【作者】 刁飞；

【导师】 卢建；

【作者基本信息】 第二军医大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 糖皮质激素(Glucocorticoid, GC)是人体内的重要激素,除了具有调节糖、脂肪和蛋白质的生物合成和代谢的作用,GC还是体内重要的抗炎激素和应激激素,随着对GC生物学作用研究的发展,人们发现GC还能够调节多种细胞的增殖、分化、凋亡和粘附等。GC在临床上被广泛用于治疗慢性非感染性炎性疾病、过敏性疾病及器官移植等,但大量的使用GC会导致骨质疏松(osteoporosis),临床上称为糖皮质激素性骨质疏松症(glucocorticoid induced osteoporosis, GIOP)。以往对GIOP的研究多放在GC影响钙稳态和对性激素的作用上,对骨细胞的直接影响也多放在GC增强破骨细胞的活性上,而GC对成骨细胞的作用研究不多；目前有许多研究发现GC能够直接抑制成骨细胞的增殖,诱导细胞分化和凋亡,但具体的分子机制还尚未阐明。深入研究GIOP的分子机制将有助于为临床治疗提供理论依据。RhoB隶属于小G蛋白家族Rho亚族,Rho亚族主要包括RhoA, RhoB和RhoC,它们参与了体内多种生理或者病理过程,比如细胞粘附、运动,增殖、存活和炎症等。RhoB虽然在蛋白质一级结构上和RhoA、RhoC相似(-90%),但RhoB却有许多特殊性。比如,大量文献表明,RhoB能够抑制细胞增殖,促进细胞凋亡；而RhoA和RhoC则分别促进细胞的恶变和肿瘤细胞的转移。另外,RhoB是个诱导型蛋白,多种因素,例如放射线、化疗药、热、低氧、多种生长因子等都能诱导细胞RhoB的表达；而RhoA和RhoC则是组成型表达的蛋白。RhoB几乎是和RhoA、RhoC同时发现的,由于和RhoA同源性太高,人们把注意力主要放在对RhoA的研究上,直到发现RhoB具有抑制肿瘤的功能，人们才开始把目光转向RhoB。总之,相对于RhoA,迄今对RhoB的研究有限,对其功能及调控机制的了解还很不够。转化生长因子β1(Transforming growth factorβ1, TGF-β1)是成骨细胞表达最多的细胞因子之一,它在细胞增殖、分化、凋亡、粘附和游走方面发挥了重要作用,许多作用与糖皮质激素相似。另外,糖皮质激素和TGF-β1激活的信号转导通路间具有复杂的相互作用,根据细胞类型和调节的靶基因不同,GC和TGF-β1往往表现为相互协同或者相互拮抗的效应。例如,已知糖皮质激素被临床广泛用于治疗肺、肾和肝等组织的纤维化。该作用的重要机制之一是抑制TGF-β的作用。研究表明,GC不仅能抑制组织TGF-β1的分泌,GC/GR还能通过与Smad的直接相互作用抑制TGF-β1的信号转导和靶基因的转录。而近年来的研究发现,GC和TGF-β1信号通路之间也具有协同效应。如有报道人工合成的糖皮质激素-地塞米松(Dex)和TGF-β1单独都能抑制前列腺癌PC-3细胞的增殖,两者共同作用时,抑增殖作用更为明显。我们前期的实验也发现,在人卵巢癌HO-8910细胞中,Dex和TGFβ1单独作用均能增加该细胞与基质的粘附能力,两者联合作用时促进粘附的效果明显大于两者单独作用的效果；对其机制的研究表明Dex在PC-3细胞和HO-8910细胞中都可以上调TGF-β1的Ⅱ型受体的表达,并增强TGF-β1的信号转导,表明两条通路间的确存在相互作用。但是对于两条通路间协同作用的很多细节以及共同影响的靶基因的表达,我们至今了解的不多。在前期研究工作中,我们发现Dex能够上调人卵巢癌HO-8910细胞RhoB的表达,RhoB的上调参与了Dex对HO-8910细胞的增殖抑制作用,并发现Dex能明显抑制骨肉瘤细胞的增殖、诱导其分化,但是RhoB是否参与了Dex对骨肉瘤细胞的上述作用还不清楚。此外,有研究表明,TGF-β1能够抑制MG-63等骨细胞的增殖。新近还有报道TGF-β1在3T3成纤维细胞和HaCaT角质细胞中能够上调RhoB的表达。因此TGF-β1对MG-63细胞RhoB的表达是否有影响,以及RhoB在骨肉瘤细胞中的作用也是令人感兴趣的问题。本实验以骨肉瘤细胞为模型,首先研究了Dex对人成骨肉瘤MG-63和HOS-8603细胞RhoB的诱导作用,在证实Dex可以上调上述细胞RhoB之后,进一步研究了Dex上调MG-63细胞RhoB的分子机制以及RhoB在Dex调节MG-63细胞增殖、分化和粘附中可能的作用。在此基础上,我们又研究了TGF-β对RhoB表达的影响及其可能的机制,以及Dex和TGF-β1联合作用对人成骨肉瘤MG-63细胞RhoB的诱导作用和对MG-63细胞增殖与粘附的影响。本课题有助于进一步阐明RhoB的作用和调节机制,以及糖皮质激素和TGF-β对骨细胞和骨肉瘤细胞的作用机制。一、糖皮质激素对人成骨肉瘤细胞RhoB的诱导作用、机制及生物学意义(一)Dex对人成骨肉瘤细胞RhoB表达的影响我们选择MG-63和HOS-8603两种人成骨肉瘤细胞作为研究对象,用Real-timePCR和Western Blot方法检测了Dex对RhoB表达的影响。结果发现Dex能够上调成骨肉瘤细胞RhoB的表达。(二)Dex诱导MG-63细胞RhoB表达的机制研究1.Dex上调RhoB的表达通过GR介导用GR的拮抗剂RU486预处理MG-63细胞,再加Dex处理,分别用Real-time PCR和Western Blot方法检测RhoB mRNA和蛋白水平的表达情况。