Maintaining telomere integrity is essential for cellular survival, and reactivation of telomerase or alternative lengthening of telomeres (ALT) represents a hallmark of cancer, ensuring replicative immortality. Osteosarcoma (OS), a malignancy in which many tumors rely on ALT for telomere maintenance, lacks effective therapeutic strategies targeting this pathway. This study aimed to identify and characterize novel molecular regulators of ALT activity and explore their potential as therapeutic targets in OS. Methods: Immunohistochemistry was performed to evaluate the expression of phosphorylated NPM1 (pT199-NPM1) in OS tissues. Functional experiments including NPM1 knockdown and rescue assays were conducted to assess the impact of NPM1 on break-induced telomere replication (BITR) and cell viability in ALT-positive cells. Mechanistic studies involving phosphorylation analysis, ubiquitination assays, and co-immunoprecipitation were used to determine how ATR-mediated phosphorylation of NPM1 regulates POLD3 stability and its interaction with the CST complex. Pharmacological screening was performed to identify compounds that inhibit ALT activity, followed by in vitro proliferation assays and in vivo mouse xenograft experiments to evaluate therapeutic efficacy and synergy with doxorubicin. Results: We identified pT199-NPM1 as a novel, highly expressed protein factor in ALT-positive OS tissues. NPM1 depletion impaired break-induced telomere replication and significantly reduced the viability of ALT-positive cells. ATR signaling phosphorylated NPM1 at Thr199, which stabilized POLD3 by preventing its ubiquitin-mediated degradation. Recruitment and function of pT199-NPM1 at telomeric damage sites required STN1, defining a CST/pT199-NPM1/POLD3 regulatory axis essential for ALT activity. Clinically, elevated Thr199 phosphorylation correlated with poor survival in OS patients, while expression of a phosphorylation-deficient T199A mutant failed to sustain ALT telomere maintenance. Pharmacological screening identified EPZ-6438, an EZH2 inhibitor, as a potent ALT suppressor that reduced NPM1 transcription, inhibited homologous recombination-mediated telomere synthesis, and suppressed OS cell proliferation. In mouse xenografts, EPZ-6438 enhanced OS cell sensitivity to doxorubicin, suggesting therapeutic synergy. Conclusions: This study uncovers a novel CST/pT199-NPM1/POLD3 regulatory module that is critical for ALT telomere maintenance in OS. Targeting NPM1 or its downstream effectors effectively suppresses ALT activity and enhances chemotherapy response. These findings provide new mechanistic insights into telomere regulation in ALT-positive tumors and highlight the therapeutic potential of NPM1-centered pathways in OS.

Human telomeres exhibit progressive shortening with each replication cycle. This phenomenon plays a critical role in the onset of senescence and the development of cancers. The measurement of absolute telomere length (TL) is not only serving as a marker of aging, but also holds substantial medical relevance. However, current TL measurement technologies face significant challenges, including limited precision, inability to resolve TL heterogeneity or distinguish telomeric signals from interstitial telomeric sequences (ITS), and data inconsistency. Single-molecule mechanical techniques have shown promise in manipulating DNA and providing precise contour length measurements of DNA, making them suitable for assessing TL quantitatively. In this study, we developed a method, named single-molecule terminal restriction fragment (smTRF) analysis, for measuring telomeres at single-molecule resolution. We applied smTRF to seven human cancer cell lines and successfully determined TL ranging from a few to tens of kilobases, highlighting the versatility and high-fidelity performance of the smTRF assay. The smTRF data were validated against results from standard TRF, qPCR, and Q-FISH analysis, demonstrating well agreement and confirming the assay's reliability in the measurement of average TL. To further test the robustness of smTRF, we measured TL distribution profiles for 48 individuals, establishing the smTRF assay as a reliable tool for the accurate and precise measurement of human TLs. The comprehensive telomere profiles obtained via the smTRF assay promise to provide in-depth insights into public health research, particularly in the study of aging, where TL serves as a critical biomarker.

