纳米二氧化硅诱导巨噬细胞焦亡对肺成纤维细胞的影响

目的 探讨纳米二氧化硅(SiNPs)诱导巨噬细胞焦亡对原代肺成纤维细胞基质金属蛋白酶及成纤维细胞向肌成纤维细胞转分化的影响。方法 构建SiNPs致巨噬细胞焦亡模型后,采用纳米高光谱定位巨噬细胞中SiNPs、光学显微镜观察巨噬细胞SiNPs暴露后形态、Western blot检测焦亡相关蛋白表达。通过胰酶和Ⅳ型胶原酶消化法提取小鼠原代肺成纤维细胞;以光学显微镜和免疫荧光技术表征并鉴定原代肺成纤维细胞;体外构建SiNPs诱导巨噬细胞焦亡与原代肺成纤维细胞共培养模型。以正常巨噬细胞上清干预原代肺成纤维细胞作为MF组,焦亡巨噬细胞上清干预原代肺成纤维细胞作为PF组,采用qRT-PCR、Western blot技术分别检测原代肺成纤维细胞中MMP2、MMP9的mRNA水平和蛋白表达,免疫荧光、免疫细胞化学染色检测肌成纤维细胞标志物α-SMA蛋白表达。结果 纳米高光谱、形态学观察和Western blot检测结果显示, SiNPs 成功诱导J774A.1 巨噬细胞发生焦亡,光学显微镜观察发现,与胰酶消化法相比,Ⅳ型胶原酶法提取小鼠原代肺成纤维细胞的纯度更高;免疫荧光结果提示Ⅳ型胶原酶法获得的成纤维细胞波形蛋白呈现典型的微丝结构,细胞骨架结构完整。qRT-PCR 结果显示,PF组MMP2 和MMP9 mRNA水平较MF组增加(P<0.05), Western blot结果显示,PF组MMP2和MMP9蛋白表达水平较MF组升高(P<0.05), 免疫荧光结果显示,与MF组相比,PF组α-SMA红色荧光强度增加,免疫细胞化学染色为强阳性。结论 SiNPs诱导巨噬细胞焦亡能够促进原代肺成纤维细胞MMP2和MMP9表达水平上调,并促进成纤维细胞向肌成纤维细胞转分化。     

氯沙坦对糖尿病大鼠肾小管上皮细胞转分化的影响

目的观察糖尿病大鼠肾小管上皮细胞转分化及氯沙坦对其的影响,探讨ARB糖尿病肾病保护作用中与TEMT有关的新机制.方法 50只雄性Wistar大鼠随机分为对照组(n=10只)和糖尿病模型组(n=40只);模型组大鼠给予腹腔内一次注射链脲佐菌素(STZ)60 mg/kg诱导糖尿病模型.模型成功后再随机分为糖尿病组和氯沙坦组.氯沙坦组给予氯沙坦20 mg/kg*d-1一次灌胃.于干预后第16周测定尿蛋白和内生肌酐清除率;光镜观察肾小管间质损害及纤维化;免疫组织化学法检测肾小管上皮细胞TGF-β1、Vimentin和α-SMA表达,并对结果进行半定量分析.结果糖尿病组大鼠24 h尿蛋白排泄增加(P＜0.01),肾小管上皮细胞α-SMA、Vimentin和TGF-β1阳性表达面积分别为0.3690±0.0587,0.1546±0.0141和0.3716±0.0657; 氯沙坦组大鼠24 h尿蛋白排泄较糖尿病组减少,肾小管间质损伤和间质纤维化程度减轻;其肾小管上皮细胞α-SMA、Vimentin及TGF-β1表达较糖尿病组显著下调(P＜0.01).结论氯沙坦能显著下调糖尿病大鼠肾小管上皮细胞α-SMA、Vimentin和TGF-β1表达,阻抑肾小管上皮细胞向肌成纤维细胞转分化.     

