Sox9基因促软骨形成作用机制研究进展

Sox9基因作为一个重要转录因子,在软骨形成中起着极其重要的调控作用,相关研究对软骨缺损修复等组织再生工程发展具有十分重要的意义。软骨形成过程中软骨细胞增殖、分化调控过程复杂,参与的细胞因子较多,目前比较明确的是Sox9结合于软骨细胞特异增强子Col2α1,直接调控其表达。L-Sox5、Sox6与Sox9共同形成蛋白复合物协同作用于Col2α1增强子序列,增强其转录,并通过结合于PTHrP基因的启动子区上调PTHrP基因启动子活性,刺激软骨细胞早期阶段增殖和发育。Sox9与Wnt/β-catenin和TGF-β/Smad信号转导通路相互作用,调控软骨细胞分化。最新研究显示,Sox9基因在软骨形成过程中还与其他物理因子和生物因子如低氧、电磁场等相互影响、相互作用。     

小分子干扰RNA诱导HTB-94软骨肉瘤细胞凋亡的研究

目的 利用小分子干扰RNA(siRNA)表达质粒转染HTB-94软骨肉瘤细胞,观察其在人体软骨肉瘤细胞HTB-94中对肿瘤细胞生长的影响.方法 设计并合成高泳动家族性别决定基因9(Sox9)siRNA,将其转染HTB-94肿瘤细胞,观察肿瘤细胞的Sox9基因的mRNA和蛋白表达、生长曲线和细胞凋亡情况.结果 Sox9 siRNA的插入片段测序结果与合成的siRNA结果一致,Sox9基因的mRNA和蛋白表达降低,HTB-94肿瘤细胞的生长繁殖受到抑制,细胞凋亡增加.结论 Sox9 siRNA表达质粒可以通过稳定转染HTB-94肿瘤细胞,抑制Sox9基因的mRNA和蛋白的表达,抑制肿瘤细胞的生长繁殖,同时使肿瘤细胞凋亡.     

高泳动家族性别决定基因9(Sox9)启动子转染软骨肉瘤细胞的实验研究

[目的]研究观察Sox9启动子在软骨肉瘤细胞中对软骨相关基因的调控与影响。[方法]将合成Sox9启动子表达质粒转染SW1535软骨肉瘤细胞,观察其在人体软骨肉瘤细胞中对Sox9基因和Ⅱ型胶原蛋白基因Col2a1蛋白及mRNA表达的影响。[结果]转染Sox9启动子后人体软骨肉瘤细胞中Sox9基因和Ⅱ型胶原蛋白基因Col2a1的蛋白表达及mRNA表达均较对照细胞强。[结论]外源性Sox9启动子可以稳定转染SW1535人体软骨肉瘤细胞,转染后可以增加Sox9基因和Ⅱ型胶原蛋白基因Col2a1蛋白及mRNA的表达。     

Sox1抑制膀胱癌增殖及转移的作用及分子机制研究

目的:研究Sox1抑制膀胱癌增殖及转移的作用及分子机制。方法:通过慢病毒系统在T24细胞中上调Sox1的表达,在激光共聚焦显微镜下观察其结果,并通过相关实验观察其反应。结果:激光共聚焦显示Sox1定位于细胞核内,可显著抑制肿瘤细胞增殖能力;细胞侵袭实验表明Sox1可显著抑制细胞的迁移和侵袭能力;荧光素酶报告基因结果显示Sox1可以抑制β-catenin转录活性。结论:Sox1是重要的抑癌基因。本研究揭示了膀胱癌发生及转移的新机制,加深了对膀胱癌发生和转移的认识。     

