【作者】 张雪萍；

【导师】 孙海基； 马晓丽；

【作者基本信息】 山东师范大学 ， 动物学， 2011， 硕士

【摘要】 目前,由于机械、外力或自身因素引起的骨及软骨损伤已成为临床治疗的主要病症之一。现阶段利用组织工程进行骨修复是骨组织缺损治疗的热点和发展方向。骨髓间充质干细胞(bone marrow mesenchy stem cell,BMSCs)作为一类存在于骨髓中的非造血多能干细胞,具有自我更新、多向分化的特性,而且来源广、取材简便、免疫排斥反应弱、遗传背景稳定,已成为组织工程中首选的种子细胞。许多研究已经证实,BMSCs可分化为成骨细胞、软骨细胞等多种细胞,因而被广泛应用于治疗骨组织缺损。但是BMSCs向成骨细胞以及软骨细胞分化的诱导条件尚无标准,常用的成骨诱导剂是地塞米松,软骨诱导剂是TGF-β1。在脊椎动物中,Hedgehog(Hh)蛋白家族包括3个成员:Sonic Hedgehog(SHH)、Indian Hedgehog(IHH)和Dersert Hedgehog(DHH)。SHH与细胞在肢体、体节、神经管发育中的分化建立有关,IHH特征性地参与软骨发育,DHH在生殖细胞的发育中起关键作用。Hedgehog信号通路不但在骨组织发育过程中起重要的调控作用,也可以促进成骨相关细胞的分化。BMSCs在骨髓中含量很少并且迄今为止仍未筛选到它的特异性分子标记,在体外培养的环境中如何提纯并富集BMSCs问题还没有解决。而且在高等动物体内,Hedgehog在骨、软骨发育中的作用机制研究较清楚,但其在骨组织工程中(由种子细胞定向分化为软骨和骨细胞的过程)的作用及机理尚不清楚。因此,本文采用贴壁分离法分离大鼠骨髓间充质干细胞,用含有地塞米松的成骨诱导液诱导其向成骨细胞分化,用含TGF-β1的软骨诱导液诱导其向软骨细胞分化。采用RT-PCR、免疫组化及Western Blotting等方法检测分析BMSCs向成骨、软骨分化过程中信号分子SHH和IHH在蛋白水平以及基因水平的表达变化,以探讨Hedgehog信号分子在体外诱导BMSCs成骨分化和软骨分化过程中的作用。本研究主要有以下研究结果:1、利用直接贴壁分离法分离大鼠骨髓间充质干细胞,比较四种培养环境发现,用进口胎牛血清和DMEM-L培养液培养于一次性塑料培养皿中,细胞生长状态最好、细胞增殖速度最快。传至第3代,即可见绝大数细胞为长梭形的成纤维样细胞,呈漩涡状生长。流式细胞术检测第3代细胞CD44和CD34的表达情况,结果发现含有CD44的细胞数占总数的90.16%,而含有CD34的细胞数占总数的2.27%。2、地塞米松诱导7d、14d、21d后,I型胶原阳性表达率逐渐升高,且从14d开始阳性率高于90%。SHH蛋白在诱导组较同时段未诱导组表达量显著增加(P<0.05),随着诱导时间的延长SHH蛋白的表达量先升后降,其中14d时SHH表达量最高。IHH蛋白在诱导组表达量显著低于未诱导组(P<0.05),随着诱导时间的延长IHH蛋白的表达量无显著变化。在MSCs诱导成成骨过程中Shh基因和Ihh基因的表达无显著性变化(P>0.05)。3、TGF-β1诱导7d、14d、21d后,Ⅱ型胶原阳性表达率逐渐升高,从14d开始阳性率高于90%。SHH蛋白在诱导组较同时段未诱导组表达量显著降低(P<0.05),但随着诱导时间的延长SHH蛋白的表达量显著增加(P<0.05)。IHH蛋白在诱导14d和21d时表达量较未诱导组显著增高(P<0.05),且随着诱导时间的延长IHH蛋白的表达量显著增加(P<0.05)。Shh基因表达量的变化趋势与SHH蛋白的相似。Ihh基因的表达量在诱导7d和14d时显著低于未诱导组(P<0.05),且Ihh基因随诱导时间的延长表达呈增加趋势,诱导21d后显著增加(P<0.05)。鉴于上述一系列新发现,可以得出以下主要结论:1、贴壁分离法分离BMSCs后,用进口胎牛血清和DMEM低糖培养液培养于一次性进口塑料培养皿的方法获得BMSCs的效率高、纯度高,可直接用于后续研究。2、诱导组Ⅰ型胶原免疫反应呈阳性结果表明,地塞米松能诱导BMSCs向成骨细胞分化。在诱导组中,SHH蛋白表达较未诱导组显著升高,IHH蛋白的表达较未诱导组显著降低,即SHH蛋白与成骨细胞分化呈正相关,IHH呈负相关,表明SHH、IHH以不同的作用方式参与间充质干细胞诱导成成骨细胞的过程。3、诱导组Ⅱ型胶原免疫反应呈阳性结果表明,TGF-β1能诱导BMSCs向软骨细胞分化。在诱导组中,SHH蛋白表达较未诱导组显著降低, IHH蛋白表达在诱导14d后较未诱导组显著升高,即SHH蛋白与成软骨细胞分化呈负相关,IHH呈正相关,表明SHH、IHH以不同的作用方式参与间充质干细胞诱导成软骨细胞的过程。综上所述,本文首次系统的研究Hedgehog信号途径主要分子(SHH、IHH)在BMSCs定向分化为软骨和骨细胞的过程中的作用及机理。研究结果显示在成骨诱导分化过程中,SHH蛋白的表达显著升高,IHH蛋白的表达显著降低;在软骨诱导分化过程中,SHH蛋白的表达显著降低,IHH蛋白的表达显著升高,表明在BMSCs定向分化为软骨和骨细胞的过程中,可能存在不同SHH信号、IHH信号表达模式,本研究将为阐明Hedgehog信号途径在骨组织工程中的作用机理提供新资料。更多还原

