端粒功能缺陷细胞分泌的蛋白质即人类衰老和疾病的预报因子

发布时间：2018-09-06 17:56

【摘要】： 端粒的缩短限制了人类细胞的分裂增殖，因此它常见于人类组织器官的衰老过程中。在第四代端粒酶敲除小鼠中，端粒功能的缺陷致使组织器官功能受损，寿命缩短。然而，端粒功能缺陷在人类衰老和疾病中的作用目前仍存在很大的争议。我们研究发现了一组蛋白质：CRAMP、EF-1α、Chitinase 3L3、Stathmin。这组蛋白质在老年端粒酶敲除小鼠的多个器官(如肝脏、肾脏、脾脏、心脏、肺、大脑等)和血清中异常高表达，但是在老年野生型端粒功能正常的小鼠中没有相似的高表达发现。我们还发现，这组人类的同源蛋白质在人类衰老过程中高表达于血清以及其他的器官中(如肝脏、脾脏等)。而且在端粒缩短相关疾病中呈过度表达(如肝脏纤维化、骨髓发育不良综合征等)。我们将进一步研究这组蛋白质与肾脏疾病的关系，探索其作用机制。端粒位于染色体末端防止由于DNA损伤激活导致得细胞衰老或者凋亡。已经有确凿的证据表明端粒缩短会导致人类衰老和慢性疾病的发生。且同时发现端粒酶基因的突变会导致端粒的缩短，组织器官修复功能的损伤，以及人类和小鼠寿命的缩短。虽然大量的遗传学和临床实验证明端粒缩短导致组织器官修复功能的损伤和寿命的缩短。然而，在端粒功能缺陷导致人类寿命缩短和疾病的机制方面仍然存在不同的争议。在人类和某些灵长类动物的衰老过程中，皮肤会有大量衰老细胞累积，，但在其他例如肌肉或者肝脏等器官中并未发现此类衰老细胞累积现象。在衰老的端粒酶敲除小鼠中，端粒功能的缺陷还与干细胞功能的损伤相关，并导致器官修复功能受损以及寿命缩短。然而在这种端粒酶敲除小鼠模型体内并没有发现衰老细胞的累积现象。在这种模型小鼠体内发现衰老的细胞被凋亡和免疫反应清除。显然衰老是受到细胞周期调控的。因此端粒功能缺陷在衰老过程中的作用可以通过对衰老细胞的实验研究来证实。而衰老的临床生物学信号的研究与发现，对于衰老的研究领域更是具有划时代的挑战意义。在人类细胞的体外培养实验研究中发现端粒功能低水平缺陷就可以诱导细胞衰老。因此如果我们能够发现鉴定一组蛋白质生物学信号，它们可以在细胞衰老前期或者疾病早期就反应细胞体内的端粒功能水平的底下和DNA损伤，那么将对人类衰老和某些衰老相关疾病的研究产生重要的影响。由于全球都面临老龄化这一重要问题，人口老龄化所带来的一系列相关疾病问题，这一组蛋白质生物学信号的发现与鉴定无疑将对此意义深远。我们的实验发现第四代端粒酶敲除小鼠(G4mTerc~(-/-))并没有呈现衰老细胞体内累积现象，但是其衰老表型仍然与端粒功能缺陷密切相关。我们有充分理由相信这种小鼠模型能够为发现和鉴定端粒功能缺陷诱导衰老前期细胞分泌的蛋白质提供完善的实验体系。而好的临床信号应当能够易于通过体液检测到。因此，我们的实验设计体外短时间培养(4小时)2月和12月龄的第四代端粒酶敲除小鼠以及端粒功能正常野生型小鼠的骨髓细胞，收集其培养液，通过蛋白质组学实验方法(CE-TOF-MS)鉴别不同的蛋白质组分泌。然后通过统计学方法建立能够鉴别这四组不同实验小鼠的蛋白质组模型(suppl．Fig．1a-e)。然后我们通过质谱技术测序鉴定了一组4个蛋白质。他们在12月龄的第四代端粒酶敲除小鼠中异常高表达，显著区别于其他三组小鼠(n=5／组，figure 1a)。我们还设计了一个单盲实验来检验这组蛋白质模型。四组共计26只小鼠用于单盲检测，敏感度为91％，特异性为60％。这四组小鼠骨髓细胞蛋白质Western blot结果进一步证实这组蛋白质在12月龄的第四代端粒酶敲除小鼠中异常高表达(figure1b)。测序结果发现这组蛋白质为：1，CRAMP-它在免疫反应早期被激活，保护机体免受细菌引起的感染侵扰。2，Chitinase 3 like protein 3(Chi3L3)-它属于chitinase家族，也在免疫反应早期被激活，已经有实验证实它与软骨细胞的衰老和关节炎相关。3，Elongation factor1α(EF-1α)，它与蛋白质的合成密切相关，也在人类纤维细胞的衰老中表达增加。4，Stathmin(OP18)，它与微小管的稳定结构，细胞活性和有丝分裂密切相关(25)。我们同时检测了实验小鼠(2月和12月龄的第四代端粒酶敲除小鼠以及端粒功能正常野生型小鼠，24月龄端粒功能正常野生型小鼠)的心脏、肝脏、肾脏、大脑、脾脏和肺脏等组织器官这组蛋白质信号的mRNA水平表达的高低(figure 1c)。发现CRAMP和Chi3L3在所有12月龄第四代端粒酶敲除小鼠检测器官mRNA水平均为高表达，而EF-1α和Stathmin则表现出器官特异性，仅在12月龄第四代端粒酶敲除小鼠心脏和肺脏中高表达。免疫荧光进一步证实这组蛋白质信号在12月龄第四代端粒酶敲除小鼠脏器中特异性高表达(figure 1d，Suppl．Table 1，Suppl．Fig．2)。在实验小鼠血清ELISA检测中我们也发现了这组蛋白质信号在12月龄第四代端粒酶敲除小鼠体内的高表达(Figure 1e)。CRAMP和Chi3L3在24月龄端粒功能正常野生型小鼠血清中还呈现中等量的高表达，但是并没有在12月龄的24月龄端粒功能正常野生型小鼠体内表达量高。这组蛋白质信号在老龄的生长激素基因缺陷小鼠体内并没有高表达(Suppl．Fig．3a-d)。这一结果说明这组蛋白质信号仅与端粒功能缺陷小鼠的衰老相关。由于人类还未发现与小鼠Chi3L3相似同源蛋白质，因此我们检测体内chitinase酶的活性来反应人类衰老和疾病过程中chitinase的变化。我们选用体外培养30代、40代、50代、60代和70代的人类纤维细胞进行体外实验，其中60代细胞是衰老前期细胞，70代的细胞已经是衰老的细胞了。mRNA水平蛋白质水平检测均发现60代和70代的细胞CRAMP、EF-1α、stathimin呈现显著高表达(figure3a，b)。