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  本文選題：活化誘導的細胞凋亡(AICD)　切入點：反同信號(reverse　出處：《華中科技大學》2006年博士論文　論文類型：學位論文

【摘要】： TNF-α(tumor necrosis factor，TNF-α)是由激活的單核／巨噬細胞，淋巴細胞等分泌的具有多種生物學活性的細胞因子，它能直接介導腫瘤細胞的凋亡和壞死，參與機體局部及全身炎癥反應，介導免疫細胞的增殖分化，發(fā)揮免疫調節(jié)作用。在體內以兩種形式存在：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。兩型TNF-α通過與兩型TNF受體(TNFR1與TNFR2)結合而發(fā)揮廣泛的生物學作用。 成熟T細胞的AICD是機體清除免疫應答中過量的效應T細胞，調節(jié)免疫反應，維持免疫穩(wěn)態(tài)和建立外周免疫耐受的重要機制。目前，已證實FasL／Fas是介導T細胞AICD重要的、但非唯一的死亡分子。關于TNF-α在AICD的作用主要限制于對sTNF-α的研究，mTNF-α正向信號在AICD中的作用則未見報道。 近來研究發(fā)現，mTNF-α除了作為配體可與TNFR結合，向靶細胞傳遞正向信號而發(fā)揮生物學效應外，還可同時作為受體向效應細胞傳遞反向信號(reverse signaling)。有學者報道Infliximab可以誘發(fā)Crohn's病患者腸相關淋巴組織固有層(lamina propria)T細胞凋亡及外周血活化的T細胞凋亡。提示mTNF-α的反向信號可以參與AICD。 本課題以PHA活化白血病CD4~+淋巴細胞系Jurkat建立體外的AICD模型，研究和比較mTNF-α和sTNF-α對T細胞AICD的影響，以期闡明兩型TNF-α正向信號及mTNF-α的反向信號在AICD中的生物學特征及生物學作用，從而深入探討AICD的分子機制，為臨床治療自身免疫病提供新的線索。一、AICD實驗模型的建立 1．PHA誘導Jurkat細胞AICD的劑量依賴性 體外以不同劑量的PHA刺激Jurkat細胞24h，借助Annexin／PI法觀察AICD。結果顯示，PHA可誘導Jurkat細胞發(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PHA為1μg／ml時其凋亡率已達50％，5μg／ml為63％，20μg／ml則超過80％。 2．PHA誘導Jurkat細胞AICD的時間動力學 選取5μg／ml的PHA，觀察其誘導Jurkat細胞AICD的時間動力學。凋亡在2h時已達22％，6h已升高至56％，隨著PHA刺激時間的延長，其凋亡率緩慢上升，24h時凋亡率為63％，而72h時則達82％。選取PHA既可有效誘導AICD，凋亡值又適中的劑量(5μg／ml)和作用時間點(24h)作為誘導體外AICD模型的條件。 3．TNF-α參與PHA誘導Jurkat細胞的AICD 用本實驗室制備的對mTNF-α無反向信號作用的抗TNF-α單克隆抗體中和內源性TNF-α，可部分抑制PHA誘導的AICD(抑制率14.01％，p＜0.05)，提示TNF-α參與PHA誘導Jurkat細胞的AICD。 二、mTNF-α和sTNF-α正向信號在AICD中的作用 1．mTNF-α正向信號在AICD中的作用 1．1 AICD中Jurkat細胞表達內源性mTNF-α 實驗證實未刺激的Jurkat表達少量mTNF-α(4.5％)，PHA活化的T細胞表達mTNF-α明顯增加，并隨著刺激時間的延長而逐漸增高。24h時mTNF-α的表達率為45.8％。 1．2 mTNF-α對未活化Jurkat的殺傷作用 用1％多聚甲醛固定PHA活化24h的Jurkat細胞，以不同的效靶比與未活化的Jurkat細胞共同孵育24h，借助WST-1法檢測活化T細胞的殺傷活性。結果顯示活化T細胞對未活化細胞具有明顯的殺傷作用，且隨效靶比的升高而增強，5：1時殺傷率為71％。應用兔抗人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細胞的殺傷作用部分是由mTNF-α介導的。 1．