【Abstract】 Recently, bone and cartilage damage due to mechanical, external force or its own factors has become one of the main symptoms of clinical treatment. Now, bone repair using of tissue engineering is on the hot of tissue defect and will become the development direction of treatment. Mesenchymal stem cells (bone marrow mesenchy stem cell, BMSCs)is one kind of non-hematopoietic multipotent in the bone marrow, which characteristics contains self-renewal and multiplex differentiation, and has become the first project organization seed cells because of source wide, drawn simple, immune rejection weak, genetic background stable. Many studies have shown that BMSCs can differentiate into cartilage cells and osteoblasts and other cells, so BMSCs are widely used for the treatment of tissue defect. However there was no induction standard of osteoblast differentiation and cartilage differentiation in BMSCs. Commonly, dexamethasone and TGF-β1 are used as osteoblast inducer and osteoblast cartilage inducer respectively.In vertebrate animals, there are three members in Hedgehog (Hh) protein family: Sonic Hedgehog (SHH), Indian Hedgehog (IHH) and Dersert Hedgehog (DHH). SHH establishes relevant with the differentiation of cells development in the body, the relationship and neural tube. IHH characteristically participates in chondrogenic differentiation. DHH plays a key role in the development of reproductive cells. Hedgehog signaling pathways not only play important readjustment function in bone growth process but also can promote osteogenesis related cell differentiation.So far, there was few molecular markers for the purification and enrichment of BMSCs. And in the advanced animals, roles of Hedgehog mechanism is more clear, but its mechanisms are unclear in osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells in vitro. Here to investigate the expressions of Hedgehog signaling molecules, Sonic Hedgehog (SHH) and Indian Hedgehog (IHH) , during osteogenic and chondrogenic differentiation in rat bone marrow mesenchymal stem cells (MSCs) induced by Dexamethasone and TGF-β1 respectively.Mainly results:1. Expressions of Hedgehog Signaling Molecules during Osteogenic and Chondrogenic Differentiation in Rat Bone Marrow Mesenchymal Cells To separate rat mesenchymal stem cells using direct stick wall separation, we found that the best way for cell growth state and cell proliferation is to culture cells in a plastics petri dishes with foreign womb bovine serum and DMEM-L liquor through comparing with four different culture enviroment. Most of cells were spindle and swirling growth after the third passage. The expression of CD44 and CD34 in the third passage cell were 90.16% and 2.27% respectively by flow cytometry detection.2. The positive expression of I type collagen were increased following osteoblastic differentiation in MSCs. After 14d Dexamethasone induction, the positive rate of I type collagen expression was higher than 90% and the staining intensity increased. Compared to non-induction group, the expression of SHH protein was significantly increased in induction group (P<0.05). With the induction time extension, the expression of SHH protein significantly increased (P<0.05). The expression of IHH protein was significantly lower in the induction group compared with non-induction group (P<0.05). There was no significantly changed of IHH among 7d, 14d and 21d induction groups(P>0.05). In the process of MSCs induction into bone, expression of Shh mRNA and Ihh mRNA had no significant change (P>0.05).3. The positive expression of II type collagen were increased following chondrogenic differentiation in MSCs. After 14d TGF-β1 induction, the positive rate of II type collagen expression was higher than 90% and the staining intensity increased. Compared to non-induction group, the expression of SHH protein was significantly decreased in induction group (P<0.05). With the induction time extension, the expression of SHH protein significantly increased (P<0.05). The expression of IHH protein was significantly higher in the induction group compared with non-induction group at 14 d and 21day(P<0.05). With the induction time extension, the expression of IHH protein significantly increased (P<0.05). The trend of Shh mRNA expression was similar to the expression of SHH. Expression of Ihh mRNA was significantly decreased in induction group compared to the non-induction group at 7d and 14d while significantly increased at 21d (P<0.05). Conclusion:1. After comparing four methods to get MSCs, the best way for cell growth state and cell proliferation is to culture cells in a plastics petri dishes with foreign womb bovine serum and DMEM-L liquor. These BMSCs can be applied directly to the following study.2. Expression ofⅠtype collagen in induction group showed that dexamethasone induced BMSCs to osteoblast. In induction group, SHH protein expression was significantly increased and IHH protein expression was significantly lower compare with non- induction group, which suggested that SHH and IHH may play different roles involving in the osteogenic differentiation of bone marrow stromal cells induced by Dexamethasone.3. Expression ofⅡtype collagen in induction group showed that TGF-β1 induced BMSCs to cartilage. In induction group, SHH protein expression was significantly decreased and IHH protein expression was significantly increased compared with non- induction group, which suggested that SHH and IHH may play different roles involving in the chondrogenic differentiation of bone marrow stromal cells induced by TGF-β1.In summary, this study first systematically investigated the expressions of hedgehog signaling molecules during osteogenic and chondrogenic differentiation in rat bone marrow mesenchymal cells. The results showed that expression of SHH protein and IHH protein were significantly increased during osteoblast differentiation and chondrogenic differentiation process respectively. These results suggests the expression pattern of SHH and IHH may be different in osteogenic and chondrogenic differentiation of rat bone marrow stromal cells.更多还原