Downregulation of MicroRNA-495 Alleviates IL-1β Responses among Chondrocytes by Preventing SOX9 Reduction

PurposeOur previous work demonstrated that miRNA-495 targets SOX9 to inhibit chondrogenesis of mesenchymal stem cells. In this study, we aimed to investigate whether miRNA-495-mediated SOX9 regulation could be a novel therapeutic target for osteoarthritis (OA) using an in vitro cell culture model.Materials and MethodsAn in vitro model mimicking the OA environment was established using TC28a2 normal human chondrocyte cells. Interleukin-1β (IL-1β, 10 ng/mL) was utilized to induce inflammation-related changes in TC28a2 cells. Safranin O staining and glycosaminoglycan assay were used to detect changes in proteoglycans among TC28a2 cells. Expression levels of COX-2, ADAMTS5, MMP13, SOX9, CCL4, and COL2A1 were examined by qRT-PCR and/or Western blotting. Immunohistochemistry was performed to detect SOX9 and CCL4 proteins in human cartilage tissues obtained from patients with OA.ResultsmiRNA-495 was upregulated in IL-1β-treated TC28a2 cells and chondrocytes from damaged cartilage tissues of patients with OA. Anti-miR-495 abolished the effect of IL-1β in TC28a2 cells and rescued the protein levels of SOX9 and COL2A1, which were reduced by IL-1β. SOX9 was downregulated in the damaged cartilage tissues of patients with OA, and knockdown of SOX9 abolished the effect of anti-miR-495 on IL-1β-treated TC28a2 cells.ConclusionWe demonstrated that inhibition of miRNA-495 alleviates IL-1β-induced inflammatory responses in chondrocytes by rescuing SOX9 expression. Accordingly, miRNA-495 could be a potential novel target for OA therapy, and the application of anti-miR-495 to chondrocytes could be a therapeutic strategy for treating OA.     

Decreased expression of microRNA-130a correlates with TNF-α in the development of osteoarthritis

Objective: Increased expression of tumor necrosis factor a (TNF-α) has emerged as an important inflammatory factor in osteoarthritis (OA) and other joint diseases. The study was performed to investigate whether the expression of TNF-α in human chondrocytes was regulated by miRNAs. Methods: MiRNA-130a and TNF-α expression in cartilage specimens was examined in patients with knee osteoarthritis, chondrocytes and osteoarthritis rat model. Chondrocytes were transfected with siRNAs as a gene silencing methods. Expression of genes and proteins were analyzed by real-time PCR and western blotting respectively. Results: Increased TNF-α and decreased miRNA-130a were observed in tissues from osteoarthritis patients. Moreover, we found a highly negitive correlation between miRNA-130a and TNF-α. Next, miRNA-130a loss-of-function increased the expression of TNF-α and promoted inflammation in chondrocytes. It was reasonable that miRNA-130a regulated a distinct underlying molecular and pathogenic mechanism of OA by forming a negative feedback loop with TNF-α. Furthermore, there were the abnormalities of bone metabolism in OA rat, which showed the miRNA-130a and TNF-α dysfunction that was one of important factors for the occurrence and development of OA. Conclusions: Our results indicated that miR-130a played an important role in regulating the expression of TNF-α in human chondrocytes and identified miR-130a as a novel therapeutic target in OA.     

Matrine inhibits IL-1β-induced expression of matrix metalloproteinases by suppressing the activation of MAPK and NF-κB in human chondrocytes in vitro

Interleukin (IL)-1β plays an important role in promoting osteoarthritis (OA) lesions by inducing chondrocytes to secrete matrix metalloproteinases (MMPs), which degrade the extracellular matrix and facilitate chondrocyte apoptosis. Matrine was shown to exert anti-inflammatory effects. However, the role of matrine in OA is still unclear. Therefore, in this study, we investigated the effects of matrine on the expression of MMPs in IL-1β-treated human chondrocytes and the underlying mechanism. The cell viability of chondrocytes was detected by MTT assay. The cell apoptosis of chondrocytes was measured by flow cytometric analysis. The protein production of MMPs was determined by ELISA. The protein expression of phosphorylation of mitogen-activated protein kinases (MAPKs) and the inhibitor of kappaB alpha (IκBα) was determined by Western blot. Matrine significantly inhibited the IL-1β-induced apoptosis in chondrocytes. It also significantly inhibited the IL-1β-induced release of MMP-3 and MMP-13, and increased the production of TIMP-1. Furthermore, matrine inhibits the phosphorylation of p-38, extracellular regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and IκBα degradation induced by IL-1β in chondrocytes. Taken together, our results show that matrine inhibits IL-1β-induced expression of matrix metalloproteinases by suppressing the activation of MAPK and NF-κB in human chondrocytes in vitro. Therefore,-matrine may be beneficial in the treatment of OA.     

