低浓度紫杉醇对大鼠肺动脉平滑肌细胞外胶原沉积的作用及其机制研究

目的 探讨低浓度紫杉醇（PTX）对转化生长因子β1（TGF-β1）促进大鼠肺动脉平滑肌细胞（PASMCs）外胶原沉积的作用及其机制。方法 原代培养大鼠PASMCs并分为空白对照组、模型组和干预组（n=3）。空白对照组不做任何处理,模型组施加终浓度为10 ng/mL的TGF-β1,干预组在模型组基础上施加终浓度为100 nmol/L的PTX。MTT比色法检测细胞增殖能力;实时荧光定量PCR法检测Ⅰ型胶原（COLⅠ）、Ⅲ型胶原（COLⅢ） mRNA相对表达量;ELISA法检测COLⅠ、COLⅢ蛋白的OD值;Western blot法检测COLⅠ、COLⅢ蛋白,以及TGF-β1/Smad3信号通路关键蛋白Smad3、p-Smad3的相对表达水平。结果 与空白对照组比较,模型组细胞增殖能力、COLⅠ、COLⅢ mRNA及其蛋白、p-Smad3蛋白相对表达水平均明显增高（P < 0.05）;干预组上述指标较模型组均有所下降,但仍高于空白对照组（P < 0.05）;各组Smad3蛋白相对表达水平比较差异均无统计学意义（P > 0.05）。结论 低浓度PTX对TGF-β1促进PASMCs外胶原合成具有明显抑制作用,该作用可能是通过调控Smad3蛋白的磷酸化来实现。     

Temporal Pattern of Stimulation of Osteoblast-Associated Genes During Mechanically-Induced Osteogenesis In Vivo: Early Responses of Osteocalcin and Type I Collagen

Mechanical loading is an essential environmental factor in skeletal homeostasis, but the response of osteoblast-associated genes to mechanical osteogenic signal is largely unknown. This study uses our recently characterized in vivo osteoinductive model to analyze the sequence of stimulation and the time course of expression of osteoblast-associated genes in mechanically loaded mouse periodontium. Temporal pattern of regulation of osteocalcin (OC), alkaline phosphatase (ALP), and type I collagen (collagen I) was determined during mechanically-induced osteoblast differentiation in vivo, using a mouse tooth movement model earlier shown to induce bone formation and cell-specific regulation of genes in osteoblasts. The expression of target genes was determined after 1, 2, 3, 4, and 6 days of orthodontic movement of the mouse first molar. mRNA levels were measured in the layer of osteoblasts adjacent to the alveolar bone surface, using in situ hybridization and a relative quantitative video image analysis of cell-specific hybridization intensity, with non-osseous mesenchymal periodontal cells as an internal standard. After 24 hours of loading, the level of OC in osteoblasts slightly decreased, followed by a remarkable 4.6-fold cell-specific stimulation between 1 and 2 days of treatment. The high level expression of OC was maintained throughout the treatment with a peak 7-fold stimulation at day 4. The expression of collagen I gene was not significantly affected after 1 day, but it was stimulated 3-fold at day 2, and maintained at a similar level through day 6. The ALP gene, which we previously found to be mechanically stimulated during the first 24 hours, remained enhanced from 1.8- to 2.2-fold throughout the 6 days of treatment. Thus, in an intact alveolar bone compartment, mechanical loading resulted in a defined temporal sequence of induction of osteoblast-associated genes. Stimulation of OC 48 h after the onset of loading (and 24 h prior to deposition of osteoid) temporally coincided with that of collagen I, and was preceded for 24 h by an enhancement of ALP. Identification of OC as a mechanically responsive gene induced in functionally active osteoblasts in this study is consistent with its potential role in limiting the rate of mechanically-induced bone modeling. Furthermore, these results show that temporal progression of mechanically-induced osteoblast phenotype in this in vivo model occurs very rapidly. This suggests that physiologically relevant mechanical osteoinductive signal in vivo is targeting a population of committed osteoblast precursor cells that are capable of rapidly responding by entering a differentiation pathway and initiating an anabolic skeletal adaptation process.     

Col1a1-GFP Transgene Expression in Developing Incisors

Previous studies have shown that terminal differentiation of odontoblasts is accompanied by dramatic increases in type I collagen synthesis. Recently transgenic mice in which green fluorescent protein (GFP) expression is under the control of the rat 3.6 (pOBCol3.6GFPtpz) and 2.3 (pOBCol2.3GFPemd) Col1a1 promoter fragments were generated. Our analysis of these GFP-expressing transgenic mice shows that the 2.3-kb promoter fragment directs strong expression of GFP only to bones and teeth, whereas the 3.6-kb fragment of promoter directs strong expression of GFP in bone and tooth, as well as in other type I collagen producing tissues. Our observations of incisors in these transgenic mice show high levels of GFP expression in functional odontoblasts and in differentiated osteoblasts. These observations show that expression of GFP reporter genes closely follow the patterns of expression of &#102 1(I) collagen in various tissues including odontoblasts.     

