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Evaluation of Stem Cell Laden Collagen + Polycaprolactone + Multi-Walled Carbon Nano-Tubes Nano-Neural Scaffold with and Without Insulin Like Growth Factor-I For Sciatic Nerve Regeneration Post Crush Injury in Wistar Rats. 干细胞负载胶原+聚己内酯+多壁碳纳米管纳米神经支架在Wistar大鼠坐骨神经挤压损伤后再生中的应用
Q1 Medicine Pub Date : 2023-11-15 DOI: 10.33594/000000670
Mamta Mishra, Swapan Kumar Maiti, Kalaiselvan Elangovan, Shivaraju Shivaramu, Karam Pal Singh, Amitha Banu S, Merlin Mamachan, Manish Arya, Divya Mishra, Jurgen Hescheler

Background/aims: All body functions are activated, synchronized and controlled by a substantial, complex network, the nervous system. Upon injury, pathophysiology of the nerve injury proceeds through different paths. The axon may undergo a degenerative retraction from the site of injury for a short distance unless the injury is near to the cell body, in which case it continues to the soma and undergoes retrograde neuronal degeneration. Otherwise, the distal section suffers from Wallerian degeneration, which is marked by axonal swelling, spheroids, and cytoskeleton degeneration. The objective of the study was to evaluate the potential of mesenchymal stem cell laden neural scaffold and insulin-like growth factor I (IGF-I) in nerve regeneration following sciatic nerve injury in a rat model.

Methods: The animals were anaesthetized and a cranio-lateral incision over left thigh was made. Sciatic nerve was exposed and crush injury was introduced for 90 seconds using haemostat at second locking position. The muscle and skin were sutured in routine fashion and thus the rat model of sciatic crush injury was prepared. The animal models were equally distributed into 5 different groups namely A, B, C, D and E and treated with phosphate buffer saline (PBS), carbon nanotubes based neural scaffold only, scaffold with IGF-I, stem cell laden scaffold and stem cell laden scaffold with IGF-I respectively. In vitro scaffold testing was performed. The nerve regeneration was assessed based on physico-neuronal, biochemical, histopathological examination, and relative expression of NRP-1, NRP-2 and GAP-43 and scanning electron microscopy.

Results: Sciatic nerve injury model with crush injury produced for 90 seconds was standardized and successfully used in this study. All the biochemical parameters were in normal range in all the groups indicating no scaffold related changes. Physico-neuronal, histopathological, relative gene expression and scanning electron microscopy observations revealed appreciable nerve regeneration in groups E and D, followed by C and B. Restricted to no regeneration was observed in group A.

Conclusion: Carbon nanotubes based scaffold provided electro-conductivity for proper neuronal regeneration while rat bone marrow-derived mesenchymal stem cells were found to induce axonal sprouting, cellular transformation; whereas IGF-I induced stem cell differentiation, myelin synthesis, angiogenesis and muscle differentiation.

背景/目的:所有的身体功能都是由一个庞大而复杂的网络——神经系统——激活、同步和控制的。损伤后,神经损伤的病理生理经过不同的途径。轴突可能从损伤部位退行性缩回一小段距离,除非损伤靠近细胞体,在这种情况下,它继续到体细胞并经历逆行神经元变性。否则,远端部分出现沃勒氏变性,其特征是轴突肿胀、球状体和细胞骨架变性。本研究的目的是评估间充质干细胞负载神经支架和胰岛素样生长因子I (IGF-I)在大鼠坐骨神经损伤后神经再生中的潜力。方法:麻醉动物,在左大腿上开颅外侧切口。暴露坐骨神经,在第二锁定位置使用止血剂进行挤压损伤90秒。采用常规方法缝合肌肉和皮肤,制备大鼠坐骨挤压损伤模型。将动物模型平均分为A、B、C、D、E 5组,分别采用磷酸缓冲盐水(PBS)、碳纳米管神经支架、IGF-I支架、干细胞负载支架和IGF-I干细胞负载支架处理。进行体外支架试验。通过物理-神经元、生化、组织病理学检查,以及NRP-1、NRP-2、GAP-43的相对表达和扫描电镜评估神经再生情况。结果:90秒挤压伤坐骨神经损伤模型标准化并成功应用于本研究。各组生化指标均在正常范围内,未见支架相关变化。物理-神经元、组织病理学、相关基因表达和扫描电镜观察显示,E组和D组有明显的神经再生,其次是C组和b组,a组没有再生。结论:碳纳米管支架为神经再生提供了电导率,而大鼠骨髓源间充质干细胞可诱导轴突萌发、细胞转化;而igf - 1诱导干细胞分化、髓磷脂合成、血管生成和肌肉分化。
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引用次数: 0
Do the Effects of Krebs Cycle Intermediates on Oxygen-Dependent Processes in Hypoxia Mediated by the Nitric Oxide System Have Reciprocal or Competitive Relationships? 由一氧化氮系统介导的缺氧中克雷布斯循环中间体对氧依赖过程的影响是否具有互惠或竞争关系?
Q1 Medicine Pub Date : 2023-11-15 DOI: 10.33594/000000669
Natalia Kurhaluk, Oleksandr Lukash, Halina Tkaczenko

