【作者】 王梓；

【导师】 齐志明；

【作者基本信息】 大连医科大学 ， 外科学， 2010， 硕士

【摘要】 目的肌腱病的治疗是临床骨科的难点,骨髓基质干细胞的应用为肌腱病的治疗提供了新思路。骨髓基质干细胞具有多向分化潜能并可以被诱导分化为多种细胞,但由于骨髓基质干细胞的分化机制及在治疗过程中的作用方式尚未明确,限制了其在肌腱病治疗中的应用。本课题通过体外构建骨髓基质干细胞与自体肌腱细胞的间接共培养体系,在单纯间接共培养环境下与间接共培养同时添加自体富血小板血浆进行干预的情况下研究间接共培养环境对骨髓基质干细胞的分化诱导作用,以探索骨髓基质干细胞的分化机制并为骨髓基质干细胞在肌腱病治疗中的应用提供参考。方法应用组织块培养法分离培养大鼠肌腱细胞。体外采集大鼠骨髓基质干细胞并扩增。采集大鼠血液制备富血小板血浆并激活以获取富血小板血浆萃取液。利用孔径0.4μm的细胞共培养嵌盒与6孔板构建培养液相通但细胞不直接接触的间接共培养体系。将生长状态良好的第三代(P3)大鼠肌腱细胞及第四代大鼠骨髓基质干细胞(P4)用于实验,实验组骨髓基质干细胞被接种于间接共培养体系外室,将同体肌腱细胞种植于内室,另外单独设置一组间接共培养组并加入富血小板血浆萃取液。于间接共培养后第3天、第7天及第10天,分别提取各组间接共培养体系内外室的细胞进行形态学观察,并利用RT-PCR及免疫组化染色技术分别对各组肌腱细胞与骨髓基质干细胞的Ⅰ型胶原与腱调蛋白(TNMD)进行基因层面与蛋白层面的检测,以分析间接共培养环境对骨髓基质干细胞分化与细胞外基质表达的影响。结果应用组织块培养法成功分离出细胞,并经鉴定证实为肌腱细胞。应用肌腱细胞与骨髓基质干细胞间接共培养3天后,间接共培养组的骨髓基质干细胞在RT-PCR检测与免疫组化染色检测中均未表达Ⅰ型胶原与腱调蛋白(TNMD)。间接共培养7天后,间接共培养组干细胞在Ⅰ型胶原的PCR电泳图像中出现模糊条带,同时Ⅰ型胶原免疫组化显示部分细胞呈弱阳性染色,单纯间接共培养组与富血小板血浆干预组之间结果无显著差异。间接共培养组干细胞在各种检测中未出现对腱调蛋白(TNMD)的表达。间接共培养10天后,间接共培养组干细胞在Ⅰ型胶原的PCR电泳图像中出现清晰条带,但亮度低于肌腱细胞对照组。同时Ⅰ型胶原免疫组化显示间接共培养组干细胞呈广泛弱阳性染色,通过测定免疫组化结果的平均光密度进行统计学分析,结果显示间接共培养组骨髓基质干细胞Ⅰ型胶原表达量显著高于干细胞阴性对照组,但低于肌腱细胞对照组。间接共培养组干细胞在腱调蛋白(TNMD)的PCR电泳图像中表现为模糊条带,但腱调蛋白(TNMD)免疫组化染色结果为阴性。结论通过组织块培养法可以在体外成功分离并培养大鼠肌腱细胞。与肌腱细胞间接共培养的培养环境可以诱导骨髓基质干细胞表达Ⅰ型胶原。间接共培养环境下骨髓基质干细胞未出现明显的腱调蛋白表达。根据实验结果,不能认为富血小板血浆的干预对间接共培养环境诱导后骨髓基质干细胞细胞外基质的分泌产生影响。尚不能认为间接共培养环境可以诱导骨髓基质干细胞向肌腱细胞分化。更多还原

【Abstract】 Objective:How to treat tendinopathy is a Clinical difficulty. The application of bone marrow mesenchymal cells(BMSCs) in treating tendinopathy is a novel method which has already lead to some positive results. BMSCs have the multipotentials of differentiation into various cell types,but both of the mechanism of differentiation and the role BMSCs plays in the therapeutic process are poorly understood and because of which the scientific and Clinical application of BMSCs has been restricted. We were trying to build an indirect coculture system to investigate differentiation of BMSCs induced by indirect coculturing with tenocytes. comparison was also made between the simple coculture group and the coculture group with platelet-rich plasma added to study the effects of platelet-rich plasma to BMSCs on secretion of extracellular matrix when being inducted by indirect coculture with tenocytes. On this basis we could explore the mechanism of differentiation and making a reference for Clinical application of BMSCs in treatment of tendinopathy.Methods:Tenocytes of SD rats were isolated using explant cuture method.BMSCs were collected using adherence screening method. Autolo-gous platelet-rich plasma was prepared. An indirect coculture system was established using transwell(0.4μm pore size) to inhibiting migration of cells and direct contact.The BMSCs(P4) and tenocytes (P3) were collected, BMSCs were placed in the outer chamber of this system and tenocytes in the inner chamber, one additional PRP added indirect coculture group was set up. The mRNA expression of TNMD and typeⅠcollagen were measured by RT-PCR and the corresponding protein levels of TNMD and typeⅠcollagen by technique of immunohistochemistry at various time points (3d,7d,10d) to study the secretion of extracellular matrix and differentiation of BMSCs induced by the coculture envioroment.Results:Cells were isolated using tendon explant cuture and proved to be tenocytes.There is no significant TNMD nor typeⅠcollagen expresssion observed 3days after coculture. Indistinc bands were observed on the PCR image detecting typeⅠcollagen expression by BMSCs in coculture enciroment and some of BMSCs were stained by immunohistochemical staining 7days later.TNMD expression of BMSCs in coculture enciroment was not observed.On the 10th day after coculture,distinct bands were observed on the PCR image detecting typeⅠcollagen expression by BMSCs in coculture enciroment and BMSCs of experimental groups were widely stained by immunohistochemical staining.Mean optical density(MOD)of the results of immunohistochemical staining were measured for statistical analysis.The statistical data showed that experimental groups had a higher typeⅠcollagen expression than the negative control but lower than the positive control.During the experimental groups, there is no marked qualitative difference between the simple coculture group and PRP group.There is indistinct bands on the PCR image detecting TNMD expression by BMSCs in coculture enciroment,the result of TNMD immuno-histochemical staining is negative.Conclusions:Tenocytes of SD rats could be successfully isolated using explant cuture method.The typeⅠCollagen expression of BMSCs could be induced by the enviroment of being cocultured with tenocytes.No significant TNMD expression of BMSCs was observed after coculture process.There is no significant effect of PRP to the secretion of extracellular matrix of BMSCs in the enviroment of coculture.There is not enough evidence to prove that BMSCs could differentiate into tenocytes after being induced by the inviroment of coculture.更多还原