跨膜型TNF-α对急性肝衰与内毒素性休克的影响

发布时间：2018-06-01 12:42

本文选题：tmTNF-α + 急性鼠肝衰　；参考：《华中科技大学》2014年博士论文

【摘要】：跨膜型TNF-α (Transmembrane tumor necrosis factor, tmTNF-α)是分泌型TNF-α (Secreted TNF-α, sTNF-α)前体,在TNF-α转换酶处理后,释放其胞外端而成为sTNF-α。两型TNF-α均具有生物学功能。sTNF-α是典型的促炎细胞因子,在急性肝衰和内毒素性休克发病机制中扮演着关键作用；然而,tmTNF-α在这两种疾病中的作用虽然有转基因动物的实验报道,但转染的是缺失酶切位点的tmTNF-α突变体。我们的前期工作证实该突变体与野生型分子在信号转导上有明显差异,且不同实验室报道结果相反。鉴于这些报道可能不能反映天然tmTNF-α在这2种疾病中的作用,本研究旨在观察内源性tmTNF-α对急性鼠肝衰及内毒素性休克的影响,并探索其作用机制。主要结果如下：第一部分跨模型TNF-α对急性肝衰的影响一、肝组织损伤变化特点给小鼠注射LPS/D-gal制备急性肝衰模型,HE染色结果显示,注射后2h可见肝细胞呈轻度水肿,4h肝细胞水肿,并有少量灶状坏死,6h可见肝窦淤血,肝细胞坏死,肝细胞索断裂；血清ALT和AST直至LPS/D-gal处理后6h才显著升高,提示这个时间点肝组织明显受损。二、急性肝衰内源性sTNF-α与tmTNF-α表达的变化与肝组织损伤的相关性 1. sTNF-α变化特点：ELISA结果显示LPS/D-gal注射后2h小鼠血清sTNF-α达峰值,随着时间延长而降低,4h后几乎消失；从LPS/D-gal处理2h小鼠分离的KCs培养上清sTNF-α含量最高,而4h与6h分离的KCs培养上清sTNF-α虽然明显降低,但仍然显著高于正常鼠分离的KCs培养上清,提示循环中sTNF-α不能代表炎症局部sTNF-α的变化。 2. tmTNF-α表达变化：用FCM分析急性肝衰小鼠KCs和PMs细胞表面tmTNF-α表达的变化,结果显示在LPS/D-gal处理后2h及4h tmTNF-α呈低表达,而在6htmTNF-α表达显著上升。 3.两型TNF-α与血清转氨酶关系：急性肝衰小鼠血清TNF-α变化与血清转氨酶AST及ALT变化相反,而KCs和PMs表达tmTNF-α水平与肝组织是否放至血清ALT或AST的量呈正相关,提示是tmTNF-α而不是sTNF-α与急性肝组织损伤相关。三、 KCs-6h与PMs-6h分泌促炎和抑炎因子失衡 1.血清促炎因子IL-6与抑炎因子IL-10的比值变化：用ELISA证实急性肝衰小鼠血清IL-6显著升高,其在4h达高峰,在6h则有所降低,为高峰水平的87.8%；而血清IL-10含量则在2h最高,随后下降,其在6h的水平仅为高峰水平的23.6%。计算IL-6与IL-10比值,发现其在6h才显著增加,与肝组织损伤变化一致,提示血清IL-6/IL-10比值增加可更好的预测急性肝损伤。 2.高表达tmTNF-α的KCs-6h和PMs-6h分泌促炎和抑炎因子失衡：将LPS/D-gal注射后不同时间分离的KCs和PMs在体外进行培养,结果显示这两种巨噬细胞培养上清IL-6与IL-10的变化与血清变化一致；IL-6/IL-10比值在急性肝衰6h显著增加,与此时间点这2种巨噬细胞高表达tmTNF-α呈正相关；提示在LPS/D-gal处理6h分离的KCs (KCs-6h)和PMs (PMs-6h)分泌促炎和抑炎因子严重失衡,进而加重肝脏的炎性损伤。四、高表达tmTNF-α的KCs-6h诱导肝细胞凋亡 1-KCs-6h通过直接接触诱导肝细胞凋亡：将肝细胞与来自LPS/D-gal处理2h或6h的KCs进行共培养,结果显示,高表达tmTNF-α的KCs-6h细胞可通过直接接触诱导肝细胞凋亡增加,导致培养上清AST升高；而低表达tmTNF-α的KCs-2h细胞则无此效应；当用Transwell膜将KCs-6h细胞与肝细胞隔离培养后,无肝细胞出现凋亡。 2. KCs-6h细胞通过tmTNF-α直接诱导肝细胞凋亡：当用TNF-α抗体预处理KCs-6h细胞30min,以中和其表达的tmTNF-α,再与肝细胞共培养,肝细胞凋亡显著降低,提示KCs-6h细胞表达的tmTNF-α可直接诱导肝细胞凋亡。 3.高表达tmTNF-α的KCs-6h细胞表达FasL增加：尽管用抗体中和tmTNF-α可显著降低肝细胞凋亡,但仍有部分凋亡细胞,提示其它凋亡途径可能参与了KCs-6h诱导肝细胞凋亡。