BACKGROUND:Osteogenesis imperfecta (OI), as a disease of congenital bone dysplasia, is often accompanied by the abnormal alteration of bone absorption and bone formation. DNA methyltransferases (Dnmts) can regulate the gene expression involved in osteogenesis and osteoclastogenesis. Dnmts changes and their effects on bone cells under OI is poorly understood. METHODS:The Dnmts expression in adipose derived mesenchymal stem cells (ADSCs), bone marrow derived pre-osteoclasts (pre-Ocs) and femurs of Col1a2oim/+ and Col1a1+/-365 mice, both modeling mild OI types, were determined. The effects of azacitidine (Aza) administration and Dnmt3a knockdown by ShRNA on the osteogenic differentiation of ADSCs together with osteoclasts (Ocs) production of pre-Ocs were studied in vitro. The synthesis and secretion of collagen fibers of OI derived ADSCs were examined. The therapeutic outcomes of intraperitoneal (i.p.) infused Aza (1 mg/kg/2d) for 30 days were evaluated in OI mice. RESULTS:Obviously elevated expression of Dnmts, especially Dnmt3a, existed in ADSCs, pre-Ocs, and femurs isolated from OI modeled mice. Much more collagen molecules of mutant ADSCs were secreted into the extracellular medium post Aza addition. Both Aza administration and Dnmt3a knockdown effectively enhanced the bone-forming capacity of affected ADSCs and reduced Ocs formation of OI mice in vitro. Aza treatment apparently improved the femora microstructure and biomechanical properties, increased bone formation and decreased the number of Ocs in mice with OI. CONCLUSION:Highly expressed Dnmt3a contributed to the impaired osteogenesis and enhanced osteoclastogenesis of collagen defect-related OI. Aza medication effectively improved the femora phenotype of the two types of OI modeled mice partly by Dnmts inhibition and modulating cell stress response. These findings facilitated understanding the role of Dnmts alteration in skeletal pathological development of mild OI and preliminary confirmed the therapeutic potential of Dnmts depressants in mild OI treatment. Still, further researches are needed to explore the specific function of Dnmts in OI bones and clarify the benefits of Aza administration in OI treatment.

Background: Bronchiolitis obliterans syndrome (BOS), induced by a chronic rejection, remains a significant obstacle for end-stage lung diseases after lung transplantation. We have previously determined that the small non-coding mRNA (miRNA) miR-27a-3p maintained the immature state of dendritic cells (DCs) via the inter-leukin 10 (IL-10)-dependent regulatory pathway. Such status helped in preventing rejection and alleviating BOS. The present study explored mechanisms how miR-27a-3p may suppress the fibrosis as well as the maturation of DCs, ultimately attenuating BOS in vitro and in vivo. Methods/Results: In our tracheal transplantation mouse model, the expression of Smad2, Smad4, and alpha SMA were significantly decreased in the miR-27a-3p-transfected DCs (p < 0.0001, p = 0.0006, andp = 0.0002 respectively). Moreover, the expression of fibrosis markers (alpha-SMA, collagen I, and Fn) were potently inhibited in the miR-27a-3p-transfected NIH-3 T3 cells (p < 0.0001, p = 0.00148, andp < 0.0001 respectively). At the same time, reversed results were observed in the inhibitor group (p = 0.0002, p < 0.0001, and p < 0.0001 respectively), indicating that miR-27a-3p could directly inhibit myofibroblast differentiation. Furthermore, in the tracheal transplanted mice, the population of Treg cells was significantly decreased (p < 0.0001). In contrast, Th17 cells were down-regulated in the miR-27a-3p-transfected DCs group (p < 0.0001), accompanied by the decreased IL-17 levels (p = 0.0007) and the induction of TGF-beta 1 and IL-10 (p < 0.0001 and p = 0.0016 respectively). Further mechanistic studies indicated that miR-27a-3p altered the maturation of DCs by targeting TLR4 and IRAK (p < 0.0001 and p = 0.0002 respectively). Conclusions: Our study suggests that miR-27a-3p selectively blocked the TGF-beta 1/Smad pathways to suppress the myofibroblast differentiation and targeted the TRL4/IRAK4 pathway to restrain DCs maturation, thus attenu-ating BOS. Our findings suggest that miR-27a-3p is a potential active molecule on BOS management after lung transplantation.

