【作者】 宋永周；

【导师】 崔慧先；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2009， 博士

【摘要】 一、骨髓间充质干细胞的培养、生物学特性及向类雪旺细胞诱导分化。目的:掌握大鼠骨髓间充质干细胞的分离、培养和扩增的方法,并探讨其生物学特性及向类雪旺细胞分化的条件。方法:无菌条件下取大鼠双侧股骨、胫骨和胸骨,并用吸取D-Hank’s液的注射器冲出骨髓细胞,置于含10%FBS、100U/ml双抗的L-DMEM培养基中,37℃、5%CO2环境下进行培养,应用全骨髓培养法结合贴壁分离的方法进行纯化。待细胞达95%融合时,用0.125%的胰酶-0.02%的EDTA消化2-3min后进行传代。倒置显微镜下观察原代及传代后的细胞生长特点。用MTT法测定大鼠骨髓间充质干细胞的生长曲线,从而进一步了解其生长特性。将骨髓间充质干细胞按顺序分别加入β-巯基乙醇、全反式视黄酸、Forskolin、神经调节蛋白、碱性成纤维细胞生长因子、血小板源性生长因子-AA向类雪旺细胞进行诱导,在倒置显微镜下观察生长情况及诱导后的形态学变化,S-100、GFAP免疫细胞化学染色并计算其阳性率,Western-blot法检测S-100、GFAP蛋白的表达。结果:原代培养显示原代细胞1-2天开始有少量细胞贴壁,形状不规则,呈梭形成纤维样细胞形态,7-9天左右细胞可长满培养瓶底,达95%以上融合,呈漩涡状,辐射状或鱼群样排列。传代后2h后部分细胞即开始贴壁,24h内即可完全贴壁,6-7天可铺满培养瓶底。传代10代以上,各代之间细胞生长均一,稳定。P2、P4、P6细胞生长曲线基本相同,分为潜伏期(第1-2天)、对数生长期(第3-5天)和平台期(第6-7天),且几代细胞间生长状况稳定。依次加入诱导剂后部分细胞向雪旺细胞形态转变,胞体呈椭圆形,有2-3个纤细的细胞突起,成纵形栅栏状排列,免疫细胞化学染色S-100和GFAP表达阳性率分别为71.34%和68.32%,Western-blot检测有S-100,GFAP表达,未诱导细胞则不表达。结论:1应用全骨髓培养法结合贴壁分离法成功建立了一种体外简单、快速的分离、纯化大鼠骨髓间充质干细胞的方法,并使其在体外迅速得到了增殖,而且性状稳定。2大鼠骨髓间充质干细胞可以在体外经诱导分化为类雪旺细胞。二、羊膜脱细胞基质的制备及生物相容性的研究目的:通过去污剂和酶消化法进行羊膜脱细胞基质的制备,并观察其作为组织工程支架材料的生物相容性。方法:取新鲜人羊膜冲洗后以1%tritonX-100溶液振荡24 h,0.25%胰蛋白酶37℃振荡4 h,充分漂洗,干燥后环氧乙烷消毒,并进行苏木精-伊红染色和扫描电镜检测,皮下埋置实验检测其组织相容性。四甲基偶氮唑盐法测定羊膜脱细胞基质浸提液对类雪旺细胞毒性。取培养的类雪旺细胞复合培养于羊膜脱细胞基质上,倒置显微镜下观察生长情况,并进行苏木精-伊红检测。结果:制备的羊膜脱细胞基质为白色菲薄、半透明的膜状物,柔韧性好,经苏木精-伊红和扫描电镜检测无细胞残留。皮下埋置实验检测其组织相容性好。羊膜脱细胞基质浸提液对类雪旺细胞毒性评分为1级,复合培养后可见羊膜脱细胞基质表面细胞黏附生长良好,细胞伸展,形态与正常培养细胞无差异。结论:经去污剂和酶消化法制备的羊膜脱细胞基质生物相容性良好,是一种理想的组织工程支架材料。三、类雪旺细胞复合羊膜脱细胞基质修复大鼠坐骨神经缺损的实验研究目的:探讨应用类雪旺细胞及羊膜脱细胞基质构建组织工程化神经修复大鼠坐骨神经缺损的实验效果,为临床研究提供资料。方法:以类雪旺细胞为种子细胞,羊膜脱细胞基质为支架材料,体外复合培养后构建组织工程化神经来修复大鼠10mm坐骨神经缺损。术后12周,通过大体观察、电生理检测、组织学、超微结构、逆行示踪及图像分析等多方面分析评价修复效果。结果:术后12周,实验组患肢溃疡基本愈合,移植段与近、远坐骨神经直径相当。组织学检查示含有大量的有髓神经纤维,且髓鞘较厚。透射电镜见再生神经纤维呈较成熟形态学表现,髓鞘板层清晰。电生理功能检测、逆行示踪检测被HRP标记的神经元及图像分析与自体神经移植组相比无显著差异。结论:类雪旺细胞复合羊膜脱细胞基质构建的组织工程化人工神经可以成功修复大鼠周围神经缺损,其效果接近于自体神经移植。四、创伤性脑组织匀浆对大鼠骨髓间充质干细胞分化为神经元样细胞的影响目的:观察创伤后24 h和正常脑组织匀浆诱导大鼠骨髓间充质干细胞向神经元样细胞分化的差别。方法:采用Gruncr改良法制作中度脑损伤大鼠模型,取伤后24 h和正常大鼠脑组织匀浆,对体外培养的第3代骨髓间充质干细胞进行诱导。在倒置显微镜下观察细胞形态学变化,并应用免疫细胞化学技术检测细胞内神经元特异性烯醇化酶的表达,比较创伤后和正常脑组织匀浆两组诱导率差别。结果:骨髓间充质干细胞经创伤性脑组织匀浆培养基诱导24 h后,细胞的胞体变大,36 h后部分细胞分化,回缩成圆形或梭形,48 h后部分细胞可见两个或多个突起伸出,类似神经元。免疫细胞化学技术检测显示,创伤性脑组织匀浆培养基诱导组神经元特异性烯醇化酶阳性表达为54.28±6.03%,正常脑组织匀浆诱导分化率较前者低,神经元特异性烯醇化酶阳性表达为32.76±3.25%,细胞生长状态略差。结论:脑组织匀浆可诱导大鼠骨髓间充质干细胞向神经元样细胞分化,创伤性脑组织匀浆可明显提高诱导率。更多还原

【Abstract】 1 The culture of bone marrow mesenchymal stromal cells and induce to differentiate into Schwann-like cellsObjective: To master the method for the isolation,culture and amplification of rat bone marrow mesenchymal stromal cells (BMSCs) in vitro and explore their biological characteristics and induce them to differentiate into Schwann-like cells.Methods: BMSCs were collected from degermed femurs, tibias and sternum of 4 to 6-week-old SD rat by flushing the shaft with D-Hank’s using a syringe. Cells were disaggregated by gentle pipetting several times and plated in culture flask and re-fed every 2-3 days (L-DMEM with 10% FBS and 1% penicillin-streptomycin).When 95% fusion, cells were digested with 0.125% trypsogen and 0.02% EDTA 2 min and passaged. After successive isolation, purification, subculture and proliferation, the morphology was observed with phase contrast microscope. The growth curve of P2, P4, P6 were drawn with MTT. In order to induce BMSCs differentiate into Schwann-like cells,subconfluent cultures of BMSCs treated with beta-mercaptoethanol (β-ME) followed by all trans retinoic acid and cultured in the presence of forskolin, basic-FGF, PDGF-AA and heregulin, The