对细胞培养液的ELISA检测均发现60代和70代的细胞CRAMP、EF-1α、stathimin和chitinase酶活性呈现显著高表达(figure3c-e)。这组实验结果揭示这组蛋白质信号亦与人类细胞端粒功能缺陷和DNA损伤相关。我们进一步进行人类衰老和疾病组的体外检测。发现这组蛋白质信号在健康老年人体内显著高表达与健康年轻人，在疾病老年人则表现为进一步的高表达(figure2a-d)。揭示这组蛋白质信号在人类血清中的表达与人类的衰老和疾病密切相关。多因素回归统计分析结果显示CRAMP和chitinase酶活性是其中作用最强的2个信号，将这二个蛋白质信号进行加权统计，可以更好的鉴别健康年轻组、健康老年组和疾病老年组(figure2e)。我们还和传统的衰老信号IL-6~(26-28)进行比较，发现我们的这组蛋白质信号比IL-6具有更高的敏感度和特异性(figure2f)。免疫荧光进一步证实这些蛋白质信号在老年器官中的高表达(Suppl．Fig．4,Suppl．Table2)。有很多人类疾病与端粒缩短相关，例如肝脏纤维化，是慢性肝脏疾病终末期功能衰竭的标志。纤维化患者肝脏组织的这组蛋白质信号mRNA表达显著高与未纤维化患者(figure3f)。血清ELISA结果办显示纤维化患者这组蛋白质信号表达显著高与未纤维化患者(figure4g-k)。免疫荧光进一步显示纤维化患者肝脏组织这组蛋白质信号的显著高表达(figure4a)．骨髓增生不良综合征MDS是另一种与端粒缩短导致造血干细胞和骨髓细胞功能衰竭相关的疾病~(29)。MDS患者血清中这组蛋白质信号表达明显高于健康对照组(figure4b-f)。 IgA肾病也是一种与端粒缩短相关的疾病。我们的实验发现这组蛋白质信号的表达与IgA肾病的进展相关，血清ELISA结果明确显示(figure5)，免疫荧光进一步证实结果(figure5d-g)。综上所述，本研究从衰老的端粒酶敲除小鼠体内发现了一组四个特异性的蛋白质。他们与非端粒缩短功能异常所致的衰老无关。本研究还发现这组蛋白质信号不仅仅是端粒缺陷小鼠的衰老信号，还是人类衰老和疾病的信号。说明了端粒的缩短在人类衰老和疾病的过程中的重要作用。这些由于端粒功能缺陷说所释放的蛋白质不仅仅是人类衰老的生物学信号，还预示着在衰老和慢性疾病过程中细胞和组织器官的损坏、功能的异常。已有的文献报道证明，端粒功能异常的间质细胞会影响临近的肿瘤细胞或者造血干细胞功能。CRAMP和Chitinase已经被证实与感染性疾病和免疫应答相关。免疫应答的启动在衰老所致的心血管疾病中起到重要作用。已有的文献报道也证实了端粒的缩短和功能缺陷能够激活免疫应答，这可以在一定程度上帮助我们理解人类衰老过程中的易感状态。
[Abstract]:Telomere shortening limits the division and proliferation of human cells, so it is common in the aging process of human tissues and organs. In the fourth generation of telomerase knockout mice, telomere dysfunction impairs tissue and organ function and shortens life span. However, the role of telomere dysfunction in human aging and disease remains controversial. We found a group of proteins: CRAMP, EF-1a, Chitinase 3L3, Stathmin. These proteins were abnormally high in many organs (such as liver, kidney, spleen, heart, lung, brain, etc.) and serum of aged telomerase-knockout mice, but no similar high expression was found in aged wild-type telomere-functioning mice. We also found that this group of human homologous proteins is highly expressed in serum and other organs (such as liver, spleen, etc.) during human aging, and is overexpressed in telomere shortening-related diseases (such as liver fibrosis, myelodysplastic syndrome, etc.). We will further study the relationship between this group of proteins and kidney diseases. To explore the mechanism of action.
