【作者】 袁源；

【导师】 杨树源；

【作者基本信息】 天津医科大学 ， 神经外科学， 2006， 博士

【摘要】 第一部分:人脐带间充质干细胞分离纯化及基本生物学特性研究 目的 探讨从人脐带组织分离、培养间充质干细胞(MSC)的方法,并对获取的MSC进行扩增、纯化,研究其基本生物学特性。 方法 无菌条件下收集剖宫产新生儿脐带,以20ml注射器分别插入两根脐静脉及一跟脐动脉内,反复冲洗,冲去血管内的残存积血。组织剪将脐带剪碎,直至1mm~3大小组织块。直接将组织块接种于含DMEM/F12培养基或将组织块经酶消化法获取单个核细胞后接种于DMEM/F12培养基。进行原代培养,观察并记录其形态学变化。细胞达到融合状态时,消化传代进行纯化和扩增培养。用流式细胞仪检测MSC的细胞表面标志。绘制细胞生长曲线,通过5-溴脱氧尿嘧啶核苷(BrdU)掺入实验检测其增殖能力。流式细胞仪测定细胞周期。RT-PCR检测OCT-4 mRNA表达。透射电镜观察脐带MSC的形态和超微结构。 结果 两种分离方法均可获得成纤维样细胞。组织块贴壁法1周后于组织块间隙已可见散在分布的长条索状纺锤型细胞,3周左右细胞达到80%融合。胶原酶消化法,细胞接种后第2天即可见有少量形态各异的贴壁细胞,散在分布,1周左右时,贴壁细胞形成集落,占优势的是成纤维样细胞,及少量鹅卵石样细胞,至2周左右可达到80%融合。经传代培养后,可得到较为纯化的成纤维样细胞,细胞约每3天即可达到90%～95%融合,需再次传代或冻存,细胞可传代20代以上。分别对第3代、第5代、第10代脐带MSC行FACS检测,结果表明各代间免疫表型无明显差异,均强烈表达CD13、CD29、CD105、CD44,弱表达CD106,不表达CD14、CD34、CD11a、CD31、CD45。对第2、6、12代UCMSCs生长曲线进行分析,表明其倍增时间分别为33.1h、34.4h、34.65h。BrdU掺入实验中,更多还原

【Abstract】 Part one: Investigation on the isolation, purification and biological character ofhuman umbilical cord-derived mesenchymal stem cells in vitro Objective To explore the isolation, purification and expansion method of human umbilical cord derived mesenchymal stem cells(UCMSC) and to investigate there biological characteristics.Method The umbilical cords from full-term section patients were collected in sterile condition immediately upon delivery. Blood clot in umbilical veins and arteries were rinsed out using a 20ml syringe. The umbilical cord was then cut into pieces of 1mm~3. Tissure fragments were then put into bottom of Petri dish, cultured in DMEM/ F12 medium. Another way was digesting the pieces by collagenase to obtain mononuclear cells. Then the mononuclear cells were cultured in DMEM/ F12 medium. We observed the cells’ morphological changes and took photos. Once adherent cells reached approximately 80-90% confluence, they were detached and re-plated under the same culture conditions to amplify. Surface antigens of UCMSCs were detected by FACS. The cell-proliferation growth curve and cell cycle of UCMSC were also analysed. We also add BrdU into the medium to detect the self-renewal capacity of UCMSCs. RT-PCR method was used to detect OCT-4 mRNA expression and the cell’s ultrastructure was recorded using electron microscope.Results Fibroblast-like cells could be harvested by both of the two methods. 1 week after transferring fragments into plate, some mesenchymal like cells with a fusiform or stellate appearance migrated from the explants. After 3 weeks of primary culture, isolated UCMSCs reached 80% confluence. In collagenase method, adherent cells with different morphology could be found 1 day after cultivation. They formedcolonies, then expanding after 1 week. Most of the colonies were fibroblast-like cells. These UCMSCs reached 80% confluence 2 weeks later. For passaging, confluent UCMSCs were detached with trypsin and reseeded at 1:3 under the same culture conditions. After approximately 3days, cells cultures reached 90%-95% confluence again, and need to passage or cryopreservation. Primary UCMSCs can expand at least 20 passages. Surface antigens of UCMSCs of P3and P5 were detected by FACS. Results showed that UCMSCs express CD 13, CD29, CD44, CD 105, and not express CD106, CD 34, CDlla, CD14, CD31, CD45. The cell-proliferation growth curve showed that doubing time of p2, p6, pl2 UCMSCs were 33.11u 34.41k 34.65h respectively. Adding BrdU into the medium and cultured for 48h, immunohistochemistry showed that 80-90% UCMSCs were BrdU positive cell. Cell cycle analysis showed that 80-90% cells were in G0-G1 phase. RT-PCR analysis showed that OCT-4, an important transcription factor of pluripotent stem cells, was expressed on UCMSCs. Visualized