Background/aims: Currently, it is proven that the cellular metabolism of nitric oxide is necessary to maintain optimal health and adaptation of the organism to the impact of various environmental factors. The aim of this work was to reveal the biological role of nitric oxide, its metabolic changes, and its mechanism of action in tissues under hypoxia, as well as the possibility of tissue metabolism correction through NO-dependent systems under the influence of Krebs cycle intermediates.

Methods: A systematic assessment of the effect of succinate (SC, 50 mg/kg b.w.) and α-ketoglutarate (KGL, 50 mg/kg b.w.) in the regulation of oxygendependent processes in rats (mitochondrial oxidative phosphorylation, microsomal oxidation, intensity of lipid peroxidation processes, and the state of the antioxidant defense system) depending on functional changes in nitric oxide production during hypoxia was evaluated. The state of the nitric oxide system was estimated spectrophotometrically by determination of the concentration of its stable nitrite anion metabolite (NO2 -). The levels of catecholamines were estimated from the content of epinephrine and norepinephrine using the differentially fluorescent method. The activity of cytochrome P450-dependent aminopyrine-N-demethylase was determined with the Nash reagent.

Results: Tissue hypoxia and metabolic disorders caused by this condition through changes in the content of catecholamines (epinephrine, norepinephrine, dopamine, DOPA) as well as the cholinesterase-related system (acetylcholine content and acetylcholinesterase activity) were the studied experimental parameters under acute hypoxia (AH, 7% O2 in N2, 30 min). The activation of lipid peroxidation and oxidatively modified proteins and an increase in the epinephrine content in AH are associated with an increased role of SC and a decrease in KGL as substrates of oxidation in mitochondria. A more pronounced effect of exogenous KGL, compared to SC, on the content of nitrite anion as a stable metabolite of nitric oxide in the liver under acute hypoxia against the background of a decrease in the intensity of lipid peroxidation processes was revealed. The activation of SC-dependent mitochondrial oxidative processes caused by AH was found to decrease in animals after an intermittent hypoxia training (IHT) course. IHT (7% O2 in N2, 15-min, 5 times daily, 14 days) prevented the activation of oxidative stress in tissues and blood after the AH impact and increased the efficiency of energy-related reactions in the functioning of hepatic mitochondria through increased oxidation of KGL.

Conclusion: The studied effects of adaptation are mediated by an increase in the role of NO-dependent mechanisms, as assessed by changes in the pool of nitrates, nitrites, carbamides, and total polyamines.

