本文選題：臍帶間充質(zhì)干細(xì)胞 + 再生障礙性貧血　； 參考：《蚌埠醫(yī)學(xué)院》2011年碩士論文

【摘要】：目的：建立從人的臍帶組織中分離和培養(yǎng)臍帶間充質(zhì)干細(xì)胞（umbilical cordmesenchymal stem cells, UC-MSCs）的方法，并探討臍帶間充質(zhì)干細(xì)胞的生物學(xué)特性。方法：1.臍帶間充質(zhì)干細(xì)胞分離培養(yǎng)及鑒定：分別采用植塊法和酶聯(lián)合消化法(膠原酶Ⅱ和胰酶)分離培養(yǎng)臍帶間充質(zhì)干細(xì)胞；瑞氏染色觀察細(xì)胞形態(tài)，流式細(xì)胞術(shù)檢測(cè)第3代以后的UC-MSCs表面特異標(biāo)志物表達(dá)。2.臍帶間充質(zhì)干細(xì)胞生長(zhǎng)和增殖能力的觀察：MTT法檢測(cè)P3、P10細(xì)胞增殖情況，流式細(xì)胞術(shù)檢測(cè)P3、P10細(xì)胞生長(zhǎng)周期，比較兩代細(xì)胞生長(zhǎng)增殖能力有無(wú)變化。3. UC-MSCs誘導(dǎo)分化：選取狀態(tài)較穩(wěn)定的第4代細(xì)胞，以2×104/mL接種于放置有多聚賴氨酸處理的無(wú)菌蓋玻片的6孔板內(nèi)，待細(xì)胞達(dá)到一定的融合度時(shí)，以相應(yīng)的誘導(dǎo)體系誘導(dǎo)其向脂肪細(xì)胞和骨細(xì)胞分化。待脂肪細(xì)胞誘導(dǎo)體系的胞質(zhì)中有脂滴形成時(shí)，以質(zhì)量分?jǐn)?shù)為0.375％油紅O染色；骨細(xì)胞誘導(dǎo)3周后，茜素紅染色，顯微鏡下觀察、拍照。4. RT-PCR法檢測(cè)UC-MSCs中SCF、G-CSF、GM-CSF、VEGF mRNA的表達(dá)。5. UC-MSCs同健康人外周血單個(gè)核細(xì)胞共培養(yǎng)后，用MTT法測(cè)定細(xì)胞增殖率，觀察UC-MSCs對(duì)健康人淋巴細(xì)胞增殖的影響；用Hoechst與PI雙染色來(lái)觀察UC-MSCs和健康人淋巴細(xì)胞共培養(yǎng)時(shí)UC-MSCs的變化。結(jié)果：植塊法培養(yǎng)1周左右可見(jiàn)成纖維樣細(xì)胞從組織塊邊緣爬出，，成簇生長(zhǎng)；酶消化法3-5天可見(jiàn)成纖維樣細(xì)胞均勻生長(zhǎng)。傳代培養(yǎng)后，臍帶間充質(zhì)干細(xì)胞均呈長(zhǎng)梭形、旋渦狀生長(zhǎng)；細(xì)胞核大，核仁清晰；免疫表型分析顯示，CD29陽(yáng)性率（95.71±2.23）%，CD31和CD34陽(yáng)性率分別為（2.47±0.54）%和（3.24±0.34）%；第10代UC-MSCs仍具有較強(qiáng)的分裂增殖能力，且第3、10代細(xì)胞周期無(wú)明顯變化；UC-MSCs在體外具有向脂肪細(xì)胞和骨細(xì)胞分化潛能，可表達(dá)SCF、VEGF、G-CSF和GM-CSF mRNA。UC-MSCs在體外對(duì)淋巴細(xì)胞的增殖反應(yīng)具有抑制作用，且呈細(xì)胞數(shù)量劑量依賴性，而淋巴細(xì) 胞對(duì)UC-MSCs的生長(zhǎng)未見(jiàn)影響。結(jié)論：成功建立臍帶間充質(zhì)干細(xì)胞的分離方法；從臍帶中分離培養(yǎng)的細(xì)胞，具有間充質(zhì)干細(xì)胞生物學(xué)特性。 目的：探討人臍帶源性間充質(zhì)干細(xì)胞（UC-MSCs）移植對(duì)藥物聯(lián)合射線誘導(dǎo)的再生障礙性貧血（aplastic anemia, AA）模型小鼠的治療作用。方法：選用第6～7周的雌性昆明鼠120只，參照孫紀(jì)元等方法60Co-γ射線照射后iP環(huán)磷酰胺與氯霉素的復(fù)合法建立小鼠AA模型。實(shí)驗(yàn)分正常對(duì)照組、模型組及MSC組。MSC組于造模后第2天從小鼠尾靜脈一次性輸注人UC-MSCs1×106/Kg，觀察各組小鼠生存率、外周血象、骨髓有核細(xì)胞數(shù)量、骨髓病理學(xué)特征等的變化。同時(shí)觀察模型鼠輸注UC-MSCs前后INF-γ量的變化以及造血細(xì)胞的集落數(shù)量的變化。結(jié)果：模型組小鼠于第8天瀕臨死亡，第10天開(kāi)始死亡，病理解剖發(fā)現(xiàn)各臟器色澤蒼白。生存率比MSC組低(P 0.05)。第10天WBC、PLT和RET明顯減少，RBC變化不明顯；16天時(shí)兩組外周血象指標(biāo)仍然很低，除RBC繼續(xù)降低外，其他都輕度回升，而MSC組回升比模型組明顯，兩組間差異有統(tǒng)計(jì)學(xué)意義。造模過(guò)程中，小鼠骨髓有核細(xì)胞數(shù)量明顯減少，晚期脂肪組織增生。輸注UC-MSCs后，INF-γ量有所下降，MSC組骨髓有核細(xì)胞數(shù)、造血細(xì)胞集落中CFU-GM數(shù)量均明顯高于模型組，而CFU-F數(shù)量沒(méi)有明顯變化。結(jié)論：UC-MSCs輸注可減輕再生障礙性貧血模型小鼠骨髓造血衰竭程度，提高存活率。
[Abstract]:Objective : To establish a method for the isolation and culture of umbilical cordmesenchymal stem cells ( UC - MSCs ) from human umbilical cord tissue , and to explore the biological characteristics of umbilical cord mesenchymal stem cells .
Cell morphology and flow cytometry were used to detect the expression of specific markers of UC - MSCs in the 3rd generation . The growth and proliferation of umbilical cord mesenchymal stem cells were observed : MTT assay was used to detect the proliferation of P3 and P10 cells . Flow cytometry was used to detect the growth cycle of P3 and P10 cells . The differentiation of UC - MSCs was : the 4th generation cells with stable status were selected , and 2 脳 104 / mL was inoculated into 6 - well plate of sterile cover glass placed with multiple lysine treatment . When the cells reached a certain degree of fusion , the cells were induced to differentiate into adipocytes and osteocytes in the corresponding induction system .
After 3 weeks of induction of osteoblasts , alizarin red staining , microscopic observation , photo . 4 . The expression of SCF , G - CSF , GM - CSF and VEGF mRNA in UC - MSCs was detected by RT - PCR . UC - MSCs were co - cultured with human peripheral blood mononuclear cells ( PBMC ) , and the proliferation of lymphocytes in healthy human lymphocytes was observed by MTT assay .
The changes of UC - MSCs in co - culture of lymphocytes with UC - MSCs and healthy human lymphocytes were observed by double - staining method .
The fiber - like cells were grown on 3 - 5 days after enzymatic digestion . After subculture , the umbilical cord mesenchymal stem cells were elongated spindle - shaped and spiral - shaped .
the nucleus is large , the nucleolus is clear ;
The positive rate of CD29 was ( 95.71 鹵 2.23 ) % , and the positive rates of CD31 and CD34 were ( 2.47 鹵 0.54 ) % and ( 3.24 鹵 0.34 ) % , respectively .
The 10th generation UC - MSCs still had stronger proliferative capacity , and there was no obvious change in the 3rd and 10th generation cell cycles .
UC - MSCs can express SCF , VEGF , G - CSF and GM - CSF mRNA in vitro and can express SCF , VEGF , G - CSF and GM - CSF mRNA .

