早产儿与足月儿脐血间充质干细胞体外分离、培养及鉴定

发布时间：2018-06-02 03:35

本文选题：脐血 + 间充质干细胞　；参考：《广州医学院》2011年硕士论文

【摘要】：【目的】观察分析早产儿与足月儿脐血间充质干细胞分离培养成功率,并研究其相关生物学特性和诱导分化潜能,寻求脐血间充质干细胞获取的最佳途径,为其进一步实验研究提供理论依据。【方法】无菌条件下共取52份脐血,其中32份为足月儿脐血,20份为早产儿脐血(孕周32～36周),枸橼酸钠抗凝。取12份足月儿脐血,采用Ficoll淋巴细胞分离分离液密度梯度离心获取单核细胞(MNCs),同等接种密度下对比DMEM/F12培养基和Mesencult~(TM)培养基对脐血间充质干细胞培养成功率的影响;取20份足月儿脐血,密度梯度离心法获取MNCs,采用Mesencult~(TM)培养基,分别以1×10~6/ml,1×10~7/ml密度接种,对比两种接种密度对脐血间充质干细胞培养成功率的影响;取20份早产儿脐血,以1×10~7/ml密度接种于Mesencult~(TM)培养基,对比早产儿与足月儿脐血间充质干细胞培养成功率,并研究其各自生物学特性及诱导分化潜能。通过倒置显微镜观察其形态、生长增殖情况并描绘细胞生长曲线;流式细胞仪检测细胞表面标记,包括CD29、CD44、CD105、CD34、CD45;分别用成骨细胞、成脂细胞和成软骨细胞完全培养基进行诱导分化,通过茜红素染色检测间充质干细胞向成骨细胞诱导分化的能力,油红O染色检测其向脂肪细胞分化能力,阿利新蓝染色检测其向软骨细胞分化能力。采用SPSS13.0统计软件进行数据统计分析。【结果】 1.采用Mesencult~(TM)培养基和DMEM/F12培养基,1×107/ml接种密度和1×106/ml接种密度培养脐血间充质干细胞成功率的比较采用Mesencult~(TM)和DMEM/F12培养基,其他条件等同的情况下,间充质干细胞培养成功率分别为50.00%(6/12)和8.33%(1/12),差异有统计学意义(P0.05);采用1×107/ml和1×106/ml接种密度,其他条件等同的情况下,间充质干细胞培养成功率分别为55.00%(11/20)和20.00%(4/20),差异有统计学意义(P0.05)。 2.早产儿与足月儿脐血间充质干细胞培养成功率的比较采用Mesencult~(TM)培养基及1×107/ml接种密度,其他条件相同,早产儿与足月儿脐血间充质干细胞培养成功率分别为85.00%(17/20)和55.00%(11/20),差异有统计学意义(P0.05)。 3.早产儿与足月儿脐血间充质干细胞生物学特性的比较 3.1早产儿与足月儿脐血间充质干细胞原代培养时间分别为(19.65±1.69 )d和(28.36±1.36)d,差异有统计学意义(P0.05);P4代早产儿与足月儿脐血MSCs传代培养时间分别为(14.94±1.75)d和(19.27±2.10)d,差异有统计学意义(P0.05)。 3.2流式细胞仪检测显示,早产儿与足月儿脐血MSCs均表达典型间充质干细胞标记CD29、CD44和CD105,均不表达造血干细胞标记CD34和CD45。 4.早产儿与足月儿脐血间充质干细胞诱导分化潜能的比较早产儿与足月儿脐血间充质干细胞成骨细胞诱导2周行茜素红染色均可见胞浆中有大量红色钙化基质沉积;成脂肪细胞诱导3周时行油红O染色均可见胞质中的脂滴被染成红色;成软骨细胞诱导3周时行阿利新蓝染色均可见细胞微团中有淡蓝色酸性蛋白多糖。早产儿与足月儿脐血间充质干细胞均具有向成骨细胞、成脂细胞及成软骨细胞分化潜能。【结论】 1.早产儿与足月儿脐血中均存在间充质干细胞,并可在体外分离、纯化与扩增。 2.采用Mesencult~(TM)培养基和1×107/ml接种密度更有利于提高脐血间充质干细胞的培养成功率。 3.早产儿较足月儿脐血间充质干细胞培养成功率更高,原代和传代培养时间更短。 4.早产儿与足月儿脐血间充质干细胞均可在体外诱导分化为成骨细胞、成脂细胞和成软骨细胞。
[Abstract]:[Objective] to observe and analyze the success rate of isolation and culture of umbilical cord blood mesenchymal stem cells (MSCs) in preterm infants and infants, and to study their related biological characteristics and induce differentiation potential, seek the best way to obtain mesenchymal stem cells from umbilical cord blood, and provide a theoretical basis for further experimental research.
[Methods] 52 umbilical cord blood were obtained under aseptic condition, of which 32 were umbilical cord blood of foot moon, 20 were umbilical blood of preterm infants (32~36 weeks of pregnancy), sodium citrate was anticoagulant. 12 full moon umbilical cord blood was taken, MNCs was obtained by density gradient centrifugation with Ficoll lymphocyte separation and separation solution, and DMEM/F12 medium and Mesencult~ were compared under the same inoculation density. (TM) the effect of culture medium on the culture success rate of umbilical cord blood mesenchymal stem cells; taking 20 full moon cord blood and density gradient centrifugation to obtain MNCs, using Mesencult~ (TM) medium, 1 x 10~6/ml and 1 x 10~7/ml density inoculation respectively, compared the effect of two inoculation density on the culture success rate of umbilical cord blood mesenchymal stem cells, 20 premature infants' umbilical blood, 1 The density of X 10~7/ml was inoculated on the Mesencult~ (TM) medium, compared with the success rate of the cultivation of mesenchymal stem cells from the premature infants and the umbilical cord blood of the foot moon, and studied their respective biological characteristics and the differentiation potential. The morphology, growth and proliferation of the cells were observed by inverted microscope, and the cell growth curve was depicted. The flow cytometry was used to detect the cell surface markers, including the cell surface markers. CD29, CD44, CD105, CD34, CD45; osteoblasts, adipocytes and chondrocytes were used to induce differentiation. The differentiation ability of mesenchymal stem cells from mesenchymal stem cells to osteoblasts was detected by Alizarin staining. Oil red O staining was used to detect the differentiation ability to adipocytes. Alcia blue staining was used to detect the differentiation ability of the chondrocytes to chondrocytes. SPSS13.0 statistical software was used for data statistical analysis.
