發(fā)布時(shí)間：2018-05-08 11:22

  本文選題：間充質(zhì)干細(xì)胞 + 免疫性肝損傷　； 參考：《河北醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:間充質(zhì)干細(xì)胞(MSC)是一類中胚層來源的多能干細(xì)胞,具有多向分化、造血支持及促進(jìn)干細(xì)胞植入等功能。隨著對其認(rèn)識的不斷深入,其免疫調(diào)節(jié)特性也越來越多地引發(fā)了研究者的關(guān)注【1,2】;近來,MSC更是被作為免疫損傷性疾病的理想治療策略而被廣泛研究【26,27,28,29】。我們的前期研究發(fā)現(xiàn),經(jīng)體外預(yù)處理的人胎兒來源MSC可有效保護(hù)伴刀豆蛋白A(concanavalin A,ConA)誘導(dǎo)的小鼠免疫性肝損傷病情進(jìn)展,其作用機(jī)制可能與白細(xì)胞介素6(interleukin-6,IL-6)表達(dá)增高有關(guān)。經(jīng)檢索文獻(xiàn),未見相關(guān)報(bào)道。本研究擬采用多種策略,調(diào)控IL-6在MSC中的表達(dá),為進(jìn)一步確證其是否是MSC保護(hù)免疫性肝損傷中的關(guān)鍵分子奠定基礎(chǔ)。 方法:采用膠原酶消化貼壁傳代法,自人胎兒臍帶Wharton層獲取細(xì)胞,經(jīng)三代以上穩(wěn)定傳代后,行形態(tài)學(xué)觀察、流式細(xì)胞術(shù)分析細(xì)胞表面標(biāo)記分子及定向誘導(dǎo)分化予以鑒定。通過慢病毒IL-6高/低表達(dá)載體轉(zhuǎn)染、腫瘤壞死因子α(tumor necrosis factor,TNF-α)和干擾素γ(interferon,IFN-γ)單獨(dú)或聯(lián)合應(yīng)用等策略,上調(diào)/下調(diào)IL-6在MSC中的表達(dá),PT-PCR、real-time PCR鑒定基因表達(dá)。 結(jié)果: 1臍帶來源MSC的分離鑒定 1.1原代細(xì)胞可在5至14天形成細(xì)胞集落,細(xì)胞最初表現(xiàn)為兩種形態(tài),多部分為成纖維細(xì)胞樣,一小部分為內(nèi)皮細(xì)胞樣,后者在傳代中將逐步消失,至第4代以后,呈現(xiàn)典型的梭形生長形態(tài)。 1.2流式細(xì)胞檢測結(jié)果顯示,間質(zhì)細(xì)胞標(biāo)志CD29、CD90及CD105陽性表達(dá);HLA-DR及造血細(xì)胞標(biāo)志CD34陰性表達(dá)。 1.3脂肪樣細(xì)胞定向誘導(dǎo)分化后,分離獲取的細(xì)胞胞質(zhì)中可出現(xiàn)大小不一的脂滴,油紅染色后呈現(xiàn)紅色;成骨樣細(xì)胞定向誘導(dǎo)分化后,細(xì)胞可逐漸變方,Von Kossa染色結(jié)果顯示,胞漿內(nèi)有棕黑色區(qū)域。提示該細(xì)胞具有脂肪樣和成骨樣細(xì)胞分化潛能。 上述結(jié)果提示,我們從臍帶獲取的細(xì)胞具有MSC典型特征,可用于下一步研究。 2慢病毒IL-6表達(dá)載體的構(gòu)建 2.1以質(zhì)粒pLXSN/IL-6為模板,PCR擴(kuò)增IL-6片段,與T載體相連接,轉(zhuǎn)化擴(kuò)增后,測序鑒定,結(jié)果顯示序列正確。提示IL-6基因與T載體連接成功。 2.2 IL-6基因與慢病毒pBPLV載體相連接,轉(zhuǎn)化擴(kuò)增后,測序鑒定,結(jié)果顯示序列正確。提示慢病毒IL-6表達(dá)載體(pBPLV/IL-6)構(gòu)建成功。 3慢病毒IL-6表達(dá)載體轉(zhuǎn)染MSC RT-PCR結(jié)果顯示,慢病毒IL-6表達(dá)載體(pBPLV/IL-6-MSC)的IL-6表達(dá)水平較慢病毒空載體轉(zhuǎn)染的MSC及未轉(zhuǎn)染MSC(pBPLV-MSC;MSC)顯著增高(p0.05); real-time PCR結(jié)果亦顯示,pBPLV/IL-6-MSC組IL-6表達(dá)水平顯著高于pBPLV/MSC組和MSC組(p0.05); 上述結(jié)果提示,pBPLV/IL-6轉(zhuǎn)染可顯著上調(diào)IL-6基因表達(dá)。4慢病毒IL-6干涉載體轉(zhuǎn)染MSC RT-PCR結(jié)果顯示,慢病毒IL-6干涉載體(pSicoR/IL-6-MSC)的IL-6表達(dá)水平較慢病毒空載體轉(zhuǎn)染的MSC及未轉(zhuǎn)染MSC(pSicoR-MSC; MSC)顯著降低(p0.05); real-time PCR結(jié)果亦顯示,pSicoR/IL-6-MSC組IL-6表達(dá)水平顯著低于pSicoR -MSC組和MSC組(p0.05); 上述結(jié)果提示,pSicoR/IL-6轉(zhuǎn)染可顯著下調(diào)IL-6表達(dá)。 5 ConA刺激的小鼠脾細(xì)胞培養(yǎng)上清對MSC IL-6表達(dá)的影響 RT-PCR、real-time PCR結(jié)果均顯示不同濃度小鼠脾細(xì)胞培養(yǎng)上清處理后UCMSC IL-6表達(dá)水平均高于對照組(p0.05); RT-PCR、real-time PCR結(jié)果均顯示同一濃度小鼠脾細(xì)胞培養(yǎng)上清處理后UCMSC于不同時(shí)間點(diǎn)IL-6表達(dá)水平均高于對照組(p0.05); 上述結(jié)果提示,ConA刺激的小鼠脾細(xì)胞培養(yǎng)上清可顯著上調(diào)IL-6表達(dá)。 6細(xì)胞因子TNF-α和IFN-γ聯(lián)合以及單獨(dú)應(yīng)用對MSC IL-6表達(dá)的影響 RT-PCR結(jié)果顯示不同濃度TNF-α和IFN-γ聯(lián)合應(yīng)用其IL-6表達(dá)量顯著高于對照組(p0.05);RT-PCR、real-time PCR結(jié)果顯示同一濃度TNF-α和IFN-γ聯(lián)合以及TNF-α單獨(dú)應(yīng)用后UCMSC各時(shí)間點(diǎn)IL-6表達(dá)水平均高于對照組(p0.05),而IFN-γ單獨(dú)應(yīng)用后各時(shí)間點(diǎn)IL-6表達(dá)水平均低于對照組(p0.05)。 上述結(jié)果提示,TNF-α和IFN-γ聯(lián)合應(yīng)用以及TNF-α單獨(dú)應(yīng)用可上調(diào)IL-6表達(dá),而IFN-γ單獨(dú)應(yīng)用則下調(diào)IL-6表達(dá)。 結(jié)論: 1采用膠原酶消化貼壁傳代法,可自人胎兒臍帶Wharton層成功獲取合格的MSC 2成功構(gòu)建了慢病毒IL-6表達(dá)載體pBPLV/IL-6 3慢病毒IL-6表達(dá)載體pBPLV/IL-6、ConA刺激的小鼠脾細(xì)胞培養(yǎng)上清、TNF-α單獨(dú)或聯(lián)合IFN-γ應(yīng)用可顯著上調(diào)MSC IL-6基因表達(dá) 4慢病毒IL-6干涉載體pSicoR/IL-6和IFN-γ單獨(dú)應(yīng)用可顯著下調(diào)IL-6基因表達(dá)
