【摘要】：目的：通過(guò)對(duì)混合酶組分、培養(yǎng)基的選擇、接種密度、首次換液時(shí)間等條件進(jìn)行優(yōu)化,探討優(yōu)選人臍帶來(lái)源間充質(zhì)干細(xì)胞(MSCs)體外培養(yǎng)純化的最佳方法,為臍帶MSCs的制備和在臨床的廣泛應(yīng)用奠定研究基礎(chǔ)。 方法： 1不同混合酶組分分離培養(yǎng)人臍帶來(lái)源間充質(zhì)干細(xì)胞 無(wú)菌條件下取正常足月剖腹產(chǎn)的臍帶近胎幾段；臍帶組織均分為9等分,每一等分臍帶長(zhǎng)度為0.5cm,仔細(xì)剔除動(dòng)靜脈,剪碎組織呈1mm×1mm×lmm大��；按不同混合酶濃度比值劃分為混合酶Ⅰ組,混合酶Ⅱ組和混合酶Ⅲ組,每組再按消化時(shí)間分為1、2、3h三個(gè)亞組：重懸細(xì)胞終體積均定位4ml并計(jì)數(shù),每個(gè)亞組計(jì)數(shù)6次,采用含血清的DMEM培養(yǎng)液體外培養(yǎng)細(xì)胞。觀察、計(jì)數(shù)并比較相同時(shí)間消化時(shí)間內(nèi)細(xì)胞總數(shù),活細(xì)胞比值以及對(duì)細(xì)胞增殖活性的影響。 2不同培養(yǎng)基、接種密度和首次換液時(shí)間培養(yǎng)人臍帶來(lái)源間充質(zhì)干細(xì)胞 將獲得的單個(gè)核細(xì)胞分別以5×105、1×106、5×106、1×107密度接種于六孔板中,觀察細(xì)胞貼壁延伸時(shí)間、原代培養(yǎng)時(shí)間；分別以間充質(zhì)專(zhuān)用培養(yǎng)基(MesencultTM),L—DMEM、DMEM/F12均含有10%胎牛血清,對(duì)分離得到的細(xì)胞進(jìn)行培養(yǎng),觀察細(xì)胞貼壁及生長(zhǎng)狀況。 3人臍帶來(lái)源間充質(zhì)干細(xì)胞的鑒定和定向誘導(dǎo)流式細(xì)胞儀檢測(cè)人臍帶來(lái)源間充質(zhì)干細(xì)胞CD34、CD44、CD45、CD29、CD105的表達(dá)情況。Vonkassa方法和油紅O染色分別檢測(cè)人臍帶來(lái)源間充質(zhì)干細(xì)胞成骨、成脂的誘導(dǎo)。 結(jié)果： 1不同混合酶組分分離培養(yǎng)人臍帶來(lái)源間充質(zhì)干細(xì)胞：采用三種不同混合酶濃度進(jìn)行消化,發(fā)現(xiàn)相同消化時(shí)間內(nèi),混合酶Ⅲ組消化細(xì)胞最多,細(xì)胞總數(shù)與其他組比較,具有顯著性差異(P0.05)。在作用時(shí)間為3h時(shí),混合酶Ⅲ組獲取的細(xì)胞中活細(xì)胞比值顯著低于混合酶Ⅰ組和混合酶Ⅱ組獲取細(xì)胞的活細(xì)胞比值,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。 2比較4種接種密度,觀察比較發(fā)現(xiàn)以5×105個(gè)/cm2密度接種細(xì)胞長(zhǎng)勢(shì)緩慢,一直無(wú)法傳代,以5×106個(gè)/cm2密度接種細(xì)胞在出現(xiàn)延伸時(shí)間(92.0±6.3h)及原代培養(yǎng)時(shí)間(21.2±4.1d)方面均短于其余接種密度,差異具有顯著性意義。 3不同培養(yǎng)基對(duì)人臍帶來(lái)源間充質(zhì)干細(xì)胞體外培養(yǎng)的影響：18份標(biāo)本中其中用間充質(zhì)專(zhuān)用培養(yǎng)基培養(yǎng)的有14份(77.8%)得到均一的間充質(zhì)干細(xì)胞。其余4份得到的異質(zhì)性貼壁細(xì)胞多,細(xì)胞不均一,形態(tài)多樣。采用DMEM/F12培養(yǎng)的18份標(biāo)本中,有11份(61.1%)得到均一的間充質(zhì)干細(xì)胞,6份得到異質(zhì)性貼壁細(xì)胞,1份細(xì)胞呈懸浮狀態(tài),未貼壁。用L-DMEM培養(yǎng)基的18份標(biāo)本中,有9份(50.0%)最終得到均一的間充質(zhì)干細(xì)胞,但生長(zhǎng)速度緩慢,7份得到異質(zhì)性貼壁細(xì)胞,2份細(xì)胞呈懸浮狀態(tài),未貼壁。 4流式細(xì)胞儀表面標(biāo)志鑒定CD31、CD34、CD45呈陰性,CD44、CD29、CD105呈陽(yáng)性,符合臍帶來(lái)源間充質(zhì)干細(xì)胞的生長(zhǎng)特性。 5臍帶來(lái)源間充質(zhì)干細(xì)胞經(jīng)成骨、成脂誘導(dǎo)后可表達(dá)骨細(xì)胞、脂肪細(xì)胞特征。 結(jié)論： 在其他條件不變的情況下,0.3%Ⅱ型膠原酶、0.1%胰酶、0.02%EDTA、0.1%透明質(zhì)酸酶和0.1%DNA酶混合液消化臍帶組織塊2h獲取間充質(zhì)干細(xì)胞,采用間充質(zhì)干細(xì)胞專(zhuān)用培養(yǎng)基體外培養(yǎng)所得細(xì)胞,5×106個(gè)/cm2為臍帶MSCs培養(yǎng)的適宜接種密度。優(yōu)化條件下培養(yǎng)的細(xì)胞符合間充質(zhì)干細(xì)胞特征。我們優(yōu)化了人臍帶來(lái)源間充質(zhì)干細(xì)胞高效的分離擴(kuò)增體系,為將來(lái)成體干細(xì)胞的臨床應(yīng)用提供了可靠的MSCs新來(lái)源。
[Abstract]:OBJECTIVE: To explore the best method of culture and purification of human umbilical cord derived mesenchymal stem cells (MSCs) in vitro by optimizing the composition of mixed enzymes, the selection of medium, the density of inoculation, and the time of first fluid exchange, so as to lay a foundation for the preparation and clinical application of umbilical cord MSCs.
Method:
1 Isolation and culture of human umbilical cord derived mesenchymal stem cells with different mixed enzyme components