The role of microRNA-23b in the differentiation of MSC into chondrocyte by targeting protein kinase A signaling

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is critical for successful cartilage regeneration. Several methods have been developed to attempt to chondrogenic differentiation, because chondrogenic differentiated cells can form stable cartilage and induce expression of a cartilage-specific phenotype. In this study, we found that both H-89 and microRNA-23b induced differentiation into chondrocyte of hMSCs through down-regulation of protein kinase A (PKA) signaling. The small molecule, H-89, was identified by PCA analysis as a potential mediator of chondrogenic differentiation. H-89 induced the expression of the chondrocyte marker, aggrecan, as well as miR-23b. We searched that miR-23b regulates protein level of PKA. When miR-23b was transfected into hMSCs, chondrogenic differentiation was induced. We confirmed the target of miR-23b using a reporter gene assay. Furthermore, not only H-89 or miR-23b-treated cells, but also cell co-treated with H-89 and miR-23b differentiated into chondrocytes. Our results indicate that H-89 induces the expression of endogenous miR-23b, thereby inducing chondrogenic differentiation by negatively inhibition of PKA signaling.     

Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9

Chondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.     

CaMKII plays a part in the chondrogenesis of bone marrow-derived mesenchymal stem cells

Aims: The purpose of the study is to observe the functions of calcium/calmodulin dependent protein kinase II (CaMKII) in the induced chondrogenic differentiation of bone marrow derived mesenchymal stem cells (BMSCs). Methods: BMSCs was in vitro isolated and cultured for induced chondrogenesis. Western blot was used to ascertain the expression of CaMKII and phosphorylated CaMKII (PCaMKII, activatory CaMKII) in chondrogenic induced BMSCs. MTT method was utilized to observe the impact of CaMKII on the proliferation of BMSCs. The generation of cartilage matrix in BMSCs cells was detected by toluidine blue staining. The levels of cartilage marker genes COL2A1, Aggrecan and SOX9 in BMSCs were gained by real-time fluorescence quantitative polymerase chain reaction (RT-QPCR). Finally, BMSCs proliferation, cartilage matrix generation and the changes of COL2A1, Aggrecan and SOX9 were surveyed after CaMKII being blocked by CaMKII inhibitor KN93. Results: Expression of CaMKII and PCaMKII could be found in chondrogenic induced BMSCs. CaMKII had no significant influence on BMSCs proliferation, but the toluidine blue staining was obviously lighter, indicating a significant decline in the expression of COL2A1, Aggrecan and SOX9. Conclusion: As one of the factors influencing the chondrogenic capacity of BMSCs, CaMKII does not impact on BMSCs proliferation, but it can inhibit the chondrogenic ability of BMSCs by influencing its differentiation.     

SOX6和SOX9基因转染对人原发性骨关节炎关节软骨间充质祖细胞增殖和成软骨分化的调控作用

目的观察SOX6和SOX9基因转染对原发性OA关节软骨MPCs的促增殖、分化作用,为通过调控关节软骨MPCs以防治原发性OA提供理论依据。方法分别以pAdTrack-CMV-SOX6、SOX9腺病毒穿梭质粒构建SOX6、SOX9基因,并感染原发性OA关节软骨MPCs,比较基因感染组和未感染组成软骨诱导分化后TB、Ⅱ型胶原以及Ⅱ型胶原mRNA表达的变化。结果SOX6和SOX9能够分别稳定感染OA关节软骨MPCs;经二者分别感染的关节软骨MPCs成软骨诱导分化后,其TB染色、Ⅱ型胶原染色呈强阳性表达,未基因感染细胞为弱阳性着色;SOX6基因感染原发性OA关节软骨MPCs的Ⅱ型胶原mRNA表达量为未基因感染细胞的3.8倍(P0.01),SOX9基因为未感染细胞的5.15倍(P0.01)。结论构建的SOX6、SOX9基因序列与基因库报道序列完全一致;SOX6和SOX9能稳定感染原发性OA关节软骨MPCs,并显著促进感染细胞成软骨分化;提示通过适宜浓度的bFGF、TGF-β1对原发性OA关节软骨MPCs的作用及SOX6和SOX9基因感染,可能具有促进原发性OA关节软骨损伤修复的作用。     