Real time PCR和Western blotting的结果显示高表达tmTNF-α的KCs-6h细胞表达FasL基因及其蛋白明显高于低表达tmTNF-α的KCs-2h细胞,提示tmTNF-α可能通过增加FasL表达而促进肝细胞凋亡。五、过继KCs-6h细胞可加重LPS/D-gal诱导小鼠肝组织损伤 1. KCs-6h细胞可持续表达tmTNF-α:为了保证过继有效性,我们首先检查KCs-6h细胞tmTNF-α表达是否稳定。结果显示：连续培养KCs-6h细胞至6天,其表达tmTNF-α仍能维持原有水平； 2.过继KCs-6h细胞加重肝组织损伤：HE结果显示：与LPS/D-gal组相比,过继KCs-6h细胞可明显加重小鼠在LPS/D-gal处理4h的肝组织损伤,表现为肝窦淤血,肝细胞索断裂,水样变性及少量坏死。 3.过继KCs-6h促进肝细胞凋亡：肝组织切片TUNEL染色显示,过继KCs-6h细胞明显促进肝细胞凋亡,血清AST也显著高于过继KCs-2h的小鼠,提示高表达tmTNF-α的KCs-6h在体内可以加重LPS/D-gal诱导的肝衰。 4.过继KCs-6h增加血清IL-6/IL-10比值：ELISA结果显示过继KCs-6h细胞导致血清促炎因子IL-6和IL-1β含量明显高于过继KCs-2h组小鼠,而抑炎因子IL-10低于KCs-2h组小鼠,IL-6/IL-10比值明显增加,提示高表达tmTNF-α的KCs-6h分泌促炎和抑炎因子平衡障碍,从而参与急性肝衰的发生发展。第二部分跨模型TNF-α对内毒素性休克的影响一、特异性tmTNF Ab的制备与鉴定： 1.tmTNFAb的制备与鉴定：针对tmTNF-α单表位成功制备了兔抗小鼠tmTNF-α抗体。ELISA显示兔血清含有高效价抗tmTNF-α单表位抗体,且只结合tmTNF-α肽段,不与sTNF-α发生交叉反应。 2. tmTNF Ab可与细胞表面tmTNF-α结合：用FCM证实该抗体可与TNF-α+NH3T3细胞表面tmTNF-α结合,而不能与TNF-α阴性的CT26与H22细胞株结合,提示该抗体可识别细胞表面完整的tmTNF-α分子。二、tmTNF Ab可有效阻断tmTNF-α转换为sTNF-α: 1. tmTNF Ab阻断tmTNF-α转换为sTNF-α:用100ng/ml LPS和tmTNF Ab分别刺激RAW264.7巨噬细胞系和PMs原代细胞4h, ELISA和FCM结果显示该抗体可有效抑制sTNF-α分泌,而显著增加tmTNF-α的表达,提示该抗体可有效阻断tmTNF-α转换为sTNF-α。 2. tmTNF Ab抑制LPS诱导RAW264.7上清sTNF-α胞毒效应：用生物活性证实LPS刺激RAW264.74h的上清对TNF-α敏感靶细胞L929具有明显胞毒效应,用TNF-α抗体预先中和上清sTNF-α后,可阻断其胞毒效应；用tmTNF Ab明显抑制LPS刺激RAW264.7上清的胞毒效应,再次证实tmTNF Ab抑制1tmTNF-α酶解,致使sTNF-α分泌减少,因而其胞毒效应受到抑制。四、tmTNF Ab对LPS诱导巨噬细胞产生促炎和抑炎因子的影响 1. tmTNF Ab抑制LPS诱导巨噬细胞产生NO：用LPS和tmTNF Ab刺激RAW264.7和PMs,结果显示该抗体可明显抑制LPS诱导这2种巨噬细胞转录iNOS基因以及分泌NO。 2. tmTNF Ab抑制LPS诱导巨噬细胞产生促炎因子IL-6和IL-1β：用LPS和tmTNF Ab刺激RAW264.7和PMs,结果显示该抗体可明显抑制LPS诱导这2种巨噬细胞转录IL-6和IL-1p基因及分泌这些细胞因子。 3. tmTNF Ab对LPS诱导巨噬细胞产生抑炎因子IL-10无影响：用LPS和tmTNFAb刺激RAW264.7和PMs,结果显示该抗体对LPS诱导这2种巨噬细胞转录IL-10基因及其表达无影响。五、tmTNF Ab保护小鼠抵抗内毒素血症 1. tmTNF Ab可阻断内毒素血症小鼠tmTNF-α转变为sTNF-α:预先给予tmTNF-α Ab明显增强LPS注射小鼠4h腹腔巨噬细胞的tmTNF-α表达,同时抑制该时间点内毒素血症小鼠血清TNF-α含量(P0.01),提示tmTNF Ab在体内可有效阻断tmTNF-α转换成sTNF-α。 2. tmTNF Ab可改善内毒素性休克临床症状,提高小鼠存活率：用tmTNF Ab可明显减轻内毒素性休克鼠唇紫绀,改善临床评分,显著提高内毒素血症小鼠的存活率。 3. tmTNF Ab抑制内毒素血症促炎因子分泌：LPS注射小鼠2h血清促炎因子IL-6、IL-1p及NO以及抑炎因子IL-10即开始升高,6h达高峰,此后下降。