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  本文選題：原始生殖細胞 + 肝細胞��； 參考：《青島大學》2014年碩士論文

【摘要】：目的：研究小鼠胚胎肝臟發(fā)育的過程和體外干細胞向肝細胞定向分化與NF-κB和UBC9表達的關系,探討干細胞向肝細胞定向分化的分子機制。 方法：分離培養(yǎng)擴增小鼠原始生殖嵴細胞(PGCs),向培養(yǎng)液中分別加入20μ/Lβ-神經(jīng)細胞生長因子(β-NGF)、0.5g/L新生鼠肝臟提取液和胎肝細胞上清液體外誘導小鼠生殖嵴來源的胚胎干細胞(PGCs)向肝樣細胞定向分化。觀察細胞的形態(tài)學的變化；免疫細胞熒光法檢測肝樣分化細胞內(nèi)α1-抗胰蛋白酶(AAT)的表達及對NF-κB的表達進行定位；免疫細胞化學法檢測肝樣分化細胞內(nèi)UBC9的表達；Real-TimePCR檢測在誘導分化過程中NF-κB和UBC9mRNA的相對表達量。免疫組化對胚胎發(fā)育過程中11.5d、14.5d、18.5d的肝臟中NF-κB和UBC9的表達進行分析。 結果：PGCs培養(yǎng)液中加入20μg/Lβ-NGF、0.5g/L新生鼠肝臟提取液和胎肝細胞上清液可見部分細胞變?yōu)閳A形或卵圓形；誘導12天后,圓形或卵圓形細胞呈現(xiàn)α1-抗胰蛋白酶陽性表達；在誘導分化過程中NF-κB的表達逐漸增強,伴隨肝細胞的成熟而呈陽性表達并由胞質(zhì)轉入胞核。Real-Time PCR結果顯示在誘導分化過程中NF-κB的表達量升高,UBC9的表達量增加。而在胎肝發(fā)育過程中隨肝原細胞向肝成熟細胞轉化,肝細胞胞質(zhì)中NF-κB的表達逐漸增強,并向細胞核內(nèi)轉移；UBC9表達在11.5d-14.5d逐漸增強,18.5d,肝細胞成熟時,UBC9在胞核中幾乎不表達。 結論：20μg/Lβ-NGF、0.5g/L新生鼠肝臟提取液和胎肝細胞上清液可以誘導小鼠PGCs分化為肝細胞,誘導分化過程中NF-κB的表達逐漸增強,UBC9的表達量升高；而在胎肝發(fā)育過程中隨肝原細胞向肝成熟細胞轉化,NF-κB的表達逐漸增強,UBC9表達先增強后減弱。
[Abstract]:Objective: to study the relationship between the development of mouse embryonic liver and the expression of NF- 魏 B and UBC9, and the relationship between the differentiation of stem cells into hepatocytes in vitro and the expression of NF- 魏 B and UBC9. To explore the molecular mechanism of the differentiation of stem cells into hepatocytes. Methods: the mouse primordial reproductive crest cells (PGCsN) were isolated and expanded, and 20 渭 / L L 尾 -neuronal growth factor (尾 -NGF) and fetal hepatocytes were added to the culture medium, respectively. The supernatant induced the differentiation of mouse embryonic stem cells derived from reproductive crest into hepatocyte-like cells in vitro. The expression of 偽 1-antitrypsin (AATA) and the expression of NF- 魏 B in hepatoid differentiation cells were detected by immunofluorescence assay. Immunocytochemistry was used to detect the expression of UBC9 in hepatoid differentiation cells. Real-time PCR was used to detect the relative expression of NF- 魏 B and UBC9 mRNA in hepatoid differentiation cells. The expression of NF- 魏 B and UBC9 in liver of 11.5d 14.5 d and 18.5d during embryonic development was analyzed by immunohistochemistry. Results some cells became round or oval in 20 渭 g / L 尾 -NGF 0.5 g / L neonatal rat liver extract and fetal liver cell supernatant after 12 days of induction. The positive expression of 偽 1-antitrypsin was observed in round or oval cells, and the expression of NF- 魏 B increased gradually during differentiation. The expression of NF- 魏 B and UBC9 increased during induction and differentiation of hepatocytes from cytoplasm to nucleus. Real-Time PCR results showed that the expression of NF- 魏 B was increased during differentiation. During the development of fetal liver, the expression of NF- 魏 B in the cytoplasm of hepatocytes increased gradually with the transformation of hepatocytes into mature hepatocytes. The expression of UBC9 was gradually increased at 18.5 days after 11.5d-14.5d was transferred into the nucleus, and almost no expression of UBC9 was found in the nucleus of hepatocytes at maturation. Conclusion the liver extract and fetal hepatocyte supernatant of 0.5 g / L of L 尾 -NGFN at 20 渭 g / L can induce the differentiation of 11.5d-14.5d into hepatocytes. During the process of differentiation, the expression of NF- 魏 B increased gradually and the expression of UBC9 increased gradually, while the expression of NF- 魏 B increased at first and then decreased with the transformation of hepatocytes into mature cells during the development of fetal liver.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575

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本文編號：1988129