发现RU486几乎完全阻断Dex对RhoB的诱导。表明Dex对RhoB表达的诱导作用是由GR受体介导的。2.Dex不能诱导含有人RhoB启动子序列(-1765/+111)的报告基因的表达为明确Dex对RhoB的诱导作用是否发生在转录水平,我们将含有人RhoB基因部分启动子的序列(-1765/+111)的荧光素酶报告基因质粒转染MG-63细胞,再用Dex处理,并利用报告基因技术来进行分析。结果表明在RhoB启动子的这段长1.9 kb的区域(-1765/+111)内没有功能性的GRE存在。用生物信息学方法检索了人RhoB基因启动子转录起始位点上游长约5kb的片段，也未发现有典型的GREs存在。3.Dex能够增强RhoB mRNA和蛋白质的稳定性我们继而观察了Dex对MG-63细胞RhoB mRNA和RhoB蛋白稳定性的影响。用放线菌素D或放线菌酮分别抑制转录和翻译,实验组加入Dex处理,Real-time PCR和Western Blot方法检测不同时间RhoB mRNA和RhoB蛋白的表达。结果发现Dex能够明显增强RhoB mRNA的稳定性和RhoB蛋白的稳定性。4. PI-3K/Akt和p38 MAPK信号转导通路参与了Dex对RhoB的诱导有文献报道,Dex能够通过激活PI-3K/Akt和MAPK信号转导通路发挥生物学功能,为了确定PI-3K/Akt和MAPK信号通路是否参与了Dex对RhoB的诱导作用,我们用Western blot的方法检测了Dex处理MG-63细胞不同时间后磷酸化Akt、总Akt、磷酸化p38、总p38、磷酸化JNK、总JNK和RhoB的变化。结果表明,Dex能激活PI-3K/Akt和p38 MAPK信号转导通路,但不激活JNK通路。我们进一步在MG-63细胞的培养液中分别加入Akt、p38、JNK和ERK的抑制剂,Dex处理后Western blot方法观察RhoB蛋白的变化。结果表明Dex上调RhoB的表达与PI-3K/Akt和p38通路的激活有关,而与JNK和ERK通路无关。(三)Dex诱导MG-63细胞RhoB表达的生物学意义1.RhoB参与了Dex对MG-63细胞的增殖抑制作用用细胞计数和MTT的方法观察了Dex对MG-63细胞的增殖的影响。结果显示,Dex能以浓度依赖性和时间依赖性方式抑制MG-63细胞增殖。为确定RhoB是否参与了Dex对MG-63细胞的增殖抑制作用,我们将MG-63细胞分别瞬时转染空载体(pcDNA3)、RhoB野生型质粒(RhoB-wt)、RhoB干扰阴性对照质粒(RhoB-neg)和RhoB干扰质粒(RhoB-RNAi),48h后加入或不加Dex处理,细胞计数结果表明,RhoB参与了Dex对MG-63细胞的增殖抑制作用,并能增强Dex的增殖抑制作用。2. RhoB不参与Dex对MG-63细胞的诱导分化作用将MG-63细胞分别瞬时转染空载体和上述表达及干扰质粒,然后加入或不加Dex处理,p-nitrophenol法检测碱性磷酸酶(AP)的活性。结果表明Dex能够诱导MG-63细胞AP的分化,但RhoB并未参与这种分化过程。3. RhoB参与了Dex增强MG-63细胞粘附活性的作用首先用细胞粘附实验检测了Dex处理后MG-63细胞与纤连蛋白之间的粘附活性。结果显示,Dex能够以剂量依赖性的方式增强MG-63细胞与纤连蛋白之间的粘附。接着将MG-63细胞分别瞬时转染pcDNA3、RhoB-wt、RhoB-neg和RhoB-RNAi质粒。然后加入或不加Dex处理,粘附实验证明,RhoB参与了Dex增强MG-63细胞粘附活性的作用。二、TGF-β1对人成骨肉瘤细胞RhoB的诱导作用、机制及生物学意义(一)TGF-β1对人成骨肉瘤细胞RhoB表达的影响用Western Blot方法检测TGF-β1对MG-63细胞RhoB蛋白表达的影响。结果发现,TGF-β1能够上调MG-63细胞RhoB蛋白的表达。(二)TGF-β1诱导MG-63细胞RhoB表达的机制研究1.TGF-β1能够诱导含有人RhoB启动子序列(-1765/+111)的报告基因的表达将含有人RhoB基因部分启动子的序列(-1765/+111)的荧光素酶报告基因质粒瞬时转染入MG-63细胞,再用TGF-β1处理,双荧光素酶法检测报告基因的活性。结果显示,TGF-β1能够直接诱导RhoB荧光素酶报告基因的活性。表明TGF-β1能够直接在转录水平诱导RhoB mRNA的转录。2.PI-3K/Akt信号转导通路参与了TGF-β1对RhoB的诱导作用用Western blot的方法检测了TGF-β1处理MG-63细胞后磷酸化Akt、总Akt、磷酸化p38、总p38和RhoB的变化。结果表明,TGF-β1处理后,PI-3K/Akt通路激活,而p38MAPK通路不激活。我们进一步在MG-63细胞的培养液中加入Akt的抑制剂,TGF-βl处理后,Western Blot方法检测RhoB表达的变化。结果表明TGF-β1上调RhoB的表达与PI-3K/Akt通路的激活有关。(三)TGF-β1抑制MG-63细胞的增殖,增强其粘附活性用细胞计数和MTT的方法证明,TGF-β1对人成骨肉瘤MG-63细胞具有增殖抑制作用,并有时间依赖性。用粘附实验证明,TGF-β1能以浓度依赖性方式增强MG-63细胞与基质的粘附活性。