Conditioned Medium from Chondrocyte/Scaffold Constructs Induced Chondrogenic Differentiation of Bone Marrow Stromal Cells

For the application of bone marrow stromal cells (BMSCs) in cartilage tissue engineering, it is imperative to develop efficient strategies for their chondrogenic differentiation. In this study, the conditioned media derived from chondrocyte/scaffold constructs were used to direct chondrogenic differentiation of BMSCs. The porcine articular chondrocytes were seeded on the PGA/PLA scaffolds to form chondrocyte/scaffold constructs and were cultured to form engineered cartilage in vitro. The culture media were collected as conditioned media and used for chondrogenic induction of BMSC pellets (experimental group, Exp.). The chondrocyte pellets and BMSC pellets were cultured routinely as positive control (PC) and negative control (NC), respectively. After 4 weeks, the wet weight and GAG content in Exp. group and PC group were significantly higher than that in NC group. Histological and immunohistochemical analysis showed that cartilaginous tissue was formed with typical cartilage lacuna structure and positive staining of collagen Type II (Col II) in the peripheral area of the BMSC pellets in Exp. group. Gene expression of Sox9, Col II, and COMP in Exp. group and PC group were significantly higher than that in NC group. The growth factors in the conditioned media derived from human costal chondrocytes‐scaffold constructs were tested by protein microassay. The conditioned media contained low levels of TGF‐β1,2,3, IGF‐1 and high levels of IGF‐2, FGF‐4, and IGFBP4,6, and so forth. The soluble factors derived from the engineered cartilage can induce chondrogenic differentiation of BMSCs independently. Many cytokines may function in chondrogenesis in a coordinated way. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

小鼠间充质干细胞三系分化中SOX9与Ⅱ型胶原mRNA的定量

背景：研究表明,软骨中的主要成分Ⅱ型胶原的基因-Col2a1在软骨细胞中的表达与SOX9 的浓度呈剂量依赖正相关关系。 目的：通过成骨、成软骨、成脂肪诱导干细胞分化,分析3种分化过程及不同时期的SOX9与Ⅱ型胶原 mRNA含量的变化,探讨SOX9在不同时空分布的表达规律及与Ⅱ型胶原的相关关系。 方法：取4周龄昆明小鼠骨髓间充质细胞,体外培养得到间充质干细胞并传达至第3代,对间充质干细胞进行流式细胞仪鉴定细胞表型,共分3组每组设3个时间段,通过成骨、成软骨、成脂肪3种诱导培养液对3组细胞进行诱导,另设不进行诱导的细胞作为对照组。分别在诱导3,7,14 d后收集提取细胞的总RNA,通过RT-PCR进行SOX9与Ⅱ型胶原的mRNA定量检测,同时对诱导后的细胞进行染色、免疫荧光染色,观察其分化状态及相关统计分析。 结果与结论：第3代骨髓间充质干细胞生长良好,流式细胞仪鉴定细胞表型证实为干细胞,对诱导后细胞进行染色、免疫荧光染色结果证实细胞分化为骨、软骨、脂肪细胞。经RT-PCR检测,在3组诱导分化细胞中SOX9 mRNA含量由高到低分别是成软骨、成骨、成脂肪,Ⅱ型胶原 mRNA含量由高到低分别是成软骨、成脂肪、成骨。在成软骨分化中SOX9在3,7 d表达不断升高,14 d呈下降趋势。Ⅱ型胶原在3,7,14 d均逐渐升高。在成骨分化中SOX9 mRNA含量随着时间推移而增加,而Ⅱ型胶原则随着时间推移而不断降低。在成脂肪分化中SOX9 mRNA表达与对照组比较差异无显著性意义(P > 0.05);而Ⅱ型胶原的表达没有规律可循,时间点的延伸及检测未观察到。结果提示,SOX9在软骨分化中作用优于成骨、成脂肪组,且软骨分化中SOX9与Ⅱ型胶原存在相关性,可能在软骨分化的早期Ⅱ型胶原随着SOX9的变化而变化;且软骨分化和成骨分化过程中SOX9可能起到了一个互相协调促进平衡的关键作用。     

Shikonin protects chondrocytes from interleukin-1beta-induced apoptosis by regulating PI3K/Akt signaling pathway

