本文選題：人臍帶間充質(zhì)干細胞 + 超順磁性氧化鐵納米顆粒　； 參考：《第三軍醫(yī)大學》2010年博士論文

【摘要】： 脊髓損傷(spinal cord injury,SCI)在全球呈高發(fā)生率、高致殘率、高耗費、發(fā)病年輕化等特點。脊髓損傷后由于廣泛的神經(jīng)元死亡、大量的軸突變性、彌漫性的脫髓鞘造成患者勞動能力喪失、生活不能自理以及各種并發(fā)癥,其后果是終身性和毀滅性的,不僅給病人造成極大的痛苦,也給家庭和社會帶來沉重的負擔。一個多世紀以來,醫(yī)學界先后采用了手術(shù)、藥物、物理、基因以及細胞治療等多種方法來治療脊髓損傷,但都不能有效地解決患者不同程度的癱瘓這一難題。因此,尋找有效而安全的脊髓損傷后的治療方法仍然是困擾醫(yī)學界的一個難題且具有非常重要的科學意義、經(jīng)濟意義和社會意義。外源性干細胞移植是近十幾年來脊髓損傷治療的研究熱點,近些年來在胎兒附屬物如臍帶中發(fā)現(xiàn)有豐富的間充質(zhì)干細胞,具有低免疫原性、高增殖能力以及來源更方便等獨特的優(yōu)越性,體外研究顯示能夠向骨、軟骨、心肌、血管內(nèi)皮以及神經(jīng)系細胞等方向分化。因此,本研究利用人源性臍帶間充質(zhì)干細胞移植治療大鼠脊髓損傷,探討其療效和機制。 第一部分人臍帶間充質(zhì)干細胞的分離、培養(yǎng)與鑒定 目的: 探討人源性臍帶間充質(zhì)干細胞的培養(yǎng)方法并研究其生物學特性。 方法: 1.獲取健康、足月、剖腹產(chǎn)胎兒臍帶,剝離臍帶wharton’s jelly膠,充分剪碎,組織塊貼壁培養(yǎng)法獲得臍帶間充質(zhì)干細胞,體外傳代、純化、擴增,倒置顯微鏡下觀察細胞形態(tài)。 2.流式細胞儀檢測細胞表面標志物CD73、CD90、CD105、CD14、CD34、CD45、HLA-DR。 結(jié)果: 1.人臍帶wharton’s jelly膠組織塊培養(yǎng)5-7 d后組織塊周圍即可見新生細胞,為長梭形或多角形,培養(yǎng)至16-20 d細胞明顯增多,達90%以上融合,類似成纖維樣細胞,放射狀或漩渦狀分布。傳代后細胞增殖迅速,生長3-4d細胞即呈80%-90%以上融合。 2.流式細胞儀檢測人臍帶間充質(zhì)干細胞高表達CD73、CD90、CD105,不表達CD14、CD34、CD45、HLA-DR。 結(jié)論: 1.應用組織塊貼壁培養(yǎng)法能夠從臍帶wharton’s jelly膠中培養(yǎng)出大量增殖能力較強的成纖維樣細胞。 2.流式細胞儀檢測顯示臍帶來源間充質(zhì)干細胞和骨髓、臍血、胎盤等其它組織來源的間充質(zhì)干細胞具有相似的表面標志。 3.臍帶能為間充質(zhì)干細胞移植提供充足的干細胞來源。 第二部分人臍帶間充質(zhì)干細胞磁標記后生物學特性及磁共振信號研究 目的: 探討人臍帶間充質(zhì)干細胞超順磁性氧化鐵(superparamagnetic iron oxide, SPIO)納米顆粒標記及磁共振示蹤的可行性。 方法: 1.細胞的SPIO標記共分為5個濃度組,分別為對照(0μg)、5.6μg、11.2μg、22.4μg和44.8μg Fe/ml,其中每個濃度有四個孵育條件,即12h-pll,24h-pll,12h+pll和24h+pll。 2.普魯士藍染色計數(shù)SPIO標記細胞,計算標記陽性率,MTT法檢測SPIO標記細胞生長和增殖活性。 3.體外磁共振GRE T2*WI和SE T2WI成像檢測標記細胞的磁共振信號。 4.標記細胞大鼠脊髓內(nèi)移植后,磁共振TSE T2WI成像追蹤體內(nèi)移植磁標記細胞。 結(jié)果: 1.細胞標記陽性率隨著孵育濃度和時間的延長而升高,在22.4μg Fe/ml濃度、24h-pll條件下,細胞的標記率達到94.1%,提高濃度到44.8μg Fe/ml、24h-pll,陽性率不再升高;在5.6μg、11.2μg、22.4μgFe/ml三個濃度下(孵育12h),增加pll可以顯著提高標記率;在24h+pll條件下,細胞生長受到明顯影響,大部分細胞壞死脫落。 2.低于22.4μgFe/ml濃度標記,細胞生長和增殖活性不受到明顯影響,濃度達到44.8μgFe/ml(孵育24h),二者均顯著減弱。 3. 22.4μg Fe/ml SPIO標記24h后,體外磁共振檢查示GRE T2*WI和SE T2WI上均呈低信號,且隨著細胞數(shù)目的增加,信號不斷降低,與未標記細胞組具有統(tǒng)計學差異,且信號強度與細胞數(shù)目呈直線相關(guān)。 4.細胞移植3d后MRI檢查發(fā)現(xiàn)標記細胞注射點呈明顯的低信號,而未標記細胞注射點信號稍有減低。14d后標記細胞注射點仍然可以追蹤到標記細胞低信號。脊髓標本普魯士藍和核固紅染色,可見標記細胞注射點有大量SPIO陽性細胞,而未標記細胞注射點則見到少量陽性細胞。 結(jié)論: 1.超順磁性氧化鐵納米顆粒能夠有效標記人源性臍帶間充質(zhì)干細胞,且不影響細胞的生長和增殖活性。 2.磁標記細胞體外磁共振GRE T2*WI和SE T2WI成像可以產(chǎn)生特征性低信號轉(zhuǎn)變,其信號強度與細胞數(shù)量成直線相關(guān)。 3.磁共振TSE T2WI成像可以追蹤體內(nèi)移植磁標記細胞,持續(xù)時間達2w以上。 第三部分人臍帶間充質(zhì)干細胞移植治療脊髓損傷療效及機制研究 目的: 研究人臍帶間充質(zhì)干細胞移植治療脊髓損傷的療效并初步探討其機制。 方法: 1. 36只SD健康成年雌性大鼠隨機分為:假手術(shù)組12只,大鼠只行T9-T11椎板切開術(shù),不行脊髓打擊傷;對照組12只,行T10段脊髓打擊傷,傷后第1d脊髓內(nèi)注射DMEM/F12;實驗組,行T10段脊髓打擊傷,傷后第1d脊髓內(nèi)注射第5代人源性臍帶間充質(zhì)干細胞。 2.于傷后1d、1w、3w、5w、7w、8w進行行為學BBB運動功能評分;細胞免疫熒光染色觀察GDNF、BDNF和NT-3的表達;于傷后3w采用ELISA檢測大鼠脊髓標本GDNF、BDNF和NT-3的含量。 3.于傷后1m、2m采用免疫熒光染色觀察臍帶間充質(zhì)干細胞在宿主脊髓內(nèi)的遷移和分化。 4.于傷后2m采用免疫組織化學染色觀察GAP-43、NF-200、GFAP在脊髓內(nèi)的表達。 結(jié)果: 1.假手術(shù)組運動功能于1w后基本正常,對照組和實驗組隨著時間的延長,運動功能逐漸恢復,在傷后3w內(nèi)恢復明顯。第5w后實驗組的BBB評分與對照組相比明顯升高。 2.