【摘要】： 目的:腦血管意外是世界范圍內(nèi)的三大死因之一,也是導(dǎo)致成人殘疾的首位病因。我國年發(fā)病率約130-300萬,死亡60-100萬,存活者中75%有不同程度的殘疾。盡管在腦血管意外的急救和早期康復(fù)方面取得了很大進(jìn)展,但對腦血管意外后期嚴(yán)重的神經(jīng)功能障礙缺乏有效的治療方法。21世紀(jì)是再生醫(yī)學(xué)的世紀(jì),干細(xì)胞的研究則是當(dāng)前生命科學(xué)的重中之重。與胚胎干細(xì)胞和神經(jīng)干細(xì)胞相比,人骨髓間充質(zhì)干細(xì)胞BMSCs具有以下優(yōu)點(diǎn):①取材方便,分離獲取容易,可從自身骨髓提取;②在體外擴(kuò)增迅速,具有多向分化潛能;③是來自中胚層的早期細(xì)胞,避免組織配型及免疫排斥等問題;④可避免倫理學(xué)方面的爭議等。因此,受到越來越多的學(xué)者關(guān)注。但BMSCs移植后在體內(nèi)微環(huán)境下分化為神經(jīng)膠質(zhì)細(xì)胞的比率較大而分化為神經(jīng)元樣細(xì)胞的比率較小,如果能將骨髓基質(zhì)細(xì)胞在體外分化為神經(jīng)元樣細(xì)胞,再行細(xì)胞移植將會有事半功倍的效果。本研究擬探討體外分離培養(yǎng)、純化鑒定hBMSCs的方法,分析其生物學(xué)特性并定向誘導(dǎo)分化為神經(jīng)元樣細(xì)胞,并通過立體定向?qū)BMSCs源性神經(jīng)元樣細(xì)胞植入腦缺血大鼠腦內(nèi),觀察移植后細(xì)胞存活、遷移、分化以及大鼠的神經(jīng)功能障礙恢復(fù)情況,并與hBMSCs移植做一比較,探討hBMSCs源性神經(jīng)元樣細(xì)胞移植對于局灶性腦缺血后神經(jīng)功能缺失的修復(fù)作用及可能機(jī)制,以期為hBMSCs源性神經(jīng)元樣細(xì)胞移植治療腦缺血提供理論依據(jù)。 方法:(1)用密度梯度離心結(jié)合貼壁培養(yǎng)法分離純化hBMSCs,在體外觀察hBMSCs生長特性、傳代擴(kuò)增,測定生長曲線,形態(tài)學(xué)觀察,免疫細(xì)胞化學(xué)及圖像分析測定細(xì)胞表面抗原表達(dá)情況;(2)將其與T淋巴細(xì)胞共培養(yǎng),通過3H-TdR摻入、β液閃計數(shù),分析其免疫原性;(3)采用全反式維甲酸(all-trans-retinoic acid,ATRA)、叔丁對甲氧酚(butylated hydroxyanisole,BHA)等對培養(yǎng)的細(xì)胞向神經(jīng)細(xì)胞方向誘導(dǎo)分化,繼而采用免疫細(xì)胞化學(xué)等方法對分化的細(xì)胞進(jìn)行鑒定;(4)采用線栓法制備大鼠局灶性腦缺血模型。將腦缺血大鼠隨機(jī)分為磷酸鹽緩沖液(PBS)組、hBMSCs組及hBMSCs源性神經(jīng)元樣細(xì)胞組,分別將PBS、BrdU標(biāo)記的hBMSCs及BrdU標(biāo)記的源性神經(jīng)元樣細(xì)胞通過立體定向植入大鼠紋狀體內(nèi)。術(shù)前、術(shù)后1、3、6及8周進(jìn)行神經(jīng)損傷嚴(yán)重程度評分(NSS)、平衡木實(shí)驗(yàn)(BBT)、抬高身體搖擺實(shí)驗(yàn)(EBST)、一次性被動回避平臺實(shí)驗(yàn)(Step-down Passive Avoidance)及水迷宮實(shí)驗(yàn)(Water Maze Test),觀察各組術(shù)后神經(jīng)功能改變情況;8周后處死大鼠取腦,HE染色、BrdU免疫組化染色以及BrdU/NSE, BrdU/GFAP免疫組化雙染,觀察植入BMSCs的存活、遷移、分化情況。 結(jié)果:(1)hBMSCs在體外可以大量增殖,原代培養(yǎng)需要2~3周,傳代后速度加快,可以獲取大量貼壁細(xì)胞。細(xì)胞呈均一的成纖維細(xì)胞樣,應(yīng)用流式細(xì)胞儀檢測MSCs的表面抗原,均一表達(dá)CD29、CD44和CD105,不表達(dá)CD34、CD45、CD19、HLA-DR和CD106;(2)與T淋巴細(xì)胞體外共培養(yǎng),無明顯的促T細(xì)胞增殖反應(yīng),提示其具有低免疫原性的特點(diǎn);(3)可誘導(dǎo)表達(dá)神經(jīng)元標(biāo)志抗原神經(jīng)元特異性烯醇化酶(neuron specific enolase, NSE)、星型膠質(zhì)細(xì)胞標(biāo)志抗原膠質(zhì)纖維酸性蛋白(glial fibrilament acidic protein,GFAP);(4)應(yīng)用線栓法短暫閉塞大鼠右側(cè)大腦中動脈后,大鼠出現(xiàn)左側(cè)肢體不同程度偏癱同時伴有右側(cè)Honner氏征。