proliferation and morphology before and after induction of BMSCs was observed with inverted microscope constantly. Immunocytochemistry of S-100 and glail fibrillary acidic protein(GFAP) ,the ratio of positively stained cells was counted. Western blot were used to identify the expressing of S-100 and GFAP.Results: The components of primarily cultured BMSCs were very complex. The marrow cells were round in the beginning and a few cells adhered to flask at 1-2 days, which were in irregular shape such as fusiform, polygon and so on. After changing half of the medium, the cell clone began to proliferate immediately. About 7-9 days later, cells might overgrow the bottom of culture flask and reached over 95% fusion. The cells arranged regularly as a whirlpool. Generated cells stuck on the wall more quickly than primary cells. From the 2nd hour, they began to adhered and completely adhered within 24h. The cell morphology was more uniform and all the cells arranged more regularly. Cells could spread the full flask bottom for 6 or 7 days. After subcultured 10 passages, the cells proliferated rapidly and kept the morphology unchanged. The growth curve of P2, P4, P6 were quite similar and the cells biological character kept stable. The result of growth curve showed that cell growth phase was composed of latency phase, logarithmic phase and platform phase. After the induction, BMSCs displayed morphologies of schwann cells, such as ellipsoidal cell bodies and fences-like array, with two or three sligh cell processes. The positive percentages of S-100 protein expression was 71.34%, GFAP was 68.32%. The expression of GFAP, S-100 protein was detected by western blot, but untreated BMSCs were not.Conclusion: 1 In this part, a simple new method which was used for the isolation, purification and cultivation in vitro of BMSCs from SD rat bone marrow by total marrow culture associating with adhering to flash has been established.The BMSCs could proliferate immediately and keep their biological character stable in vitro. 2 BMSCs could be induced into Schwann-like cells in vitro.2 Preparation and biocompatibility of human acellular amniotic membrane matrixObjective: In this experiment, human acellular amniotic membrane matrix (HAAM) was prepared and its biocompatibility was investigated as a scaffold in tissue engineering. Methods: Fresh human amnion was shaken in 1% tritonX-100 for 24 hours,and then treated with 0.25% trypsin for 4 hours at 37℃. The production was sterilized using ethylene oxide.The HAAM was stained with hematoxylin-eosin (HE) and observed with scanning electron microscope. The HAAM was implanted in the back of SD rats to investigate its histocompatibility.The cytotoxicity of HAAM to Schwann-like cells was measured by methyl thiazolyl tetrazolium method (MTT).The Schwann-like cells were seeded in HAAM,the specimen was observed under inverted microscope and stained with HE.Results: There were no residues of cells in the HAAM, which was a white, thin and semitransparent membrane with good flexibility. The biocompatibility of HAAM in the subcutaneous implant test was good. The cytotoxicity score was graded as one. The Schwann-like cells could be seeded and adhered on the surface of HAAM, then proliferated. Their appearances were the same as those cultured in normal conditions.Conclusion: The HAAM prepared by detergent-enzymatic approach has good biocompatibility. It is an ideal scaffold for tissue engineering.3 