Telomeres are located at the end of chromosomes to prevent cell senescence or apoptosis due to activation of DNA damage. Confirmed evidence has shown that telomere shortening leads to aging and chronic diseases in humans. Despite numerous genetic and clinical studies that have shown that telomere shortening causes damage to tissue and organ repair and shortens life span, there are still disputes about the mechanisms by which telomere dysfunction leads to shortened life span and disease in humans and in some primates. In aging telomerase knockout mice, defective telomere function is also associated with damage to stem cell function, resulting in impaired organ repair and shortened life span. Aging is apparently regulated by the cell cycle. Therefore, the role of telomere dysfunction in the aging process can be confirmed by experimental studies on aging cells. The research and discovery of physical signals is of epoch-making significance in the field of aging research.
So if we can identify a set of proteomic signals that can reflect the level of telomere function and DNA damage in cells at the early stages of cell senescence or disease, then The discovery and identification of this group of protein biological signals will undoubtedly be of far-reaching significance to the study of human aging and some aging-related diseases.
Our experiment found that the fourth generation of telomerase knockout mice (G4mTerc ~(-/-)) did not show in vivo accumulation of aging cells, but their aging phenotype is still closely related to telomere dysfunction. Quality provides a complete experimental system, and good clinical signals should be easily detected by body fluids. Therefore, our experimental design is to culture bone marrow cells of 2nd and 12th generation telomerase knockout mice and wild type mice with normal telomere function in vitro for a short period of time (4 hours), and collect their culture medium through proteomic experiments. Methods CE-TOF-MS was used to identify the secretion of different proteomes, and then a proteomic model (suppl.Fig.1a-e) was established to identify these four groups of mice.
Then we sequenced a group of four proteins by mass spectrometry. They were overexpressed in the fourth generation of 12-month-old telomerase knockout mice, significantly different from the other three groups of mice (n=5/group, figure 1a). We also designed a single-blind experiment to test the protein model. A total of 26 mice in the four groups were used for single-blind detection. The sensitivity and specificity were 91% and 60% respectively. Western blot results of bone marrow cell proteins from these four groups of mice further confirmed the abnormally high expression of these proteins (figure 1b) in the fourth generation of 12-month-old telomerase knockout mice.
