本文選題：臍血 + 間充質(zhì)干細(xì)胞��； 參考：《廣州醫(yī)學(xué)院》2011年碩士論文

【摘要】：【目的】觀察分析早產(chǎn)兒與足月兒臍血間充質(zhì)干細(xì)胞分離培養(yǎng)成功率,并研究其相關(guān)生物學(xué)特性和誘導(dǎo)分化潛能,尋求臍血間充質(zhì)干細(xì)胞獲取的最佳途徑,為其進(jìn)一步實(shí)驗(yàn)研究提供理論依據(jù)。 【方法】無菌條件下共取52份臍血,其中32份為足月兒臍血,20份為早產(chǎn)兒臍血(孕周32～36周),枸櫞酸鈉抗凝。取12份足月兒臍血,采用Ficoll淋巴細(xì)胞分離分離液密度梯度離心獲取單核細(xì)胞(MNCs),同等接種密度下對比DMEM/F12培養(yǎng)基和Mesencult~(TM)培養(yǎng)基對臍血間充質(zhì)干細(xì)胞培養(yǎng)成功率的影響;取20份足月兒臍血,密度梯度離心法獲取MNCs,采用Mesencult~(TM)培養(yǎng)基,分別以1×10~6/ml,1×10~7/ml密度接種,對比兩種接種密度對臍血間充質(zhì)干細(xì)胞培養(yǎng)成功率的影響;取20份早產(chǎn)兒臍血,以1×10~7/ml密度接種于Mesencult~(TM)培養(yǎng)基,對比早產(chǎn)兒與足月兒臍血間充質(zhì)干細(xì)胞培養(yǎng)成功率,并研究其各自生物學(xué)特性及誘導(dǎo)分化潛能。通過倒置顯微鏡觀察其形態(tài)、生長增殖情況并描繪細(xì)胞生長曲線;流式細(xì)胞儀檢測細(xì)胞表面標(biāo)記,包括CD29、CD44、CD105、CD34、CD45;分別用成骨細(xì)胞、成脂細(xì)胞和成軟骨細(xì)胞完全培養(yǎng)基進(jìn)行誘導(dǎo)分化,通過茜紅素染色檢測間充質(zhì)干細(xì)胞向成骨細(xì)胞誘導(dǎo)分化的能力,油紅O染色檢測其向脂肪細(xì)胞分化能力,阿利新藍(lán)染色檢測其向軟骨細(xì)胞分化能力。采用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)統(tǒng)計(jì)分析。 【結(jié)果】 1.采用Mesencult~(TM)培養(yǎng)基和DMEM/F12培養(yǎng)基,1×107/ml接種密度和1×106/ml接種密度培養(yǎng)臍血間充質(zhì)干細(xì)胞成功率的比較 采用Mesencult~(TM)和DMEM/F12培養(yǎng)基,其他條件等同的情況下,間充質(zhì)干細(xì)胞培養(yǎng)成功率分別為50.00%(6/12)和8.33%(1/12),差異有統(tǒng)計(jì)學(xué)意義(P0.05);采用1×107/ml和1×106/ml接種密度,其他條件等同的情況下,間充質(zhì)干細(xì)胞培養(yǎng)成功率分別為55.00%(11/20)和20.00%(4/20),差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 2.早產(chǎn)兒與足月兒臍血間充質(zhì)干細(xì)胞培養(yǎng)成功率的比較 采用Mesencult~(TM)培養(yǎng)基及1×107/ml接種密度,其他條件相同,早產(chǎn)兒與足月兒臍血間充質(zhì)干細(xì)胞培養(yǎng)成功率分別為85.00%(17/20)和55.00%(11/20),差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 3.早產(chǎn)兒與足月兒臍血間充質(zhì)干細(xì)胞生物學(xué)特性的比較 3.1早產(chǎn)兒與足月兒臍血間充質(zhì)干細(xì)胞原代培養(yǎng)時間分別為(19.65±1.69 )d和(28.36±1.36)d,差異有統(tǒng)計(jì)學(xué)意義(P0.05);P4代早產(chǎn)兒與足月兒臍血MSCs傳代培養(yǎng)時間分別為(14.94±1.75)d和(19.27±2.10)d,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 3.2流式細(xì)胞儀檢測顯示,早產(chǎn)兒與足月兒臍血MSCs均表達(dá)典型間充質(zhì)干細(xì)胞標(biāo)記CD29、CD44和CD105,均不表達(dá)造血干細(xì)胞標(biāo)記CD34和CD45。 4.早產(chǎn)兒與足月兒臍血間充質(zhì)干細(xì)胞誘導(dǎo)分化潛能的比較 早產(chǎn)兒與足月兒臍血間充質(zhì)干細(xì)胞成骨細(xì)胞誘導(dǎo)2周行茜素紅染色均可見胞漿中有大量紅色鈣化基質(zhì)沉積;成脂肪細(xì)胞誘導(dǎo)3周時行油紅O染色均可見胞質(zhì)中的脂滴被染成紅色;成軟骨細(xì)胞誘導(dǎo)3周時行阿利新藍(lán)染色均可見細(xì)胞微團(tuán)中有淡藍(lán)色酸性蛋白多糖。早產(chǎn)兒與足月兒臍血間充質(zhì)干細(xì)胞均具有向成骨細(xì)胞、成脂細(xì)胞及成軟骨細(xì)胞分化潛能。 【結(jié)論】 1.早產(chǎn)兒與足月兒臍血中均存在間充質(zhì)干細(xì)胞,并可在體外分離、純化與擴(kuò)增。 2.采用Mesencult~(TM)培養(yǎng)基和1×107/ml接種密度更有利于提高臍血間充質(zhì)干細(xì)胞的培養(yǎng)成功率。 3.早產(chǎn)兒較足月兒臍血間充質(zhì)干細(xì)胞培養(yǎng)成功率更高,原代和傳代培養(yǎng)時間更短。 4.早產(chǎn)兒與足月兒臍血間充質(zhì)干細(xì)胞均可在體外誘導(dǎo)分化為成骨細(xì)胞、成脂細(xì)胞和成軟骨細(xì)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.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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