本文選題：脊髓損傷 + 細(xì)胞移植�。� 參考：《天津醫(yī)科大學(xué)》2012年碩士論文

【摘要】：[目的] 觀察人雪旺細(xì)胞(human Schwann cells, hSCs)對(duì)人臍帶間充質(zhì)干細(xì)胞(human umbilical cord-mesenchymal stem cells, hUC-MSCs)在脊髓損傷(spinal cord injury, SCI)大鼠體內(nèi)存活、分化的影響；描述膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein, GFAP)、髓磷脂堿性蛋白(myelin basic protein, MBP)與高分子量神經(jīng)絲蛋白(high molecular weight neurofilament, NF-H)表達(dá)的時(shí)間特征,并探討hSCs促進(jìn)hUC-MSCs向神經(jīng)方向分化的可能機(jī)制。 [方法] 無(wú)菌條件下取出健康、因外傷行截肢治療患者的坐骨神經(jīng),應(yīng)用雙酶消化組織塊結(jié)合機(jī)械分離法分離培養(yǎng)hSCs、采用低濃度胰蛋白酶消化和差速貼壁法對(duì)細(xì)胞進(jìn)行純化,傳至第4代(P4)的hSCs用S-100抗體對(duì)其進(jìn)行鑒定,本實(shí)驗(yàn)植入的hSCs為P5；實(shí)驗(yàn)所用的hUC-MSCs由天津協(xié)和干細(xì)胞基因工程有限公司提供,細(xì)胞經(jīng)過(guò)流式細(xì)胞儀鑒定符合干細(xì)胞標(biāo)準(zhǔn),植入的hUC-MSCs亦為P5。取8周齡雌性Wistar大鼠80只,以Impactor Model-Ⅱ型打擊器制作脊髓胸10(T10)損傷模型。實(shí)驗(yàn)隨機(jī)均分為4組：DMEM對(duì)照組(A), hSCs移植組(B),hUC-MSCs移植組(C), hUC-MSCs與hSCs聯(lián)合移植組(D)。移植后2w,4w,各組實(shí)驗(yàn)動(dòng)物隨機(jī)取出2只經(jīng)心臟灌注后取材固定(脊髓長(zhǎng)度包括損傷中心頭尾端長(zhǎng)約1.5cm),制成石蠟切片后行GFAP, MBP與NF-H免疫熒光染色,植入的細(xì)胞行MAB1281免疫熒光染色進(jìn)行體內(nèi)示蹤。各組分別于移植后1W,2w,3w,4w以同樣方法取材,分別行Western-Blot, Real-Time PCR檢測(cè)。上述數(shù)據(jù)用SPSS16.0軟件方差分析進(jìn)行統(tǒng)計(jì)分析。 [結(jié)果] hSCs在原代培養(yǎng)第3天即可從組織塊邊緣爬出,至第9天時(shí)即可觀察到有大量的細(xì)胞長(zhǎng)出,2w后進(jìn)行細(xì)胞傳代,該細(xì)胞可以在體外穩(wěn)定傳至5代以上；P5代的hUC-MSCs在鏡下觀察可見(jiàn)其形態(tài)規(guī)則、大小均一、呈漩渦狀生長(zhǎng)。 免疫熒光染色顯示：A組未見(jiàn)到MAB1281陽(yáng)性染色細(xì)胞,而其它各組為陽(yáng)性染色；D組可觀察到MBP與NF-H陽(yáng)性染色細(xì)胞,其它各組為陰性染色。 Western-Blot, Real-Time PCR檢測(cè)結(jié)果顯示：A組3種神經(jīng)標(biāo)志物在蛋白與mRNA水平的表達(dá)量隨時(shí)間延長(zhǎng)均逐漸增高,且GFAP的表達(dá)量在移植后第2w明顯增高(Western-blot所測(cè)第2w與第1w的GFAP條帶灰度值比值為1.34而PCR所測(cè)mRNA比值為2.86±0.33),差異有統(tǒng)計(jì)學(xué)意義(P0.05)；而MBP與NF-H的表達(dá)量在移植后第3w明顯增高(Western-blot所測(cè)第3w與第1w的MBP條帶灰度值比值為3.43而PCR所測(cè)mRNA比值為6.47±0.62,NF-H所測(cè)結(jié)果的比值則分別為4.12,6.78±0.77),差異有統(tǒng)計(jì)學(xué)意義(P0.05)。與A組相對(duì)應(yīng)時(shí)間點(diǎn)比較,細(xì)胞移植組MBP,NF-H的表達(dá)量較高而GFAP的表達(dá)量較低,其中D組的MBP, NF-H的表達(dá)量最高(D組與A組MBP在第3w時(shí)條帶灰度值比值為2.11而NF-H在第3w時(shí)條帶灰度值比值為2.11),差異有統(tǒng)計(jì)學(xué)意義(P0.05)；雖然D組GFAP的表達(dá)在第2w較第1w增高,但其在第2,3與4w時(shí)間點(diǎn)之間的表達(dá)量差異(各周所測(cè)條帶灰度比值依次為1：1.12：1.13：1.14)無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 [結(jié)論] 在hSCs影響下,hUC-MSCs至少可在SCI動(dòng)物體內(nèi)存活4周；hSCs能促進(jìn)hUC-MSCs向神經(jīng)元與少突膠質(zhì)細(xì)胞方向分化；hSCs與‘(?)UC-MSCs在SCI大鼠體內(nèi)共同作用后,能夠抑制膠質(zhì)瘢痕的形成：GFAP, MBP,與NF-H等3種神經(jīng)標(biāo)志物的表達(dá)有一定的時(shí)間特征。
[Abstract]:Purpose of the project