There was no effect on the growth of UC - MSCs . Conclusion : The isolation method of umbilical cord mesenchymal stem cells was successfully established .
The cultured cells are isolated from the umbilical cord and have the biological characteristics of mesenchymal stem cells .

Objective : To investigate the therapeutic effect of human umbilical cord - derived mesenchymal stem cells ( UC - MSCs ) in the treatment of aplastic anemia ( AA ) mice induced by combined radiation . Methods : 120 female Kunming mice were selected from 6 to 7 weeks . The changes of the survival rate , peripheral blood image , the number of bone marrow nucleated cells and the pathological characteristics of hematopoietic cells were observed . The results showed that the survival rate , peripheral blood image , the number of bone marrow nucleated cells and the pathological characteristics of hematopoietic cells were observed in the normal control group , the peripheral blood image , the bone marrow nucleated cells and the pathological characteristics of the hematopoietic cells . On the 10th day , WBC , PLT and RET decreased significantly and RBC changes were not obvious .
Compared with the model group , the number of CFU - GM in the bone marrow of the MSC group decreased significantly , the number of CFU - GM in the MSC group was significantly higher than that in the model group , but the CFU - F quantity did not change significantly . Conclusion : The UC - MSCs can alleviate the degree of bone marrow hematopoietic failure in the model mice with aplastic anemia and improve the survival rate .
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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