白细胞介素1对椎间盘细胞软骨特异性基因Sox9 mRNA的调节作用

目的探讨白细胞介素1（interleukin-1,IL-1）对人椎间盘细胞软骨特异性基因Sox9和Ⅱ型胶原基因表达的调节作用。方法应用RT—PCR技术检测IL-1对培养的椎间盘细胞中软骨特异性基因so西和Ⅱ型胶原基因mRNA表达的调节作用。结果在IL-1浓度为0．1ng／ml、1ng／ml和10ng／ml培养24h时,其对椎间盘细胞Sox9和Ⅱ型胶原基因mRNA可起到显著的负向调控作用;10ng／ml的IL-1随着培养时间的延长对椎间盘细胞中Sox9和Ⅱ型胶原基因mRNA出现显著的负向调控作用。结论IL-1可以按照剂量及时间依赖方式负向调节椎间盘细胞Sox9和Ⅱ型胶原基因的表达。     

mir-638和Sox2在原发性肝癌中的表达及临床意义

目的检测mi R-638和Sox2在原发性肝细胞癌中的表达情况,探讨其与肝癌患者临床病理特征的关系。方法采用实时定量PCR(q RT-PCR)对78例原发性肝癌患者癌组织及对应癌旁组织中mi R-638和Sox2 m RNA表达水平进行检测。免疫组化法检测所有癌组织及其相应的癌旁正常组织Sox2蛋白的表达情况。分析mir-638和Sox2与肝癌患者临床病理参数间的关系。结果 (1)与癌旁正常组织相比,mi R-638在肝癌组织中的表达明显降低(P0.05),而Sox2 m RNA在癌组织中的表达显著升高(P0.05);(2)Spearman相关分析显示,在肝癌组织中mi R-638与Sox2蛋白的表达呈显著负相关(r=-0.441,P0.05);(3)临床病理相关性分析表明mi R-638和Sox2的表达与肿瘤TNM分期(χ~2=10.617,P=0.001;χ~2=9.939,P=0.002)及门静脉侵犯与否明显相关(χ~2=7.885,P=0.005;χ~2=6.370,P=0.012),而与患者年龄、性别、肿瘤大小、肿瘤分化程度、肝炎状况等其他临床病理参数无关(均P0.05)。结论肝癌中mi R-638表达水平的下调及SOX2表达水平的上升可能与肝癌的发生、发展密切相关。     

Sox9诱导人脐血干细胞向软骨细胞分化的实验研究

目的 探讨Sox9基因对脐血干细胞向软骨细胞分化的影响。方法 体外分离培养人脐血干细胞,采用脂质体转染Sox9载体质粒,观察转染后细胞形态的变化,免疫组化染色观察collagenⅡ、aggrecan的表达,RT-PCR检测collagenⅠ、collagenⅡ、aggrecan的m RNA水平变化,Western blot检测collagenⅡ的表达变化。结果 Sox9对脐血干细胞形态无明显影响,与传统方法诱导组相比,Sox9转染后可明显促进脐血干细胞向软骨细胞分化。结论 Sox9对脐血干细胞的软骨分化有很强的调控作用,脐血干细胞可作为组织工程软骨一种良好的种子细胞。     

骨形态发生蛋白-2与椎间盘细胞Sox9 和Ⅱ型胶原基因的调控关系

目的：探讨骨形态发生蛋白-2（BMP-2）对椎问盘细胞Sox9和Ⅱ型胶原基因表达的调节作用。方法：应用逆转录聚合酶链反应和Western印迹检测不同浓度BMP-2对培养的人椎间盘细胞中软骨特异性基因Sox9和Ⅱ型胶原基因表达的调节作用。结果：BMP-2浓度为100ng／ml和1000ng／ml时，椎间盘细胞中Sox9和Ⅱ型胶原基因mRNA表达与0n∥ml组（对照组）相比明显增加（P〈0．05）；在蛋白水平的检测中，也得到了一致的结果。结论：BMP-2可以按照剂量依赖方式正向调节椎间盘细胞Sox9和Ⅱ型胶原基因的表达，提示BMP-2可能对退变早期的椎间盘具有修复功能。     