by TEM, UCMSCs has a large and irregular nucleus and prominent nucleolus, with much euchromatin and little heterochromatin. It has little cytoplasm, with some organelle scattered, mainly rough endoplasmic reticulum and mitochondria and relatively more free ribosome. Conclusion Fibroblast-like cells could be harvested by both of tissue fragment adherent method and collagenase digesting method. Umbilical cord derived fibroblast-like cells have strong proliferation capacity and immature ultrastructure, and they express stem cell marker OCT-4, indicating there stem cell characteristic. Umbilical cord derived fibroblast-like cells have the same immunophenotype with bone marrow mesenchymal stem cell, indicating that they are new member of MSC family. As a kind of stem cell isolated from off-fall of delivery, umbilical cord, UCMSCs eliminate the ethical or social concerns and are easily obtainable. Our research pave the way for therapeutic and biotechnological study of stem cells.Part two: study on neuron- differentiation of human umbilical cord derivedmesenchymal stem cells(UCMSC) Objective To discuss the condition and methods of neuron- differentiation ofhuman umbilical cord derived mesenchymal stem cells(UCMSCs) and purify there potency of neuron- differentiation and therapeutic use for neural transplantation. Method UCMSCs of P3, P5 and P10 were induced to differentiate into neural like cells using three different methods. Chemical methods: UCMSCs were put into pre-induction medium (DMEM/ F12+20%FBS+10ng/mlbFGF) for 24 hours, then the pre-induction medium were replaced by induction medium(DMEM/F12+2%DMSO+200uM BHA+25uM KCL+2mM Valproic acid+10|iM Forskolin+luM Hydrocortisone)and the cells were induced for 5hours. Salvia miltiorrhiza method: UCMSCs were put into pre-induction medium for 24 hours just like chemical method, while the induction medium was change to DMEM/F12+2% salvia miltiorrhiza and the cells were cultivate in for 5 hours. Neurotrophic factor method: UCMSCs were induced for 7 days in DMEM/F12 plug 10% FBS, 0.5umol/L Ratinoic acid, 20ng/ml bFGF and 20ng/ml EGF. Morphological changes were monitored during the differentiating procedure. The cells were then fixed with 4% paraformaldehyde and stained for neural specific marker Nestin, NSE, NeuN, NF-M, P-tubulin III, and GFAP. Neuron-differentiation capacity of different passages was compared. RT-PCR method was used to analyse NSE and GFAP mRNA expression of UCMSCs pre and post differentiation. Results In chemical induction group, UCMSCs began to contract and and became irregularly shaped at 0.5~lh after induction. Then the cell morphology changed more obviously, the cell become triangular or cone-shaped with multipolar ling processes. The processes continued to elaborate and to display many branches, some branch of different cells connected with each other, just like dendrite, forming a net. While some cells floated and died 8-10 hours later. In salvia miltiorrhiza group, some cytoplasm contract and extend long process, just like a neural cell, but there morphlogical changes were much more moderate. In neurotrophic factor group, the cell body gradually prolonged and extend silkness process. No cells were floated or dead. Immunohistochemistry showed that chemical means induced UCMSCs express Nestin, NSE, NeuN and NF-M, but don’t express GFAP. Immunofluorescence assay showed that salvia miltiorrhiza means or neurotrophic factors induced UCMSCswere positive for Nestin, NSE, NF-M and p-tubuhn III, and a few cells expressed GFAP. RT-PCR results showed NSE mRNA expression coult be seen in both induced and untreated