Telomere length (TL) shortening is a pivotal indicator of biological aging and is associated with many human diseases. The genetic determinates of human TL have been widely investigated, however, most existing studies were conducted based on adult tissues which are heavily influenced by lifetime exposure. Based on the analyses of terminal restriction fragment (TRF) length of telomere, individual genotypes, and gene expressions on 166 healthy placental tissues, we systematically interrogate TL-modulated genes and their potential functions. We discover that the TL in the placenta is comparatively longer than in other adult tissues, but exhibiting an intra-tissue homogeneity. Trans-ancestral TL genome-wide association studies (GWASs) on 644,553 individuals identify 20 newly discovered genetic associations and provide increased polygenic determination of human TL. Next, we integrate the powerful TL GWAS with placental expression quantitative trait locus (eQTL) mapping to prioritize 23 likely causal genes, among which 4 are functionally validated, including MMUT, RRM1, KIAA1429, and YWHAZ. Finally, modeling transcriptomic signatures and TRF-based TL improve the prediction performance of human TL. This study deepens our understanding of causal genes and transcriptomic determinants of human TL, promoting the mechanistic research on fine-grained TL regulation. Variation in human telomere length has been well studied, but most previous studies have used adult telomere length. Here, the authors explore the genetic basis of telomere length in the placenta and find suggestive causal genes modulating human telomere length.

Osteogenesis imperfecta (OI) is an inherited congenital disorder, characterized primarily by decreased bone mass and increased bone fragility. Bone morphogenetic protein-2 (BMP-2) is a potent cytokine capable of stimulating bone formation, however, its rapid degradation and unanticipated in vivo effects restrict its application. The sustained release characteristic of silk fibroin (SF) microspheres may potentially address the aforementioned challenges, nevertheless they have not previously been tested in OI treatment. In the current investigation, recombinant BMP-2 (rBMP-2) loaded SF (rBMP-2/SF) microspheres-based release carriers were prepared by physical adsorption. The SF microparticles were characterized by scanning electron microscopy (SEM) and were investigated for their cytotoxicity behavior as well as the release profile of rBMP-2. The rBMP-2/SF microspheres were administered via femoral intramedullary injection to two genotypes of OI-modeled mice daily for two weeks. The femoral microstructure and histological performance of OI mice were evaluated 2 weeks later. The findings suggested that rBMP-2/SF spheres with a rough surface and excellent cytocompatibility demonstrated an initial rapid release within the first three days (22.15 ± 2.88% of the loaded factor), followed by a transition to a slower and more consistent release rate, that persisted until the 15th day in an in vitro setting. The factor released from rBMP-2/SF particles exhibited favorable osteoinductive activity. Infusion of rBMP-2/SF microspheres, as opposed to blank SF spheres or rBMP-2 monotherapy, resulted in a noteworthy enhancement of femoral microstructure and promoted bone formation in OI-modeled mice. This research may offer a new therapeutic approach and insight into the management of OI. However, further investigation is required to determine the systematic safety and efficacy of rBMP-2/SF microspheres therapy for OI.

目的 探索替格瑞洛(Ticagrelor)促进Ⅰ型成骨不全小鼠(Col1a1+/-365)骨形成的效果及作用机制.方法 选取 6 只 8周龄野生C57/BL小鼠作为正常对照组(WT),选取 12 只 8 周龄Col1a1+/-365小鼠,随机分为DMSO对照组和替格瑞洛治疗组,每组6 只,通过灌胃给药[2 mg/(kg·d)]进行治疗,DMSO对照组每天灌胃同等剂量DMSO.10 周后分离各组小鼠的股骨,通过Micro-CT检测股骨骨体积分数(BV/TV)、骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)、骨小梁模式因子(Tb.Pf)等参数的变化;采用HE染色检测股骨形态学变化;采用Trap染色检测破骨细胞的数量变化;采用Real-time PCR法检测股骨破骨特异基因Mmp9、Ctsk、Nfatc1 的表达情况.结果 ①与DMSO对照组比较,Ticagrelor治疗组BV/TV、Tb.N、Tb.Th和骨面积(B.Ar)均增高(P＜0.05),而Tb.Pf降低(P＜0.05).Ticagrelor治疗组小鼠与WT组小鼠BV/TV、Tb.N和Tb.Th差异无统计学意义(P＞0.05);②与DMSO对照组比较,Ticagrelor治疗组破骨细胞数量减少(P＜0.05),破骨特异基因Mmp9、Nfatc1 表达水平降低(P＜0.05).结论 替格瑞洛可以有效改善Col1a1+/-365小鼠的股骨微观结构,促进骨形成,其机制可能与抑制破骨细胞分化相关.

