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Quantitative coherent anti-Stokes Raman scattering imaging of lipid distribution in coexisting domains 下载免费PDF全文
We demonstrate quantitative vibrational imaging of specific lipid molecules in single bilayers using laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy with a lateral resolution of 0.25 mum. A lipid is spectrally separated from other molecules by using deuterated acyl chains that provide a large CARS signal from the symmetric CD(2) stretch vibration around 2100 cm(-1). Our temperature control experiments show that d62-DPPC has similar bilayer phase segregation property as DPPC when mixing with DOPC. By using epi-detection and optimizing excitation and detection conditions, we are able to generate a clear vibrational contrast from d62-DPPC of 10% molar fraction in a single bilayer of DPPC/d62-DPPC mixture. We have developed and experimentally verified an image analysis model that can derive the relative molecular concentration from the difference of the two CARS intensities measured at the peak and dip frequencies of a CARS band. With the above strategies, we have measured the molar density of d62-DPPC in the coexisting domains inside the DOPC/d62-DPPC (1:1) supported bilayers incorporated with 0-40% cholesterol. The observed interesting changes of phospholipid organization upon addition of cholesterol to the bilayer are discussed. 相似文献
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Jiabin Zhu Bonggi Lee Kimberly K. Buhman Ji-Xin Cheng 《Journal of lipid research》2009,50(6):1080-1089
The absorptive cells of the small intestine, enterocytes, are not generally thought of as a cell type that stores triacylglycerols (TGs) in cytoplasmic lipid droplets (LDs). We revisit TG metabolism in enterocytes by ex vivo and in vivo coherent anti-Stokes Raman scattering (CARS) imaging of small intestine of mice during dietary fat absorption (DFA). We directly visualized the presence of LDs in enterocytes. We determined lipid amount and quantified LD number and size as a function of intestinal location and time post-lipid challenge via gavage feeding. The LDs were confirmed to be primarily TG by biochemical analysis. Combined CARS and fluorescence imaging indicated that the large LDs were located in the cytoplasm, associated with the tail-interacting protein of 47 kDa. Furthermore, in vivo CARS imaging showed real-time variation in the amount of TG stored in LDs through the process of DFA. Our results highlight a dynamic, cytoplasmic TG pool in enterocytes that may play previously unexpected roles in processes, such as regulating postprandial blood TG concentrations. 相似文献
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Yanping Li Binglin Shen Gengjin Zou Shiqi Wang Junle Qu Rui Hu Liwei Liu 《Journal of biophotonics》2021,14(8):e202100080
Stimulated Raman scattering (SRS) microscopy is a nonlinear optical imaging method for visualizing chemical content based on molecular vibrational bonds. However, the imaging speed and sensitivity are currently limited by the noise of the light beam probing the Raman process. In this paper, we present a fast non-average denoising and high-precision Raman shift extraction method, based on a self-reinforcing signal-to-noise ratio (SNR) enhancement algorithm, for SRS spectroscopy and microscopy. We compare the results of this method with the filtering methods and the reported experimental methods to demonstrate its high efficiency and high precision in spectral denoising, Raman peak extraction and image quality improvement. We demonstrate a maximum SNR enhancement of 10.3 dB in fixed tissue imaging and 11.9 dB in vivo imaging. This method reduces the cost and complexity of the SRS system and allows for high-quality SRS imaging without use of special laser, complicated system design and Raman tags. 相似文献