乳腺癌组织Ras相关区域家族1A基因启动子甲基化状态及临床意义

目的 探讨Ras相关区域家族1A基因(RASSF1 A)启动子甲基化在乳腺癌发生发展中的作用.方法 甲基化特异性聚合酶链反应(MS-PCR)法分析44例乳腺癌石蜡包埋切片组织中RASSF 1A基因启动子甲基化状况,分析甲基化与临床病理特征之间的关系.结果 44例乳腺癌组织切片中有25例RASSF1A基因启动子发生了超甲基化,甲基化率为56.8％,启动子甲基化与患者年龄明显相关(P＜0.05),与患者月经初潮年龄和生育次数无相关,与肿瘤大小、淋巴结转移、TNM分期及雌激素受体(ER)、孕激素受体(PR)之间无相关.结论 RASSF1A启动子甲基化为乳腺癌中的频发事件,其发生率随着年龄增长而升高,在乳腺癌发生中起重要作用.     

乳腺癌组织中survivin，p53蛋白的表达与预后的关系

目的探讨乳腺癌组织中survivin基因和p53基因的表达与预后的关系。方法用免疫组化S P法检测80例乳腺癌组织中survivin、p53的表达,分析其与腋淋巴结转移和5年无病生存率之间的关系。结果survivin基因在乳腺癌中阳性表达率为68.75%(55/80)、p53基因阳性表达率为46.25%(37/80),均与腋淋巴结转移相关(P<0.05),与5年无病生存率相关(P<0.05)。survivin和p53表达与肿瘤病理类型、发病年龄、临床分期无明显相关性(P>0.05);survivin和p53基因表达相关(P=0.0025)。结论survivin和p53基因可能在乳腺癌的发生、发展过程中起重要作用,联合检测能更好地判断乳腺癌的预后。     

Sox‐9 facilitates differentiation of adipose tissue‐derived stem cells into a chondrocyte‐like phenotype in vitro

The purpose of this study is to test whether ectopic expression of Sox‐9 can induce adipose tissue‐derived stem cells (ASCs) to function as real nucleus pulposus (NP) cells in vitro. Adenoviral vectors expressing Sox‐9 were reported to infect the chondroblastic and human disc cells, which resulted in increased Sox‐9 and type II collagen production. ASCs were isolated from rat inguinal adipose pad, characterized, and transduced in vitro with a retroviral vector encoding the Sox‐9 gene. Sox‐9‐engineered ASCs (ASCs/Sox‐9) were induced for the chondrocyte‐like cell differentiation by 3D cultured in alginate beads and TGF‐β3 for 2 weeks. Expression of exogenous Sox‐9 protein was detected. Type II collagen and Aggrecan gene expressions of induced ASCs/Sox‐9 were measured using real‐time PCR; proteoglycans expressions were measured by checking the glycosaminoglycan content and type II collagen production by enzyme‐linked immunosorbent assay. Isolated ASCs were CD 29⁺/CD44⁺/C‐Kit^?/Lin^?/CD34^?/CD45^?. ASCs/Sox‐9 expressed marked increase in exogenous Sox‐9 protein. After induction, type II collagen gene expression was sevenfold higher in mRNA levels, with an approximately twofold increase in protein levels of ASCs/Sox‐9 compared to ASCs. Type II collagen and proteoglycan productions were significantly increased in the ASCs/Sox‐9 compared to the ASCs. In addition, co‐culture of induced ASCs/Sox‐9 with matured NP cells resulted in enhanced increase in proteoglycan and type II collagen production. Constitutive retroviral expression of Sox‐9 could efficiently induce ASCs differentiation into chondrocyte‐like cells. This novel approach may provide a practicable system for a simple and rapid differentiation of ASCs into chondrocyte‐like cells which may be potentially used as a stem cell‐based therapeutic tool for the treatment of degenerative disc diseases. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1291–1297, 2011     