背景/目的:目前已经证明,一氧化氮的细胞代谢是维持机体最佳健康和适应各种环境因素影响所必需的。本研究旨在揭示缺氧条件下一氧化氮的生物学作用、代谢变化及其在组织中的作用机制,以及在克雷布斯循环中间体的影响下,一氧化氮依赖系统对组织代谢进行校正的可能性。方法:系统评估琥珀酸盐(SC, 50 mg/kg b.w.)和α-酮戊二酸盐(KGL, 50 mg/kg b.w.)对缺氧时一氧化氮生成的功能变化对大鼠氧依赖过程(线粒体氧化磷酸化、微粒体氧化、脂质过氧化过程强度和抗氧化防御系统状态)的调节作用。用分光光度法测定其稳定的亚硝酸盐阴离子代谢物(NO2 -)的浓度来估计一氧化氮体系的状态。儿茶酚胺的水平是用差异荧光法从肾上腺素和去甲肾上腺素的含量来估计的。采用Nash试剂测定细胞色素p450依赖性氨基吡啶- n -去甲基化酶的活性。结果:研究急性缺氧(AH, 7% O2 in N2, 30min)下组织缺氧及由此引起的儿茶酚胺(肾上腺素、去甲肾上腺素、多巴胺、多巴胺)含量变化及胆碱酯酶相关系统(乙酰胆碱含量和乙酰胆碱酯酶活性)代谢紊乱的实验参数。AH中脂质过氧化和氧化修饰蛋白的激活以及肾上腺素含量的增加与SC作为线粒体氧化底物的作用增加和KGL的减少有关。与SC相比,外源性KGL对急性缺氧下肝脏中一氧化氮的稳定代谢物亚硝酸盐阴离子含量的影响更为明显,其背景是脂质过氧化过程的强度降低。间歇性缺氧训练(IHT)后,发现AH引起的sc依赖性线粒体氧化过程的激活减少。IHT (7% O2在N2中,15分钟,每天5次,14天)阻止AH冲击后组织和血液中氧化应激的激活,并通过增加KGL的氧化提高肝线粒体功能中能量相关反应的效率。结论:通过硝酸盐、亚硝酸盐、氨酰胺和总多胺的变化来评估,所研究的适应效应是由no依赖机制的作用增加介导的。
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引用次数: 0
Docosahexaenoic Acid (DHA) Reduces LPS-Induced Inflammatory Response Via ATF3 Transcription Factor and Stimulates Src/Syk Signaling-Dependent Phagocytosis in Microglia. 二十二碳六烯酸(DHA)通过ATF3转录因子减少脂多糖诱导的炎症反应并刺激小胶质细胞Src/Syk信号依赖性吞噬。
Q1 Medicine Pub Date : 2023-11-13 DOI: 10.33594/000000668
Katarzyna Wieczorek-Szukala, Monika Markiewicz, Anna Walczewska, Emilia Zgorzynska

Background/aims: Microglial cells play a crucial role in the development of neuroinflammation in response to harmful stimuli, such as infection, ischemia or injury. Their chronic activation, however, is associated with a progression of neurodegenerative diseases. Therefore, looking for potential factors limiting microglial activation, the effect of docosahexaenoic acid (DHA) on the inflammatory response and TREM2-dependent phagocytic activity in microglia was investigated.

Methods: In LPS-induced primary microglia preincubated with DHA, or without preincubation the expression of ATF3 and TREM2 genes and TREM2, Syk, Akt proteins were determined by RT-PCR and WB, respectively. Cell viability was assayed by MTT and cytokine and chemokine expression was determined by the Proteome Profiler assay. Moreover, the phagocytic activity of microglia was assayed using immunofluorescence.

Results: We found that DHA significantly increased the expression of ATF3 , and decreased the levels of CINC-1, CINC-2αβ, CINC-3 chemokines, IL-1α and IL-1β cytokines, and ICAM-1 adhesion protein. Additionally, preincubation of microglia with DHA resulted in increased Src/Syk kinases activation associated with increased phagocytic microglia activity.

Conclusion: These findings indicate that DHA efficiently inhibits ATF3-dependent release of proinflammatory mediators and enhances phagocytic activity of microglia. The study provides a new mechanism of DHA action in reactive microglia, which may help limit neuronal damage caused by the pro-inflammatory milieu in the brain.