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目的: 探讨大鼠肝纤维化病理过程中肝组织及在体肝星状细胞 (HSC)的含SH2结构域的蛋白酪氨酸磷酸酶1 (SHP1)表达变化与在体HSC活化及增殖的关系。方法: 随机将50只健康雄性SD大鼠分为对照组(10只)、模型组(40只),采用腹腔注射四氯化碳法建立大鼠肝纤维化模型,Masson三色染色及HE染色检测大鼠肝脏组织的病理组织学变化,SHP1与α-平滑肌肌动蛋白 (α-SMA)免疫荧光双标记检测大鼠肝组织中活化HSC的SHP1表达,免疫组织化学染色检测大鼠肝组织的α-SMA及SHP1表达,并分别对大鼠肝组织的SHP1表达及大鼠肝组织中活化HSC的SHP1表达与大鼠肝组织的α-SMA表达进行Pearson’s相关性分析。结果: 大鼠肝纤维化模型成功构建,随着造模时间延长,大鼠肝纤维化逐渐加重。与对照组大鼠肝组织的SHP1阳性表达平均光密度值 (MOD) (0.08±0.01)比较,造模不同时间(2周、4周、6周、8周)大鼠纤维化肝组织的SHP1阳性表达MOD (0.11±0.01、0.14±0.01、0.16±0.01、0.19±0.01)显著增加(P<0.05),并逐渐升高(P<0.05)。与对照组大鼠肝组织的α-SMA阳性表达MOD (0.04±0.01)比较,造模不同时间(2周、4周、6周、8周)大鼠纤维化肝组织的α-SMA阳性表达MOD (0.06±0.01、 0.09±0.01、0.12±0.01、0.16±0.02)明显增加(P<0.05),并逐渐升高(P<0.05),即在体HSC的活化及增殖逐渐加快(α-SMA是HSC的活化标志)。SHP1与α-SMA免疫荧光双标记检测显示,造模2周、4周、6周、8周大鼠纤维化肝组织中表达SHP1的活化HSC占总的活化HSC的百分比(26.49%±3.44%、37.14%±4.57%、44.90%±2.94%、58.09%±5.33%)逐渐升高(P<0.05)。上述大鼠纤维化肝组织的SHP1表达及大鼠纤维化肝组织中表达SHP1的活化HSC占总的活化HSC的百分比均与大鼠纤维化肝组织的α-SMA表达呈显著正相关(r值为0.926, 0.984,P<0.05)。结论: 在大鼠肝纤维病理过程中,肝组织及在体HSC 的SHP1表达与在体HSC的活化及增殖呈显著正相关。 相似文献
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Katia Bourd-Boittin Laetitia Basset Dominique Bonnier Annie L'Helgoualc'h Michel Samson Nathalie Théret 《Journal of cellular and molecular medicine》2009,13(8A):1526-1535
Chemokines are the inflammatory mediators that modulate liver fibrosis, a common feature of chronic inflammatory liver diseases. CX3CL1/fractalkine is a membrane-associated chemokine that requires step processing for chemotactic activity and has been recently implicated in liver disease. Here, we investigated the potential shedding activities involved in the release of the soluble chemotactic peptides from CX3CL1 in the injured liver. We showed an increased expression of the sheddases ADAM10 and ADAM17 in patients with chronic liver diseases that was associated with the severity of liver fibrosis. We demonstrated that hepatic stellate cells (HSC) were an important source of ADAM10 and ADAM17 and that treatment with the inflammatory cytokine inter-feron-γ induced the expression of CX3CL1 and release of soluble peptides. This release was inhibited by the metalloproteinase inhibitor batimastat; however, ADAM10/ADAM17 inhibitor GW280264X only partially affected shedding activity. By using selective tissue metalloprotease inhibitors and overexpression analyses, we showed that CX3CL1 was mainly processed by matrix metalloproteinase (MMP)-2, a metalloprotease highly expressed by HSC. We further demonstrated that the CX3CL1 soluble peptides released from stimulated HSC induced the activation of the CX3CR1-dependent signalling pathway and promoted chemoattraction of monocytes in vitro . We conclude that ADAM10, ADAM17 and MMP-2 synthesized by activated HSC mediate CX3CL1 shedding and release of chemotactic peptides, thereby facilitating recruitment of inflammatory cells and paracrine stimulation of HSC in chronic liver diseases. 相似文献
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Giorgio Nanni Claudio Canepa Federica Majorani Anna Casu 《Cell biochemistry and function》1998,16(3):203-209
The liver sinusoids, that are considered as a functional unit, harbour four types of sinusoidal cells (Ito, Kupffer, endothelial and pit cells). Dolichol content has been determined in many tissues and subcellular compartments, alteration has been reported in many types of liver injury, but until now no data are available on its content in every type of sinusoidal non-parenchymal liver cells. Dolichol and retinol metabolism might intersect in their traffic in biological membranes. Intercellular as well as intracellular exchange of retinoids is an essential element of important processes occurring in liver cells. It has been suggested that the role of dolichol, besides being a carrier of oligosaccharides in the biosynthesis of N-linked glycoproteins, may be to modify membrane fluidity and permeability, and facilitate fusion of membranes. Dolichol in the membrane is intercalated between the two halves of the phospholipid bilayer, but its exact disposition is not known and the movement and distribution of retinoid in membranes may vary with the geometry of the membranes. Therefore the aim of this study is to obtain a global understanding of the sinusoidal system regarding dolichol and retinol content in each type of isolated rat liver sinusoidal cell, in normal