三、Dex和TGF-β1联用对MG-63细胞RhoB的诱导作用及生物学意义(一)Dex和TGF-β1对MG-63细胞RhoB的表达有协同作用用WesternBlot方法检测了Dex和TGF-β1单独及联合作用对MG-63细胞RhoB表达的变化。结果表明,Dex和TGF-β1能够协同上调MG-63细胞RhoB蛋白的表达。(二)Dex和TGF-β1联用诱导MG-63细胞RhoB的生物学意义1.Dex和TGF-β1联用能够协同上调MG-63细胞的粘附活性用细胞粘附实验观察Dex和TGF-β1联用对MG-63细胞粘附活性的影响。结果发现,Dex联用TGF-β1能够协同增强MG-63细胞的粘附活性。2.Dex和TGF-β1联用对MG-63细胞增殖的影响采用细胞计数和MTT的方法观察Dex和TGF-β1联用对MG-63细胞增殖的影响。结果发现,Dex和TGF-β1联用对MG-63细胞的增殖没有协同作用。我们推测,Dex和TGF-β1通过激活不同的信号转导通路增强MG-63细胞的粘附、通路间有协同作用；而通过相同的信号转导通路抑制细胞增殖。(三)Dex对MG-63细胞TGF-β1的分泌没有影响为明确Dex和TGF-β1的协同作用是否是因为Dex诱导TGF-β1分泌增加,用ELISA的方法进行了检测,发现Dex对MG-63细胞TGF-β1的分泌没有影响。综上所述,我们获得以下结论：1.Dex能够通过增强RhoB mRNA和RhoB蛋白的稳定性上调人骨肉瘤MG-63细胞RhoB的表达,Dex激活PI-3K/Akt和p38 MAPK信号转导通路参与了Dex对RhoB的上调作用；2. RhoB参与了Dex对MG-63细胞的增殖抑制作用和增强细胞粘附的作用,但对Dex的诱导细胞分化作用没有明显影响；3.TGF-β1也能在转录水平上诱导MG-63细胞RhoB的表达；PI-3K/Akt信号转导通路的激活参与了TGF-β1对RhoB的诱导作用；4.Dex和TGF-β1联用能够协同上调MG-63细胞RhoB的表达,在促进细胞粘附方面两者也具有明显的协同作用。但Dex不影响MG-63细胞对TGF-β1的分泌。更多还原

【Abstract】 Glucocorticoids (GCs) regulate a variety of biological processes, including cell growth, differentiation and apoptosis. GCs have well-documented effects on bone metabolism. Physiological concentrations of glucocorticoids promote the development or maturation of osteoblast cells, and continued exposure of the skeletal tissue to pharmacological dose of glucocorticods can cause osteoporosis. GC-induced osteoporosis is characterized histologically by a decreased bone formation rate, decreased trabecular wall thickness, and depleted osteoblast numbers, all indicators of a deficient osteoblast population. It has been known that GCs exert antiproliferative effect in most osteoblast cell contexts including G-292, osteoblast-like cancer cells through activating the glucocorticoid receptor (GR), which is a ligand-dependent transcriptional regulator that transduces the hormonal signal into the nucleus to alter the expression of target genes. But the down-stream effector proteins of GR mediated the antiproliferative action of these compounds on osteoblast cells, however, is not fully understood.Small GTPases of the Rho subfamily have been implicated in many physiological and pathological cellular processes, including cell adhesion, motility, proliferation, survival and inflammation. The Rho subfamily mainly includes RhoA, RhoB and RhoC proteins. RhoB