背景/目的:小胶质细胞在对有害刺激(如感染、缺血或损伤)的神经炎症反应的发展中起着至关重要的作用。然而,它们的慢性激活与神经退行性疾病的进展有关。因此,为了寻找限制小胶质细胞激活的潜在因素,我们研究了二十二碳六烯酸(DHA)对小胶质细胞炎症反应和trem2依赖性吞噬活性的影响。方法:采用RT-PCR和WB分别检测脂多糖诱导的原代小胶质细胞中ATF3、TREM2基因和TREM2、Syk、Akt蛋白的表达。MTT法检测细胞活力,Proteome Profiler法检测细胞因子和趋化因子表达。免疫荧光法检测小胶质细胞的吞噬活性。结果:我们发现DHA显著提高了ATF3的表达,降低了cnc -1、cnc -2αβ、cnc -3趋化因子、IL-1α和IL-1β细胞因子以及ICAM-1粘附蛋白的水平。此外,用DHA预孵育小胶质细胞导致Src/Syk激酶活化增加,这与吞噬小胶质细胞活性增加有关。结论:DHA能有效抑制atf3依赖性促炎介质的释放,增强小胶质细胞的吞噬活性。该研究为DHA在反应性小胶质细胞中的作用提供了一种新的机制,这可能有助于限制大脑中促炎环境引起的神经元损伤。
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引用次数: 0
Erratum. 勘误表。
Q1 Medicine Pub Date : 2023-10-31 DOI: 10.33594/000000666
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引用次数: 0
Retraction Statement. 撤回声明。
Q1 Medicine Pub Date : 2023-10-31 DOI: 10.33594/000000667
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引用次数: 0
Emerging Roles of SPT5 in Transcription. SPT5在转录中的新作用
Q1 Medicine Pub Date : 2023-10-23 DOI: 10.33594/000000665
Vivek Pandey, Shirani Punniyamoorthy, Yuba Raj Pokharel

Suppressor of Ty homolog-5 (SPT5) discovered in the yeast mutant screens as a suppressor of mutation caused by the insertion of the Transposons of yeast (Ty) element along with SPT4, with which it forms a holoenzyme complex known as DRB sensitivity-inducing factor (DSIF) and plays an essential role in the regulation of transcription. SPT5 is a highly conserved protein across all three domains of life and performs critical functions in transcription, starting from promoter-proximal pausing to termination. We also highlight the emerging role of SPT5 in other non-canonical functions, such as the regulation of post-translational modifications (PTM) and the transcriptional regulation of non-coding genes. Also, in brief, we highlight the clinical implications of SPT5 dysregulation.

在酵母突变体中发现的Ty同源物抑制剂-5(SPT5)筛选为酵母转座子(Ty)元件与SPT4一起插入引起的突变的抑制剂,与之形成称为DRB敏感性诱导因子(DSIF)的全酶复合物,并在转录调控中发挥重要作用。SPT5是一种高度保守的蛋白质,横跨生命的所有三个结构域,在转录中发挥关键功能,从启动子近端暂停到终止。我们还强调了SPT5在其他非经典功能中的新作用,如翻译后修饰(PTM)的调节和非编码基因的转录调节。此外,简而言之,我们强调了SPT5失调的临床意义。
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引用次数: 0
Immunogenicity and Protection by DnaK and SpaA Recombinant Proteins Against Erysipelothrix Rhusiopathiae in a Murine Model. DnaK和SpaA重组蛋白在小鼠模型中对赤藓病的免疫原性和保护作用。
Q1 Medicine Pub Date : 2023-10-09 DOI: 10.33594/000000664
Naiane Lima Godoy, Jonathan Ballico de Moraes, Cynthia Aparecida de Castro, Jhonne Pedro Pedott Santana, Teresa Cristina Zangirolami, Adilson José da Silva, Maria Teresa Marques Novo-Mansur, Fernanda de Freitas Anibal

Background/aims: Swine erysipelas is a disease caused by Erysipelothrix rhusiopathiae, a Gram-positive bacillus, which has great economic importance because it leads to the loss of the swine herd. To control this disease, animals are immunized with a cellular vaccine of killed or attenuated E. rhusiopathiae, but even with herd vaccination, cases of swine erysipelas outbreaks have been reported in the United States, China and Japan, leading to the search for other antigenic components of the bacteria that may promote greater protection against E. rhusiopathiae. The surface protein SpaA from E. rhusiopathiae has been shown to be a candidate to constitute a subunit vaccine, since it has already been reported to induce a host immune response against the bacterium. DnaK, a hsp70 molecular chaperone, also seems to be a good candidate in the composition of a vaccine, as it has been demonstrated to be an antigenic protein of the bacteria.