Alterations of Collagen Matrix in Weight-Bearing Bones during Skeletal Unloading

Skeletal unloading induces loss of bone mineral density in weight-bearing bones. The objectives of this study were to characterize the post-translational modifications of collagen of weight-bearing bones subjected to hindlimb unloading for 8 weeks. In unloaded bones, tibiae and femurs, while the overall amino acid composition was essentially identical in the unloaded and control tibiae and femurs, the collagen cross-link profile showed significant differences. Two major reducible cross-links (analyzed as dihydroxylysinonorleucine and hydroxylysinonorleucine) were increased in the unloaded bones. In addition, the ratios of the former to the latter as well as pyridinoline to deoxypyridinoline were significantly decreased in the unloaded bones indicating a difference in the extent of lysine hydroxylation at the cross-linking sites between these two groups. These results indicate that upon skeletal unloading the relative pool of newly synthesized collagen is increased and it is post-translationally altered. The alteration could be associated with impaired osteoblastic differentiation induced by skeletal unloading that results in a mineralization defect.     

A novel tissue engineered three-dimensional in vitro colorectal cancer model

The interactions of cancer cells within a solid mass with the surrounding reactive stroma are critical for growth and progression. The surrounding vasculature is recruited into the periphery of the growing tumour to supply cancer cells with nutrients and O₂. This study focuses on developing a novel three-dimensional (3-D) in vitro biomimetic colorectal cancer model using colorectal cancer cells and connective tissue cells. The 3-D model comprises a dense artificial cancer mass, created by partial plastic compression of collagen type I containing HT29 colorectal cancer cells, nested in a non-dense collagen type I gel populated by fibroblasts and/or endothelial cells. HT29 cells within the dense mass proliferate slower than when cultured in a two-dimensional system. These cells form tumour spheroids which invade the surrounding matrix, away from the hypoxic conditions in the core of the construct, measured using real time O₂ probes. This model is also characterized by the release of vascular endothelial growth factor (VEGF) by HT29 cells, mainly at the invading edge of the artificial cancer mass. This characterization is fundamental in establishing a reproducible, complex model that could be used to advance our understanding of cancer pathology and will facilitate therapeutic drug testing.     

Cholinergic hyperactivity in patients with myasthenia gravis with MuSK antibodies: A neurophysiological study

ObjectivePatients with myasthenia gravis associated with muscle-specific tyrosine kinase antibodies (MuSK-MG) often manifest signs of cholinergic hyperactivity with standard doses of acetylcholinesterase inhibitors (AChE-Is). Aim of the study was to investigate whether repetitive compound muscle action potential (R-CMAP), the neurophysiological correlate of cholinergic hyperactivity, was present in MuSK-MG irrespective of AChE-I treatment.MethodsPatients with confirmed diagnosis of MuSK-MG were consecutively enrolled during follow-up visits, from January 2019 to April 2020. All these subjects underwent the same neurophysiological protocol, including motor nerve conduction studies and repetitive nerve stimulation. In patients taking pyridostigmine, neurophysiological testing was performed at least 12 hours after the last dose. For comparison, the presence of R-CMAP was investigated in 20 consecutive acetylcholine receptor antibody positive myasthenia gravis (AChR-MG) patients.ResultsWe enrolled 25 MuSK-MG patients (20 females), aged 16–79 years at the study time, with disease duration ranging 0.6–48.8 years (median: 17.7 years). R-CMAP was detected in 12/25 (48%) MuSK-MG cases and in none of the AChR-MG controls (p = 0.0003). In the MuSK-MG population, a history of muscle cramps and fasciculations, during low-dose pyridostigmine therapy, was significantly more frequent in R-CMAP positive than in R-CMAP negative patients (100% vs 31%, p = 0.001). At the time of the study, the proportion of patients still symptomatic for MG was higher among R-CMAP positive cases (92% vs 23%, p = 0.0005).ConclusionsCholinergic hyperactivity is a relatively common finding in MuSK-MG patients, independent of AChE-I treatment, and may constitute an intrinsic feature of the disease.SignificanceR-CMAP detection can represent a useful diagnostic clue for MuSK-MG and predicts poor tolerance to AChE-Is.     

Tissue-engineered dermo-epidermal skin grafts prevascularized with adipose-derived cells

The major problem in skin grafting is that tissue-engineered skin grafts after their transplantation are initially entirely dependent on diffusion. Since this process is slow and inefficient, nutrients, growth factors, and oxygen will insufficiently be supplied and the regenerating graft will undergo a physiological crisis, resulting in scar-like dermal structures and shrinkage. The tissue-engineering of a vascular network in human dermo-epidermal skin substitutes (DESS) is a promising approach to overcome this limitation. Here we report, for the first time, on the use of the adipose stromal vascular fraction (SVF)-derived endothelial cell population to tissue-engineer DESS containing a highly efficient capillary plexus. To develop vascular networks in vitro, we employed optimized 3D fibrin or collagen type I hydrogel systems. Upon transplantation onto immune-deficient rats, these pre-formed vascular networks anastomosed to the recipient's vasculature within only four days. As a consequence, the neo-epidermis efficiently established tissue homeostasis, the dermis underwent almost no contraction, and showed sustained epidermal coverage in vivo. Overall, the here described rapid and efficient perfusion of SVF-based skin grafts opens new perspectives for the treatment of hitherto unmet clinical needs in burn/plastic surgery and dermatology.