is quite different from RhoA and RhoC in many aspects although it shares-90% homology to RhoA and RhoC. For example, RhoB has a tumor-suppressive role, including inhibiting cell proliferation and inducing apoptosis in several human cancer cells, and inhibiting tumor growth in a nude mouse xenograft model, while RhoA activation promotes cell malignant transformation, cell proliferation, invasion and metastasis. Furthermore, RhoB, unlike RhoA which is constitutively expressed, has been shown to be induced by genotoxic stress, such as UV, chemotherapeutic drugs (e.g. cisplatin and 5-FU), and some growth factors such as EGF, PDGF.We have demonstrated previously that RhoB is also upregulated by Dexmethasone (a synthesis glucocorticoid, Dex), and RhoB signaling is involved in Dex-induced proliferation inhibition of human ovarian cancer HO-8910 cells. However, RhoB is not induced by Dex in human fibrosarcoma cell HT-AR1, indicating that the effect of glucocorticoid on RhoB expression is cell specific. So, we want to know whether RhoB is regulated by Dex in human osteosarcoma cells and plays a role in Dex-induced cell growth inhibition and differentiation.We demonstrated that Dex could induce both mRNA transcription and protein expression of RhoB in osteosarcoma MG-63 cell line. The up-regulation of RhoB mRNA by Dex may mainly due to Dex’s effect on the stabilization of RhoB mRNA and RhoB protein instead of enhancement of transcripts because RhoB promoter (-1765 to+111) contains no functional GRE. Induction of RhoB expression by Dex depend on new protein synthesis, both PI-3K/Akt and p38 MAPK signaling are involved in the RhoB expression by Dex, but JNK and ERK signaling are not. Once again we confirmed the inhibition effect of Dex to MG-63 cells time and dose dependently. Overexpression of RhoB repressed the growth of osteosarcoma cell line MG-63 and enhanced Dex-induced cell growth inhibition, but has no effect on cell differentiation. While interfering of RhoB expression facilitated cell growth and reversed partially Dex-induced proliferation inhibition. Furthermore, we reported that Dex can enhance the adhesive activity of MG-63 cells to fibronection and RhoB signaling is involved in the adhesion enhancement of MG-63 cells by Dex.Transforming