To investigate the effects of human Schwann cells ( hSCs ) on survival and differentiation of human umbilical cord - mesenchymal stem cells ( hUC - MSCs ) in spinal cord injury ( SCI ) rats .
This paper describes the time characteristics of glial fiber acidic protein ( GFAP ) , myelin basic protein ( MBP ) and high molecular weight neurofilament ( NF - H ) expression , and discusses the possible mechanism of hSCs to promote the differentiation of hUC - MSCs into nerve .

Methodology

The hSCs were isolated and cultured under the condition of aseptic condition . The hSCs were isolated by double enzyme digestion and mechanical separation , and the cells were purified by low concentration trypsin digestion and differential adherent method . The hSCs transferred to 4th generation ( P4 ) were identified by S - 100 antibody , and the hSCs implanted in this experiment were P5 ;
The experimental animals were divided into four groups : DMEM control group ( A ) , hSCs transplantation group ( B ) , hUC - MSCs transplantation group ( C ) , hUC - MSCs and hSCs combined transplantation group ( D ) . The experimental animals were randomly divided into 4 groups : DMEM control group ( A ) , hSCs transplantation group ( B ) , hUC - MSCs transplantation group ( C ) , hUC - MSCs and hSCs .

The result is not valid .

The hSCs can be isolated from the edge of the tissue mass on the third day of primary culture , and a large number of cells can be observed at the 9th day , and cell passages can be carried out after 2w , and the cells can be stably transferred to more than 5 generations in vitro ;
The expression of hUC - MSCs in P5 was observed under the microscope , and its size was uniform and vortex - like growth was observed .

Immunofluorescence staining showed that MAB1281 positive staining cells were not seen in group A , while other groups were positive staining .
In group D , MBP and NF - H positive staining cells were observed , and the other groups were negative staining .

The results of Western - Blot and Real - Time PCR showed that the expression level of three kinds of nerve markers in group A was gradually increased with time , and the expression of GFAP increased significantly after transplantation ( 2 . 86 鹵 0.33 ) .
Compared with group A , the expression of MBP and NF - H was 4.12 , 6.78 鹵 0.77 respectively . The expression of MBP and NF - H in group D was higher than that in group A . The ratio of MBP and NF - H was 2.11 in group D and 2.11 in group A , and the difference was significant ( P0.05 ) .
Although the expression of GFAP in group D was higher at 2w compared to 1w , there was no significant difference in the expression of GFAP ( 1 : 1.12 : 1.13 : 1.14 ) between the 2nd , 3rd and 4w time points ( P0.05 ) .

Conclusion

Under the influence of hSCs , hUC - MSCs could survive at least 4 weeks in SCI animals .
hSCs can promote the differentiation of hUC - MSCs into neurons and less glial cells ;
hSCs and ' ( ? ) UC - MSCs can inhibit glial scar formation in SCI rats . The expression of GFAP , MBP and NF - H has a certain time characteristic .
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R329

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