)，且抗TNF pAb和mAb可以部分阻斷AICD(p＜0.01)。提示mTNF-α可能是AICD的效應(yīng)分子之一。 2．sTNF-α正向信號(hào)在AICD中的作用 2．1活化Jurkat細(xì)胞分泌內(nèi)源性sTNF-α 收集PHA作用24小時(shí)的Jurkat細(xì)胞培養(yǎng)上清，以ELISA法未能檢出sTNF-α的分泌。 2．2 PHA誘導(dǎo)Jurkat細(xì)胞表達(dá)TACE 進(jìn)一步觀察TNF轉(zhuǎn)換酶(TACE)的表達(dá)是否受到抑制，流式細(xì)胞術(shù)結(jié)果顯示PHA可以明顯刺激Jurkat細(xì)胞表達(dá)TACE(表達(dá)率44.39％)。推測(cè)TACE可能導(dǎo)致大量可溶性TNFR的產(chǎn)生，從而影響sTNF-α的免疫學(xué)檢測(cè)。 2．3外源性sTNF-α在AICD中的作用 為探索sTNF-α對(duì)T細(xì)胞是否具有致凋亡作用，用外源性sTNF-α(50U／ml)作用Jurkat細(xì)胞24h，結(jié)果顯示sTNF-α可殺傷未活化(凋亡率23.81％，p＜0.01)和活化Jurkat細(xì)胞(凋亡率78.13％，p＜0.01)。 三、mTNF-α反向信號(hào)在AICD中的作用 1．mTNF-α反向信號(hào)促進(jìn)PHA誘導(dǎo)的AICD 1．1抗TNF-α多克隆抗體可促進(jìn)PHA誘導(dǎo)的AICD 用抗TNF-α多克隆抗體激活mTNF-α反向信號(hào)，結(jié)果顯示，，其不能誘導(dǎo)未活化Jurkat細(xì)胞發(fā)生凋亡，但可明顯提高活化Jurkat細(xì)胞的凋亡率，增強(qiáng)率為20.11％(p＜0.01)。提示mTNF-α反向信號(hào)可增強(qiáng)AICD效應(yīng)。 1．2 sTNFR1也可促進(jìn)AICD 為進(jìn)一步確認(rèn)mTNF-α反向信號(hào)促進(jìn)AICD的效應(yīng)，我們用可溶性TNFRI(sTNFR1 20μg／ml)刺激mTNF-α反向信號(hào)，結(jié)果sTNFR1不能誘導(dǎo)未活化的Jurkat細(xì)胞凋亡，但可明顯提高活化Jurkat細(xì)胞的凋亡率(凋亡率86.82％)，且呈劑量依賴性(p＜0.01)。提示mTNF-α反向信號(hào)確可促進(jìn)AICD。 2．mTNF-α反向信號(hào)增強(qiáng)AICD的機(jī)制 2．1 mTNF-α反向信號(hào)促進(jìn)PHA誘導(dǎo)的Fas和FasL的表達(dá) 實(shí)驗(yàn)證實(shí)未經(jīng)活化的Jurkat細(xì)胞低表達(dá)Fas／FasL；PHA可誘導(dǎo)Jurkat細(xì)胞表達(dá)Fas(33.28％)和FasL(34.98％)。用抗TNF-α多克隆抗體激活mTNF-α反向信號(hào)，可明顯促進(jìn)PHA誘導(dǎo)的Fas(52.19％)和FasL(47.74％)的表達(dá)(p＜0.01)，其增強(qiáng)率分別為56.82％，36.47％。 2．2 mTNF-α反向信號(hào)促進(jìn)PHA誘導(dǎo)的mTNF-α表達(dá) 實(shí)驗(yàn)證實(shí)未活化Jurkat細(xì)胞低表達(dá)mTNF-α，PHA誘導(dǎo)其表達(dá)增加(45.51％)，而mTNF-α反向信號(hào)則可促進(jìn)PHA誘導(dǎo)的mTNF-α表達(dá)率增至61.15％，與PHA刺激組相比，增強(qiáng)率為34.36％(p＜0.01)。 2．3 mTNF-α反向信號(hào)促凋亡中TNFR1和TNFR2的變化 實(shí)驗(yàn)證實(shí)未活化Jurkat細(xì)胞低表達(dá)TNFR1和TNFR2，PHA主要誘導(dǎo)Jurkat細(xì)胞表達(dá)TNFR1(45.29％)，而TNFR2表達(dá)則輕度上升(僅6.54％)，提示TNF-α在PHA誘導(dǎo)的AICD中主要通過含有死亡結(jié)構(gòu)域的TNFR1介導(dǎo)凋亡。mTNF-α反向信號(hào)可明顯上調(diào)TNFR1(59.32％％)和TNFR2的表達(dá)(55.66％，p＜0.01)。 結(jié)論：上述結(jié)果提示①mTNF-α可能為AICD的效應(yīng)分子之一，其正向和反向信號(hào)均參與AICD。②PHA主要誘導(dǎo)Jurkat細(xì)胞表達(dá)TNFR1，提示TNF-α主要通過含有死亡結(jié)構(gòu)域的TNFR1介導(dǎo)凋亡。③mTNF-α反向信號(hào)促進(jìn)PHA誘導(dǎo)AICD的主要分子機(jī)制：上調(diào)Fas和FasL、mTNF-α、TNFR1和TNFR2的表達(dá)。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

【參考文獻(xiàn)】

中國(guó)期刊全文數(shù)據(jù)庫(kù) 前2條

1 辛利軍,李卓婭;反向信號(hào)傳遞[J];國(guó)外醫(yī)學(xué)(分子生物學(xué)分冊(cè));2003年06期

2 辛利軍,石文芳,李清芬,姜曉丹,馮瑋,熊平,徐勇,龔非力,李卓婭;TM-TNF-α介導(dǎo)的反向信號(hào)協(xié)同增強(qiáng)sTNF-α對(duì)U937細(xì)胞的激活作用[J];中華微生物學(xué)和免疫學(xué)雜志;2005年01期

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