3 mTNF-α對活化Jurkat的殺傷作用 用多聚賴氨酸使將PHA活化24h的Jurkat細胞貼壁，以效靶比5∶1加入PHA活化2h的Jurkat細胞(作為靶細胞)共同孵育48h。結果顯示，活化Jurkat細胞對活化Jurkat細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的效應分子之一。 2．sTNF-α正向信號在AICD中的作用 2．1活化Jurkat細胞分泌內源性sTNF-α 收集PHA作用24小時的Jurkat細胞培養(yǎng)上清，以ELISA法未能檢出sTNF-α的分泌。 2．2 PHA誘導Jurkat細胞表達TACE 進一步觀察TNF轉換酶(TACE)的表達是否受到抑制，流式細胞術結果顯示PHA可以明顯刺激Jurkat細胞表達TACE(表達率44.39％)。推測TACE可能導致大量可溶性TNFR的產生，從而影響sTNF-α的免疫學檢測。 2．3外源性sTNF-α在AICD中的作用 為探索sTNF-α對T細胞是否具有致凋亡作用，用外源性sTNF-α(50U／ml)作用Jurkat細胞24h，結果顯示sTNF-α可殺傷未活化(凋亡率23.81％，p＜0.01)和活化Jurkat細胞(凋亡率78.13％，p＜0.01)。 三、mTNF-α反向信號在AICD中的作用 1．mTNF-α反向信號促進PHA誘導的AICD 1．1抗TNF-α多克隆抗體可促進PHA誘導的AICD 用抗TNF-α多克隆抗體激活mTNF-α反向信號，結果顯示，，其不能誘導未活化Jurkat細胞發(fā)生凋亡，但可明顯提高活化Jurkat細胞的凋亡率，增強率為20.11％(p＜0.01)。提示mTNF-α反向信號可增強AICD效應。 1．2 sTNFR1也可促進AICD 為進一步確認mTNF-α反向信號促進AICD的效應，我們用可溶性TNFRI(sTNFR1 20μg／ml)刺激mTNF-α反向信號，結果sTNFR1不能誘導未活化的Jurkat細胞凋亡，但可明顯提高活化Jurkat細胞的凋亡率(凋亡率86.82％)，且呈劑量依賴性(p＜0.01)。提示mTNF-α反向信號確可促進AICD。 2．mTNF-α反向信號增強AICD的機制 2．1 mTNF-α反向信號促進PHA誘導的Fas和FasL的表達 實驗證實未經活化的Jurkat細胞低表達Fas／FasL；PHA可誘導Jurkat細胞表達Fas(33.28％)和FasL(34.98％)。用抗TNF-α多克隆抗體激活mTNF-α反向信號，可明顯促進PHA誘導的Fas(52.19％)和FasL(47.74％)的表達(p＜0.01)，其增強率分別為56.82％，36.47％。 2．2 mTNF-α反向信號促進PHA誘導的mTNF-α表達 實驗證實未活化Jurkat細胞低表達mTNF-α，PHA誘導其表達增加(45.51％)，而mTNF-α反向信號則可促進PHA誘導的mTNF-α表達率增至61.15％，與PHA刺激組相比，增強率為34.36％(p＜0.01)。 2．3 mTNF-α反向信號促凋亡中TNFR1和TNFR2的變化 實驗證實未活化Jurkat細胞低表達TNFR1和TNFR2，PHA主要誘導Jurkat細胞表達TNFR1(45.29％)，而TNFR2表達則輕度上升(僅6.54％)，提示TNF-α在PHA誘導的AICD中主要通過含有死亡結構域的TNFR1介導凋亡。mTNF-α反向信號可明顯上調TNFR1(59.32％％)和TNFR2的表達(55.66％，p＜0.01)。 結論：上述結果提示①mTNF-α可能為AICD的效應分子之一，其正向和反向信號均參與AICD。②PHA主要誘導Jurkat細胞表達TNFR1，提示TNF-α主要通過含有死亡結構域的TNFR1介導凋亡。③mTNF-α反向信號促進PHA誘導AICD的主要分子機制：上調Fas和FasL、mTNF-α、TNFR1和TNFR2的表達。
[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.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R392

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