用tmTNF Ab可明显降低内毒素血症小鼠上述血清促炎因子的水平,却轻度增加IL-10含量,但无统计学意义。综上所述,tmTNF-α在不同的病理状态下发挥不同的、甚至相反的作用。在LPS/D-gal诱导的急性鼠肝衰中KCs和PMs表达的tmTNF-α可直接诱导肝细胞凋亡以及通过分泌促炎/抗炎因子失衡加重肝脏的炎性损伤。反之,用tmTNF Ab促进tmTNF-α表达,抑制sTNF-α分泌,在体外可抑制巨噬细胞对LPS的应答,在体内保护小鼠抵抗内毒素性休克。该研究不但证实tmTNF-α在不同疾病中的作用,还为治疗TNF相关疾病提供新的策略和线索。
[Abstract]:Transmembrane type TNF- alpha (Transmembrane tumor necrosis factor, tmTNF- a) is the precursor of secretory TNF- alpha (Secreted TNF- alpha, sTNF- alpha). After TNF- alpha converting enzyme treatment, it releases its extracellular end and becomes sTNF- alpha. Type two alpha has biological function, which is a typical proinflammatory cytokine, in acute liver failure and endotoxic shock pathogenesis. However, although the role of tmTNF- alpha in these two diseases has been reported in transgenic animals, the transfection is a tmTNF- alpha mutant with the deletion of the enzyme cut site. Our previous work proved that the mutant and the wild type molecules have obvious differences in signal transduction, and the results of different laboratory reports are opposite. Given that these reports may not reflect the role of natural tmTNF- alpha in these 2 diseases, this study aims to observe the effect of endogenous tmTNF- a on acute hepatic failure and endotoxic shock in rats and explore its mechanism. The main results are as follows:
Part one the effect of trans model TNF- alpha on acute liver failure
The characteristics of the changes of liver tissue injury
The mice were injected with LPS/D-gal to prepare the acute liver failure model. The results of HE staining showed that the liver cells showed mild edema, 4H hepatocyte edema, and a small amount of focal necrosis, 6h showed hepatic sinusoid congestion, hepatocyte necrosis, and hepatic cell cord fracture, and the serum ALT and AST until LPS/ D-gal treatment was significantly elevated, suggesting this time point liver group. The fabric was obviously damaged.