TTC和HE染色均顯示缺血區(qū)域位于右側(cè)紋狀體、額頂區(qū)皮層,位置和范圍較為穩(wěn)定;神經(jīng)功能行為學(xué)檢測顯示:hBMSCs移植大鼠NSS、BBT、EBST在術(shù)后1、3、6、8周與PBS對照組比較有顯著好轉(zhuǎn)(P 0.01),在術(shù)后1、3周與hBMSCs源性神經(jīng)元樣細(xì)胞移植組比較有好轉(zhuǎn)(P 0.05),6、8周NSS、BBT、EBST評分兩組比較無統(tǒng)計學(xué)差異(P 0.05);hBMSCs源性神經(jīng)元樣細(xì)胞移植組大鼠NSS、BBT、EBST在術(shù)后3、6、8周較PBS對照組明顯改善(P 0.01),在術(shù)后1、3周較hBMSCs移植組差(P 0.05),6、8周NSS、BBT、EBST評分兩組比較無統(tǒng)計學(xué)差異(P 0.05);hBMSCs和hBMSCs源性神經(jīng)元樣細(xì)胞組較對照組一次性被動回避平臺實(shí)驗(yàn)及水迷宮實(shí)驗(yàn)明顯改善(P 0.01);hBMSCs源性神經(jīng)元樣細(xì)胞較hBMSCs組效果更好(P 0.05)。移植8周后,hBMSCs組和hBMSCs源性神經(jīng)元樣細(xì)胞組均可見較多BrdU表達(dá)陽性的細(xì)胞,細(xì)胞集中在移植位點(diǎn)及周圍區(qū)域,有向缺血灶遷移的趨勢,局部無膠質(zhì)增生和淋巴細(xì)胞浸潤,hBMSCs組Brdu陽性細(xì)胞群中,11.5-19.3%為GFAP陽性細(xì)胞,1.5-4.8%為NSE陽性細(xì)胞,hBMSCs源性神經(jīng)元樣細(xì)胞組Brdu陽性細(xì)胞群中,23.5-39.1%為NSE陽性細(xì)胞,9.8-17.6%為GFAP陽性細(xì)胞。 結(jié)論:hBMSCs屬骨髓中單個核細(xì)胞,具有免疫原性低、可塑性好和擴(kuò)增能力強(qiáng)等特性,密度梯度離心結(jié)合貼壁培養(yǎng)法能有效分離純化hBMSCs;全反式維甲酸(ATRA)、叔丁對甲氧酚(BHA)等對培養(yǎng)的細(xì)胞向神經(jīng)細(xì)胞方向誘導(dǎo)分化,可誘導(dǎo)表達(dá)神經(jīng)元標(biāo)志抗原神經(jīng)元特異性烯醇化酶(NSE)的hBMSCs源性神經(jīng)元樣細(xì)胞;hBMSCs和hBMSCs源性神經(jīng)元樣細(xì)胞腦內(nèi)移植均能有效改善局灶性腦缺血大鼠的神經(jīng)功能癥狀,hBMSCs移植作用起效更快,hBMSCs源性神經(jīng)元樣細(xì)胞移植在學(xué)習(xí)、記憶功能方面改善更好;hBMSCs源性神經(jīng)元樣細(xì)胞在體內(nèi)大部分表達(dá)NSE陽性,優(yōu)于hBMSCs,局部無腫瘤生成及明顯炎癥反應(yīng)。hBMSCs源性神經(jīng)元樣細(xì)胞腦內(nèi)移植是治療局灶性腦缺血的一種可能措施。
[Abstract]:Objective:Cerebrovascular accident is one of the three leading causes of death worldwide and the first cause of disability in adults.The annual incidence of cerebrovascular accident in China is about 130-3 million,the death rate is 600-1 million,75% of the survivors have different degrees of disability.Although great progress has been made in the first aid and early rehabilitation of cerebrovascular accident,it is serious in the late stage of cerebrovascular accident. Compared with embryonic stem cells and neural stem cells, human bone marrow mesenchymal stem cells (BMSCs) have the following advantages: (1) easy extraction, easy isolation and acquisition, and can be extracted from their own bone marrow; In vitro amplification is rapid and has the potential of multidirectional differentiation; (3) early cells from the mesoderm, avoiding tissue matching and immune rejection; (4) avoiding ethical controversy and so on. In order to reduce the ratio of neuron-like cells, bone marrow stromal cells can be differentiated into neuron-like cells in vitro, and then transplanted with cells. This study aims to