conditions and after vitamin A administration. The information that can be drawn from the present results is that with normal vitamin A status of the animal, the dolichol content is almost uniform in all liver cells. After vitamin A supplementation, a great increase of dolichol, together with the known increase of retinol, can be measured only in a subpopulation of the Ito cells, the Ito-1 subfraction. Therefore in the cells that are present in the hepatic sinusoid, different pools of dolichol may have separate functions. Because retinol traffic among cells, membranes and plasma still remains to be fully understood, roles of dolichol in the exchange of vitamin A among sinusoidal liver cells are discussed. © 1998 John Wiley & Sons, Ltd. 相似文献
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Yu Peng Zedong Li Sheng Chen Jun Zhou 《Journal of cellular and molecular medicine》2021,25(21):10049-10060
Liver fibrogenesis is a dynamic cellular and tissue process which has the potential to progress into cirrhosis of even liver cancer and liver failure. The activation of hepatic stellate cells (HSCs) is the central event underlying liver fibrosis. Besides, hepatic macrophages have been proposed as potential targets in combatting fibrosis. As for the relationship between HSCs and hepatic macrophages in liver fibrosis, it is generally considered that macrophages promoted liver fibrosis via activating HSCs. However, whether activated HSCs could in turn affect macrophage polarization has rarely been studied. In this study, mRNAs with significant differences were explored using exosomal RNA-sequencing of activated Lx-2 cells and normal RNA-sequencing of DHFR loss-of-function Lx-2 cell models. Cell functional experiments in both Lx-2 cells and macrophages animal model experiments were performed. The results basically confirmed exosomes secreted from activated HSCs could promote M1 polarization of macrophages further. Exosome harbouring DHFR played an important role in this process. DHFR silence in HSCs could decrease Lx-2 activation and M1 polarization of M0 macrophages and then alleviate the development of liver fibrosis both in vitro and vivo. Our work brought a new insight that exosomal DHFR derived from HSCs had a crucial role in crosstalk between HSCs activation and macrophage polarization, which may be a potential therapeutic target in liver fibrosis. 相似文献
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Hang Yu Wang Can Li Wei Hua Liu Feng Mei Deng Yan Ma Li Na Guo De Hua Kong Kang An Hu Qin Liu Jiang Wu Jing Sun Yi Lun Liu 《Journal of cellular physiology》2020,235(3):2722-2737
Liver fibrosis (LF) is the result of a vicious cycle between inflammation-induced chronic hepatocyte injury and persistent activation of hepatic stellate cells (HSCs). Mesenchymal stem cell (MSC)-based therapy may represent a potential remedy for treatment of LF. However, the fate of transplanted MSCs in LF remains largely unknown. In the present study, the fate and antifibrotic effect of MSCs were explored in a LF model induced by CCl4 in mouse. Additionally, MSCs were stimulated in vitro with LF-associated factors, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and transforming growth factor-β1 (TGF-β1), to mimic the LF microenvironment. We unveiled that MSCs exhibited autophagy in response to the LF microenvironment through Becn1 upregulation both in vivo and in vitro. However, autophagy suppression induced by Becn1 knockdown in MSCs resulted in enhanced antifibrotic effects on LF. The improved antifibrotic potential of MSCs may be attributable to their inhibitory effects on T lymphocyte infiltration, HSCs proliferation, as well as production of TNF-α, IFN-γ, and TGF-β1, which may be partially mediated by elevated paracrine secretion of PTGS2/PGE2. Thus, autophagy manipulation in MSCs may be a novel strategy to promote their antifibrotic efficacy. 相似文献
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Assessing the efficacy of coherent anti‐Stokes Raman scattering microscopy for the detection of infiltrating glioblastoma in fresh brain samples 下载免费PDF全文
