微小RNA-27b-3p与基质金属蛋白酶13在人软骨细胞的对应关系

目的探讨微小RNA-27b-3p(miR-27b-3p)与基质金属蛋白酶-13(MMP-13)在人软骨细胞表达及其靶向对应关系。 方法运用蛋白质印迹法(WB)与实时定量PCR技术(qRT-PCR)明确miR-27b-3p与MMP13在正常和骨关节炎(OA)人软骨细胞的表达。利用不同浓度的白介素(IL)1β干预原代人软骨细胞24 h,或利用不同时间点的IL-1β(10 ng/ml)干预原代人软骨细胞。利用原位杂交、转染及双荧光素酶报告技术确定miR-27b-3p与MMP13的靶向对应关系;结合运用核转录因子-κB(NF-κB)和丝裂原活化蛋白激酶(MAPK)信号通路抑制剂评估其作用机制。两组资料比较采用独立样本t检验,多组资料比较采用单因素方差分析,LSD法多重比较检验。 结果WB、qRT-PCR和原位杂交检测结果显示,与正常软骨相比,OA软骨中miR-27b-3p表达降低(t＝5.07,P<0.01),MMP13表达升高(t＝－6.31,P<0.01)。IL-1β干扰后的结果显示miR-27b-3p表达降低(F＝129.54,P<0.05),MMP-13表达升高(F＝394.50,P<0.05)。通过TargetScan数据库和荧光素酶报告基因检测结果分析,野生型-MMP13组荧光素酶活性降低(F＝55.27,P<0.001),突变型-MMP-13荧光素酶活性变化没有统计学意义(P＝0.654)。利用特异性MAPK信号抑制剂和NF-kB抑制剂干预IL-1β诱导软骨细胞模型结果提示,与对照组相比,抑制剂组的MMP13表达水平降低(F＝28.43,P<0.001),miR-27b-3p表达水平增高(F＝35.04,P<0.001)。 结论miR-27b-3p在OA软骨细胞呈现低表达,并负向调控MMP13的表达,其作用机制可能是通过NF-κB和MAPK信号通路,这结果提示这miR-27b-3p可能作为OA诊断与治疗的潜在靶点。     

Decrease of the insulin‐like growth factor‐1 bioavailability in spontaneously hypertensive rats with erectile dysfunction

We investigated the role of insulin‐like growth factor‐1 (IGF‐1) in spontaneously hypertensive rats with erectile dysfunction. Firstly, we evaluated intracavernous pressure. The bioavailability of IGF‐1 at both mRNA and protein levels were measured by quantitative real‐time PCR and Western blot respectively. Then, cavernous cyclic guanosine monophosphate concentrations were detected by enzyme‐linked immunosorbent assay. The cavernosal pressure was significantly decreased in the hypertensive and the propranolol treatment groups compared to the normal control group (P < 0.01). Cavernous IGF‐1 bioavailability and the concentrations of cavernous cyclic guanosine monophosphate were both significantly decreased in the hypertensive and the propranolol treatment groups compared to the normal control group (P < 0.01). This study suggests that an obvious decrease in cavernous IGF‐1 levels might play an important role in spontaneously hypertensive rats with erectile dysfunction.     

Targeting formyl peptide receptor 1 of activated macrophages to monitor inflammation of experimental osteoarthritis in rat

Macrophages play a crucial role in the pathogenesis of osteoarthritis (OA). In this study, the feasibility of a formyl peptide receptor 1 (Fpr1)‐targeting peptide probe cFLFLF‐PEG‐⁶⁴Cu via positron emission tomography (PET) imaging was investigated for detection of macrophage activity during development of OA. Monoiodoacetate (MIA) was intraarticularly injected into the knee joint of Sprague–Dawley rats to induce OA. Five days later, cFLFLF‐PEG‐⁶⁴Cu (～7,400 kBq/rat) was injected into the tail vein and microPET/CT imaging was performed to assess the OA inflammation by detecting infiltration of macrophages by Fpr1 expression. In addition, a murine macrophage cell line RAW264.7 and two fluorescent probes cFLFLF‐PEG‐cyanine 7 (cFLFLF‐PEG‐Cy7) and cFLFLF‐PEG‐cyanine 5 (cFLFLF‐PEG‐Cy5) were used to define the binding specificity of the peptide to macrophages. It was found with the MIA model that the maximal standard uptake values (SUV_max) for right (MIA treated) and left (control) knees were 17.96 ± 5.45 and 3.00 ± 1.40, respectively. Histological evaluation of cryomicrotome sections showed that Fpr1 expression, cFLFLF‐PEG‐Cy5 binding, and tartrate‐resistant acid phosphatase activity were elevated in the injured synovial membranes. The in vitro experiments demonstrated that both fluorescent peptide probes could bind specifically to RAW264.7 cells, which was blocked by cFLFLF but not by the scramble peptide. The findings highlighted the use of cFLFLF‐PEG‐⁶⁴Cu/PET as an effective method potentially applied for detection and treatment evaluation of OA. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1529–1538, 2016.     