explore the methods of isolation, culture, purification and identification of hBMSCs in vitro, analyze their biological characteristics, and induce differentiation into neuron-like cells, and establish a new method. Neuron-like cells derived from hBMSCs were implanted into the brain of rats with focal cerebral ischemia in vivo. The survival, migration, differentiation and recovery of neurological dysfunction were observed after transplantation. The effects of transplantation of hBMSCs-derived neuron-like cells on the repair of neurological deficits after focal cerebral ischemia were compared with that of transplantation of hBMSCs. The mechanism provides a theoretical basis for the treatment of cerebral ischemia by hBMSCs derived neuron like cell transplantation.
Methods: (1) hBMSCs were isolated and purified by density gradient centrifugation combined with adherent culture, and the growth characteristics of hBMSCs were observed in vitro, subcultured and amplified, the growth curve, morphological observation, immunocytochemistry and image analysis were used to determine the expression of cell surface antigen; (2) hBMSCs were co-cultured with T lymphocytes, and were analyzed by 3H-TdR incorporation and beta liquid scintillation counting. Its immunogenicity; (3) All-trans-retinoic acid (ATRA), butylated hydroxyanisole (BHA) were used to induce the differentiation of cultured cells into neural cells, and then the differentiated cells were identified by immunocytochemical methods; (4) Focal cerebral ischemia model was prepared by thread embolization in rats. Rats with cerebral ischemia were randomly divided into phosphate buffer (PBS) group, hBMSCs group and hBMSCs-derived neuron-like cells group. PBS, BrdU-labeled hBMSCs and BrdU-labeled neuron-like cells were implanted into the striatum by stereotactic implantation. The nerve injury severity score (NSS) and balance beam were evaluated before operation, 1, 3, 6 and 8 weeks after operation. The changes of neurological function were observed by BBT, EBST, Step-down Passive Avoidance and Water Maze Test, and the rats were sacrificed 8 weeks later for brain harvesting, HE staining, BrdU immunohistochemical staining and BrdU/NSE, BrdU/GFAP immunohistochemical double staining. The survival, migration and differentiation of SCs.
