本文選題：負壓 + 骨髓間充質(zhì)干細胞��； 參考：《石河子大學》2017年碩士論文

【摘要】：目的:探討體外不同負壓對于骨髓間充質(zhì)干細胞(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進行鑒定:倒置顯微鏡對細胞進行形態(tài)學觀察,間充質(zhì)干細胞的三系分化鑒定,流式細胞儀鑒定細胞表面標志物;3.設(shè)置實驗組,分別是0mmhg組(對照組)、50mmhg組、100mmhg組、200mmhg組,誘導時間為30min/h;4.負壓下常規(guī)培養(yǎng)第三代BMSCs,分別于1d、3d、5d和7d進行細胞計數(shù)繪制細胞生長曲線,于3d、5d和7d使用CCK8檢測細胞增殖情況;5.誘導BMSCs向表皮細胞分化,使用免疫熒光法檢測BMSCs向表皮細胞分化誘導的結(jié)果;6.負壓條件下誘導BMSCs向表皮細胞分化14d后,RT-PCR檢測CK5、CK10m RNA的表達,western-blot檢測廣譜角蛋白的表達情況。結(jié)果:1.倒置顯微鏡下可見原代BMSCs呈魚群樣、漩渦樣生長,另可見大量淋巴細胞與血細胞,第三代BMSCs呈規(guī)則長梭形,雜細胞較少;2.第三代BMSCs可向骨細胞、脂肪細胞、軟骨細胞分化;3.第三代BMSCs高表達骨髓間充質(zhì)干細胞表面抗原CD29、CD44,不表達骨髓造血干細胞表面抗原CD34;4.隨著負壓的逐漸增高,細胞的增殖能力逐漸受抑制,細胞生長曲線及CCK8實驗在第7d時,四個組比較兩兩比較均有統(tǒng)計學意義;5.隨著負壓的逐漸增高,表皮樣細胞CK5、CK10m RNA的表達逐漸增高;6.隨著負壓的逐漸增高,表皮樣細胞廣譜角蛋白的表達逐漸增高。結(jié)論:隨著負壓增高,BMSCs向表皮分化的能力逐漸增強,但是細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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R61

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