【摘要】： 神經(jīng)干細胞(Neural stem cells,NSCs)因其來源于神經(jīng)組織、具有分化為神經(jīng)元和神經(jīng)膠質(zhì)潛能、能達到損傷或疾病部位等特點,成為脊髓等中樞神經(jīng)系統(tǒng)損傷的細胞替代的理想移植材料。成年動物的骨髓間質(zhì)干細胞(bone marrow mesenchymal stemcells,BMSCs)是一種成體干細胞,具有與胚胎干細胞相當?shù)姆只瘽撃?在一定條件下可誘導分化NSCs,繼而分化為神經(jīng)元,而且可用于自體移植。因此BMSCs可能成為NSCs的廣泛的來源,將能克服從腦組織獲取NSCs的危險性和局限性,同時又可避免胎兒腦組織移植存在的倫理和免疫排斥問題,可解決脊髓損傷等疾病的臨床個體化治療問題。 目的建立適合本實驗室體外分離培養(yǎng)骨髓間質(zhì)干細胞的方法,誘導骨髓間質(zhì)干細胞向神經(jīng)干細胞分化,將獲得的神經(jīng)干細胞用于脊髓損傷的細胞移植,探討骨髓間質(zhì)干細胞源性的神經(jīng)干細胞(BMSCs derived NSCs,BMSCs-D-NSCs)對脊髓損傷(spine cord injury,SCI)的修復作用。 方法取大鼠骨髓,原代貼壁培養(yǎng),通過多向分化能力鑒定細胞。取第3代BMSCs加入含bFGF及EGF的無血清培液中分化,采用免疫細胞化學法以nestin鑒定神經(jīng)干細胞,加入血清培養(yǎng)后,以MAP2、GFAP及MBP鑒定分化的神經(jīng)元樣、星形膠質(zhì)細胞樣及少突膠質(zhì)細胞樣細胞。80只成年SD大鼠,隨機分為4組:A組為正常組,不行SCI手術,與其他3組同期飼養(yǎng)檢測指標;其他三組均行T9脊髓左半側(cè)離斷手術,B組為損傷對照組,脊髓損傷區(qū)內(nèi)植入吸附生理鹽水的明膠海棉;C組為BMSCs-D-NSCs組,損傷處植入細胞懸液;D組,腦源性NSCs組。移植術后7、14、28d各組以BBB(the Basso,Beattie,Bresnahan locomotor rating scale)評定法進行神經(jīng)功能評定,并進行BrdU免疫組織化學標記等,觀察BrdU在各組脊髓組織中的表達。 結(jié)果在體外擴增培養(yǎng)的BMSCs,增殖速度快,擴增能力強,在體外能分化為成骨細胞、脂肪細胞及神經(jīng)元樣細胞。BMSCs在含bFGF及EGF的無血清培液中能分化為神經(jīng)干細胞,加入血清后能進一步分化為神經(jīng)元樣細胞、星形膠質(zhì)細胞樣細胞及少突膠質(zhì)細胞樣細胞,相應表達神經(jīng)元、星形膠質(zhì)細胞與少突膠質(zhì)細胞的標記物MAP2、GFAP及MBP。SCI細胞移植術后1—4 w,B、C、D組動物后肢神經(jīng)功能均有不同程度的恢復,C組與B組間有顯著性差異(P＜0.05),C與D組之間無顯著性差異(P＞0.05),A組BBB功能評分為21分。C組、D組BrdU標記的細胞移植后7d在損傷脊髓可見,14d陽性細胞有所減少,28d時BrdU標記的陽性細胞幾乎檢測不到。 結(jié)論體外擴增純化的骨髓間質(zhì)干細胞具有分化為神經(jīng)干細胞的生物學功能,而且獲得的BMSCs-D-NSCs具有進一步分化能力;BMSCs-D-NSCs移植可以促進脊髓損傷后的功能恢復,可成為治療SCI的細胞來源。
[Abstract]:Neural stem cells (NSCs) have the potential to differentiate into neurons and glia because they are derived from nerve tissue and can reach the injury or disease location. NSCs can be used as an ideal substitute for central nervous system injury (CNS) such as spinal cord. Adult animal bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells) is a kind of adult stem cells, which has the same differentiation potential as embryonic stem cells. Under certain conditions, it can induce the differentiation of NSCs and then differentiate into neurons, and can be used for autologous transplantation. Therefore, BMSCs may become a broad source of NSCs, which will overcome the dangers and limitations of obtaining NSCs from brain tissue, and at the same time avoid the ethical and immune rejection of fetal brain tissue transplantation. It can solve the problem of individualized treatment of spinal cord injury and other diseases. Objective to establish a method suitable for isolation and culture of bone marrow mesenchymal stem cells (BMSCs) in our laboratory in vitro, to induce the differentiation of BMSCs into neural stem cells (NSCs), and to apply the obtained neural stem cells (NSCs) to transplantation of spinal cord injury cells. To investigate the repair effect of bone marrow mesenchymal stem cell derived neural stem cell (BMSCs derived) on spinal cord injury (spine cord injury-induced sci). Methods Rat bone marrow was harvested and primary adherent culture was used to identify the cells by multi-directional differentiation. The third generation of BMSCs was added to the serum-free culture medium containing bFGF and EGF. The neural stem cells were identified by nestin by immunocytochemistry, and the differentiated neurons were identified by MAP2GFAP and MBP after the addition of serum. Astrocyte-like and oligodendrocyte-like cells. 80 adult SD rats were randomly divided into 4 groups: group A as normal group, no SCI operation, and the other three groups were fed and tested at the same time. The other three groups were treated with T9 spinal cord left hemisection. Group B was the control group, group C was BMSCs-D-NSCs group, group C was implanted with normal saline, group D was implanted with cell suspension and group D was implanted with brain-derived NSCs. The nerve function was assessed by BBB (the Basso Beattiean locomotor rating scale) method and BrdU immunohistochemical staining was used to observe the expression of BrdU in spinal cord tissues in each group on the 7th day and 28th day after transplantation. Results BMSCs could differentiate into osteoblasts in vitro, adipocytes and neuron-like cells. BMSCs could be differentiated into neural stem cells in serum-free culture containing bFGF and EGF. After addition of serum, neurons were further differentiated into neuron-like cells, astrocyte-like cells and oligodendrocyte like cells. MAP2GFAP, a marker of astrocytes and oligodendrocytes, and MBP.SCI cell transplantation showed different degrees of recovery of nerve function in hind limbs of rats in group C and group B (P < 0. 05). There was no significant difference between group C and group D (P < 0. 05). The difference was significant (P > 0. 05) the BBB function score of group A was 21. The BrdU labeled cells in group C were almost undetectable 7 days after transplantation. The positive cells of BrdU could not be detected on the 14th day after the spinal cord injury and the number of BrdU positive cells decreased on the 28th day after transplantation. Conclusion the purified bone marrow mesenchymal stem cells have the biological function of differentiation into neural stem cells, and the BMSCs-D-NSCs obtained has further differentiation ability. BMSCs-D-NSCs transplantation can promote the functional recovery after spinal cord injury. Can be used as a cell source for the treatment of SCI.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329

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本文編號：2186829