Chondrocyte apoptosis is mostly responsible for the development and progression of osteoarthritis. IL-1β is generally served as an agent that induces chondrocyte apoptosis. Shikonin exerts its anti-inflammatory effect on cartilage protection in vivo. We aimed to explore the protective effect of shikonin on interleukin-1beta (IL-1β)-induced chondrocyte apoptosis and the potential molecular mechanisms. Chondrocytes were isolated from the joints of newborn Sprague-Dawley rats. The MTT assay and LDH cell death assay were used to determine the cell viability and chondrocyte apoptosis was detected by Annexin-V/PI staining and nucleosomal degradation. The contents of phosphorylated-PI3K (p-PI3k), phosphorylated-Akt (p-Akt), Bcl-2, Bax, and cytochrome c were detected by Western blotting. A quantitative colorimetric assay was used to detect the caspase-3 activity. Our results showed that pretreatment with shikonin (4 μM) inhibited cytotoxicity and apoptosis induced by IL-1β (10 ng/ml) in chondrocytes. Shikonin pretreatment also decreased the activity of IL-1β that decreased Bcl-2 expression and levels of p-PI3K and p-Akt, and increased Bax expression, cytochrome c release, and caspase-3 activation. It also reversed the activity of IL-1β that promoted the synthesis of matrix metalloproteinase-13 and inhibited the expression of tissue inhibitor of metalloproteinase-1 expression, with the net effect of suppressing extracellular matrix degradation. These data suggested that shikonin may protect chondrocytes from apoptosis induced by IL-1β through the PI3K/Akt signaling pathway, by deactivating caspase-3.     

Xenogeneic chondrocytes promote stable subcutaneous chondrogenesis of bone marrow-derived stromal cells

The local microenvironment may change the ultimate fate of engineered cartilage differentiated from bone marrow stromal cells (BMSCs) after subcutaneous implantation. Chondrogenically differentiated BMSCs directed by growth factors or low-intensity ultrasound are apt to fibrose or vascularize in the subcutaneous environment, while BMSCs implanted in articular cartilage defects can form stable cartilage. We hypothesized that chondrocytes would provide an ideal chondrogenic environment, and thus promote the maintenance of the chondrocytic phenotype in ectopia. To test this hypothesis, we developed a new method to promote chondrocyte development from BMSCs in a chondrogenic environment produced by xenogeneic chondrocytes and compared the subcutaneous chondrogenesis of BMSCs mediated by xenogeneic chondrocytes with that produced by growth factors. These results indicate that subcutaneous chondrogenesis of BMSCs directed by xenogeneic chondrocytes is more effective than that induced by growth factors. BMSCs induced by xenogeneic chondrocytes formed relatively mature cartilage before or after implantation, following 4 weeks of culture, which reduced the induction time in?vitro and led to maintenance of a stable cartilage phenotype after subcutaneous implantation.     

Effects of co-culturing BMSCs and auricular chondrocytes on the elastic modulus and hypertrophy of tissue engineered cartilage

Co-culture of BMSCs and chondrocytes is considered as a promising strategy to generate tissue engineered cartilage as chondrocytes induce the chondrogenesis of BMSCs and inhibit the hypertrophy of engineered cartilage. Because the tissue specific stem/progenitor cells have been isolated from mature tissues including auricular cartilage, we hypothesized that adding stem cells to auricular chondrocytes in co-culture would also enhance the quality of engineered cartilage. In the present study, using the histological assay, biomechanical evaluation, and quantitative analysis of gene expression, we compared different strategies of auricular chondrocytes, BMSCs induction, and co-culture at different ratios on PGA/PLA scaffolds to construct tissue engineered elastic cartilage in vitro and in vivo. The up-regulation of RUNX2 and down-regulation of SOX9 were found in BMSCs chondrogenic induction group, which might imply a regulatory mechanism for the hypertrophy and potential osteogenic differentiation. Engineered cartilage in co-culture 5:5 group showed the densest elastic fibers and the highest Young's modulus, which were consistent with the expression profile of cartilage matrix-related genes including DCN and LOXL2 genes. Moreover, the better proliferative and chondrogenic potentials of engineered cartilage in co-culture 5:5 group were demonstrated by the stronger expression of Ki67 and Dlk1.     