Sequencing results showed that this group of proteins is: 1, CRAMP - it is activated in the early immune response to protect the body from infection caused by bacteria. 2, Chitinase 3 like protein 3 (Chi3L3) - it belongs to the chitinase family, is also activated in the early immune response, has been proved to be associated with chondrocyte aging and arthritis. 3, Elongat Iofactor1alpha (EF-1alpha), which is closely related to protein synthesis, is also increased in the aging of human fibroblasts. 4, Stathmin (OP18), which is closely related to the stable structure of microtubules, cell activity and mitosis (25).
We also examined the expression of protein signaling mRNA (figure 1c) in heart, liver, kidney, brain, spleen and lung of experimental mice (2nd and 12th generation telomerase knockout mice and wild type mice with normal telomere function, and wild type mice with normal telomere function at 24th month old). Both Chi3L3 and EF-1a were highly expressed in the detectors of all 12-month-old fourth-generation telomerase knockout mice, while EF-1a and Stathmin were organ-specific, and only overexpressed in the heart and lungs of 12-month-old fourth-generation telomerase knockout mice. High specific expression (figure 1d, Suppl. Table 1, Suppl. Fig. 2) was also found in the serum of 12-month-old fourth generation telomerase knockout mice (Figure 1e). CRAMP and Chi3L3 also appeared in the serum of 24-month-old wild type mice with normal telomere function. Moderately overexpressed, but not overexpressed in 12-month-old wild-type mice with normal telomere function at 24 months of age. This protein signal was not overexpressed in the aged mice with growth hormone deficiency (Suppl. Fig. 3a-d). This result suggests that this protein signal is only associated with the aging of mice with telomere deficiency. Relevant.
Since no homologous protein has been found to be similar to mouse Chi3L3, we tested the activity of chitinase in vivo to reflect the changes of chitinase during human aging and disease. The expression of CRAMP, EF-1a and stathimin was significantly higher in both 60 and 70 generations of cells (figure 3a, b). The activity of CRAMP, EF-1a, stathimin and chitinase was significantly higher in 60 and 70 generations of cells (figure 3c-e) by ELISA. These results reveal that this protein signal is also related to telomere dysfunction and DNA damage in human cells.
We further examined the human aging and disease group in vitro. We found that these proteins were highly expressed in healthy old people and healthy young people, but further high expressed in disease old people (figure 2a-d). Correlation. Multivariate regression analysis showed that CRAMP and chitinase activity were the two strongest signals. Weighted statistics of these two protein signals could better differentiate the healthy young group, the healthy old group and the sick old group (figure 2e). We also compared them with the traditional aging signal IL-6~ (26-28) and found that CRAMP and chitinase activity were the two strongest signals. Our set of protein signals are more sensitive and specific than IL-6 (figure 2f). Immunofluorescence further confirms the high expression of these protein signals in older organs (Suppl.Fig.4, Suppl.Table2).
There are many human diseases associated with telomere shortening, such as liver fibrosis, which is a marker of end-stage failure in chronic liver disease. This group of protein signaling mRNA expression in fibrotic liver tissue is significantly higher than that in non-fibrotic liver tissue (figure 3f). Serum ELISA results show that this group of protein signaling expression in fibrotic patients is significantly higher than that in non-fibrotic liver tissue. Immunofluorescence further demonstrated a significant overexpression of this group of protein signals (figure 4a) in fibrotic liver tissue.
Myelodysplastic syndrome (MDS) is another disease associated with telomere shortening leading to hematopoietic stem cell and bone marrow cell failure.
IgA nephropathy is also a disease associated with telomere shortening. Our experiments found that the expression of these proteins was associated with the progression of IgA nephropathy. The serum ELISA results clearly showed (figure 5), and immunofluorescence further confirmed the results (figure 5d-g).
In summary, this study found four specific proteins in aging telomerase knockout mice. They were not associated with aging caused by abnormal telomere shortening. This study also found that these proteins are not only aging signals in telomere-deficient mice, but also signals of aging and disease in humans. The important role of shortening in the process of human aging and disease.
These proteins released by telomere dysfunction theory are not only biological signals of aging in humans, but also indicate damage to cells and tissues and organs during aging and chronic diseases, and dysfunction. Previous reports have shown that telomere dysfunctional stromal cells affect adjacent tumor cells or hematopoietic stem cells. Cellular function. CRAMP and Chitinase have been shown to be associated with infectious diseases and immune responses. Initiation of the immune response plays an important role in cardiovascular diseases caused by aging. The susceptible state of aging.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

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