Two, the correlation between the expression of endogenous sTNF- alpha and tmTNF- alpha in acute liver failure and liver tissue injury.
1. sTNF- alpha change characteristics: ELISA results showed that sTNF- alpha reached peak value in 2H mice after LPS/D-gal injection, decreased with time and almost disappeared after 4H. STNF- alpha content was the highest in KCs culture supernatant from LPS/D-gal treated 2H mice, while 4H and 6h isolated culture supernatant decreased significantly, but still significantly higher than normal mice. The isolated KCs culture supernatant indicated that sTNF- alpha in the circulation could not represent the change of local sTNF- alpha in inflammation.
2. tmTNF- alpha expression changes: the changes in the expression of tmTNF- alpha on the surface of KCs and PMs cells in acute liver failure mice were analyzed with FCM. The results showed that the expression of 2H and 4h tmTNF- alpha was low after LPS/D-gal treatment, and the expression of 6htmTNF- a was significantly increased.
The relationship between 3. type two TNF- alpha and serum transaminase: the changes of serum TNF- a in mice with acute liver failure are contrary to the changes of serum aminotransferase AST and ALT, while the level of KCs and PMs expression tmTNF- A is positively correlated with the amount of ALT or AST in the liver tissue, suggesting that tmTNF- a rather than sTNF- alpha is associated with acute liver tissue injury.
Three, KCs-6h and PMs-6h secrete an imbalance between pro inflammatory and anti inflammatory factors.
1. the change of the ratio of serum proinflammatory factor IL-6 and anti inflammatory factor IL-10: the serum IL-6 in mice with acute liver failure was significantly increased with ELISA, which was at the peak of 4h, and decreased in 6h to the peak level, while the serum IL-10 content was highest in 2H, then decreased, and the ratio of 23.6%. IL-6 to IL-10 in 6h was only at the peak level of 23.6%.. It was found that it increased significantly in 6h, which was consistent with the change of liver tissue injury, suggesting that the increase of serum IL-6/IL-10 ratio can better predict acute liver injury.
2. high expression of KCs-6h and PMs-6h secretion of tmTNF- A and the imbalance of proinflammatory and anti inflammatory factors: KCs and PMs, isolated at different time after LPS/D-gal injection, were cultured in vitro. The results showed that the changes of IL-6 and IL-10 in the supernatant of the two macrophages were in accordance with the serum changes; the IL-6/IL-10 ratio in acute liver failure 6h increased significantly, and this time point was found. These 2 macrophages have a positive correlation with high expression of tmTNF- - alpha, suggesting that KCs (KCs-6h) and PMs (PMs-6h) secreted by 6h and PMs (PMs-6h) secretes a serious imbalance of proinflammatory and anti inflammatory factors in LPS/D-gal, and thus aggravates the inflammatory injury of the liver.
Four, high expression of tmTNF- alpha KCs-6h induces hepatocyte apoptosis.
