RCEES OpenIR  > 环境化学与生态毒理学国家重点实验室
基于人源间充质干细胞的代表性全氟化合物毒性评价
Alternative TitleEffects of Perfluorinated Compounds on Proliferation and Differentiation of Human Mesenchymal Stem Cells
刘抒羽
Subtype硕士
Thesis AdvisorFrancesco Faiola
2018-06
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学硕士
Degree Discipline环境科学
Keyword全氟化合物,间充质干细胞,干细胞毒理学 Perfluorinated Compounds, Mesenchymal Stem Cell, Stem Cell Toxicology
Abstract

      全氟化合物是一类人工合成的全氟取代有机物,具有不可燃性、稳定性和不可挥发性,在工业生产和日常商品中有广泛应用。同时,全氟化合物因其持久性和稳定性而在环境、野生动物和人体内积累,尤其是血液、乳汁、羊水等人体样品中都检测出了全氟化合物。因此全氟化合物的人体健康效应受到了流行病学和毒理学研究的广泛关注。

      全氟辛磺酸和全氟辛酸是典型的全氟化合物,是蓄积于人体的最主要的全氟化合物。流行病学研究表明,这两种化合物的人体半衰期长达数年,能够导致脂肪代谢紊乱和肥胖症的发生,同时对骨骼健康也有所影响。而一些毒理学研究也证实全氟化合物通过激活成脂分化重要转录因子PPAR,从而在体外及体内水平上促进脂肪细胞的形成。此外一些全氟化合物对骨骼发育有所干扰。

       随着人们认识到全氟辛磺酸和全氟辛酸的毒性,它们的短链同系物作为替代物,大量生产并广泛使用。而流行病学研究发现短链同系物在人体中的水平有显著上升,毒理学实验则发现短链同系物仍可能导致明显的毒性效应。

      由此,我们以人源间充质干细胞作为实验模型,探究全氟化合物的健康效应。间充质干细胞是广泛存在于人体器官和组织中的成体干细胞,蓄积在人体内的全氟化合物可能对间充质干细胞产生潜在的毒性;间充质干细胞在体内进行成脂分化时,决定性因素之一是转录因子PPAR的激活;由于前人已经证实,全氟化合物能够对该过程产生干扰,因此间充质干细胞成脂分化模型适用于研究全氟化合物的促成脂作用;此外间充质干细胞成骨分化模型则可以用于探讨全氟化合物是否干扰骨骼发育和骨细胞健康。间充质干细胞在体外可以维持多代,其分化过程也相对比较缓慢,因此可以用于污染物长期健康效应的研究。

      本论文研究内容主要包含以下四个部分:

      1. 全氟化合物急性毒性检测。这部分实验关注高剂量的急性细胞毒性,以获得基础的急性数据。高剂量全氟化合物的暴露导致细胞活力下降,后续实验表明这有可能是因为污染物暴露促使细胞产生过量的活性氧,细胞内游离钙离子水平持续升高,线粒体膜电位下降,细胞内抗氧化能力逐渐耗竭,从而细胞死亡。

      2. 全氟化合物低剂量亚慢性毒性检测。这部分实验关注人体内全氟化合物的剂量对细胞的潜在影响。低剂量全氟化合物的暴露对细胞活力没有显著干扰,但后续实验发现非细胞毒性剂量的全氟化合物下调间充质干细胞必要表面标志分子CD90的表达,从而损害间充质干细胞的自我更新能力。

      3. 全氟化合物对间充质干细胞成脂分化的影响。这部分实验关注全氟化合物暴露是否促进间充质干细胞的成脂分化。结果显示,PFOS、PFOA和其替代物均能够在不同程度上促进脂肪细胞的形成,而PPAR的激活可能是全氟化合物促进成脂发生的分子机制。

       4. 全氟化合物对间充质干细胞成骨分化的影响。这部分实验关注全氟化合物暴露是否促进间充质干细胞的成骨分化。结果显示,PFOA对成骨分化过程产生了一定干扰而其他全氟化合物作用不明显。综上所述,本研究为研究全氟化合物人体健康效应提供了重要的实验依据,证实了低剂量下全氟化合物的长期暴露能够减弱间充质干细胞细胞的自我更新能力,全氟化合物促进间充质干细胞成脂分化,而PFOA有可能干扰正常的骨骼发育。

Other Abstract

         Perfluorinated compounds (PFCs) are artificial compounds consist of a fully fluorinated carbon backbone, which are inflammable, non-volatile and stable. PFCs have widespread utilization in industry and commercialized products, including firefighting foam, lubricants, coatings and surfactants. Yet, PFCs are persistent and less biodegradable, and therefore measured in environmental samples and in tissue, blood of wildlife and in tissue, blood, breast milk and amniotic water of humans. Therefore, PFCs in the natural environments, wildlife and humans raised concerns of adverse environmental and health impacts.

         Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are pro-dominant PFCs. Epidemiological studies showed that half-lives of the compounds tended to be up to years, and they were associated with the occurrence of the dysfunction of lipid metabolism and adiposity, and potentially bone health. Toxicology studies, on the other hand, indicated that PFCs were able to activated key transcription factor of adipogenesis, PPARr, and thus promoted the process. Besides, it was shown that PFCs might perturb bone development.

        Due to the realization of the toxicities of PFOS and PFOA, shorter chain PFCs were introduced to replace PFOS and PFOA. However, epidemiological studies found out the potential accumulation of the shorter chain PFCs in human; meanwhile, toxicological studies demonstrated that the alternatives rendered toxic effects.

       Therefore, we here employed human mesenchymal stem cells (hMSCs) as the model to study health impact of PFCs. Human MSCs exist in multiple organs and tissues in human body, which are prone to the PFCS accumulated in human body. During adipogenic differentiation of hMSCs in vivo, activation of PPARγis essential. Therefore, it is suitable to use adipogenic differentiation of hMSCs to study the effect of PFCs, same as the osteogenic differentiation of hMSCs. Since hMSCs can be maintained for passages, and their differentiation processes take time, chronic effect of PFCs can be studied.

      To sum up, this research consists of four parts:

       1. Acute cytotoxicity test of the PFCs. This part focused on acute toxicity of various doses PFCs. Results showed that the exposure caused significant decrease in cell viability, which involved with the generation of reactive oxygen species, increased intracellular calcium ion flux, decreased mitochondrial membrane potential, depletion of antioxidation capacity.

         2. Sub-chronic toxicity test of the PFCs. This part focused on the toxicity of non-lethal doses PFCs that exit in real environmental or human body samples. Results showed that the exposure did not affect cell viability but impaired the proper expression of CD90, which is a surface antigen of hMSCs for the maintenance of undifferentiated status.

       3. Effect of PFCs on adipogenic differentiation. This part focused on whether PFC exposure promoted adipogenesis. Results showed that PFOS, PFOA and their alternatives were capable to stimulate adipogenesis, presumably due to their interaction with PPAR.

         4. Effect of PFCs on osteogenic differentiation. This part focused on whether PFC exposure disturbed osteogenesis. Results showed that PFOA had significant effect yet further exploration is required to give better explanation.In summary, this research provided with acute cytotoxicity data of PFCs, proved that chronic exposure to PFCs compromised self-renewal of hMSCs, displayed that adipogenesis was enhanced and PFOA might interfered bone development.

Pages94
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/41492
Collection环境化学与生态毒理学国家重点实验室
Recommended Citation
GB/T 7714
刘抒羽. 基于人源间充质干细胞的代表性全氟化合物毒性评价[D]. 北京. 中国科学院生态环境研究中心,2018.
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