白细胞介素6在人脐带来源间充质干细胞中的表达调控

发布时间：2018-05-08 11:22

本文选题：间充质干细胞 + 免疫性肝损伤　；参考：《河北医科大学》2011年硕士论文

【摘要】：目的:间充质干细胞(MSC)是一类中胚层来源的多能干细胞,具有多向分化、造血支持及促进干细胞植入等功能。随着对其认识的不断深入,其免疫调节特性也越来越多地引发了研究者的关注【1,2】;近来,MSC更是被作为免疫损伤性疾病的理想治疗策略而被广泛研究【26,27,28,29】。我们的前期研究发现,经体外预处理的人胎儿来源MSC可有效保护伴刀豆蛋白A(concanavalin A,ConA)诱导的小鼠免疫性肝损伤病情进展,其作用机制可能与白细胞介素6(interleukin-6,IL-6)表达增高有关。经检索文献,未见相关报道。本研究拟采用多种策略,调控IL-6在MSC中的表达,为进一步确证其是否是MSC保护免疫性肝损伤中的关键分子奠定基础。方法:采用胶原酶消化贴壁传代法,自人胎儿脐带Wharton层获取细胞,经三代以上稳定传代后,行形态学观察、流式细胞术分析细胞表面标记分子及定向诱导分化予以鉴定。通过慢病毒IL-6高/低表达载体转染、肿瘤坏死因子α(tumor necrosis factor,TNF-α)和干扰素γ(interferon,IFN-γ)单独或联合应用等策略,上调/下调IL-6在MSC中的表达,PT-PCR、real-time PCR鉴定基因表达。结果: 1脐带来源MSC的分离鉴定 1.1原代细胞可在5至14天形成细胞集落,细胞最初表现为两种形态,多部分为成纤维细胞样,一小部分为内皮细胞样,后者在传代中将逐步消失,至第4代以后,呈现典型的梭形生长形态。 1.2流式细胞检测结果显示,间质细胞标志CD29、CD90及CD105阳性表达;HLA-DR及造血细胞标志CD34阴性表达。 1.3脂肪样细胞定向诱导分化后,分离获取的细胞胞质中可出现大小不一的脂滴,油红染色后呈现红色;成骨样细胞定向诱导分化后,细胞可逐渐变方,Von Kossa染色结果显示,胞浆内有棕黑色区域。提示该细胞具有脂肪样和成骨样细胞分化潜能。上述结果提示,我们从脐带获取的细胞具有MSC典型特征,可用于下一步研究。 2慢病毒IL-6表达载体的构建 2.1以质粒pLXSN/IL-6为模板,PCR扩增IL-6片段,与T载体相连接,转化扩增后,测序鉴定,结果显示序列正确。提示IL-6基因与T载体连接成功。 2.2 IL-6基因与慢病毒pBPLV载体相连接,转化扩增后,测序鉴定,结果显示序列正确。提示慢病毒IL-6表达载体(pBPLV/IL-6)构建成功。 3慢病毒IL-6表达载体转染MSC RT-PCR结果显示,慢病毒IL-6表达载体(pBPLV/IL-6-MSC)的IL-6表达水平较慢病毒空载体转染的MSC及未转染MSC(pBPLV-MSC;MSC)显著增高(p0.05); real-time PCR结果亦显示,pBPLV/IL-6-MSC组IL-6表达水平显著高于pBPLV/MSC组和MSC组(p0.05); 上述结果提示,pBPLV/IL-6转染可显著上调IL-6基因表达。4慢病毒IL-6干涉载体转染MSC RT-PCR结果显示,慢病毒IL-6干涉载体(pSicoR/IL-6-MSC)的IL-6表达水平较慢病毒空载体转染的MSC及未转染MSC(pSicoR-MSC; MSC)显著降低(p0.05); real-time PCR结果亦显示,pSicoR/IL-6-MSC组IL-6表达水平显著低于pSicoR -MSC组和MSC组(p0.05); 上述结果提示,pSicoR/IL-6转染可显著下调IL-6表达。 5 ConA刺激的小鼠脾细胞培养上清对MSC IL-6表达的影响 RT-PCR、real-time PCR结果均显示不同浓度小鼠脾细胞培养上清处理后UCMSC IL-6表达水平均高于对照组(p0.05); RT-PCR、real-time PCR结果均显示同一浓度小鼠脾细胞培养上清处理后UCMSC于不同时间点IL-6表达水平均高于对照组(p0.05); 上述结果提示,ConA刺激的小鼠脾细胞培养上清可显著上调IL-6表达。 6细胞因子TNF-α和IFN-γ联合以及单独应用对MSC IL-6表达的影响 RT-PCR结果显示不同浓度TNF-α和IFN-γ联合应用其IL-6表达量显著高于对照组(p0.05);RT-PCR、real-time PCR结果显示同一浓度TNF-α和IFN-γ联合以及TNF-α单独应用后UCMSC各时间点IL-6表达水平均高于对照组(p0.05),而IFN-γ单独应用后各时间点IL-6表达水平均低于对照组(p0.05)。上述结果提示,TNF-α和IFN-γ联合应用以及TNF-α单独应用可上调IL-6表达,而IFN-γ单独应用则下调IL-6表达。结论: 1采用胶原酶消化贴壁传代法,可自人胎儿脐带Wharton层成功获取合格的MSC 2成功构建了慢病毒IL-6表达载体pBPLV/IL-6 3慢病毒IL-6表达载体pBPLV/IL-6、ConA刺激的小鼠脾细胞培养上清、TNF-α单独或联合IFN-γ应用可显著上调MSC IL-6基因表达 4慢病毒IL-6干涉载体pSicoR/IL-6和IFN-γ单独应用可显著下调IL-6基因表达
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【共引文献】