[Abstract]:Objective: mesenchymal stem cells (MSC) are a kind of pluripotent stem cells derived from mesoderm, which have multiple differentiation, hematopoietic support and stem cell implantation. With the deepening of their understanding, the immunoregulatory characteristics of the mesenchymal stem cells have attracted more and more attention from researchers [1,2]; recently, MSC has been used as an immune injury disease. The ideal treatment strategy has been widely studied [26,27,28,29]. Our previous study found that human fetal source MSC, which was pretreated in vitro, could effectively protect the progression of immune liver injury induced by concanavalin A (concanavalin A, ConA) in mice, and its mechanism may be related to the increased expression of interleukin 6 (interleukin-6, IL-6). This study intends to use a variety of strategies to regulate the expression of IL-6 in MSC and to confirm whether it is the key element in MSC protection of immune liver injury.
Methods: collagenase digestion and adherence were used to obtain cells from human fetal umbilical cord Wharton layer. After more than three generations of stable passages, morphological observation was performed. Flow cytometry was used to identify the cell surface markers and directional differentiation. The tumor necrosis factor alpha (tumor necrosis fact) was transfected through the IL-6 high / low expression vector of the lentivirus. Or, TNF- alpha) and interferon gamma (interferon, IFN- IFN-) alone or in combination with other strategies, upregulated / downregulated IL-6 expression in MSC, and PT-PCR, real-time PCR identified gene expression.
Result:
Isolation and identification of MSC from 1 umbilical cord
1.1 primary cells could form cell colonies from 5 to 14 days. The cells were initially displayed in two forms, mostly fibroblast like, and a small part of the endothelial cells. The latter gradually disappeared in the passage, and after the fourth generation, it showed a typical spindle shape.
1.2 the results of flow cytometry showed that interstitial cells showed positive expression of CD29, CD90 and CD105, while HLA-DR and hematopoietic cells showed negative CD34 expression.
After directed differentiation of 1.3 adipose like cells, different fat droplets could appear in the cytoplasm of the isolated cells and red in the oil red. After directed differentiation of osteoblast like cells, the cells could gradually change square. The results of Von Kossa staining showed that there were brown and black regions in the cytoplasm. It suggested that the cells have the differentiation of adipose like and osteoblast like cells. Potential.
These results suggest that the cells obtained from the umbilical cord have typical MSC characteristics and can be used for further research.
Construction of 2 lentivirus IL-6 expression vector
2.1 the plasmid pLXSN/IL-6 was used as a template, PCR amplified IL-6 fragment, and was connected with the T vector. After the transformation and amplification, the sequence was identified. The results showed that the sequence was correct. It suggested that the IL-6 gene was connected successfully with the T carrier.
The 2.2 IL-6 gene was connected with the lentivirus pBPLV vector and was transformed and amplified by sequencing and identified. The results showed that the sequence was correct. It suggested that the IL-6 expression vector (pBPLV/IL-6) of the lentivirus was constructed successfully.
3 lentivirus IL-6 expression vector transfected to MSC