TRANSDIFFERENTIATION OF PROSTATE CANCER CELLS TO A NEUROENDOCRINE CELL PHENOTYPE IN VITRO AND IN VIVO

PURPOSE: To better understand the source of neuroendocrine cells associated with human prostate cancer progression, we studied the ability of a cultured prostate cancer cell line, LNCaP, to transdifferentiate into neuroendocrine-like cells in vitro and in vivo. MATERIALS AND METHODS: Cyclic AMP concentrations were measured in extracts of LNCaP cells cultured in the presence of normal or hormone-deficient medium (containing charcoal-stripped serum) with the use of an immunoassay. Quantitative RT-PCR procedures were used to determine whether hormone depletion affects TGF-beta2 mRNA expression. Western blotting procedures (for neuron specific enolase [NSE]) were used to determine whether TGF-beta2 supplementation or antibody neutralization might affect the ability of cultured LNCaP cells to transdifferentiate to neuroendocrine-like cells. Finally, tumors formed from LNCaP cells xenografted into male nude mice were evaluated for the presence of neuroendocrine cells (prior and subsequent to castration of the host mouse) using an immunohistochemical stain for chromogranin A. RESULTS: LNCaP cells cultured in a hormone-deficient medium have a mean 9-fold increase in cyclic AMP (p = 0.02) and a significant decline in the expression of TGF-beta2 mRNA when compared with cells grown in normal medium. Supplementation or depletion of TGF-beta2 did not affect the neuroendocrine conversion of LNCaP cells as assessed by NSE expression patterns. LNCaP tumors growing in castrated male nude mice were found to have significantly increased numbers of chromogranin A positive neuroendocrine cells (46/high powered field) when compared with tumors growing in intact male mice (3/high powered field) (p = 0.0038). CONCLUSIONS: Exposure of LNCaP cells to a hormone deficient medium drastically increased cyclic AMP production and this may identify the biochemical pathway through which hormone depletion induces a neuroendocrine conversion of prostate cancer cells. Hormone depletion also reduced TGF-beta2 mRNA expression and this finding was consistent with our inability to demonstrate any effect of TGF-beta2 on neuroendocrine conversion in vitro. Finally, our demonstration of increased neuroendocrine cells found in LNCaP tumors growing in castrated immunodeficient mice suggests that the neuroendocrine cells associated with advanced human prostate tumors in vivo, arise from prostate cancer cells through the transdifferentiation process.     

马兜铃酸I诱导人肾小管上皮细胞转分化的作用及机制

目的：探讨马兜铃酸Ｉ（Ａｒｉｓｔｏｌｏｃｈｉｓ ａｃｉｄ Ｉ,ＡＡＩ）在人类肾小管上皮细胞（ＨＫＣ）转分化中的可能作用,了解ＡＡＩ引起肾小管间质损害的机制。方法：将体外培养ＨＫＣ细胞分为无血清对照组（Ｃ组）和ＡＡＩ实验组两组,波形蛋白和α－平滑肌肌动蛋白（α－ｓｍｏｏｔｈ ｍｕｓｃｌｅ ａｃｔｉｎ,α－ＳＭＡ）表达的变化;应用流式细胞技术检测表达α－ＳＭＡ阳性（＋）的ＨＫＣ细胞百分率;采用免疫酶标     

假上皮瘤样增生病变中上皮细胞向间质细胞转化的不稳定性

目的探讨上皮细胞向间质细胞转分化(EMT)与外伤继发假上皮瘤样增生(PEH)损害之间的关系。方法收集11例创伤后继发PEH病变的标本及其边缘正常皮肤(PEH-N),采用组织病理学和间接免疫荧光双标记方法观察Ⅰ／Ⅲ型胶原(CoⅡ／Ⅲ)、α-平滑肌肌动蛋白(α-SMA)、波形蛋白(Vim)、结蛋白(Des)、转化生长因子-β1(TGF-β1)及其受体(TGFRI)、广谱角蛋白(CKp)和Ⅳ型胶原(ColⅣ)在PEH异常分化的上皮细胞中的表达水平和分布特征。结果组织学观察发现,与PEH—N组相比,PEH呈现鳞状上皮不典型增生．上皮基底部细胞顶一基极性紊乱、结构崩溃,细胞向间质迁移,在该部位的表皮基底细胞和部分附件上皮细胞中可检测到CoⅡ／Ⅲ、α-SMA、Vim和Des等间质细胞物,但这些上皮细胞CKp、ColⅣ、TGF-β1和TGFRI表达水平明显减弱甚至消失,并且在深层间质内发现大量游离的CKp阳性上皮细胞。结论PEH病变存在不稳定性EMT现象,上皮细胞去分化后再分化以及TGF-β1信号通路作用微弱可能赋予了PEH病变肉芽组织化而非形成增生性瘢痕的病理特征。     

Dietary ω-3 polyunsaturated fatty acids are protective for myopia

Myopia is a leading cause of visual impairment and blindness worldwide. However, a safe and accessible approach for myopia control and prevention is currently unavailable. Here, we investigated the therapeutic effect of dietary supplements of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on myopia progression in animal models and on decreases in choroidal blood perfusion (ChBP) caused by near work, a risk factor for myopia in young adults. We demonstrated that daily gavage of ω-3 PUFAs (300 mg docosahexaenoic acid [DHA] plus 60 mg eicosapentaenoic acid [EPA]) significantly attenuated the development of form deprivation myopia in guinea pigs and mice, as well as of lens-induced myopia in guinea pigs. Peribulbar injections of DHA also inhibited myopia progression in form-deprived guinea pigs. The suppression of myopia in guinea pigs was accompanied by inhibition of the “ChBP reduction–scleral hypoxia cascade.” Additionally, treatment with DHA or EPA antagonized hypoxia-induced myofibroblast transdifferentiation in cultured human scleral fibroblasts. In human subjects, oral administration of ω-3 PUFAs partially alleviated the near-work–induced decreases in ChBP. Therefore, evidence from these animal and human studies suggests ω-3 PUFAs are potential and readily available candidates for myopia control.