β-catenin基因敲减与Sox9过表达联合对人间充质干细胞早期成软骨分化作用的实验研究

背景:β-catenin基因抑制人间充质干细胞(hMSCs)成软骨分化,而Sox9基因促进hMSCs成软骨分化,但两者联合在hMSCs早期成软骨分化中的作用及其可能机制目前尚不明确。目的:探究β-catenin基因敲减与Sox9过表达联合对hMSCs早期成软骨分化的作用。方法:将骨髓来源hMSCs分为4组:阴性对照组,β-catenin基因敲减组,Sox9过表达组,β-catenin基因敲减与Sox9过表达联合组,加入成软骨分化培养基培养7 d。转染24 h,RT-PCR检测β-catenin基因敲减效率与Sox9过表达效率,RT-PCR检测COL2A1和ACAN的m RNA表达量,番红固绿染色、甲苯胺蓝染色、免疫细胞化学检测COL2A1和Aggrecan蛋白。结果:RT-PCR结果显示转染成功,β-catenin基因敲减效率与Sox9过表达效率较高(P<0.05)。β-catenin基因敲减与Sox9过表达联合组中COL2A1和ACAN的mRNA表达量显著高于其他3组(P<0.05),软骨成分染色更深(P<0.05)。免疫组化结果显示β-catenin基因敲减与Sox9过表达联合组的COL2A1和Aggrecan蛋白表达量显著高于其他3组(P<0.05)。结论:β-catenin基因敲减与Sox9过表达联合对hMSCs早期成软骨分化有促进作用。     

The Sox2 high mobility group transcription factor inhibits mature osteoblast function in transgenic mice

We have previously shown that in osteoblasts Sox2 expression can be induced by Fgfs, and can inhibit Wnt signaling and differentiation. Furthermore, in mice in which Sox2 is conditionally deleted in the osteoblastic lineage, bones are osteopenic, and Sox2 inactivation in cultured osteoblasts leads to a loss of proliferative ability with a senescent phenotype. To help understand the role of Sox2 in osteoblast development we have specifically expressed Sox2 in bone from a Col1α1 promoter, which extended Sox2 expression into more mature osteoblasts. In long bones, trabecular cartilage remodeling was delayed and the transition from endochondral to cortical bone was disrupted, resulting in porous and undermineralized cortical bone. Collagen deposition was disorganized, and patterns of osteoclast activity were altered. Calvarial bones were thinner and parietal bones failed to develop the diploic space. Microarray analysis showed significant up- or downregulation of a variety of genes coding for non-collagenous extracellular matrix proteins, with a number of genes typical of mature osteoblasts being downregulated. Our results position Sox2 as a negative regulator of osteoblast maturation in vivo.     

膀胱癌组织Cripto-1和Sox2表达及血清CA125水平及其与临床分期分级的相关性

目的 研究膀胱癌组织Cripto-1、Sox2的表达及血清CA125水平与膀胱癌临床分期和分级的相关性.方法 收集84例膀胱癌患者血液及癌组织标本,分别用免疫组织化学法和化学发光法检测膀胱癌组织Cripto-1、Sox2表达和血清CA125水平,分析与膀胱癌临床分期的相关性.结果 Cripto-1和Sox2在膀胱癌组织的高表达率分别为54.8％和58.3％,两者均与膀胱癌临床分期存在显著的相关性(P＜0.05);膀胱癌患者血清CA125水平与临床分期和分级无显著相关性(P＞0.05).进一步分层分析发现,与血清CA125低水平组相比,血清CA125高水平患者的膀胱癌组织Cripto-1表达与临床分期和分级显著相关(P＜0.05).结论 膀胱癌组织Cripto-1表达与膀胱癌临床分期和分级相关,在膀胱癌恶性潜能及预后判断中可能具有潜在的临床应用价值.     