RT-PCR results showed that the IL-6 expression level of the lentivirus IL-6 expression vector (pBPLV/IL-6-MSC) was significantly higher than that of the MSC and the untransfected MSC (pBPLV-MSC; MSC) transfected by the slow virus (P0.05).
The results of real-time PCR also showed that the expression level of IL-6 in pBPLV/IL-6-MSC group was significantly higher than that in pBPLV/MSC group and MSC group (P0.05).
These results suggest that pBPLV/IL-6 transfection can significantly increase IL-6 gene expression,.4 lentiviral IL-6 interference vector transfection MSC
The results of RT-PCR showed that the IL-6 expression level of lentivirus IL-6 interference carrier (pSicoR/IL-6-MSC) was significantly lower than that of MSC and MSC (pSicoR-MSC; MSC) transfected by slow virus.
The results of real-time PCR also showed that the expression level of IL-6 in group pSicoR/IL-6-MSC was significantly lower than that in pSicoR -MSC group and MSC group (P0.05).
These results suggest that pSicoR/IL-6 transfection can significantly reduce IL-6 expression.
Effect of 5 ConA stimulated splenocytes supernatant on MSC IL-6 expression in mice
RT-PCR and real-time PCR results showed that the expression level of UCMSC IL-6 in splenocytes supernatant treated with different concentrations was higher than that in the control group (P0.05).
The results of RT-PCR and real-time PCR showed that the expression level of UCMSC at different time points after treatment with the same concentration of splenocytes supernatant was higher than that in the control group (P0.05).
These results suggest that ConA stimulated splenocytes supernatant can significantly increase IL-6 expression.
6 cytokine TNF- alpha and IFN- gamma combined with single application on the expression of MSC IL-6
The RT-PCR results showed that the IL-6 expression of different concentrations of TNF- A and IFN- y was significantly higher than that of the control group (P0.05); RT-PCR, real-time PCR results showed that the expression level of the same concentration TNF- alpha and IFN- gamma and TNF- alpha was higher than that of the control group. The level of the group was lower than that of the control group (P0.05).
These results suggest that the combination of TNF- alpha and IFN- gamma and TNF- alone can increase IL-6 expression, while IFN- gamma alone can downregulate IL-6 expression.
Conclusion:
1 by collagenase digestion and adherent passage, the qualified MSC can be obtained from the human fetal umbilical cord Wharton layer.
2 the lentiviral IL-6 expression vector pBPLV/IL-6 was successfully constructed.
3 the lentivirus IL-6 expression vector pBPLV/IL-6 and ConA stimulated the splenocytes supernatant of mice, and TNF- alpha alone or combined with IFN- gamma significantly increased the expression of MSC IL-6 gene.
4 lentivirus IL-6 interference vector pSicoR/IL-6 and IFN- gamma alone can significantly reduce the expression of IL-6 gene.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

【共引文獻(xiàn)】

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2 楊舟鑫;CD106是具有強(qiáng)免疫調(diào)節(jié)功能的胎盤絨毛膜間充質(zhì)干細(xì)胞的標(biāo)志[D];北京協(xié)和醫(yī)學(xué)院;2013年

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6 呂自明;重組人腫瘤壞死因子α影響人臍靜脈內(nèi)皮細(xì)胞活性及PD-L1蛋白表達(dá)的研究[D];南方醫(yī)科大學(xué);2013年

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