藏系绵羊MSX2基因皮肤特异表达载体构建及功能分析

发布时间：2018-06-29 13:31

本文选题：藏系绵羊 + MSX2基因　；参考：《西南民族大学》2015年硕士论文

【摘要】：本研究对藏系绵羊同源异型基因家族成员MSX2基因的序列、组织表达谱及其对细胞内信号分子表达的影响进行分析,为进一步研究MSX2基因对藏系绵羊毛囊发育等过程的影响奠定基础。试验采集了母藏系绵羊(n=5)的心、肝、脾、肺、肾、皮肤和脂肪等组织,从皮肤中克隆了MSX2基因编码区序列,并分析了其在不同组织的表达;构建了该基因的皮肤特异表达载体,并转染体外培养的人毛囊内根鞘细胞(Human hair inner root sheath cells,HHIRSC),再利用荧光定量PCR及Western blot技术检测该基因下游靶基因在mRNA和蛋白质水平上的表达情况。本研究获得的主要结果如下:1、克隆得到藏系绵羊MSX2基因编码区序列,长度为804 bp。与预测的绵羊MSX2基因序列相比,编码区有6个差异碱基,匹配序列相似性为99.25%,另外还有6个碱基缺失。藏系绵羊MSX2基因推导的氨基酸序列包含267个氨基酸,理论分子量为28.98 kD,预测的理论等电点与普通绵羊相同,均为10.32,但存在3个氨基酸差异,并缺失了连续2个氨基酸,序列相似性为98.88%。2、利用荧光定量PCR分析了MSX2基因在藏系绵羊心、肝、脾、肺、肾、皮肤和脂肪的表达情况。结果显示,皮肤中MSX2基因的表达量极显著高于其他组织(P0.01),暗示MSX2基因可能与藏系绵羊毛发生长或性状相关。3、以pCDsRed2-1真核载体为骨架,构建了含KAP6.1皮肤特异启动子的MSX2基因皮肤特异表达载体pCDsR-KM。将该真核表达载体瞬时转染进入人毛囊内根鞘细胞,转染效率为10%左右;用流式细胞术分选转染成功的细胞,获得了转染MSX2基因的HHIRSC。4、荧光定量PCR检测表明,在流式细胞仪筛选后的HHIRSC中,MSX2基因超表达约9000倍,而MSX2基因下游靶基因Lef1、Foxn1、Notch1和Notch2基因的mRNA水平均显著上调。进一步用Western blot检测Notch1和Notch2蛋白的表达情况,证实这两种蛋白质的表达量亦显著上调。这些结果提示,MSX2基因有可能通过Wnt、Notch和MSX2/Foxn1/毛发角蛋白等信号通路,影响皮肤毛囊发生和周期性变化。
[Abstract]:In this study, the sequence, tissue expression profile of MSX2 gene member of homologous gene family of Tibetan sheep and its effect on intracellular signal molecule expression were analyzed. The results laid a foundation for further study on the effect of MSX2 gene on the development of Tibetan sheep hair follicles. The heart, liver, spleen, lung, kidney, skin and fat of female Tibetan sheep were collected, the sequence of MSX2 gene coding region was cloned from the skin, the expression of MSX2 gene in different tissues was analyzed, and the skin specific expression vector of the gene was constructed. Human hair inner root sheath cells (HHIRSC) were transfected into human hair follicle cells in vitro. The expression of the downstream target gene of HHIRSC was detected by fluorescence quantitative polymerase chain reaction and Western blot. The main results of this study were as follows: 1. The sequence of MSX2 gene coding region of Tibetan sheep was cloned and the length of MSX2 gene was 804 BP. Compared with the predicted sheep MSX2 gene sequence, there were 6 different bases in the coding region, the similarity of the matching sequence was 99.25 and there were 6 base deletions. The deduced amino acid sequence of MSX2 gene of Tibetan sheep contained 267 amino acids with a theoretical molecular weight of 28.98 KD. The predicted theoretical isoelectric point was the same as that of common sheep (10.32), but there were three amino acid differences and two successive amino acids were missing. The sequence similarity was 98.880.The expression of MSX2 gene in heart, liver, spleen, lung, kidney, skin and fat of Tibetan sheep was analyzed by fluorescence quantitative PCR. The results showed that the expression of MSX2 gene in skin was significantly higher than that in other tissues (P0.01), suggesting that MSX2 gene might be related to hair growth or traits of Tibetan sheep, and was based on pCDsRed2-1 eukaryotic vector. The skin specific expression vector pCDsR-KMof MSX2 gene containing KAP6.1 skin specific promoter was constructed. The eukaryotic expression vector was transiently transfected into human hair follicle root sheath cells, and the transfection efficiency was about 10%. HHIRSC.4 transfected MSX2 gene was obtained by flow cytometry and detected by fluorescence quantitative PCR. The overexpression of MSX2 gene in HHIRSC by flow cytometry was about 9000 fold, while the mRNA levels of Lef1Foxn1n1 Notch1 and Notch2 genes were significantly up-regulated in the downstream target genes of MSX2 gene, Lef1, Foxn 1, Notch1 and Notch2 genes. Furthermore, the expression of Notch1 and Notch2 proteins was detected by Western blot, and the expression of Notch1 and Notch2 proteins was also significantly up-regulated. These results suggest that the MSX2 gene may affect the occurrence and periodic changes of the skin hair follicles through signal pathways such as WnttNotch and MSX2 / Foxn 1 / hair keratin.
【学位授予单位】：西南民族大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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