UCMSCs, while neural-induction can really increase the expression and OCT-4 mRNA expression disappeared after induction. Conclusion UCMSCs can differentiate into neuron like cells in vitro by different methods and express neural specific marker, indicating there capacity to differentiate into a neurophenotype and revealed there potential use for neural transplantation.Part 3 The experimental research of human umbilical cord derivedmesenchymal stem cells transplantation into adult rat brain and theretherapeutic effect on rat brain fluid percussion injuryObjective To investigate the survival, migration and transdifferentiation ofumbilical cord derived mesenchymal stem cells(UCMSCs) after transplantation intoadult rat brain and to investigate the effects and mechanism of human umbilical cordderived mesenchymal stem cell (UCMSC) transplantation on the repair of rat brainfluid percussion injury.Methods1? Mesenchymal stem cells were derived from human umbilical cord. UCMSCsbefore the 6th passage were marked with BrdU or Hoechst33258. 2, Marked UCMSCs(10ul, 1 xl06>h)were injected to hippocampal Cl region of adult rats with the injection speed of l(il/min. After 1, 2, 4, 6w, transplanted animals were sacrificed respectively by intracardiac perfusion under deep anesthesia. The perfusion was with icecold PBS, followed by 4% buffered paraformaldehyde. Sequential sections of 5um were cut aroud the transplant site. NSE, GFAP, NF-M expression was detected by immunohistochemical method. 3^ All rats underwent fluid percussion brain injury. Animals divided into 4 groups: sham group, TBI group, TBI+DF group, and TBI +UCMSCs group. UCMSCs, labeled with BrdU, transplanted into rat brain nearby injury area 24h after fluid percussion injury. Control animals underwent the same injury but injected with only vehicle. The motor function was evaluated before and 24h, 48h, lw, 2w, 3w,4w after injury. 4weeks after injury, animals were sacrificed by intracardiac perfusion under deep anesthesia. The perfusion was with icecold PBS, followed by 4% buffered paraformaldehyde. sequential sections of 5 urn were cut aroud the transplant site. Immunohistochemical stain was used to detect BrdU positive cell, nearby sections were used to make immunohistochemical stain to detect NSE, GFAP and VEGF expression. Fitc—Bandeiraea simplicifolia lectin-1 stain was used to identify microvessel density. (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) were performed to identify apoptotic cells in the brain. Results1 > After transplanted into hippocampal Cl region of adult rats, some UCMSCs survived for more than 6 weeks. The engraftments were mainly distributed around the injection site, but some of them migrated much farther, even migrate to subependymal zone. Part of transplanted cells expressed NSE, NF and GFAP by immunohistochemistry.2^ Significant recovery of behavior was found in UCMSC-treated rats at l-3w after injury, compared with control animals (P<.05).But all animals’ motor function recovered to normal level at 4w.3 -. NSE, NF and GFAP expression of transplanted UCMSCs were detected by Immunohistochemistry. Animals that received UCMSC transplantation showed more VEGF expression, higher microvessel density and less apoptotic cell in injured area than that of control group.Conclusion HUCMSCs can survived and migrated to a long distance after engrafted into rat brain, some of them can differentiated into neuron. UCMSC transplantation can stimulate the host to secrete VEGF and increase microvessel density and inhibit apoptosis, thereby improving functional recovery after tramatic brain injury in rat.更多还原