Umbilical cord tissues were divided into 9 equal parts, each equal length of umbilical cord was 0.5 cm, the arteries and veins were carefully removed, and the shredded tissues were 1 mm 65 The cells were divided into three subgroups at 1, 2, and 3 h: the final volume of suspended cells was positioned at 4 ml and counted. Each subgroup was counted 6 times. Cells were cultured in serum-containing DMEM medium in vitro.
2 culture of human umbilical cord derived mesenchymal stem cells with different culture medium, inoculation density and first exchange time.
Mononuclear cells were inoculated into six-well plate at the density of 5 105, 1 106, 5 106, 1 Condition.
The expression of CD34, CD44, CD45, CD29 and CD105 in human umbilical cord-derived mesenchymal stem cells was detected by flow cytometry. The osteogenesis and adipogenic induction of human umbilical cord-derived mesenchymal stem cells were detected by Vonkassa method and oil red O staining respectively.
Result:
1 Isolation and culture of human umbilical cord-derived mesenchymal stem cells with different enzyme mixtures: three different enzyme mixtures were used for digestion. It was found that the number of digestive cells in group III was the largest in the same digestive time, and the total number of cells in group III was significantly different from that in other groups (P 0.05). Cell ratio was significantly lower than that of mixed enzyme group I and mixed enzyme group II (P 0.01).
2. Comparing the four inoculation densities, it was found that the growth rate of the cells inoculated with 5 105 / cm 2 density was slower than that of the other inoculation densities. The cells inoculated with 5
3 Effects of different media on human umbilical cord-derived mesenchymal stem cells in vitro culture: 14 (77.8%) of the 18 specimens were cultured in special mesenchymal medium to obtain homogeneous mesenchymal stem cells. Homogeneous mesenchymal stem cells were obtained in 61.1%. Heterogeneous adherent cells were obtained in 6 samples. One cell was suspended and not adhered to the wall.
Flow cytometry showed that CD31, CD34 and CD45 were negative, CD44, CD29 and CD105 were positive, which accorded with the growth characteristics of umbilical cord derived mesenchymal stem cells.
5 umbilical cord derived mesenchymal stem cells can express osteoblasts and adipocytes after osteogenic and adipogenic induction.
Conclusion:
Under the same conditions, 0.3% collagenase type II, 0.1% trypsin, 0.02% EDTA, 0.1% hyaluronidase and 0.1% DNA enzyme mixture were used to digest umbilical cord tissue blocks for 2 hours to obtain mesenchymal stem cells. The cells were cultured in vitro using the special medium of mesenchymal stem cells, and 5 *106/cm 2 was the suitable density for umbilical cord MSCs culture. The cultured cells conform to the characteristics of mesenchymal stem cells. We optimized the efficient isolation and amplification system of human umbilical cord-derived mesenchymal stem cells and provided a reliable new source of MSCs for the clinical application of adult stem cells in the future.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 武爽;魯西黃牛臍帶間充質(zhì)干細(xì)胞的分離培養(yǎng)及生物學(xué)特性研究[D];中國(guó)農(nóng)業(yè)科學(xué)院;2012年

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本文編號(hào)：2226159