【作者】 刘辉；

【导师】 井玲；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 结缔组织生长因子(connective tissue growth factor,CTGF)可促进软骨细胞和成骨细胞(osteoblast,OB)的增殖和分化,在骨骼发育以及骨量维持方面发挥重要作用。转化生长因子β(transforming growth factor-β,TGF-β)具有与CTGF相似的生物学作用,又是CTGF表达的有效刺激因子。本研究将重点观察氟化物对体外培养成纤维细胞中CTGF mRNA和蛋白表达的特点,尝试分析氟化物作用下TGF-β和CTGF之间的相互关系,从而探讨在氟化物诱导成纤维细胞(fibroblast,FB)成骨功能表达过程中CTGF的作用和作用特点,为解释氟骨症骨周化骨的发生机制提供重要依据。以小鼠成纤维细胞株(L929)和小鼠颅骨成骨样细胞为实验对象,细胞培养方法采用二维和三维两种方法。应用ELISA、免疫组化等方法,检测染氟成纤维细胞CTGF和TGF-β蛋白的表达。结果表明:(1)染氟可刺激二维和三维培养体系中成纤维细胞CTGF蛋白的表达,后者在高氟剂量组明显;(2)PCR检测结果显示,氟化物对两种培养体系中成纤维细胞CTGF mRNA表达均有一定程度的刺激作用,以高氟剂量组(20mg/L)增加幅度大;(3)染氟对二维培养的FB中TGF-β蛋白的表达无明显增强作用,但在染氟早期即明显增强三维培养FB中TGF-β蛋白的表达;(4)相同染氟条件下,二维培养中成骨细胞CTGF mRNA表达与染氟FB的变化趋势相似,但增加程度更为明显。综合分析上述结果,氟化物对FB中CTGF从mRNA到蛋白的表达的刺激作用,提示在氟诱导FB成骨过程中,CTGF可能发挥一定作用,在氟骨症骨周化骨的发生机制的研究中是值得探讨的细胞因子。在氟化物刺激FB中CTGF和TGF-β蛋白的表达方面,未发现明显规律,可能是二者在氟化物的刺激中缺少内在联系,但更可能是由于本实验条件所限,将在进一步的研究中探索改进加氟剂量和时间。氟化物仅对三维培养体系中FB TGF-β蛋白表达具有刺激作用,说明FB在不同的培养条件下,对氟化物的反应可能会有所不同,也提示我们在染氟FB的体外研究中,应用多种培养方法可能得到比较全面的实验结果。更多还原

【Abstract】 Connective tissue growth factor ( CTGF ) can promote proliferation and differentiation of chondrocyte and osteoblast ( OB ) . It is of great importance in bone development and bone mass maintenance . Investigations have proved that CTGF can also stimulate cell proliferation and collogen secretion of fibroblast . TGF-βhas the same usage with CTGF in biological research and it is also the factor which stimulate the expression of CTGF effectively . We observe the characteristics of FB cultured in vitro and protein expression , we attempt to analyze the interrelation between TGF-βand CTGF in the effect of fluorid. By doing that we can provide an important evidence to explain the cause of the non-physiological osteogenesis leading to extraperiosteal ossification .Fluoride can stimulate the enhancement of osteogenic phenotype and osteogenic function. Based on inferences above , we investigated the effect of fluorid on FB line of mouse ( L929 ) and mouse’s osteoplastic cell of skull ,we may observe on FB CTGF mRNA and protein expression affected by fluoride in the 2-dimensional ( 2D ) and 3-dimensional ( 3D ) cultured system . We investigate simultaneously the expression of TGF-βprotein in FB following the treatment with fluoride . We expect the relationship between TGF-βand CTGF , which produce the effect of the expression in FB exposed to fluoride . We investigated the effects and importance of fluoride on FB .1、The expression of CTGF mRNA and protein in fluoride-treated FB(1) By using the method of Elisa , we detected the protein level of CTGF in supernatant fluid in fluoride-treated FB . Compared with the group of 0 mg F-/L , either in 2D or 3D cultured system , the content of CTGF protein was increased obviously in the group of higher fluoride dosage and later stimulation of fluoride .(2) By using the method of IHC , we detected the expression of the masculine protein in cytoplasm of FB , as a result , we found that fluoride can stimulate the masculine protein of CTGF . Compared with the CTGF protein expression in the group of 0 mg F-/L , it in the group of fluoride-treated FB in 96h enhanced apparently in 3D cultured system .(3) By using the method of RT-PCR , we detected the expression of the fluoride-treated FB CTGF mRNA in 48h . The result showed that the expression of CTGF mRNA had been enhanced in the group of higher fluoride dosage in 2D and 3D cultured system . It , however , did not rich the apparent degree in statistics .Above-mentioned result shows that the expression of CTGF in FB has been stimulated by fluoride in 2D and 3D cultured system . CTGF stimulated the function of osteogenesis and it may have an important usage in osteoplastic potency of FB .2、Effect of the expression of TGF-βin fluoride-treated FB and OB(1) We used the method of Elisa to check TGF-βprotein level in supernatant fluid in fluoride-treated FB . The result shows that there is a different tendency in the expression of TGF-βprotein between the 2D and 3D cultured-FB . The expression of TGF-βprotein was decreased or reduced obviously in 2D culture . In 3D culture , compared with 0 mg F-/L the expression of TGF-βprotein in group of 0.0001 mg F-/L was more increased obviously than in other group .(2) By using the method of Elisa , we detected the protein level of TGF-βin supernatant fluid in fluoride-treated OB . According to the experiment , the protein level affected by fluoride was not changeable obviously in protophase , was fortified in intermediate stage and was depressed obviously in last period .Above-mentioned result shows that in two cultured systems reaction of FB TGF-βprotein expression affected by fluoride differs a little . It reminds us to use different cultured methods in the future research so that we can gain allround results .3、The expression of TGF-βand CTGF in FB affected by fluoride and the interrelation between themWe did not find obvious connection between the expressions of FB TGF-βand CTGF in the experiment . This is probably because two factors work respectively or because of limitation of the experimental condition . It is necessary for us to make a further research .4、The expression of CTGF mRNA in OB affected by fluoride is similar to FB affected by fluoride , but different from expression of proteinThe expressive tread of CTGF mRNA in FB and OB is extremely similar and it is in agreement with the expression of bone growth factor mRNA which we have research before . The fluoride has little effect on the expression of CTGF protein in OB ,we still need a further research .We , in the project , will find the effect of the expression of CTGF on different cultured condition induced by fluoride , and contrast the expression of CTGF in fluoride-treated FB with that in OB under the same condition . The studies above will demonstrate that the fluoride could stimulate the expression of CTGF in FB with two cultured condition . The expressive tread of CTGF in FB with 2D culture and OB is extremely similar . The interrelationship of TGF-βand CTGF in fluoride-treated FB is not coincident with our hypothesis . Several improvement need to be put forward in our further study . CTGF may be an important factor in the project which fluoride promotes osteogenesis of FB . It can give clues to thestudies of other similar diseases .更多还原