Roberta Galli Ortrud Uckermann Achim Temme Elke Leipnitz Matthias Meinhardt Edmund Koch Gabriele Schackert Gerald Steiner Matthias Kirsch 《Journal of biophotonics》2017,10(3):404-414
Coherent anti‐Stokes Raman scattering (CARS) microscopy is an emerging technique for identification of brain tumors. However, tumor identification by CARS microscopy on bulk samples and in vivo has been so far verified retrospectively on histological sections, which only provide a gross reference for the interpretation of CARS images without matching at cellular level. Therefore, fluorescent labels were exploited for direct assessment of the interpretation of CARS images of solid and infiltrative tumors. Glioblastoma cells expressing green fluorescent protein (GFP) were used for induction of tumors in mice (n = 7). The neoplastic nature of cells imaged by CARS microscopy was unequivocally verified by addressing two‐photon fluorescence of GFP on fresh brain slices and in vivo. In fresh unfixed biopsies of human glioblastoma (n = 10), the fluorescence of 5‐aminolevulinic acid‐induced protoporphyrin IX was used for identification of tumorous tissue. Distinctive morphological features of glioblastoma cells, i.e. larger nuclei, evident nuclear membrane and nucleolus, were identified in the CARS images of both mouse and human brain tumors. This approach demonstrates that the chemical contrast provided by CARS allows the localization of infiltrating tumor cells in fresh tissue and that the cell morphology in CARS images is useful for tumor recognition.
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CXCL6‐EGFR‐induced Kupffer cells secrete TGF‐β1 promoting hepatic stellate cell activation via the SMAD2/BRD4/C‐MYC/EZH2 pathway in liver fibrosis 下载免费PDF全文
Xiaobo Cai Zhenghong Li Qidi Zhang Yin Qu Mingyi Xu Xinjian Wan Lungen Lu 《Journal of cellular and molecular medicine》2018,22(10):5050-5061
Liver fibrosis is the excessive accumulation of extracellular matrix proteins in response to the inflammatory response that accompanies tissue injury, which at an advanced stage can lead to cirrhosis and even liver failure. This study investigated the role of the CXC chemokine CXCL6 (GCP‐2) in liver fibrosis. The expression of CXCL6 was found to be elevated in the serum and liver tissue of high stage liver fibrosis patients. Furthermore, treatment with CXCL6 (100 ng/mL) stimulated the phosphorylation of EGFR and the expression of TGF‐β in cultured Kupffer cells (KCs). Although treatment with CXCL6 directly did not activate the hepatic stellate cell (HSC) line, HSC‐T6, HSCs cultured with media taken from KCs treated with CXCL6 or TGF‐β showed increased expression of α‐SMA, a marker of HSC activation. CXCL6 was shown to function via the SMAD2/BRD4/C‐MYC/EZH2 pathway by enhancing the SMAD3‐BRD4 interaction and promoting direct binding of BRD4 to the C‐MYC promoter and CMY‐C to the EZH2 promoter, thereby inducing profibrogenic gene expression in HSCs, leading to activation and transdifferentiation into fibrogenic myofibroblasts. These findings were confirmed in a mouse model of CCl4‐induced chronic liver injury and fibrosis in which the levels of CXCL6 and TGF‐β in serum and the expression of α‐SMA, SMAD3, BRD4, C‐MYC, and EZH2 in liver tissue were increased. Taken together, our results reveal that CXCL6 plays an important role in liver fibrosis through stimulating the release of TGF‐β by KCs and thereby activating HSCs. 相似文献
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Ho-Joon Lee Seon Ju Mun Cho-Rok Jung Hyun-Mi Kang Jae-Eun Kwon Jae-Sung Ryu Hyo-Suk Ahn Ok-Seon Kwon Jiwon Ahn Kyung-Sik Moon Myung Jin Son Kyung-Sook Chung 《Biotechnology and bioengineering》2023,120(5):1241-1253