Methods: This work evaluated the immunogenicity and protection induced by the E. rhusiopathiaee SpaA and DnaK recombinant proteins in a murine model, by intramuscular administration to mice with two doses of 100 µg at 21-day interval between them. The candidate proteins were tested either separately and together, compared with the commercial vaccine and the non-vaccination condition, and mice were challenged with a virulent strain of E. rhusiopathiae. Serum was collected to assess the produced antibodies and peripheral blood cells, whereas spleen and kidney tissues were assayed for E. rhusiopathiae presence by colony counting.

Results: A survival curve of the animals was performed, which confirmed the protection induced by the proteins. IgG antibodies increased in the animal serum inoculated with the proteins when compared to the control, and a significant delay in disease symptoms was observed.

Conclusion: These results suggest that E. rhusiopathiae DnaK and SpaA are immunogenic in mice and interfere with the disease development.

背景/目的:猪丹毒是由一种革兰氏阳性杆菌——猪赤藓引起的一种疾病,由于它会导致猪群的损失,因此具有重要的经济意义。为了控制这种疾病,用杀死或减毒的蛇颈杆菌细胞疫苗对动物进行免疫,但即使进行群体疫苗接种,美国、中国和日本也报告了猪丹毒爆发的病例,这导致了对该细菌的其他抗原成分的寻找,这些成分可能会促进对蛇颈杆菌的更大保护。已经证明,来自蛇颈杆菌的表面蛋白SpaA是构成亚单位疫苗的候选蛋白,因为已经有报道称它可以诱导宿主对该细菌的免疫反应。DnaK,一种hsp70分子伴侣,似乎也是疫苗组成中的一个很好的候选者,因为它已被证明是细菌的抗原蛋白。方法:本工作评估了E.rhusopathiee-SPA和DnaK重组蛋白在小鼠模型中诱导的免疫原性和保护作用,方法是在21天间隔两次给药100µg。与商业疫苗和非疫苗接种条件相比,对候选蛋白进行单独或一起测试,并用蛇颈杆菌的毒力菌株攻击小鼠。收集血清以评估产生的抗体和外周血细胞,而通过菌落计数来检测脾脏和肾脏组织中是否存在蛇颈杆菌。结果:绘制了动物的存活曲线,证实了蛋白质的保护作用。与对照组相比,接种蛋白质的动物血清中的IgG抗体增加,并且观察到疾病症状的显著延迟。结论:上述结果表明,蛇颈草杆菌DnaK和SpaA在小鼠体内具有免疫原性,并干扰疾病的发展。
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引用次数: 0
Activity of Lysosomal ABCC3, ABCC5 and ABCC10 is Responsible for Lysosomal Sequestration of Doxorubicin and Paclitaxel-Oregongreen488 in Paclitaxel-Resistant Cancer Cell Lines. 溶体ABCC3、ABCC5和ABCC10的活性负责多柔比星和紫杉醇-Oregongreen488在紫杉醇耐药癌症细胞系中的溶体滞留。
Q1 Medicine Pub Date : 2023-09-27 DOI: 10.33594/000000663
Karolina Gronkowska, Sylwia Michlewska, Agnieszka Robaszkiewicz

Background/aims: Cancer cell multidrug resistance induced by paclitaxel contributes to the high failure rates of chemotherapy and relapse of the disease. Several mechanisms have been described that underlie the observed resistance, including the overexpression of ABCB1 (P-glycoprotein), which represents an ATP-binding cassette (ABC) transmembrane protein, and its functional occurrence in lysosomal membranes is linked to drug accumulation in these organelles.

Methods: Using clinically-relevant models of paclitaxel-resistant triple-negative breast cancer and non-small cell lung cancer cell lines, we provide evidence for the role of ABCC subfamily members in the lysosomal sequestration of drugs in multidrug resistant phenotypes. Proteins expression level and its cellular localisation was measured using Western Blot and confocal microscopy. Drug accumulation was analysed by confocal microscopy and flow cytometry. Drug cytotoxicity was tested using resasurin assay and anexin V propidium iodide staining.