Osteogenesis imperfecta (OI) is a congenital bone dysplasia mainly caused by either defective production or assembly of type I collagen. The skeletal phenotypes especially fractures are often seen in OI adolescents. Studies have found that an increased number of osteoclasts and excessive bone resorption existed in collagen-related OI, which has not been well understood. Emerging evidence has suggested that inflammation may be associated with OI. We speculated that the bone marrow (BM) niche had similar inflammatory changes and performed RNA-sequencing (RNA-seq) in BM cells derived from young male mice to analyze the related differentially expressed genes (DEGs) and pathways. Data showed that there were 117 shared DEGs (Q ≤ 0.05, |log2FC| ≥ 1) in BM cells isolated from two types of OI murine models that respectively simulate different OI types. Gene Ontology (GO) (Q ≤ 0.05) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) (Q ≤ 0.05) analysis and real-time PCR validation indicated the dysregulated biology process of cellular response to interferon (Ifn) together with upregulated IL-17 signaling, tumor necrosis factor (Tnf) signaling and osteoclast differentiation in OI BM niche. Either defective collagen production or abnormal collagen assembly shared similar alterations in gene profiles and pathways involving inflammation and osteoclast activation. Data presented here not only contributed to understanding of the mechanism of the enhanced bone absorption in the bones of OI, but also provided more evidence to develop potential anti-inflammation therapies.

Short stature is the second conspicuous characteristic of osteogenesis imperfecta (OI), but the etiological mechanism is unclear. The proliferation of growth plate chondrocytes (GPCs) plays an essential role in longitudinal bone growth, and chondrocyte division deficiency can cause shortened limbs. However, few studies have reported the abnormal changes of growth plate and GPCs in OI. In this study, the cell proliferative performance of GPCs in heterozygous Col1a2oim/+ mice were studied and the underlying mechanism was explored by RNA-Sequencing. The results indicated that chondrocytes of Col1a2oim/+ background displayed impaired cell division when compared with cells of wild-type littermates. A group of differentially expressed genes involving chondrocyte proliferation related pathways including cell cycle, TGF-β signaling pathway and Hedgehog signaling pathway were identified. These dysregulated genes and pathways in GPCs of Col1a2oim/+ mice are likely to play an important role in their shortened long bones. Further investigations to reveal the effect of these genes on bone elongation not only facilitate the understanding of OI short stature, but also contribute to developing new treatments.

目的:对细胞生物学实验教学改革需求进行问卷调查.方法:应用“医学生对医学细胞生物学实验课程教学评估问卷”进行问卷调查.结果:71.2％的学生支持实验课教学改革,实验课程设置分值(t=2.518,P=0.012)和学生学习效果分值(t=2.108,P=0.036)是教学改革的影响因素.结论:学生具有较强的实验课教学改革意愿和需求.

ABSTRACT Osteogenesis imperfecta (OI) is a congenital genetic disorder mainly manifested as bone fragility and recurrent fracture. Mutation of COL1A1/COL1A2 genes encoding the type I collagen are most responsible for the clinical patients. Allogenic mesenchymal stem cells (MSCs) provide the potential to treat OI through differentiation into osteoblasts. Autologous defective MSCs have not been utilized in OI treatment mainly because of their impaired osteogenesis, but the latent mechanism has not been well understood. Here, the relative signaling abnormality of adipose-derived mesenchymal stem cells (ADSCs) isolated from OI type I mice (Col1a1+/−365 mice) was explored. Autologous ADSCs transfected by retrovirus carrying human COL1A1 gene was first utilized in OI therapy. The results showed that decreased activity of Yes-associated protein (YAP) due to hyperactive upstream Hippo kinases greatly contributed to the weakened bone-forming capacity of defective ADSCs. Recovered collagen synthesis of autologous ADSCs by COL1A1 gene modification normalized Hippo/YAP signaling and effectively rescued YAP-mediated osteogenesis. And the COL1A1 gene engineered autologous ADSCs efficaciously improved the microstructure, enhanced the mechanical properties and promoted bone formation of Col1a1+/−365 mice after femoral bone marrow cavity delivery and might serve as an alternative source of stem cells in OI treatment. © 2021 American Society for Bone and Mineral Research (ASBMR).