Hepatic stellate cells (HSCs) play an important role in liver fibrosis; however, owing to the heterogeneity and limited supply of primary HSCs, the development of in vitro liver fibrosis models has been impeded. In this study, we established and characterized a novel human HSC line (LSC-1), and applied it to various types of three-dimensional (3D) co-culture systems with differentiated HepaRG cells. Furthermore, we compared LSC-1 with a commercially available HSC line on conventional monolayer culture. LSC-1 exhibited an overall upregulation of the expression of fibrogenic genes along with increased levels of matrix and adhesion proteins, suggesting a myofibroblast-like or transdifferentiated state. However, activated states reverted to a quiescent-like phenotype when cultured in different 3D culture formats with a relatively soft microenvironment. Additionally, LSC-1 exerted an overall positive effect on co-cultured differentiated HepaRG, which significantly increased hepatic functionality upon long-term cultivation compared with that achieved with other HSC line. In 3D spheroid culture, LSC-1 exhibited enhanced responsiveness to transforming growth factor beta 1 exposure that is caused by a different matrix-related protein expression mechanism. Therefore, the LSC-1 line developed in this study provides a reliable candidate model that can be used to address unmet needs, such as development of antifibrotic therapies. 相似文献
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Hepatic stellate cells (HSCs) in the perisinusoidal space are surrounded by hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, and other resident immune cells. In the normal liver, HSCs communicate with these cells to maintain normal liver functions. However, after chronic liver injury, injured hepatocytes release several proinflammatory mediators, reactive oxygen species, and damage-associated molecular patterns into the perisinusoidal space. Consequently, such alteration activates quiescent HSCs to acquire a myofibroblast-like phenotype and express high amounts of transforming growth factor-β1, angiopoietins, vascular endothelial growth factors, interleukins 6 and 8, fibril forming collagens, laminin, and E-cadherin. These phenotypic and functional transdifferentiation lead to hepatic fibrosis with a typical abnormal extracellular matrix synthesis and disorganization of the perisinusoidal space of the injured liver. Those changes provide a favorable environment that regulates tumor cell proliferation, migration, adhesion, and survival in the perisinusoidal space. Such tumor cells by releasing transforming growth factor-β1 and other cytokines, will, in turn, activate and deeply interact with HSCs via a bidirectional loop. Furthermore, hepatocellular carcinoma-derived mediators convert HSCs and macrophages into protumorigenic cell populations. Thus, the perisinusoidal space serves as a critical hub for activating HSCs and their interactions with other cell types, which cause a variety of liver diseases such as hepatic inflammation, fibrosis, cirrhosis, and their complications, such as portal hypertension and hepatocellular carcinoma. Therefore, targeting the crosstalk between activated HSCs and tumor cells/immune cells in the tumor microenvironment may also support a promising therapeutic strategy. 相似文献
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Pei Wang Yu Jie Lin Yao Yi-meng Sun Da-peng Jiang Shi-qi Zhang Xiao-yan Wang Yu Fan 《Cell biochemistry and function》2024,42(2):e3969
The research of liver metastasis is a developing field. The ability of tumor cells to invade the liver depends on the complicated interactions between metastatic cells and local subpopulations in the liver (including Kupffer cells, hepatic stellate cells, liver sinusoidal endothelial cells, and immune-related cells). These interactions are mainly mediated by intercellular adhesion and the release of cytokines. Cell populations in the liver microenvironment can play a dual role in the progression of liver metastasis through different mechanisms. At the same time, we can see the participation of liver parenchymal cells and nonparenchymal cells in the process of liver metastasis of different tumors. Therefore, the purpose of this article is to summarize the relationship between cellular components of liver microenvironment and metastasis and emphasize the importance of different cells in the occurrence or potential regression of liver metastasis. 相似文献