Results: Regardless of the alteration in gene expression, paclitaxel induced the intracellular redistribution of ABCC3, ABCC5 and ABCC10 and their enrichment in lysosomes. The use of ABCC inhibitors and transient silencing of these three genes limited the accumulation of doxorubicin and paclitaxel-OregonGreen488 in lysosomes, while having little impact on the total drug level inside cells. The cancer cells were also sensitized to various structurally unrelated chemotherapeutics of differing acidity.

Conclusion: The results suggest that lysosome membranes anchored ABCC proteins which remained functionally active and were capable to load chemotherapeutics into lysosomes in paclitaxel-resistant cancer cells. Therefore, targeting of lysosomal ABCC transporters may help to overcome paclitaxel-induced resistance by reducing the accumulation of drugs in lysosomes.

背景/目的:紫杉醇诱导的癌症细胞多药耐药性导致化疗失败率和疾病复发。已经描述了几种机制,这些机制是观察到的耐药性的基础,包括ABCB1(P-糖蛋白)的过度表达,其代表ATP结合盒(ABC)跨膜蛋白,其在溶酶体膜中的功能发生与这些细胞器中的药物积累有关。方法:利用多药耐药的癌症和癌症非小细胞肺癌细胞系的临床相关模型,我们为ABCC亚家族成员在多药耐药表型的药物溶酶体固存中的作用提供了证据。蛋白质表达水平及其细胞定位使用蛋白质印迹和共聚焦显微镜进行测量。通过共聚焦显微镜和流式细胞术分析药物积聚。用雷沙苏林法和安烯新V碘化丙啶染色法检测药物的细胞毒性。结果:无论基因表达的改变如何,紫杉醇都能诱导ABCC3、ABCC5和ABCC10在细胞内的重新分布,并在溶酶体中富集。ABCC抑制剂的使用和这三个基因的短暂沉默限制了阿霉素和紫杉醇-OregonGreen488在溶酶体中的积累,而对细胞内的总药物水平几乎没有影响。癌症细胞也对不同酸度的各种结构无关的化疗药物敏感。结论:在抗紫杉醇癌症细胞中,溶酶体膜锚定ABCC蛋白,该蛋白保持功能活性,并能够将化疗药物加载到溶酶体中。因此,靶向溶酶体ABCC转运蛋白可能有助于通过减少药物在溶酶体中的积累来克服紫杉醇诱导的耐药性。
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引用次数: 0
Induced Pluripotent Stem Cells (Ipscs) Based Liver Organoid: the Benefits and Challenges. 基于诱导多能干细胞(Ipscs)的肝类器官:益处和挑战。
Q1 Medicine Pub Date : 2023-09-26 DOI: 10.33594/000000662
Wahyunia Likhayati Septiana, Ariyani Noviantari, Radiana Dhewayani Antarianto

The liver is the main metabolic organ and functions to regulate many physiological functions in the human body. Approximately 70% of liver mass consists of hepatic cells (hepatocytes), which execute the liver's metabolic processes. When liver damage progresses to a chronic condition, such as end-stage liver disease (ESLD) or cirrhosis of the liver, the patient's only option for therapy is organ transplantation if the supply of available transplanted organs is insufficient to meet the patient's needs. The fundamental objective of the search for alternatives to organ transplantation has been to make liver tissue replacement more accessible and to produce hepatic and bioartificial liver tissue. Multiple hepatic cell lineages can be formed from human-induced pluripotent stem cells (hiPSCs) from embryoid bodies to become mature hepatocytes. hiPSCs also show a promising source for manufacturing human liver spheroids and are made to produce three-dimensional hepatobiliary organoids, and in some ways, it also briefly highlights important features of early hepatogenesis. Unquestionably, the art of cell culture has evolved to include the use of organoid technology as a resource for learning human biology in the context of health and illness. Organoids are essentially miniature organs that can grow in a three-dimensional matrix to resemble genuine organs in terms of both structure and function. This review summarized alternative protocols to differentiate hepatocytes from iPSC and to produce liver organoids based on iPSC in various ways. The growth of human iPSCs into liver organoids has been accomplished using several procedures.