Electroporation-mediated transfer of SOX trio genes (SOX-5, SOX-6, and SOX-9) to enhance the chondrogenesis of mesenchymal stem cells

The purpose of this study was to test the hypothesis that the SOX trio genes (SOX-5, SOX-6, and SOX-9) have a lower level of expression during the chondrogenic differentiation of mesenchymal stem cells (MSCs) compared with chondrocytes and that the electroporation-mediated gene transfer of SOX trio promotes chondrogenesis from human MSCs. An in vitro pellet culture was carried out using MSCs or chondrocytes at passage 3 and analyzed after 7 and 21 days. Then, MSCs were transfected with SOX trio genes and analyzed for the expression of chondrogenic markers after 21 days of in vitro culture. Without transforming growth factor-β1, the untransfected MSCs had a lower level of SOX trio gene and protein expression than chondrocytes. However, the level of SOX-9 gene expression increased in MSCs when treated with transforming growth factor-β1. GAG level significantly increased 7-fold in MSCs co-transfected with SOX trio, which was corroborated by Safranin-O staining. SOX trio co-transfection significantly increased COL2A1 gene and protein and decreased COL10A1 protein in MSCs. It is concluded that the SOX trio have a significantly lower expression in human MSCs than in chondrocytes and that the electroporation-mediated co-transfection of SOX trio enhances chondrogenesis and suppresses hypertrophy of human MSCs.     

Effect of dynamic loading on MSCs chondrogenic differentiation in 3-D alginate culture

Mesenchymal stem cells (MSCs) are regarded as a potential autologous source for cartilage repair, because they can differentiate into chondrocytes by transforming growth factor-beta (TGF-β) treatment under the 3-dimensional (3-D) culture condition. In addition to these molecular and biochemical methods, the mechanical regulation of differentiation and matrix formation by MSCs is only starting to be considered. Recently, mechanical loading has been shown to induce chondrogenesis of MSCs in vitro. In this study, we investigated the effects of a calibrated agitation on the chondrogenesis of human bone MSCs (MSCs) in a 3-D alginate culture (day 28) and on the maintenance of chondrogenic phenotypes. Biomechanical stimulation of MSCs increased: (i) types 1 and 2 collagen formation; (ii) the expression of chondrogenic markers such as COMP and SOX9; and (iii) the capacity to maintain the chondrogenic phenotypes. Notably, these effects were shown without TGF-β treatment. These results suggest that a mechanical stimulation could be an efficient method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering in a 3-D environment. Additionally, it appears that MSCs and chondrocyte responses to mechanical stimulation are not identical.     

microRNA-25-3p suppresses osteogenic differentiation of BMSCs in patients with osteoporosis by targeting ITGB3

This study was conducted to investigate the impact of the microRNA (miR)-25–3p/ITGB3 axis on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) from patients with osteoporosis (OP). BMSCs isolated from the bone marrow of healthy controls and OP patients were identified by flow cytometry, in which ITGB3 mRNA and miR-25–3p expression was detected by RT-qPCR and ITGB3, Runx2, OPN, ALP, and OSX protein expression by western blot. The binding between ITGB3 and miR-25–3p was assessed by dual-luciferase reporter gene and Ago2-RIP assays. BMSC osteogenic differentiation was observed by alizarin red staining and ALP activity. The differentiation of BMSCs to adipocytes and chondrocytes was measured by oil red O staining and alcian blue staining, respectively. BMSCs were successfully isolated from the bone marrow of healthy controls (normal-BMSCs) and OP patients (OP-BMSCs). ITGB3, Runx2, OPN, ALP, and OSX expression was poorer and miR-25–3p expression was higher in OP-BMSCs than in normal-BMSCs. Mechanistically, ITGB3 was negatively targeted by miR-25–3p. After osteogenic, adipogenic, and chondrogenic differentiation of BMSCs were successfully induced, adipogenic differentiation increased and osteogenic and chondrogenic differentiation decreased in OP-BMSCs compared with normal-BMSCs. Overexpression of ITGB3 facilitated mineralized nodule formation and elevated ALP activity and Runx2, OPN, and ALP expression in OP-BMSCs. miR-25–3p upregulation diminished mineralized nodule formation, ALP activity, and Runx2, OPN, and ALP expression in OP-BMSCs and normal-BMSCs, which was annulled by additional ITGB3 overexpression. miR-25–3p targets ITGB3, thereby suppressing osteogenic differentiation of BMSCs from OP patients.     

