本文選題：骨髓間充質(zhì)干細(xì)胞 + 肝細(xì)胞��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的:探究信號(hào)傳導(dǎo)分子核因子-кB(NF-кB)和P38MAPK在參與調(diào)控肝細(xì)胞生長(zhǎng)因子(Hepatocyte Growth Factor,HGF)為誘導(dǎo)劑的條件下體外培養(yǎng)骨髓間充質(zhì)干細(xì)胞(bone mesenchymal stem cell,BMSC)向肝樣細(xì)胞定向分化的進(jìn)程中,NF-кB信號(hào)通路和P38MAPK信號(hào)通路對(duì)分化的調(diào)控是否存在著某種聯(lián)系。P38MAPK作為NF-кB的上游通路,在分化的過(guò)程中是參與了NF-кB的激活。方法:本實(shí)驗(yàn)中所用到的骨髓間充質(zhì)干細(xì)胞來(lái)源于四周齡的雄性SD大鼠。細(xì)胞的獲取通過(guò)采用全骨髓貼壁法,利用不同細(xì)胞貼壁時(shí)間的差異,篩選出所需要的目標(biāo)細(xì)胞。分離后的細(xì)胞在含10%胎牛血清的DMEM低糖培養(yǎng)液中進(jìn)行培養(yǎng),第一次換液為全換液,可以使得培養(yǎng)瓶中的大部分還沒(méi)有貼壁或者貼壁不牢的非目的細(xì)胞或狀態(tài)不佳的細(xì)胞沖出瓶外。首次換液時(shí)間為取材24小時(shí)后,之后每隔3天換一次培養(yǎng)液,當(dāng)培養(yǎng)瓶中細(xì)胞融合率達(dá)到70%至80%時(shí),使用膠原蛋白酶消化,1:2傳代至新的培養(yǎng)瓶中。實(shí)驗(yàn)使用傳代至第三代的骨髓間充質(zhì)干細(xì)胞作為實(shí)驗(yàn)對(duì)象,使用肝細(xì)胞生長(zhǎng)因子誘導(dǎo)細(xì)胞向肝樣細(xì)胞定向分化。實(shí)驗(yàn)分為四大組:誘導(dǎo)組、NF-кB抑制組、P38MAPK抑制組以及陰性對(duì)照組。誘導(dǎo)組細(xì)胞的誘導(dǎo)環(huán)境為含有肝細(xì)胞生長(zhǎng)因子的完全培養(yǎng)液,抑制組分為NF-кB抑制組和P38抑制組,這兩組細(xì)胞是在HGF誘導(dǎo)組的基礎(chǔ)上分別添加了NF-кB信號(hào)分子的抑制劑BAY 11-7082和P38MAPK信號(hào)分子的抑制劑SB203580,其他培養(yǎng)環(huán)境和培養(yǎng)時(shí)間相同。陰性對(duì)照組細(xì)胞培養(yǎng)環(huán)境為含有10%胎牛血清的DMEM低糖培養(yǎng)基。取培養(yǎng)20天后的細(xì)胞進(jìn)行靛青綠(ICG)攝取實(shí)驗(yàn)檢測(cè)已分化的肝細(xì)胞,蛋白質(zhì)印跡檢測(cè)NF-кB、p-P38和α1-抗胰蛋白酶(α1-antitrypsin,AAT)的表達(dá),取培養(yǎng)第7天和第20天的細(xì)胞進(jìn)行免疫組化染色檢測(cè)NF-кB蛋白。結(jié)果:誘導(dǎo)20天后,HGF誘導(dǎo)組中的大部分細(xì)胞呈圓形或卵圓形肝樣細(xì)胞狀生長(zhǎng),靛氰綠實(shí)驗(yàn)發(fā)現(xiàn)這些細(xì)胞可攝取ICG,并在一定時(shí)間內(nèi)將ICG排出胞體外,免疫印跡實(shí)驗(yàn)結(jié)果顯示有特異的肝細(xì)胞標(biāo)志物α1-抗胰蛋白酶AAT的表達(dá),免疫組化結(jié)果顯示,細(xì)胞培養(yǎng)7天時(shí)和20天時(shí),細(xì)胞核內(nèi)都有NF-кB的聚集,NF-кB在此過(guò)程中激活轉(zhuǎn)移至細(xì)胞核內(nèi);NF-кB抑制組和P38抑制組中細(xì)胞與對(duì)誘導(dǎo)組比,NF-кB抑制劑BAY 11-7082和P38抑制劑SB203580的抑制效果明顯,細(xì)胞對(duì)ICG的攝取和表達(dá)的α1-抗胰蛋白酶蛋白表和誘導(dǎo)組相比來(lái)看都有比較明顯的降低。免疫印跡結(jié)果顯示,P38抑制組中NF-кB蛋白表達(dá)量降低。這兩組抑制組細(xì)胞的免疫組化結(jié)果顯示NF-кB在細(xì)胞核中只有少量存在,NF-кB的核轉(zhuǎn)移被抑制。結(jié)論:在肝細(xì)胞生長(zhǎng)因子誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向肝細(xì)胞分化的過(guò)程中,NF-κB信號(hào)通路和P38MAPK信號(hào)通路均參與了肝樣細(xì)胞定向分化的調(diào)控。P38MAPK通路可能通過(guò)激活下游NF-κB通路發(fā)揮作用。
[Abstract]:Objective: to explore the process of differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro under the condition of signal transduction molecular nuclear factor-BNF-NF-B) and P38MAPK participating in the regulation of Hepatocyte Growth FactorHGF (HGFs) as inducers, during the process of differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro. Whether there is some connection between signaling pathway and P38MAPK signaling pathway in the regulation of differentiation. P38 MAPK is the upstream pathway of NF- FB. In the process of differentiation is involved in the activation of NF- B. Methods: the bone marrow mesenchymal stem cells (BMSCs) used in this study were derived from four-week old male SD rats. The target cells were selected by using the whole bone marrow adherent method and the different adherence time of different cells. The isolated cells were cultured in DMEM low glucose medium containing 10% fetal bovine serum. Most of the cells in the