[results]
1. using Mesencult~ (TM) medium and DMEM/F12 medium, 1 * 107/ml inoculation density and 1 * 106/ml inoculation density to compare the successful rate of umbilical cord blood mesenchymal stem cells.
With Mesencult~ (TM) and DMEM/F12 medium, the success rates of mesenchymal stem cells were 50% (6/12) and 8.33% (1/12), respectively. The difference was statistically significant (P0.05). The success rate of mesenchymal stem cells was 55% (11/20) using 1 x 107/ml and 1 x 106/ml inoculation density and other conditions. And 20% (4/20), the difference was statistically significant (P0.05).
2. comparison of the successful rate of umbilical cord blood mesenchymal stem cells between preterm infants and full-term infants
Using Mesencult~ (TM) medium and 1 x 107/ml inoculation density, the other conditions were the same. The success rate of umbilical cord blood mesenchymal stem cells in premature infants and foot moon was 85% (17/20) and 55% (11/20) respectively, and the difference was statistically significant (P0.05).
3. comparison of biological characteristics of umbilical cord blood mesenchymal stem cells between preterm infants and full-term infants
The primary culture time of 3.1 preterm and full moon umbilical cord blood mesenchymal stem cells was (19.65 + 1.69) D and (28.36 + 1.36) d respectively, the difference was statistically significant (P0.05). The P4 generation preterm and the MSCs passage culture time of the umbilical blood of the P4 generation were (14.94 + 1.75) D and (19.27 + 2.10) d respectively, the difference was statistically significant (P0.05).
3.2 flow cytometry showed that both preterm and foot moon cord blood MSCs expressed CD29, CD44 and CD105 of typical mesenchymal stem cells, and none of the hematopoietic stem cell markers were expressed as CD34 and CD45..
4. comparison of differentiation potential of umbilical cord blood mesenchymal stem cells between preterm infants and full-term infants
A large number of red calcified matrix deposits in the cytoplasm were observed in the 2 weeks of alizarin red staining for 2 weeks induced by the osteoblast induced by umbilical cord blood mesenchymal stem cells in preterm infants and human umbilical cord blood cells. The lipid droplets in the cytoplasm were dyed red in the 3 weeks of induction of adipocytes, and the chondrocytes were stained in the cell micromass for 3 weeks when the chondrocytes were induced. There are pale blue acid proteoglycan. Umbilical cord blood mesenchymal stem cells from preterm and full-term infants have the potential to differentiate into osteoblasts, adipocytes and chondrocytes.
[Conclusion]
1. mesenchymal stem cells exist in umbilical cord blood of preterm infants and full-term infants, and can be isolated, purified and amplified in vitro.
2. using Mesencult~ (TM) medium and 1 * 107/ml inoculation density is more conducive to improving the success rate of umbilical cord blood mesenchymal stem cells.
3. preterm infants had higher success rate of umbilical cord blood mesenchymal stem cells than the term infants, and the primary and subculture time was shorter.
4. umbilical cord blood mesenchymal stem cells from preterm infants and full-term infants can be induced to differentiate into osteoblasts, adipocytes and chondrocytes in vitro.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【参考文献】