細胞免疫熒光染色結(jié)果顯示BDNF、nestin無表達,GDNF、NT-3弱表達。ELISA檢測可見實驗組GDNF、NT-3含量比對照組明顯增多,而BDNF兩組間無明顯差別。 3.移植后2m,人臍帶間充質(zhì)干細胞在宿主脊髓內(nèi)存活并且呈縱向遷移,距離達到5mm,損傷區(qū)可見大量hNu染色陽性細胞匯集。移植后第1m、2m未見到臍帶間充質(zhì)干細胞向神經(jīng)元、少突膠質(zhì)細胞和星形膠質(zhì)細胞方向分化。 4.移植后2m,GFAP免疫組化染色結(jié)果顯示對照組脊髓損傷區(qū)周圍灰質(zhì)和白質(zhì)GFAP表達明顯比實驗組和假手術(shù)組增強,對照組脊髓損傷程度比實驗組重,GFAP形成致密的膠質(zhì)瘢痕;NF-200免疫組化染色結(jié)果顯示對照組脊髓損傷區(qū)周圍NF-200陽性神經(jīng)纖維長度明顯比實驗組縮短;GAP-43免疫組化染色結(jié)果顯示實驗組脊髓損傷區(qū)周圍GAP-43陽性細胞比對照組明顯增多,并且有較多典型的再生軸突生長錐樣結(jié)構(gòu),對照組未見到典型的生長錐。 結(jié)論: 1.臍帶間充質(zhì)干細胞移植后能夠在宿主脊髓內(nèi)存活,并且沿著脊髓縱軸遷移。但是不能見到其向神經(jīng)元、少突膠質(zhì)細胞和星形膠質(zhì)細胞方向分化。 2.臍帶間充質(zhì)干細胞移植后能夠分泌GDNF和NT-3促進大鼠脊髓損傷后后肢運動功能評分增加,從而改善行為學功能。 3.臍帶間充質(zhì)干細胞移植后能夠抑制大鼠脊髓損傷后膠質(zhì)瘢痕的形成,促進神經(jīng)纖維再生。
[Abstract]:Spinal cord injury (SCI) is characterized by high incidence, high disability, high consumption, and younger onset in the world. After spinal cord injury, extensive neuronal death, a large number of axons, and diffuse demyelinating causes the loss of labor, life and various complications. The consequences are life-long and destruction. For more than a century, the medical community has used a variety of methods, such as surgery, medicine, physics, gene and cell therapy, to treat spinal cord injury, but it can not effectively solve the problem of the patient's paralysis to varying degrees. The treatment of effective and safe spinal cord injury remains a difficult problem in the medical field and is of great scientific significance, economic significance and social significance. Exogenous stem cell transplantation is a hot spot in the recent decade for the treatment of spinal cord injury. In recent years, there are abundant mesenchymal stem cells found in fetal appendages such as umbilical cord. Cells have unique advantages such as low immunogenicity, high proliferation ability and more convenient sources. In vitro studies have shown that the cells can differentiate into bone, cartilage, myocardium, vascular endothelial cells and nerve cells. Therefore, human umbilical cord mesenchymal stem cells are used to transplant spinal cord injury in rats and explore its therapeutic effect and mechanism.
Part one: isolation, culture and identification of human umbilical cord mesenchymal stem cells
Objective:
Objective to explore the culture methods of human umbilical cord mesenchymal stem cells and study their biological characteristics.
Method:
1. to obtain health, full-term, fetal umbilical cord by caesarean section, umbilical cord Wharton 's jelly glue, full scissors, tissue block wall culture method to obtain umbilical cord mesenchymal stem cells, external generation, purification, amplification, and inverted microscope observation of cell morphology.
2. cell surface markers CD73, CD90, CD105, CD14, CD34, CD45, HLA-DR. were detected by flow cytometry.
Result:
After the 1. human umbilical cord Wharton 's jelly glue tissue mass was cultured for 5-7 D, the new cells were seen around the tissue block, which was long spindle or polygon, and the culture to 16-20 D cells increased obviously, up to 90% fusion, similar to fibroblast like cells, radiate or whirlpool. The cells proliferated rapidly after the passage, and the growth of 3-4d cells was more than 80%-90% fusion.
2. flow cytometry showed that human umbilical cord mesenchymal stem cells were highly expressed CD73, CD90, CD105, and did not express CD14, CD34, CD45, HLA-DR..
Conclusion:
1. the tissue block adherent culture method can produce a large number of fibroblast like cells with strong proliferative ability from the umbilical cord Wharton 's Jelly gel.
2. flow cytometry showed that mesenchymal stem cells from umbilical cord derived mesenchymal stem cells and bone marrow, umbilical cord blood, placenta and other tissue derived mesenchymal stem cells have similar surface markers.
3. umbilical cord can provide sufficient stem cell source for mesenchymal stem cell transplantation.
The second part of human umbilical cord mesenchymal stem cells after magnetic labeling biological characteristics and magnetic resonance signals
Objective:
Objective to investigate the feasibility of iron oxide (SPIO) nanoparticle labeling and magnetic resonance tracing in human umbilical cord mesenchymal stem cells (superparamagnetic).
Method:
The SPIO markers of 1. cells were divided into 5 concentration groups, the control (0 mu g), 5.6 mu g, 11.2 mu g, 22.4 mu g and 44.8 micron g Fe/ml, with four incubation conditions for each concentration, namely 12h-pll, 24h-pll, 12h+pll and 24h+pll..
2. Prussian blue staining was used to count SPIO labeled cells, the positive rate of markers was calculated, and the growth and proliferation activity of SPIO labeled cells were detected by MTT.
3. the magnetic resonance signals of labeled cells were detected by external magnetic resonance GRE T2*WI and SE T2WI imaging.
4. labeled cells were transplanted into the spinal cord of rats, and magnetic resonance TSE T2WI imaging was used to track the transplanted magnetic labeled cells in vivo.
Result:
The positive rate of 1. cell markers increased with the prolongation of incubation concentration and time. Under the condition of 22.4 mu g Fe/ml concentration and 24h-pll, the labeling rate of cells reached 94.1%, the concentration increased to 44.8 mu Fe/ml, 24h-pll, and the positive rate no longer increased; at 5.6 mu g, 11.2 mu g, 22.4 mu gFe/ml three concentration (incubating 12h), increasing PLL could significantly increase the labeling rate; in 24h+ Under the condition of PLL, cell growth was obviously affected, and most of the cells were necrotic and shedding.
2. below 22.4 mu gFe/ml concentration, cell growth and proliferation activity were not significantly affected, with a concentration of 44.8 gFe/ml (incubation of 24h), and two of them decreased significantly.