1-KCs-6h induced hepatocyte apoptosis through direct contact: co culture of hepatocytes with KCs from LPS/D-gal to treat 2H or 6h. The results showed that KCs-6h cells with high expression of tmTNF- alpha could induce apoptosis in liver cells by direct contact, leading to the increase of AST in culture supernatant, while KCs-2h cells with low level of tmTNF- alpha did not have this effect; T After ranswell membrane isolated KCs-6h cells from hepatocytes, no hepatocytes apoptosis.
2. KCs-6h cells directly induced the apoptosis of hepatocytes through tmTNF- alpha: when the KCs-6h cell 30min was pretreated with TNF- alpha antibody, the expression of tmTNF- a was neutralized, and the hepatocytes were co cultured with the hepatocytes, and the apoptosis of the liver cells decreased significantly. It suggested that the tmTNF- a expressed by KCs-6h cells could directly induce the apoptosis of the liver cells.
3. the expression of FasL in KCs-6h cells with high expression of tmTNF- alpha was increased: Although there was a significant decrease in apoptosis of liver cells with antibodies and tmTNF- a, there were still some apoptotic cells, suggesting that other apoptotic pathways may be involved in KCs-6h induced apoptosis of hepatocytes,.Real time PCR and Western blotting, showing the expression of KCs-6h cells expressing high expression tmTNF- alpha. The gene and its protein were significantly higher than those of KCs-2h cells with low expression of tmTNF- alpha, suggesting that tmTNF- alpha might promote hepatocyte apoptosis by increasing FasL expression.
Five, adoptive KCs-6h cells can aggravate LPS/D-gal induced liver injury in mice.
1. KCs-6h cells express tmTNF- alpha sustainably: in order to ensure the adoptive effectiveness, we first examine the stability of the expression of tmTNF- alpha in KCs-6h cells. The results show that the expression of tmTNF- a still can maintain the original level of the KCs-6h cells in continuous culture to 6 days.
2. adoptive KCs-6h cells aggravated liver tissue damage: HE results showed that adoptive KCs-6h cells significantly aggravated the liver tissue damage in LPS/D-gal treatment of 4H in mice, manifested as hepatic sinusoid congestion, hepatic cell cord rupture, water like degeneration and a small amount of necrosis.
3. adoptive KCs-6h promoted hepatocyte apoptosis: liver tissue section TUNEL staining showed that adoptive KCs-6h cells obviously promoted hepatocyte apoptosis, and serum AST was significantly higher than that of adoptive KCs-2h mice, suggesting that KCs-6h with high expression of tmTNF- alpha could aggravate LPS/D-gal induced liver failure in the body.
4. adoptive KCs-6h increased the serum IL-6/IL-10 ratio: ELISA results showed that the serum proinflammatory factor IL-6 and IL-1 beta in the adoptive KCs-6h cell was significantly higher than that of the adoptive KCs-2h group, while the anti inflammatory factor IL-10 was lower than the KCs-2h group, and the IL-6/IL-10 ratio was significantly increased, suggesting that the KCs-6h secreting proinflammatory and anti inflammatory factor balance barrier of high expression tmTNF- alpha was high. It is involved in the development of acute liver failure.
The second part is the effect of trans model TNF- alpha on endotoxic shock.
1. Preparation and identification of specific tmTNF Ab:
Preparation and identification of 1.tmTNFAb: a Rabbit anti mouse tmTNF- alpha antibody.ELISA was successfully prepared for the single epitope of tmTNF- a to show that the rabbit serum contains high effective anti tmTNF- alpha epitopes antibody and only tmTNF- alpha peptide segment, and no cross reaction with sTNF- alpha.
2. tmTNF Ab can bind to tmTNF- alpha on cell surface: it is confirmed by FCM that the antibody can bind to tmTNF- alpha on the surface of TNF- alpha +NH3T3 cells, but can not be combined with TNF- alpha negative CT26 and H22 cell lines, suggesting that the antibody can identify the intact tmTNF- alpha molecule on the surface of the cell.
