本文關(guān)鍵詞：人臍血間充質(zhì)干細胞分離培養(yǎng)及向神經(jīng)細胞定向誘導(dǎo)分化的研究　出處：《濰坊醫(yī)學(xué)院》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 臍血 間充質(zhì)干細胞 細胞培養(yǎng) 分化 神經(jīng)細胞

【摘要】： 目的: 1、探討人臍血間充質(zhì)干細胞(MSCs)體外分離培養(yǎng)的最佳方法。 2、探討人臍血MSCs向神經(jīng)細胞定向誘導(dǎo)分化的最佳方案。 方法: 1、無菌條件下采集足月分娩和早產(chǎn)兒(不足37周)臍血,密度梯度離心法分離臍血單個核細胞(MNCs),比較不同條件對臍血MSCs原代培養(yǎng)過程的影響:(1)培養(yǎng)基:L-DMEM培養(yǎng)基、DMEM/F12培養(yǎng)基和Mesencult~(TM)培養(yǎng)基;(2)胎齡:足月妊娠和早產(chǎn)兒;(3)接種密度:5×10~5/cm~2、1×10~6/cm~2、5×10~6/cm~2、1×10~7/cm~2;(4)首次換液時間:2、3、4、5、6、7d。通過免疫熒光方法檢測表面標(biāo)記物CD29、CD34、CD44和CD90的表達情況,觀察臍血MSCs的生物學(xué)特性。 2、第3代臍血MSCs,胰酶消化傳代,待長至約80%融合時進行誘導(dǎo),分三組:(1)化學(xué)誘導(dǎo)劑組:3mmol/Lβ-ME+20g/L DMSO+200mmol/L BHA;(2)生長因子誘導(dǎo)組:20ng/ml EGF+20ng/ml bFGF;(3)丹參素+生長因子誘導(dǎo)組:3%香丹注射液(有效成分丹參素)+20ng/ml EGF+20ng/ml bFGF。采用免疫細胞化學(xué)方法和(或)免疫熒光方法檢測誘導(dǎo)前后神經(jīng)元特異性標(biāo)志(NeuN、β-TubulinⅢ)和星形膠質(zhì)細胞特異性標(biāo)志GFAP表達的變化。 結(jié)果: 1、足月分娩臍血,采用Mesencult~(TM)培養(yǎng)基,以5×10~6/cm~2的密度接種,首次換液時間為7d時,臍血MSCs原代培養(yǎng)成功率較高。相同培養(yǎng)條件下,早產(chǎn)兒臍血MSCs培養(yǎng)成功率高于足月分娩臍血。人臍血MSCs強表達CD29、CD44和CD90,不表達造血干細胞表面標(biāo)志CD34。 2、免疫細胞化學(xué)方法檢測結(jié)果:誘導(dǎo)前三組臍血MSCs的NeuN、β-TubulinⅢ、GFAP表達均為陰性;化學(xué)誘導(dǎo)后上述三種標(biāo)志物的陽性率分別為(71.6±4.6)%、(73.8±2.3)%和(12.6±2.0)%;生長因子誘導(dǎo)后分別為(43.6±3.9)%、(54.6±4.1)%和(31.8±5.0)%;丹參素+生長因子誘導(dǎo)后分別為(78.6±4.5)%、(82.6±4.3)%和(15.6±3.6)%。免疫熒光方法檢測發(fā)現(xiàn):誘導(dǎo)前三組臍血MSCsβ-TubulinⅢ表達均為陰性,誘導(dǎo)后三組β-TubulinⅢ的陽性率分別為(72.6±2.7)%、(54.5±1.9)%和(79.8±3.0)%。 結(jié)論: 1、足月妊娠臍血采用Mesencult~(TM)培養(yǎng)基,以5×10~6/cm~2的密度接種,首次換液時間為7d,臍血MSCs原代培養(yǎng)成功率較高。相同培養(yǎng)條件下,早產(chǎn)兒臍血MSCs培養(yǎng)成功率高于足月分娩臍血。 2、丹參素聯(lián)合生長因子EGF、bFGF在體外可誘導(dǎo)臍血MSCs分化為神經(jīng)元樣細胞,誘導(dǎo)效果最佳。
[Abstract]:Objective: 1. To investigate the best method for isolation and culture of human umbilical cord blood mesenchymal stem cells (MSCs) in vitro. 2. To explore the best way to induce the differentiation of human umbilical cord blood MSCs into neuronal cells. Methods: 1. Cord blood was collected from full-term delivery and premature infants (less than 37 weeks) under aseptic condition, and MNCswere isolated by density gradient centrifugation. The effects of different conditions on the primary culture process of umbilical cord blood MSCs were compared. (2) gestational age: term pregnancy and premature infants; (3) the inoculation density is 1 脳 10 / 5 / cm ~ (-1) / cm ~ (-1) / 10 ~ (6) / cm ~ (2) / cm ~ (2) ~ (2) / (1 脳 10 ~ (7) / cm ~ (2)); The expression of CD29, CD34, CD44 and CD90 was detected by immunofluorescence method. The biological characteristics of MSCs in umbilical cord blood were observed. 2, the third generation of cord blood MSCs, trypsin digestion passage, waiting to grow to about 80% fusion for induction. Chemical inducer group: 3 mmol / L 尾 -ME 20 g / L DMSO 200mmol / L BHA; (2) growth factor induction group: 20 ng / ml EGF 20ng / ml bFGF; 3) Danshensu growth Factor Induction Group: 3% Xiangdan injection (Danshensu) 20ng / ml EGF 20ng / ml bFGF.Immunocytochemical method and (. Or) Neun, a neuron specific marker, was detected by immunofluorescence before and after induction. The expression of 尾-Tubulin 鈪，

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