Cartilage degeneration and excessive subchondral bone formation in spontaneous osteoarthritis involves altered TGF‐β signaling

Transforming growth factor‐β (TGF‐β) has been demonstrated as a potential therapeutic target in osteoarthritis. However, beneficial effects of TGF‐β supplement and inhibition have both been reported, suggesting characterization of the spatiotemporal distribution of TGF‐β during the whole time course of osteoarthritis is important. To investigate the activity of TGF‐β in osteoarthritis progression, we collected knee joints from Dunkin–Hartley (DH) guinea pigs at 3, 6, 9, and 12‐month old (n = 8), which develop spontaneous osteoarthritis in a manner extraordinarily similar to humans. Via histology and micro‐computed tomography (CT) analysis, we found that the joints exhibited gradual cartilage degeneration, subchondral plate sclerosis, and elevated bone remodeling during aging. The degenerating cartilage showed a progressive switch of the expression of phosphorylated Smad2/3 to Smad1/5/8, suggesting dual roles of TGF‐β/Smad signaling during chondrocyte terminal differentiation in osteoarthritis progression. In subchondral bone, we found that the locations and age‐related changes of osterix⁺ osteoprogenitors were in parallel with active TGF‐β, which implied the excessive osteogenesis may link to the activity of TGF‐β. Our study, therefore, suggests an association of cartilage degeneration and excessive bone remodeling with altered TGF‐β signaling in osteoarthritis progression of DH guinea pigs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:763–770, 2016.     

Multi‐lineage differentiation and angiogenesis potentials of pigmented villonodular synovitis derived mesenchymal stem cells ‐ pathological implication

Pigmented villonodular synovitis (PVNS) is a benign tissue proliferation characterized by its hyper‐vascularity within the lesion. The true etiology and cell source of this disease entity still remain unclear. Mesenchymal stem cells (MSCs) exist in various tissues of human body. However, it has not been clarified whether MSCs could be isolated from tissue of PVNS. Here, we isolated MSCs from PVNS (PVNS‐SCs), and by comparing to the MSCs from normal synovium (Syn‐SCs) of the same individual, we investigated whether PVNS‐SCs differed in the capacity for multi‐differentiation and inducing angiogenesis. We first demonstrated that PVNS‐SCs existed in the lesion of PVNS of three individuals. Moreover, we showed PVNS‐SCs had better osteogenic differentiation potential than Syn‐SCs, whereas Syn‐SCs had better capacity for adipogenic and chondrogenic differentiation. By genome–wide analysis of gene expression profile using a complementary DNA microarray and comparing to Syn‐SCs, we identified in PVNS‐SCs a distinct gene expression profile characterized by up‐regulation of genes involved in angiogenesis. In vitro and in vivo studies further confirmed that PVNS‐SCs had better capacities for promoting angiogenesis. In summary, the identification of PVNS‐SCs in PVNS tissue and their distinct angiogenic potential may help elucidate the underlying etiology of this disease. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:395–403, 2016.     

Hemilaminoplasty for the treatment of lumbar intraspinal synovial cysts (LISCs) and literature review

Purpose

The present report intended to introduce the hemilaminoplasty technique and evaluate the efficacy of our surgical procedure for LISCs.

Methods

This retrospective study was conducted to analyze the results in 24 LISCs who had undergone our hemilaminoplasty between 2000 and 2012 in two hospitals. All were confirmed by pathological histology and mid- to long-term follow-up had been performed in all cases with a mean of 4.9 years. Using the Japanese Orthopedic Association scoring system (JOA score) and visual analog scale (VAS), symptoms resulting from cyst compression were quantified at various stage for statistical analysis.

Results

The JOA score and VAS of back/leg pain following surgery were improved significantly (P < 0.01). At final follow-up, with normal aging there was a little decrease in JOA score and VAS of back/leg pain, but still significantly improved (P < 0.01). Similarly, mean improvement rate of JOA was 83.5 % at 1 year after surgery while 75.6 % at final visit. Successful bone healing was obtained at a mean of 3.8 months after surgery. No cyst reformation and recurrent back/leg pain were observed around the surgical sites.

Conclusions

These lesions could be regarded as a result of facet arthrosis/instability and repetitive facet minor trauma with herniation of synovium through the defective joint capsule. Improvement in lumbago and leg pain may be a consequence of complete cyst resection via hemilaminoplasty plus partial facetectomy with anatomical reconstruction of the posterior spinal elements.