culture bottle that have not adhered or are not adherent to the wall can be rushed out of the bottle by non-target cells or cells in poor condition. When the cell fusion rate in the culture bottle reached 70% to 80%, 1: 2 was digested with collagenase to be passed to the new culture bottle. Bone marrow mesenchymal stem cells (BMSCs) from passage to generation 3 were used in the experiment. Hepatocyte growth factor (HGF) was used to induce the differentiation of cells into hepatoid cells. The experiment was divided into four groups: induction group and negative control group. The inducing environment of the cells in the induction group was a complete medium containing hepatocyte growth factor, and the inhibitory group was divided into NF- and P38 inhibition groups. BAY 11-7082 and SB203580 were added to the two groups on the basis of HGF induction, respectively. The other culture environment and the culture time were the same. The culture environment of negative control group was DMEM low glucose medium containing 10% fetal bovine serum. After 20 days of culture, the differentiated hepatocytes were detected by ICG uptake assay, the expression of 偽 1-antitrypsin (AATA) and the expression of NF-BP38 were detected by Western blot, and the NF-B protein was detected by immunohistochemical staining on the 7th and 20th day of culture. Results: after 20 days of induction, most of the cells in the HGF-induced group showed round or oval hepatocyte-like growth. It was found by indigo green experiment that these cells could absorb ICG and expel ICG out of the cell body within a certain period of time. The results of immunoblotting showed that there was a specific hepatocyte marker 偽 1-antitrypsin AAT expression. The immunohistochemical results showed that the cells were cultured at 7 days and 20 days after culture. During this process, NF-NF-B was activated and transferred to the cells in the NF-NF-B inhibition group and the P38 inhibitory group, and the inhibitory effect of NF-B inhibitor BAY 11-7082 and P38 inhibitor SB203580 was more obvious than that of the induced group, and the inhibitory effect of NF-B inhibitor BAY 11-7082 and P38 inhibitor SB203580 was significantly higher than that of the induction group. The uptake and expression of 偽 1-antitrypsin protein in cells were significantly lower than those in the induction group. The results of Western blot showed that the expression of NF- FB protein was decreased in P38 inhibition group. The immunohistochemical results of these two groups showed that only a small number of nuclear metastases of NFNF-B were inhibited in the nuclei of these two groups. Conclusion: both NF- 魏 B signaling pathway and P38MAPK signaling pathway are involved in the regulation of hepatocyte-like differentiation by activating downstream NF- 魏 B pathway in the process of hepatocyte growth factor inducing mesenchymal stem cells to differentiate into hepatocytes.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R329.2

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相關(guān)期刊論文 前2條

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本文編號(hào)：1915655