人脐带源性间充质干细胞的分离培养及其移植对再生障碍性贫血小鼠治疗作用的研究

发布时间：2018-06-01 03:36

本文选题：脐带间充质干细胞 + 再生障碍性贫血　；参考：《蚌埠医学院》2011年硕士论文

【摘要】：目的：建立从人的脐带组织中分离和培养脐带间充质干细胞（umbilical cordmesenchymal stem cells, UC-MSCs）的方法，并探讨脐带间充质干细胞的生物学特性。方法：1.脐带间充质干细胞分离培养及鉴定：分别采用植块法和酶联合消化法(胶原酶Ⅱ和胰酶)分离培养脐带间充质干细胞；瑞氏染色观察细胞形态，流式细胞术检测第3代以后的UC-MSCs表面特异标志物表达。2.脐带间充质干细胞生长和增殖能力的观察：MTT法检测P3、P10细胞增殖情况，流式细胞术检测P3、P10细胞生长周期，比较两代细胞生长增殖能力有无变化。3. UC-MSCs诱导分化：选取状态较稳定的第4代细胞，以2×104/mL接种于放置有多聚赖氨酸处理的无菌盖玻片的6孔板内，待细胞达到一定的融合度时，以相应的诱导体系诱导其向脂肪细胞和骨细胞分化。待脂肪细胞诱导体系的胞质中有脂滴形成时，以质量分数为0.375％油红O染色；骨细胞诱导3周后，茜素红染色，显微镜下观察、拍照。4. RT-PCR法检测UC-MSCs中SCF、G-CSF、GM-CSF、VEGF mRNA的表达。5. UC-MSCs同健康人外周血单个核细胞共培养后，用MTT法测定细胞增殖率，观察UC-MSCs对健康人淋巴细胞增殖的影响；用Hoechst与PI双染色来观察UC-MSCs和健康人淋巴细胞共培养时UC-MSCs的变化。结果：植块法培养1周左右可见成纤维样细胞从组织块边缘爬出，，成簇生长；酶消化法3-5天可见成纤维样细胞均匀生长。传代培养后，脐带间充质干细胞均呈长梭形、旋涡状生长；细胞核大，核仁清晰；免疫表型分析显示，CD29阳性率（95.71±2.23）%，CD31和CD34阳性率分别为（2.47±0.54）%和（3.24±0.34）%；第10代UC-MSCs仍具有较强的分裂增殖能力，且第3、10代细胞周期无明显变化；UC-MSCs在体外具有向脂肪细胞和骨细胞分化潜能，可表达SCF、VEGF、G-CSF和GM-CSF mRNA。UC-MSCs在体外对淋巴细胞的增殖反应具有抑制作用，且呈细胞数量剂量依赖性，而淋巴细胞对UC-MSCs的生长未见影响。结论：成功建立脐带间充质干细胞的分离方法；从脐带中分离培养的细胞，具有间充质干细胞生物学特性。目的：探讨人脐带源性间充质干细胞（UC-MSCs）移植对药物联合射线诱导的再生障碍性贫血（aplastic anemia, AA）模型小鼠的治疗作用。方法：选用第6～7周的雌性昆明鼠120只，参照孙纪元等方法60Co-γ射线照射后iP环磷酰胺与氯霉素的复合法建立小鼠AA模型。实验分正常对照组、模型组及MSC组。MSC组于造模后第2天从小鼠尾静脉一次性输注人UC-MSCs1×106/Kg，观察各组小鼠生存率、外周血象、骨髓有核细胞数量、骨髓病理学特征等的变化。同时观察模型鼠输注UC-MSCs前后INF-γ量的变化以及造血细胞的集落数量的变化。结果：模型组小鼠于第8天濒临死亡，第10天开始死亡，病理解剖发现各脏器色泽苍白。生存率比MSC组低(P 0.05)。第10天WBC、PLT和RET明显减少，RBC变化不明显；16天时两组外周血象指标仍然很低，除RBC继续降低外，其他都轻度回升，而MSC组回升比模型组明显，两组间差异有统计学意义。造模过程中，小鼠骨髓有核细胞数量明显减少，晚期脂肪组织增生。输注UC-MSCs后，INF-γ量有所下降，MSC组骨髓有核细胞数、造血细胞集落中CFU-GM数量均明显高于模型组，而CFU-F数量没有明显变化。结论：UC-MSCs输注可减轻再生障碍性贫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 卤 2.23 ) % , and the positive rates of CD31 and CD34 were ( 2.47 卤 0.54 ) % and ( 3.24 卤 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 .
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【参考文献】