本文選題：負(fù)壓 + 骨髓間充質(zhì)干細(xì)胞�。� 參考：《石河子大學(xué)》2017年碩士論文

【摘要】：目的:探討體外不同負(fù)壓對(duì)于骨髓間充質(zhì)干細(xì)胞(Bone mesenchymal stem cells,BMSCs)表皮分化的影響。方法:1.采用密度梯度離心法分離培養(yǎng)4-6月齡新西蘭大白兔BMSCs;2.培養(yǎng)至第三代BMSCs進(jìn)行鑒定:倒置顯微鏡對(duì)細(xì)胞進(jìn)行形態(tài)學(xué)觀察,間充質(zhì)干細(xì)胞的三系分化鑒定,流式細(xì)胞儀鑒定細(xì)胞表面標(biāo)志物;3.設(shè)置實(shí)驗(yàn)組,分別是0mmhg組(對(duì)照組)、50mmhg組、100mmhg組、200mmhg組,誘導(dǎo)時(shí)間為30min/h;4.負(fù)壓下常規(guī)培養(yǎng)第三代BMSCs,分別于1d、3d、5d和7d進(jìn)行細(xì)胞計(jì)數(shù)繪制細(xì)胞生長(zhǎng)曲線,于3d、5d和7d使用CCK8檢測(cè)細(xì)胞增殖情況;5.誘導(dǎo)BMSCs向表皮細(xì)胞分化,使用免疫熒光法檢測(cè)BMSCs向表皮細(xì)胞分化誘導(dǎo)的結(jié)果;6.負(fù)壓條件下誘導(dǎo)BMSCs向表皮細(xì)胞分化14d后,RT-PCR檢測(cè)CK5、CK10m RNA的表達(dá),western-blot檢測(cè)廣譜角蛋白的表達(dá)情況。結(jié)果:1.倒置顯微鏡下可見原代BMSCs呈魚群樣、漩渦樣生長(zhǎng),另可見大量淋巴細(xì)胞與血細(xì)胞,第三代BMSCs呈規(guī)則長(zhǎng)梭形,雜細(xì)胞較少;2.第三代BMSCs可向骨細(xì)胞、脂肪細(xì)胞、軟骨細(xì)胞分化;3.第三代BMSCs高表達(dá)骨髓間充質(zhì)干細(xì)胞表面抗原CD29、CD44,不表達(dá)骨髓造血干細(xì)胞表面抗原CD34;4.隨著負(fù)壓的逐漸增高,細(xì)胞的增殖能力逐漸受抑制,細(xì)胞生長(zhǎng)曲線及CCK8實(shí)驗(yàn)在第7d時(shí),四個(gè)組比較兩兩比較均有統(tǒng)計(jì)學(xué)意義;5.隨著負(fù)壓的逐漸增高,表皮樣細(xì)胞CK5、CK10m RNA的表達(dá)逐漸增高;6.隨著負(fù)壓的逐漸增高,表皮樣細(xì)胞廣譜角蛋白的表達(dá)逐漸增高。結(jié)論:隨著負(fù)壓增高,BMSCs向表皮分化的能力逐漸增強(qiáng),但是細(xì)胞增殖能力逐漸受抑制。
[Abstract]:Aim: to investigate the effect of different negative pressure on epidermal differentiation of bone mesenchymal stem cells (BMSCs) from bone marrow mesenchymal stem cells (BMSCs) in vitro. Method 1: 1. BMSCs 2 was isolated from 4-6 month old New Zealand white rabbits by density gradient centrifugation. Culture to the third generation of BMSCs for identification: inverted microscope to observe the morphology of mesenchymal stem cells, identification of the three line differentiation, flow cytometry identification of the cell surface marker. The experimental group was divided into 0mmhg group (control group, 50mmhg group) and 100mmhg group (200mmhg group). The third generation BMSCs were cultured under negative pressure, and the cell growth curves were plotted at 1 d ~ 3 d ~ 5 d and 7 d, respectively. At 3 d ~ 5 d and 7 d, CCK8 was used to detect the proliferation of BMSCs. BMSCs was induced to differentiate into epidermal cells. Immunofluorescence assay was used to detect the differentiation of BMSCs into epidermal cells. The expression of CK5CK10m RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) after 14 days of differentiation of BMSCs into epidermal cells induced by negative pressure. Western blot was used to detect the expression of broad-spectrum keratin. The result is 1: 1. Under the inverted microscope, the primary BMSCs appeared as a fish group, swirl like growth, and a large number of lymphocytes and blood cells could be seen. The third generation of BMSCs had a regular long spindle shape and less miscellaneous cells. The third generation of BMSCs could differentiate into osteocytes, adipocytes and chondrocytes. The third generation of BMSCs overexpressed bone marrow mesenchymal stem cell surface antigen CD29 and CD44, but did not express bone marrow hematopoietic stem cell surface antigen CD344. With the increase of negative pressure, the cell proliferation was inhibited gradually. At the 7th day, the cell growth curve and CCK8 test showed significant difference between the four groups. With the increase of negative pressure, the expression of CK5 + CK10 m RNA in epidermoid cells increased gradually. With the increase of negative pressure, the expression of broad spectrum keratin in epidermoid cells increased gradually. Conclusion: with the increase of negative pressure, the ability of BMSCs to differentiate into epidermis is gradually enhanced, but the ability of cell proliferation is gradually inhibited.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R61

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