The myopia epidemic is now becoming a significant public health concern to modern society (1, 2). The percentage of the global population having myopia was predicted to increase from 28.3% in 2010 to 49.8% in 2050 (3). In East Asia, this percentage could even reach 90%, with up to 20% of the cases potentially developing into high myopia (refraction ≤−6 diopters [D]) (4, 5), which is one of the leading causes of irreversible blindness (6). The dramatic increase in the incidence of myopia, due in part to COVID-19 home confinement and increased online-viewing time, further highlights the importance of identifying a safe and effective approach to myopia control (7, 8).Behavioral, pharmacological, and optical interventions are the current approaches for myopia control, all of which have unique limitations. Behavioral intervention (such as increasing the time spent outdoors) retards the progression of myopia into high myopia (9). However, competitive educational systems, as well as lifestyles that incorporate increasingly more electronic products (the prevalence of which tends to reduce the amount of time spent outdoors), are hard to avoid. Pharmacological intervention, such as the use of atropine drops, is effective in limiting myopia progression. Its use, however, is off-label in most areas, and widespread acceptance is restricted because of the potential side effects such as chronic pupillary dilatation, loss of accommodation, and declining long-term effectiveness for sustained myopia control (10–12). Mori et al. reported that the dietary intake of crocetin, a naturally occurring apocarotenoid dicarboxylic acid found in crocus and other plants, could prevent myopia development in a mouse model and in children (13, 14), but more clinical studies are needed to prove the efficacy and safety of this agent. Optical corrections such as orthokeratology and peripheral defocusing lenses suffer from risks of infectious keratitis (15) and the requirement of professional support (16). Such limitations are critical, considering the implications of employing these approaches in a larger population, especially where the availability of medical services is limited.Thus, targeting multiple signaling cascades that promote the development of myopia, from retinal image processing to scleral growth, may be an effective strategy for myopia control. Previous studies have highlighted the significance of the cascade of events wherein the reduction of choroidal blood perfusion (ChBP, refers to the amount of choroidal blood flow) induces scleral hypoxia and myopia (17–20). The cascade begins when visual stimulus–induced myopic blur causes a reduction of choroidal thickness (ChT) and ChBP (20). In turn, these physiological changes induce hypoxia in the sclera, which is dependent on oxygen delivered by the choroidal vasculature (21). Hypoxia activates the hypoxia-induced factor-1α (HIF-1α) signaling pathway, which subsequently promotes scleral fibroblast-myofibroblast transdifferentiation and extracellular matrix (ECM) remodeling and in turn the development and progression of myopia (17).In the absence of effective treatments, and given the additional safety considerations when administering therapeutic agents to children (who are most vulnerable to myopia development), investigation into a dietary supplement for a safe and accessible approach for myopia control and prevention becomes compelling. However, research in this area is still new. Because of the beneficial effect of supplemental dietary omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on cardiovascular health, daily supplements have been recommended for intake by the Food and Agriculture Organization of the United Nations (2010), which endorses the use of two types of ω-3 PUFAs, videlicet, 250 mg/day each of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) for adults (22). Mammalian brains are invariably rich in DHA (23), and numerous studies have defined the importance of ω-3 PUFAs, particularly DHA, in human neuronal development in different developmental stages (24–26). Recently, an untargeted mass spectrometric assay reported that the amounts of serum fatty acid metabolites were reduced in myopic human subjects compared to nonmyopic subjects (27). In particular, the levels of serum DHA were significantly lower in the myopic group (27). DHA and EPA can promote relaxation of vascular smooth muscle cells and vasodilation (28–31), inhibit cancerous cell growth and survival by reducing HIF-1α expression (32–34), and suppress transforming growth factor (TGF)-β1–mediated myofibroblast transdifferentiation (35–37). These effects of DHA and EPA are also known to be associated with the inhibition of myopia development.The effects of ω-3 PUFAs on systemic conditions and on myopic subjects, combined with their involvement in mediating blood perfusion, HIF-1α, and cellular proliferation, prompted us to investigate if ω-3 PUFAs could suppress myopia development through modulating ChBP and scleral hypoxia. Thus, in this study, we first assessed the ability of ω-3 PUFAs supplementation to suppress myopia development in different animal models. We then examined the effects of DHA and EPA on ChBP and scleral hypoxia. We also determined if DHA or EPA could antagonize the effects of hypoxia on cultured human scleral fibroblasts (HSFs). Finally, we administered ω-3 PUFAs supplements to humans and observed their influence on near-work–induced ChBP reduction.     

细胞外基质对前脂肪细胞分化转归的影响

前脂肪细胞是存在于脂肪组织中的一种未分化细胞,它可以依照能量需求增殖分化为成熟脂肪细胞.在特定的条件下,它也可以向血管内皮细胞、巨噬细胞及成骨细胞转分化.细胞外基质(ECM)作为一种重要的细胞外环境,其对前脂肪细胞的分化及转归具有重要的影响.脂肪组织受到创伤后,细胞外基质质与量的改变将对前脂肪细胞产生怎样的影响,它会不会朝着与修复相关的方向转分化,对这一问题的深入研究将有利于揭示脂肪组织参与创伤修复的内在机制.