Background Polychlorinated biphenyls (PCBs) are persistent pollutants with carcinogenesis and mutagenesis effects which have been closely associated with PCBs-induced DNA damage. However, the detailed DNA damage events and corresponding pathway alterations under PCBs poisoning is still not well understood. Methods Whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) were used to explore genome wide variations and related pathway changes in HEK293T cells that challenged by 15 µM PCB153 for 96 h in vitro. Double strand breaks (DSBs) were measured by 53BP1 foci detection, altered pathways were confirmed by quantitative real-time PCR (qPCR). Results The results indicated that abundant copy number variations (CNVs), including four duplications and 30 deletions, occurred in PCB153-exposed HEK293T cells. Multiple large fragment deletions (>1 Mb) involving up to 245 Mb regions on many chromosomes. Missense mutations were found in six tumor susceptibility genes, two of which are key members participating in homologous recombination (HR) repair response, BRCA1 and BRCA2. RNA-seq data showed that PCB153 poisoning apparently suppressedHR repairing genes. Besides, 15 µM PCB153 exposure significantly increased 53BP1 foci formation and effectively reduced BRCA1, RAD51B and RAD51C expression, indicating an elevated DSBs and impaired HR repairing. Conclusion This study firstly reported multiple large chromosomal deletions and impaired HR repairing in PCB153-exposed HEK293T cells, which provided a new insight into the understanding of early response and the mechanism underlying PCB153 genotoxicity. The chromosomal instabilities might be related to the impaired HR repairing that induced by PCB153; however, further investigations, especially on actual toxic effects of human body, are needed to confirm such speculation.

BACKGROUND:Osteogenesis imperfecta (OI) is a rare genetic disorder characterized by bone fragility and deformity. Mesenchymal stem cells (MSCs) infusion can improve bone performance mainly due to their differentiation into osteoblasts in OI therapy. The osteoinductive activity of NELL1 have benefited various bone defect and osteoporotic models by promoting bone formation. The present study investigated the efficacy of combined use of NELL1 and adipose-derived mesenchymal stem cells (ADSCs) in OI treatment.METHODS:Lentiviral vector carrying mouse Nell1 gene was constructed and lentivirus were used to infect ADSCs. The osteogenic capacity of MC3T3-E1 and ADSCs stimulated by recombinant mouse NELL1 protein (rmNELL1) and Nell1 gene genetically modified ADSCs (lenti-Nell1-ADSCs) were estimated by real-time quantitative PCR. Thirty adult male OI type I mice with single Col1a1 gene knockout were randomly divided into five groups and received intravenously injected PBS, rmNELL1 (1.25 mg/Kg), ADSCs (2 × 105 cells per mice), rmNELL1 (1.25 mg/Kg) combined with ADSCs (2 × 105 cells per mice), or lenti-Nell1-ADSCs (2 × 105 cells per mice) respectively. Six wildtype (WT) mice served as positive control. Bone formation was examined after 4 weeks using micro-CT, histological and immunohistochemical methods.RESULTS:Three osteoblast related genes of MC3T3-E1 and ADSCs were significantly up-regulated by rmNELL1 in vitro. Lenti-Nell1-ADSCs showed greatly enhanced osteogenic differentiation capacity. The infused lenti-Nell1-ADSCs could migrate to femur and differentiate into ALPL-positive cells. Systemic administration of rmNELL1 combined with ADSCs or lenti-Nell1-ADSCs markedly improved the femoral microstructure and promoted bone formation through increasing the ALPL and osteocalcin (OCN) expression, much better than mice that received single rmNELL1 or ADSCs. And Nell1 gene engineered ADSCs achieved slightly better outcomes than that of combinative use of rmNELL1 and ADSCs.CONCLUSIONS:NELL1 and ADSCs exhibited synergistic effect on stimulating bone formation of OI mice, which might provide an alternative strategy in OI treatment. Compared with dose escalation or multiple administration of rmNELL1, lentivirus-mediated long term expression of NELL1 might be more feasible and convenient. However, further studies are needed to confirm the safety and optimize the therapeutic regime.