Study on using the Schwann-like cells and human acellular amniotic membrane matrix into a tissue -engineered nerve to restore the injured rat sciatic nerve defectObjective:Make of a tissue-engineered peripheral nerve by the Schwann-like cells and human acellular amniotic membrane matrix.Study of the effect on the sciatic nerve defect,so that provide proof for clinical research.Methods: The Schwann-like cells induced from BMSCs as seed cells and HAAM as scaffold material were co-cultured in vitro to construct tissue-engineered artificial nerve for reparing 10mm defect of rat sciatic nerve.Pure HAAM conduit and autografting were as control groups. Twelve weeks after operation,the recovery results and mechanism were analyzed and evaluated by means of gross observation, histology, electrophysiology, image analysis, retrograde tracing, etc.Results: The results showed the ulcer of affected limb healed at 12w after operation in the HAAM compound of schwann-like cells group,the diameter and appearance of graft is similar to the proximal and distal segment of sciatic nerve.Histological examination revealed that a great quantity of myelined nerve fibers with thick myelin regenerated.TEM showed that regenerative nerve fibres demonstrated mature morphology with distinct layers of myelin sheath. Electrophysiological examination,positive neurons labelled with HRP and image analysis showed there was no statistical difference between the HAAM compound of Schwann-like cells group with autografting group.Conclusion: The tissue-engineered peripheral nerve can restore the defect of rat sciatic nerve effectively and there was no marked difference of nerve regeneration when compared with autogenous nerve.4 Effects of brain homogenate on the differentiation of rat bone mesenchymal stormal cells into neuron-like cells following traumatic brain injuryObjective: In this study,the different effects between traumatic brain tissue extracts and normal brain tissue extracts on the differentiation of rat bone mesenchymal stormal cells were observed.Methods: Rat models of moderate brain injury were established by Modified Gruncr Method. The traumatic brain tissue extracts acquired on the point about 24 hours after the injury and normal brain tissue extracts were used to induce 3rd passage of bone mesenchymal stormal cells in vitro. The morphological changes of the cells were observed with inverted phase microscope.The expression of neuron-specific enolase was identified by immunocytochemical technique. The different effects between traumatic brain tissue extracts and normal brain tissue extracts on the differentiation of bone mesenchymal stormal cells were observed.Results: After 24-hours induction with traumatic brain tissue extracts, the cellular bodies changed large,36 hours later, part of the bone mesenchymal stormal cells body contracted into round or spindle shape. 48 hours later, neuron-like cells with two or more prominence could be seen.The immunocytochemical method showed that the ratio of neuron-specific enolase expressing was 54.28±6.03%. The differential ratio of bone mesenchymal stormal cells induced with normal brain tissue extracts was lower, and ratio of neuron-specific enolase expressing was 32.76±3.25%.Conclusion: Bone mesenchymal stormal cells can be induced differentiating into neuron-like cells. The induction proportion induced with traumatic brain tissue extracts is higher than with normal brain tissue extracts.更多还原