Expression of Sox10 and c-kit in Sinonasal Mucosal Melanomas Arising in the Chinese Population

Sinonasal mucosal melanomas (SNMM) of the head and neck regions are rare and aggressive malignancies. Although they can affect patients of any ethnicity, they are more numerous in Chinese patients. The diagnosis and treatment of these tumors can be challenging. Recent studies have reported that Sox10 is a sensitive melanocytic marker for cutaneous melanoma (Nonaka et al. in Am J Surg Pathol 32:1291–1298, 2008). In addition, a CD117 (c-kit) gene mutation has been identified in cutaneous melanomas, indicating that there may be potential therapeutic benefits of tyrosine kinase inhibitors, such as Imatinib. The purpose of this study was to detect and test the immunohistochemical expression of Sox10 and c-kit in mucosal melanomas (MM) arising in the nasal cavities of Chinese patients. Twenty eight patients with mucosal melanomas of the nasal cavity were treated in two major hospitals in China. All cases had been locally diagnosed as primary SNMM. We confirmed all diagnoses with positive immunohistochemical stains for S100 and HMB-45. Additionally, automated immunohistochemistry was performed using a goat polyclonal Sox10 antibody and a monoclonal c-kit antibody counterstained using a standard avidin–biotin complex method. Immunohistochemical positive expression of Sox10 was defined by nuclear stain; and positivity for c-kit resulted in a distinct membranous staining. The extent of nuclear positivity for Sox10 and membranous stain for c-kit was graded by 4 board certified pathologists as follows: 1+, 1–25 % of positive tumor cells; 2+, 25–50 %; 3+, 50–75 %; and 4+, ≥75 %. Sox10 nuclear expression was found in all cases (100 %), with 4+ staining in 26 out of 28 cases (92.8 %) and 3+ staining in two cases with (7.1 %). The overall positivity for S100 staining was 23 out of 28 (82.1 %), with 1+ staining in 10 cases, 2+ staining in 6 cases, 3+ staining in 7 cases, and no staining in 5 cases. The sensitivity and intensity of Sox10 immunohistochemistry were both higher than with S100 immunohistochemistry. Immunopositivity of membranous stain for c-kit (CD117) was seen in 24 out of 28 cases (85.7 %), including 6 tumors that were 4+, eight that were 3+, six that were 2+, and four that showed 1+ staining. Our results demonstrate that Sox10 is a sensitive marker for SNMM and it may possess diagnostic value in addition to that of S100 protein. The expression of c-kit in the majority of MMs suggests that it may be useful in the assessment of these tumors for potential treatment with tyrosine kinase inhibitors.     

Intrajoint comparisons of gene expression patterns in human osteoarthritis suggest a change in chondrocyte phenotype.

Osteoarthritis (OA) is a degenerative cartilage disease with varying degrees of severity within a given joint. The purpose of this study was to define a sampling procedure for comparing human minimal and advanced OA cartilage in the same patient and to determine basic patterns of gene expression in these regions. A specific hypothesis under study was that the expression level of Bcl-2 would correlate with Sox9 and aggrecan mRNA expression in vivo as has been demonstrated in vitro. Femoral condylar advanced OA cartilage was located within 1cm of overt lesions, and minimal cartilage was taken from areas with no obvious surface defects. Histological sections were scored for disease severity and chondroitin sulfate and hydroxyproline content was determined. The expression level of nine specific genes (aggrecan, collagen type II, Bcl-2, Sox9, Link protein, osteopontin, and MMP-13, -3, and -9) was determined by quantitative real time PCR. The scores for fibrillation, chondrocyte cloning, and proteoglycan depletion were significantly different between advanced and minimal OA cartilage. The advanced OA cartilage had significantly less chondroitin sulfate than the minimal OA cartilage. Osteopontin mRNA expression showed a 3.6-fold increase in advanced compared to minimal OA cartilage. In contrast, the level of mRNA coding for aggrecan, link protein, Bcl-2, Sox9 and MMP-3, -9, -13 were all decreased in advanced compared to minimal cartilage in the majority of the patients studied. Collagen type II mRNA expression displayed a wide-range of variation. A statistically significant correlation was observed both between Bcl-2 and Sox9 mRNA level, and between Bcl-2 and aggrecan mRNA expression. The patient matched comparison of minimal and advanced OA cartilage revealed differences in cellular and tissue characteristics, and changes in gene expression that may be involved in OA progression. In addition, Bcl-2 may also play a role in regulating the expression of aggrecan through Sox9 in vivo as well as in vitro.