After 3. 22.4 g Fe/ml SPIO labeling 24h, in vitro magnetic resonance imaging showed that both GRE T2*WI and SE T2WI showed low signal, and as the number of cells increased, the signal decreased continuously, and there was a statistical difference with the unlabeled cell group, and the signal intensity was linearly related to the number of cells.
After the 4. cell transplantation, the MRI examination showed that the marked cell injection point was obviously low signal, while the unlabeled cell injection point signal slightly reduced.14d could still be traced to the low signal of the labeled cells. The spinal specimen Prussian blue and nuclear solid red staining, and the marked cell injection point had a large number of SPIO positive cells, but not marked by the marked cell injection point. A small number of positive cells were observed at the point of cell injection.
Conclusion:
1. superparamagnetic iron oxide nanoparticles can effectively label human umbilical cord mesenchymal stem cells and do not affect cell growth and proliferation activity.
2. in vitro magnetic resonance imaging of GRE T2*WI and SE T2WI can produce characteristic low signal transformation, and its signal intensity is linearly correlated with cell number.
3. magnetic resonance TSE T2WI imaging can track transplanted magnetic labeled cells in vivo, lasting more than 2W.
The effect and mechanism of third human umbilical cord mesenchymal stem cells transplantation on spinal cord injury
Objective:
Objective to study the effect of human umbilical cord mesenchymal stem cells transplantation on spinal cord injury and to explore its mechanism.
Method:
1.36 SD healthy adult female rats were randomly divided into 12 rats in the sham operation group. The rats were only treated with T9-T11 laminectomy and no spinal cord injury; 12 rats in the control group were treated with T10 segment spinal cord injury and DMEM/F12 in the spinal cord after the injury; the experimental group was treated with T10 segment spinal cord injury, and fifth generation of human umbilical cord mesenchymal stem cells were injected into the spinal cord after the injury in the spinal cord.
2. after injury, 1D, 1W, 3W, 5W, 7W, 8W were used to evaluate the behavioral function of BBB, and the expression of GDNF, BDNF and NT-3 were observed by cell immunofluorescence staining.
3. immunofluorescence staining was used to observe the migration and differentiation of umbilical cord mesenchymal stem cells in the spinal cord of 1m after 2m.
4. the expression of GAP-43, NF-200 and GFAP in spinal cord was observed by immunohistochemical staining after 2m.
Result:
1. the exercise function of the sham operation group was basically normal after 1W, and the control group and the experimental group were gradually restored with the extension of time, and recovered obviously in the 3W after the injury. The BBB score of the experimental group was significantly higher than that of the control group after 5W.