Results: (1) hBMSCs could proliferate in vitro, and need 2-3 weeks for primary culture. After passage, a large number of adherent cells could be obtained. The cells were homogeneous fibroblast-like, and the surface antigens of MSCs were detected by flow cytometry. CD29, CD44 and CD105 were uniformly expressed, but CD34, CD45, CD19, HLA-DR and CD106 were not expressed; (2) and T lymphocyte bodies were detected by flow cytometry. In vitro co-culture, there was no obvious T cell proliferation response, suggesting that it has low immunogenicity; (3) neuron specific enolase (NSE), astrocyte marker antigen glial fibrillary acidic protein (GFAP) could be induced to express; (4) thread embolism was used. TTC and HE staining showed that the ischemic area was located in the right striatum, and the frontal-parietal cortex, and its location and range were stable. Neurological and behavioral tests showed that NSS, BBT, EBST were transplanted with hBMSCs in rats at 1, 3, 6 after operation. Compared with the PBS control group, the NSS, BBT and EBST scores were significantly improved at 8 weeks (P 0.01) and 1, 3 weeks after operation (P 0.05) and 6, 8 weeks after operation (P 0.05), but the NSS, BBT and EBST scores were not significantly different between the two groups (P 0.05). There was no significant difference in NSS, BBT and EBST scores between the two groups at 1 and 3 weeks after operation (P 0.05), but the effect of hBMSCs and hBMSCs-derived neuron-like cells was better than that of the control group in one-off passive avoidance platform test and water maze test (P 0.01); the effect of hBMSCs-derived neuron-like cells was better than that of hBMSCs-derived neuron-like cells (P 0.05). After 8 weeks of transplantation, a large number of BrdU-positive cells were found in both hBMSCs and hBMSCs-derived neuron-like cells. The cells were concentrated in the transplantation site and surrounding areas, and migrated to ischemic focus. There was no glial proliferation and lymphocyte infiltration in the region. In the hBMSCs group, 11.5-19.3% of Brdu-positive cells were GFAP-positive cells, and 1.5-4.8% were NSE-positive cells. Among the Brdu-positive cells in sex cells and hBMSCs-derived neuron-like cells, 23.5-39.1% were NSE-positive cells and 9.8-17.6% were GFAP-positive cells.
CONCLUSION: HBMSCs are mononuclear cells in bone marrow, which have the characteristics of low immunogenicity, good plasticity and strong amplification ability. Density gradient centrifugation combined with adherent culture can effectively isolate and purify hBMSCs. All-trans retinoic acid (ATRA) and tert-butylcresol (BHA) can induce the differentiation of cultured cells into neural cells and induce the expression of neurons. Neuron-like cells derived from hBMSCs, labeled with neuron-specific enolase (NSE), and neuron-like cells derived from hBMSCs and hBMSCs can effectively improve the neurological symptoms of rats with focal cerebral ischemia. The transplantation of hBMSCs has a faster effect, and the transplantation of neuron-like cells derived from hBMSCs can improve the learning and memory functions. Most of the neuron-like cells derived from hBMSCs expressed NSE positive in vivo, which was superior to that of hBMSCs. There was no local tumor formation and obvious inflammatory reaction. Intracerebral transplantation of hBMSCs-derived neuron-like cells was a possible treatment for focal cerebral ischemia.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R329

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本文編號：2238362