Abstract Bronchiolitis obliterans (BO), is a chronic rejection phenotype characterized by chronic small airway fibrous obliteration, hinders the patients who suffer from lung transplanting for surviving longer. Cell‐based therapies using dendritic cells (DCs) and T regulatory cells (Tregs) have been developed to regulate allograft rejection, and to induce and maintain immune tolerance. In the present study, the effects of mir‐27a‐3p on regulating DCs as well as resulting effects on BO attenuation have been investigated. According to our reporter assays, the potential targets of mir‐27a‐3p were Smad2, sprouty2, and Smad4, respectively. Furthermore, sprouty2 inhibition by mir‐27‐3p indirectly activated extracellular regulated protein kinases (ERK) and increased IL‐10 production in DCs. This led to a positive feedback loop that maintained the immature state of DCs via IL‐10/JAK/STAT3 pathway, and caused an increase in Foxp3+CD4+ T cells amount as well as TGF‐β level. Furthermore, mir‐27a‐3p regulated TGF‐β function, inhibited TGF‐β/Smad pathway, and suppressed myofibroblast differentiation through influencing the function of Smad2 and Smad4. In short, the study indicated the effect of mir‐27a‐3p on suppressing DC maturation, which implicated the potential clinical application in treating postlung transplant BO.

This paper elaborates the specific implementation process of the "research-based learning" teaching reform of genetic experiment in medical undergraduate education, including the change of teaching philosophy among teachers, the integration and expansion of experimental contents, the innovation of classroom-teaching model, the compilation of proper textbook, the update of assessment methods and the establishment of evaluation mechanism for teaching and so on. Preliminary research shows that RBL teaching reform can stimulate medical students' interest and potential in learning, and improve their practical and scientific research innovation ability.

目的 医学遗传学是一门实验为基础的必修课,本文对遗传学实验教学效果及改革需求进行问卷调查,为遗传学实验课进行改革,提高实验课教学效率和医学生的创新思维与实验操作技能提供参考依据.方法 根据调查目的自编“医学生对医学遗传学实验课程教学评估问卷”,对医学遗传学实验课的认知情况、实验课程设置评价、学习效果评价、实验课教学效果评价、实验课教学改革评价等内容进行问卷调查.结果 对147名同学进行问卷调查,对课程设置(4～20分)、学习效果(8 ～40分)、教学效果(4～20分)、教学改革(7～35分)进行评分,总体分值分别为8.82±1.60、14.39±3.10、6.22+1.71、14.37±3.77.不同综合测评成绩间学生的比较结果显示课程设置和教学效果分值差别无统计学意义(P＞0.05),学习效果和教学改革分值差别具有统计学意义(P＜0.05).综合成绩为≥90分,80～90分两组学习效果和教学改革分值明显低于后两组.同时,调查结果显示有98.6％的同学认为有必要开设遗传学实验课程,60.5％的同学支持对实验课内容进行教学改革.结论 学生对医学遗传学实验课学习效果、教学效果均较满意.学习成绩更好的学生对学习效果的自我评价更好,接受调查的学生具有较强的教学改革意愿.