The results of 2. cell immunofluorescence staining showed that BDNF, nestin was not expressed, GDNF, and NT-3 weak expression.ELISA detected the experimental group GDNF, NT-3 content was significantly higher than the control group, but there was no significant difference between the two groups of BDNF.
3. 2m after transplantation, human umbilical cord mesenchymal stem cells live in the host spinal cord and migrate longitudinally, the distance reaches 5mm, and a large number of hNu staining positive cells can be found in the injured area. After transplantation, 1M, 2m does not see the direction differentiation of umbilical cord mesenchymal stem cells to neurons, oligodendrocytes and astrocytes.
4. after transplantation, 2m, GFAP immunohistochemical staining showed that the expression of gray matter and white matter GFAP in the area around the spinal cord injury of the control group was significantly higher than that of the experimental group and the sham operation group. The degree of spinal cord injury in the control group was heavier than the experimental group, and the GFAP formed a dense glial scar, and the NF-200 immunohistochemical staining fruit showed the NF-200 positive God around the spinal cord injury area of the control group. The fiber length was significantly shorter than the experimental group, and the results of GAP-43 immunohistochemical staining showed that the GAP-43 positive cells around the spinal cord injury area in the experimental group were significantly higher than those in the control group, and there were more typical conical structure of the regeneration axon growth, and the control group did not see the typical growth cone.
Conclusion:
1. umbilical cord mesenchymal stem cells can survive in the host spinal cord after transplantation and migrate along the longitudinal axis of the spinal cord. But they can not be seen in the direction of the neuron, oligodendrocytes and astrocytes.
2. after transplantation, umbilical cord mesenchymal stem cells can secrete GDNF and NT-3 to promote the motor function score of rats with spinal cord injury and improve their behavioral function.
3. the transplantation of umbilical cord mesenchymal stem cells can inhibit the formation of glial scar after spinal cord injury and promote the regeneration of nerve fibers.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R329

【引證文獻】

相關(guān)期刊論文 前2條

1 劉明濤;蔡力;陶劍虹;;人臍帶間充質(zhì)干細胞向心肌樣細胞定向分化及其臨床移植研究進展[J];心血管病學進展;2012年02期

2 高健偉;魏開斌;;人臍帶間充質(zhì)干細胞治療脊髓損傷的研究進展[J];中國矯形外科雜志;2013年06期

相關(guān)碩士學位論文 前2條

1 劉明濤;人臍帶間充質(zhì)干細胞體外向心肌細胞樣分化及HLA-G在人臍帶間充質(zhì)干細胞中的表達[D];遵義醫(yī)學院;2012年

2 宋泓;釓噴酸葡胺標記人臍帶間充質(zhì)干細胞的體外MR成像轉(zhuǎn)化實驗研究[D];暨南大學;2013年

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本文編號：1880578