本文選題：骨髓間充質(zhì)干細胞 + 肝細胞��； 參考：《中南大學》2009年碩士論文

【摘要】： 目的通過不同濃度肝細胞生長因子(hepatocyte growth factor,HGF)和表皮細胞生長因子(epidermal cell growth factor,EGF)體外誘導(dǎo)兔骨髓間充質(zhì)干細胞(bone mesenchymal stem cells,BMSCs)向肝細胞分化,以探討HGF和EGF的最適濃度組合。 方法密度梯度離心法分離兔BMSCs,體外培養(yǎng),傳代純化。建立以HGF(30.0ug/L)+EGF(1.5mg/L)、HGF(40.0ug/L)+EGF(2.5mg/L)、HGF(50.0ug/L)+EGF(3.5mg/L)、HGF(60.0ug/L)+EGF(4.5mg/L)為誘導(dǎo)的培養(yǎng)體系。觀察誘導(dǎo)細胞形態(tài)學變化,于7、14、21、28d采用免疫細胞化學及免疫熒光分別檢測肝細胞表面標志甲胎蛋白(alphafetoprotein AFP)及白蛋白(albumin ALB),留取不同時段培養(yǎng)上清液,用溴甲酚綠法(bromcresol green BCG)檢測上清液中ALB含量,用尿素合成試驗檢測尿素含量。 結(jié)果14d開始可觀察到多極性的肝細胞樣細胞,呈短梭形和多角形,隨誘導(dǎo)時間延長(21d)形成多細胞集落,各組形態(tài)學比較無明顯差異。免疫細胞化學檢測,7d時AFP表達陽性,14d達最大值后即開始下降(P＜0.05),各濃度組間表達無差別(P＞0.05)。免疫熒光檢測,14d開始ALB表達陽性,1組、4組各時間點比較無差別(P＞0.05),2組及3組隨誘導(dǎo)時間延長,陽性表達細胞數(shù)增加(P＜0.05)。縱向比較:總體上各組之間有顯著性差異,且濃度越高,陽性細胞數(shù)越高(P＜0.05)。兩兩比較,在14d時1組與2組無差別,在21d時2組與3組表達無差別,28d時3組與4組表達無差別(p＞0.05)。溴甲酚綠法檢測,12d細胞培養(yǎng)上清液中可檢測到ALB,ALB含量在早期(9-15d)表現(xiàn)為濃度越高含量越高((P＜0.05),18或21d達高峰后下降,此時各濃度組間含量無差別(p＞0.05),最高峰值在第四組(HGF60ug/L、EGF4.5ug/L)。9d時可檢測到尿素含量,各濃度組間表達無差別(p＞0.05)。 結(jié)論HGF及EGF能誘導(dǎo)兔BMSCs分化為肝細胞,誘導(dǎo)時間約2-3周。誘導(dǎo)劑濃度與誘導(dǎo)分化率之間在誘導(dǎo)早期呈劑量依賴關(guān)系,高濃度的HGF及EGF可提高BMSCs向肝細胞的分化率。誘導(dǎo)劑最適組合為HGF60ug/L、EGF4.5mg/L。
[Abstract]:Objective to induce the differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) into hepatocytes by different concentrations of hepatocyte growth factor (HGF) and epidermal cell growth factor (EGF), and to explore the optimal concentration combination of HGF and EGF. Methods BMSCs were isolated by density gradient centrifugation and cultured in vitro. We set up a culture system that was induced by HGF 30.0ugr / L) EGF1.5mg / L) HGFF 40.0ugP / L) EGF2.5mg / L HGF50.0ugP / L) EGF3.5mg / L) EGF3.5mg / L) EGF4.5mg / L). The morphological changes of induced cells were observed. The hepatocyte surface marker alphafetoprotein AFPand albumin albumin albumin were detected by immunocytochemistry and immunofluorescence for 28 days. The content of ALB in supernatant was determined by bromcresol green method and urea content was detected by urea synthesis test. Results Multi-polarity hepatocyte-like cells were observed on the 14th day, which were spindle-shaped and polygonal, and formed multicellular colonies with the prolongation of induction time (21 d). There was no significant difference in morphology among the groups. Immunocytochemistry showed that the expression of AFP reached the maximum at day 14 and then decreased (P < 0.05). There was no difference in the expression of AFP between different concentration groups (P > 0.05). Immunofluorescence assay showed that there was no difference at each time point between group 1 and group 1 (P > 0.05) and group 3 (P > 0.05). The number of positive cells increased with the time of induction (P < 0.05). Longitudinal comparison: there was significant difference among the groups, and the higher the concentration, the higher the number of positive cells (P < 0.05). There was no difference between group 1 and group 2 at day 14, and group 2 and group 3 on day 21. There was no difference in expression between group 3 and group 4 on day 28 at day 2 and group 4 (P > 0.05). The concentration of ALB in the supernatant of cell culture for 12 days was detected by bromocresol green method. The results showed that the higher the concentration of ALB was, the higher the concentration was, the higher the concentration was, the higher the concentration was, P < 0.05, P < 0.05, or 21 days after reaching the peak. At the same time, there was no difference in the content of urea between the different concentration groups. The highest peak value of urea was detected at day 9 of HGF 60ugr / L, EGF 4.5ugP / L, and there was no difference in the expression of urea among the different concentration groups (P > 0.05). Conclusion HGF and EGF can induce rabbit BMSCs to differentiate into hepatocytes for about 2-3 weeks. There was a dose-dependent relationship between the concentration of inducer and the rate of induced differentiation at the early stage of induction. High concentrations of HGF and EGF could increase the differentiation rate of BMSCs into hepatocytes. The optimal combination of inducers was HGF 60ugr LnEGF4.5 mg / L 路L ~ (-1) 路L ~ (-1) 路L ~ (-1).
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

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