肝脏是人体的主要代谢器官,具有调节人体许多生理功能的功能。大约70%的肝脏由肝细胞组成,肝细胞执行肝脏的代谢过程。当肝损伤发展为慢性疾病,如终末期肝病(ESLD)或肝硬化时,如果可用的移植器官供应不足以满足患者的需求,患者唯一的治疗选择是器官移植。寻找器官移植替代品的基本目标是使肝组织替代品更容易获得,并生产肝脏和生物人工肝组织。人类诱导多能干细胞(hiPSC)可以从胚胎体形成多个肝细胞谱系,成为成熟的肝细胞。hiPSC也显示出制造人类肝脏球体的一个很有前途的来源,并被制造成三维肝胆类器官,在某些方面,它也简要地突出了早期肝发生的重要特征。毫无疑问,细胞培养艺术已经发展到包括使用类器官技术作为在健康和疾病背景下学习人类生物学的资源。类器官本质上是一种微型器官,可以在三维基质中生长,在结构和功能上与真正的器官相似。这篇综述总结了从iPSC中分化肝细胞和以各种方式在iPSC基础上产生肝类器官的替代方案。人类iPSC向肝脏类器官的生长已经通过几种程序完成。
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引用次数: 0
Tyrosine-Mutant AAV8 Vector Mediated Efficient and Safe Gene Transfer of Pigment Epithelium-Derived Factor to Mouse Lungs. 酪氨酸突变体AAV8载体介导的色素上皮衍生因子向小鼠肺的有效和安全的基因转移。
Q1 Medicine Pub Date : 2023-09-18 DOI: 10.33594/000000660
Débora P Ferreira, Sabrina V Martini, Helena A Oliveira, Adriana L Silva, Siddharth Shenoy, Daiqin Chen, Valentina Simon, Eric Han, Natalie E West, Jung Soo Suk, Patricia R M Rocco, Hilda Petrs-Silva, Marcelo M Morales, Fernanda F Cruz

Background/aims: Recombinant adeno-associated viruses (rAAV) are an important tool for lung targeted gene therapy. Substitution of tyrosine with phenylalanine residues (Y-F) in the capsid have been shown to protect the AAV vector from ubiquitin/proteasome degradation, increasing transduction efficiency. We tested the mutant Y733F-AAV8 vector for mucus diffusion, as well as the safety and efficacy of pigment epithelium-derived factor (PEDF) gene transfer to the lung.

Methods: For this purpose, Y733F-AAV8-PEDF (1010 viral genome) was administered intratracheally to C57BL/6 mice. Lung mechanics, morphometry, and inflammation were evaluated 7, 14, 21, and 28 days after injection.

Results: The tyrosine-mutant AAV8 vector was efficient at penetrating mucus in ex vivo assays and at transferring the gene to lung cells after in vivo instillation. Increased levels of transgene mRNA were observed 28 days after vector administration. Overexpression of PEDF did not affect in vivo lung parameters.

Conclusion: These findings provide a basis for further development of Y733F-AAV8-based gene therapies for safe and effective delivery of PEDF, which has anti-angiogenic, anti-inflammatory and anti-fibrotic activities and might be a promising therapy for lung inflammatory disorders.

背景/目的:重组腺相关病毒(rAAV)是肺靶向基因治疗的重要工具。衣壳中用苯丙氨酸残基(Y-F)取代酪氨酸已被证明可以保护AAV载体免受泛素/蛋白酶体降解,提高转导效率。我们测试了突变体Y733F-AAV8载体的粘液扩散,以及将色素上皮衍生因子(PEDF)基因转移到肺部的安全性和有效性。方法:C57BL/6小鼠气管内注射Y733F-AAV8-PEDF(1010病毒基因组)。在注射后7、14、21和28天评估肺力学、形态计量学和炎症。结果:酪氨酸突变体AAV8载体在离体分析中能有效穿透粘液,并在体内滴注后将基因转移到肺细胞。载体给药28天后,观察到转基因mRNA水平增加。PEDF的过表达不影响体内肺参数。结论:这些发现为进一步开发基于Y733F-AAV8的基因疗法提供了基础,用于安全有效地递送PEDF,PEDF具有抗血管生成、抗炎和抗纤维化活性,可能是治疗肺部炎症性疾病的一种有前景的疗法。
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引用次数: 1
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Cellular Physiology and Biochemistry
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