Two, tmTNF Ab can effectively block tmTNF- to sTNF- alpha.
1. tmTNF Ab blocked tmTNF- alpha to sTNF- alpha: 100ng/ml LPS and tmTNF Ab stimulated the RAW264.7 macrophage system and PMs primary cell 4H. ELISA and results showed that the antibody could effectively inhibit the secretion of alpha, which significantly increased the expression of alpha alpha, suggesting that the antibody could effectively obstruct the conversion of alpha to alpha.
2. tmTNF Ab inhibited the cytotoxic effect of sTNF- alpha induced by LPS in RAW264.7 supernatant: using bioactivity to confirm that LPS stimulated RAW264.74h in the supernatant of RAW264.74h had obvious cytotoxic effect on TNF- alpha sensitive target cell L929, and the cytotoxic effect could be blocked by using TNF- alpha antibody in advance to neutralize sTNF- alpha, and the cytotoxic effect of stimulating supernatant was inhibited by tmTNF. It is confirmed that tmTNF Ab inhibits 1tmTNF- alpha enzymolysis, resulting in a decrease in sTNF- alpha secretion, thus inhibiting its cytotoxic effect.
Four, the effect of tmTNF Ab on LPS induced macrophages producing proinflammatory and anti-inflammatory factors.
1. tmTNF Ab inhibited LPS induced macrophage to produce NO: LPS and tmTNF Ab were used to stimulate RAW264.7 and PMs. The results showed that the antibody inhibited LPS to induce the iNOS gene of the 2 macrophages and secreted NO..
2. tmTNF Ab inhibited LPS induced macrophages to produce proinflammatory cytokines IL-6 and IL-1 beta: LPS and tmTNF Ab stimulated RAW264.7 and PMs. The results showed that the antibody inhibited LPS induction of the 2 macrophages transcriptional IL-6 and genes and secreting these cytokines.
3. tmTNF Ab had no effect on LPS induced macrophage induced anti inflammatory factor IL-10: RAW264.7 and PMs were stimulated with LPS and tmTNFAb. The results showed that the antibody had no effect on LPS induced 2 macrophage transcription IL-10 gene and its expression.
Five, tmTNF Ab protects against endotoxemia in mice
1. tmTNF Ab could block the transformation of tmTNF- alpha into sTNF- alpha in mice with endotoxemia: pre administration of tmTNF- alpha Ab significantly enhanced the tmTNF- alpha expression of 4H peritoneal macrophages in LPS injected mice, and inhibited the content of TNF- alpha in serum of mice with endotoxemia at the same time (P0.01).
2. tmTNF Ab can improve the clinical symptoms of endotoxic shock and improve the survival rate of mice: using tmTNF Ab can significantly reduce cyanosis in endotoxic shock rats, improve the clinical score, and significantly improve the survival rate of endotoxemia mice.
3. tmTNF Ab inhibited the secretion of proinflammatory cytokines from endotoxemia: LPS injection of 2H serum proinflammatory factor IL-6, IL-1p and NO and anti inflammatory factor IL-10 began to rise, and 6h reached the peak and decreased thereafter. TmTNF Ab could significantly reduce the levels of serum proinflammatory factors in mice with endotoxemia, but slightly increased IL-10 content, but no statistical significance.
To sum up, tmTNF- alpha plays different, even opposite effects in different pathological conditions. In the acute rat liver failure induced by LPS/D-gal, the tmTNF- alpha expressed by KCs and PMs can directly induce hepatocyte apoptosis and aggravate the inflammatory damage of the liver by secreting proinflammatory / anti-inflammatory factor imbalance. On the other hand, tmTNF Ab promotes the expression of tmTNF- A and inhibits sT. NF- alpha secretion, which inhibits the response of macrophages to LPS in vitro, protects mice against endotoxic shock in the body. This study not only confirms the role of tmTNF- alpha in different diseases, but also provides new strategies and clues for the treatment of TNF related diseases.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R459.7;R575.3

【共引文献】