Acute lung injury (ALI)-triggered pulmonary injury has been associated with high mortality, despite advances in drug treatment and supportive therapy. Remarkable progress has been made in attenuating the inflammatory injury associated with ALI using mesenchymal stem cells (MSCs)-based cell and gene therapy. However, to date, the benefits of interleukin-35 (IL-35)-modified MSCs in ALI intervention have not been investigated. In the present study, adult male C57BL/6 mice randomly received intravenous infusion of adipose-derived mesenchymal stem cells (ADSCs) constitutively expressing IL-35 (IL-35-GFP-ADSCs) or GFP (GFP-ADSCs) via retrovirus-mediated transduction (8 × 10 5 cells per mice) or isotonic saline 7 days before ALI modeling to investigate the effect and related mechanism. ALI was performed by lipopolysaccharide (LPS) inhalation for 24 h. Normal mice served as the sham group. The results indicated that compared with GFP-ADSCs, IL-35-modified ADSCs significantly increased cellular and pulmonary IL-10 and IL-35 production. Pretreatment with IL-35-ADSCs markedly reduced body weight loss, pulmonary wet/dry weight ratio and pathological injury. The PO 2 was rescued to normal levels in mice that received IL-35-ADSCs. IL-35-ADSCs infusion apparently inhibited IL-6 release, protein leakage and MPO activity but greatly elevated IL-35 level in the bronchoalveolar lavage fluid (BALF). Splenic regulatory T cells in IL-35-ADSCs-pretreated mice got effective increase. Moreover, IL-35-ADSCs pretreatment remarkably inhibited neutrophil and macrophage infiltration and greatly decreased IL-6, tumor necrosis factor α (TNF-α) and Toll-like receptor 4 (TLR4) expression. In conclusion, pretreatment with IL-35-engineered ADSCs provided effective protection against LPS-induced ALI through suppression of pulmonary inflammation and, thus, might be a promising strategy to improve outcomes after ALI. The enhanced paracrine and immunosuppressive capacity of IL-35-ADSCs might contribute to their beneficial effects. However, further studies are needed to illuminate the detailed mechanism.

Osteogenesis imperfecta (OI) type I caused by the null allele of COL1A1 gene is in the majority in clinical OI cases. Currently, heterozygous Mov-13 mice generated by virus insertion in the first intron of col1a1 is the exclusive model to modulate OI type I, in spite of the gradually recovered bone mineral and mechanical properties. A newly designed heterozygous col1a1±365 OI mouse was produced in the present study by partial exons knockout (exon 2-exon 5, 365 nt of mRNA) using CRISPR/Cas9 system. The deletion resulted in generally large decrease in type I collagen synthesis due to frameshift mutation and premature chain termination, closely mimicking the pathogenic mechanism in affected individuals. And the strain possessed significantly sparse mineral scaffolds, bone loss, lowered mechanical strength and broken bone metabolism by 8 and 20 weeks compared to their littermates, suggesting a sustained skeletal weakness. Notably, the remarkable down-regulation of Yes-associated protein (YAP), one of the key coactivator in Hippo signaling pathway, was first found both in the femur and adipose derived mesenchymal stem cells (ADSCs) under osteogenic differentiation of col1a1±365 mice, which might be responsible for the reduced osteogenic potential and brittle bones. Still, further research was needed in order to illuminate the underlying mechanism.

Objective The benefits of IL-35 treatment have been verified in multiple animal models of diseases, while its influence on T cells immunity under normal condition still needs to be elucidated. The present study was designed to investigate the effects modulating IL-35 levels in vivo and in vitro on T cells, response and also the effects on T cells subsets in normal mice. Methods A plasmid pMSCV-IL-35-GFP carrying mouse linear IL-35 fragment with two subunits joint together was constructed and the heterodimer expression was confirmed. Normal mice were randomly divided into three groups and received an intravenous injection of PBS, pMSCV-GFP and pMSCV-IL-35-GFP respectively. After 72 h, spleen tissues and peripheral blood were harvested for following analysis. Meanwhile, splenic T cells were isolated and incubated with 10, 30, or 50 ng/mL recombinant IL-35 factor for 24 h with the addition of anti-CD3/CD28 in vitro. T-cell subsets were assessed by Fluorescence activated cell sorting (FACS) and related cytokines together with effector molecules were determined by real time PCR. Results Western blotting confirmed a 52 kDa band in the cell lysate of HEK 293T transducted with pMSCV-IL-35-GFP plasmid, indicating a successful expression of IL-35. Ebi3 and IL-12A, two subunits of IL-35, could be identified 72 h post DNA injection. IL-35 upregulation in vivo effectively inhibit CD4+ and CD8+ T cell proliferation and Th1 cytokine secretion. Effector molecules of CD8+ T cells were also remarkably suppressed. On the contrary, high level of IL-35 significantly induced CD4+ CD25+ Tregs and Th2 enhancement. The in vitro study provided similar results. Conclusion The results indicated Th1 and CD8+ T cell inhibition and Th2 and Tregs bias in the presence of IL-35 under a normal state which partly contributed to its therapeutic potential.