growth factorβ1 (TGF-β1) is one of the most highly expressed cytokines in osteoblast cells. Like Dex, TGFβalso have multiply biology effects in cell growth, migration, differentiation and apoptosis. It has been reported that TGF-β1 can inhibit the growth of osteoblast cells including MG-63, G-292, and osteoblast-like cancer cells. On the other hand, articles declared that TGF-β1 induce the expression of RhoB in many cell types. We wonder whether TGF-β1 can induce the expression of RhoB in MG-63 cells and whether RhoB signaling are involved in the grow inhibition and adhesion of MG-63 cells by TGF-β1.We found that TGF-β1 treatment could also increases the expression of RhoB in MG-63 cells like Dex. However, TGF-β1 can enhance the transcriptional activity of the human RhoB promoter (-1765/+111) in MG-63 cells. We also demonstrate that PI-3K/Akt but not p38 MAPK signaling is involved in the RhoB expression by TGF-β1. Moreover, we confirmed the inhibition effect of TGF-β1 to MG-63 cells and demonstrated the enhancement of adhesive ability of MG-63 cells to TGF-β1.The relationship between Dex and TGF-β1 is complex. Our previous study showed that the co-treatment of Dex with TGF-β1 could significantly enhance the adhesion of HO-8910 cells to ECM and increased the synthesis of extracellular matrix (ECM). Given that both Dex and TGF-β1 can induce the expression of RhoB, and both Dex and TGF-β1 can inhibit the grow and enhance the adhesion of MG-63 cells through the involvement of RhoB, whether co-treatment of Dex with TGF-β1 has synergetic effect on the expression of RhoB, adhesion and proliferation of MG-63 cells, and whether RhoB is involved in these processes are ready to be elucidated.We found the synergetic effect of Dex and TGF-β1 to upregulate the expression of RhoB in MG-63 cells, and the synergetic effect of Dex and TGF-β1 to the adhesive ability of MG-63 cells on fibronectin. However, no synergetic effect of Dex and TGF-β1 was found to the growth inhibition effect of MG-63 cells. Furthermore, we found the secretion of TGF-β1 was not facilitated by Dex on MG-63 cells with ELISA method. Maybe different singnaling pathways are involved in the adhesion process by Dex and TGF-β1 and same singnaling pathway in the growth inhibition effect.更多还原