骨髓间充质干细胞向软骨细胞分化过程中miR130α的表达

目的诱导骨髓间充质干细胞向软骨细胞分化,初步探索miR130a在骨髓间充质干细胞向软骨细胞分化过程中的调控作用。方法体外TGF-β1诱导骨髓间充质干细胞向软骨细胞分化,免疫荧光法和免疫组化染色鉴定Ⅱ型胶原,阿辛兰染色鉴定氨基葡聚糖。用Real-time PCR法检测细胞miR130a的表达。结果骨髓间充质干细胞在TGF-β1诱导下可分化为软骨细胞。培养7 d后,诱导培养组细胞miR130a表达的水平显著低于培养前的水平(P<0.05)。结论骨髓间充质干细胞体外诱导可以分化为软骨细胞,在向软骨细胞分化的早期,miR130a表达降低伴随着软骨细胞的分化,提示与软骨形成的过程有关。     

p53/miR-502-5p/TRAF2通路对骨关节炎软骨细胞损伤的影响

目的:探究微小RNA-502-5p(miR-502-5p)对白细胞介素-1β(IL-1β)诱导的骨关节炎(OA)软骨细胞损伤的影响。方法:Western blot和RT-q PCR检测骨关节炎患者软骨组织和IL-1β诱导的软骨细胞中p53和miR-502-5p的表达;分离培养软骨,并分组处理细胞,分别用MTT法检测细胞活力,流式细胞术检测细胞凋亡,Western blot检测炎性因子和细胞外基质(ECM)相关蛋白的表达,另外,萤光素酶报告实验检测miR-502-5p对p53及肿瘤坏死因子受体相关因子2(TRAF2)的调控作用。结果:与正常软骨组织相比,OA患者软骨组织中miR-502-5p的水平显著降低,p53和TRAF2水平则明显升高(P0.05)。IL-1β处理软骨细胞后,细胞活力下降、细胞凋亡率增加、IL-6、IL-8和TNF-α的蛋白表达水平显著上升;Ⅱ型胶原和蛋白聚糖的蛋白表达显著下调,基质金属蛋白酶(MMP)-3、MMP-9和MMP-13等的蛋白表达显著上调,miR-502-5p mimic转染反转了IL-1β对软骨细胞的这些作用。此外,p53与miR-502-5p之间存在负反馈调节,同时miR-502-5p能够靶向抑制TRAF2的水平。TRAF2沉默同样反转了IL-1β对软骨细胞增殖、凋亡、炎症反应以及ECM相关蛋白表达的影响。结论:调节p53/miR-502-5p/TRAF2通路能够减轻IL-1β诱导的骨关节软骨细胞损伤。     

共沉默miR-221-3p/222-3p表达抑制骨髓间充质干细胞增殖及促成软骨分化

背景：基于干细胞的组织工程技术作为治疗骨关节损伤修复的有效手段,具有广泛的应用前景。研究表明miRNA在调控干细胞向软骨分化过程中具有重要的作用。 目的：探讨共感染慢病毒介导的反义miR-221-3p/222-3p(microRNA-221-3p,microRNA-222-3p)对人骨髓间充质干细胞成软骨分化的作用,为临床软骨损伤修复提供新的策略。 方法：利用miRNA基因芯片技术筛选转化生长因子β3诱导人骨髓间充质干细胞成软骨分化过程中不同阶段表达差异的miRNA,并利用荧光实时定量PCR(RT-qPCR)验证;构建慢病毒人反义miR-221-3p/222-3p载体(Lv-miR-221-3p-inhibition,Lv-miR-222-3p-inhibition)并共转染人骨髓间充质干细胞,空载体组(Lv-GFP)以及未转染组作为对照。利用CCK-8法检测沉默miR-221-3p/222-3p 6 d后细胞的增殖情况;通过番红O染色法、免疫组织化学方法以及RT-PCR验证各组软骨诱导21 d后软骨分化相关标志物的表达。 结果与结论：构建的Lv-miRNA-221-3p/222-3p inhibition 共转染人骨髓间充质干细胞,成功沉默了细胞中的miR-221-3p/222-3p表达水平;miRNA基因芯片与RT-qPCR验证结果显示miR-221-3p/222-3p在软骨分化后期表达明显降低;转染组与未转染组以及空载体组相比：①细胞增殖明显受到抑制。②番红Ｏ染色以及免疫组织化学显示软骨分化特征标志物硫酸软骨素以及Ⅱ型胶原表达增强。③RT-qPCR也证实硫酸软骨素以及Ⅱ型胶原的mRNA表达也明显上调。结果显示沉默人骨髓间充质干细胞miRNA-221-3p/222-3p,抑制了细胞增殖并促进了成软骨分化。  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程