人HT036基因检测、功能研究及蛋白表达

发布时间：2018-06-10 21:03

本文选题：HT036 + α-SMA　；参考：《第三军医大学》2007年硕士论文

【摘要】： 深度烧伤常常导致患者发生增生性瘢痕,临床表现为色红、突出和质硬。往往给患者身心带来严重伤害。增生性瘢痕的发病机制已成为烧伤修复领域研究的重点。我们在前期研究中选用含4096个人类基因的表达谱芯片对5例增生性瘢痕患者及自身正常皮肤进行差异表达基因的筛选,发现其中一个基因P311引人注目,它在烧伤病人早期增生性瘢痕组织中表达显著增高[1-2]。P311蛋白不属于任何一种已知蛋白家族,对其功能少有研究。Pan等发现P311基因可以诱导TGF-β1非依赖的肌成纤维细胞表型样改变[3]。Fujitani等证实腺病毒介导的P311基因上调可以促进大鼠面神经损伤侧轴突再生[4]。为进一步探讨P311基因在增生性瘢痕中的潜在分子机制,我们利用酵母双杂交系统,以融合Gal4 DNA结合域的P311为诱饵蛋白,筛选了成人肝cDNA文库,获得了与P311相互作用的候选蛋白HT036[5]。 HT036基因做为一功能未知新基因,其序列由Xu等在2001年首次提交基因库,编码一胞内蛋白。为探讨HT036基因在增生性瘢痕形成中的可能机制,我们首先检测了该基因在增生性瘢痕和同体正常皮肤中的表达差异,随后观察了HT036基因在成纤维细胞转分化中的作用,最后在原核表达系统中表达该蛋白,为后续研究做准备。研究内容和方法一、HT036基因在增生性瘢痕和同体正常皮肤中表达差异。样本经PBS清洗,提取组织总RNA,通过RT-PCR试剂盒检测HT036mRNA表达量,为保证RT-PCR在线性范围,扩增选取30个循环。采用β-actin为内对照,并用软件Quantity One分析表达量。二、HT036和P311在成纤维细胞转分化中的作用研究。通过PCR获得人HT036编码基因,经EcoRⅠ和SaLⅠ双酶切,克隆至真核表达载体pEGFP-N2,经酶切和测序鉴定正确。人胚肺成纤维细胞株(MRC-5)购自美国ATCC,采用含10%小牛血清、100U/ml青霉素G、100ug/ml链霉素DMEM培养。采用LipofectamineTM2000转染MRC-5细胞,48小时提取细胞总蛋白,通过Western blot检测α-SMA和内参GAPDH,并用软件Quantity One分析表达量。采用同样的方法转染293细胞,收集72小时培养上清,用ELISA检测纤维化相关指标TGF-β1,MMP-2,MMP-9。三、HT036蛋白原核表达、鉴定和诱导动力学研究我们按前述方法构建原核表达载体pET30a(+)-HT036,经酶切和测序鉴定正确后,重组载体转化至大肠杆菌DE3(BL21)pLysS菌株。通过IPTG诱导蛋白表达,并用特异性His抗体鉴定,并且对IPTG最佳诱导时间和诱导浓度进行分析。研究结果一、HT036基因在增生性瘢痕和同体正常皮肤中表达差异。 3例患者的HT036表达在增生性瘢痕组织中都降低,与同体正常皮肤相比,分别降低70%,75%和46%。二、HT036和P311在成纤维细胞转分化中的作用研究。 HT036抑制MRC-5细胞α-SMA表达,而P311诱导其表达。α-SMA相对表达量在空白组、HT036组、P311组和共转染组分别为0.13,0.03,0.18,0.05。HT036降低293细胞TGF-β1,MMP-2,MMP-9的分泌。与P311组相比,分别降低46.4%,33%和29%。三、HT036蛋白原核表达、鉴定和诱导动力学研究成功构建了原核表达载体pET30a(+)-HT036,并在大肠杆菌中成功表达。SDS-PAGE分析显示在29KDa分子量处出现强的蛋白带,达总蛋白的20%表左右。Western blot检测出单一的抗His阳性条带。最佳诱导时间为加入IPTG后4h,各IPTG诱导浓度间表达量无明显差别。研究结论一、增生性瘢痕组织中HT036表达降低。二、HT036能抑制成纤维细胞转分化。三、成功表达了HT036蛋白。
[Abstract]:Deep burn often causes hypertrophic scar in patients. The clinical manifestations are color red, protrusion and hard mass. It often causes serious injury to the patients. The pathogenesis of hypertrophic scar has become the focus of the research in the field of burn repair.
In the previous study, we selected the expression gene chip containing 4096 human genes to select the differentially expressed genes in 5 cases of hypertrophic scar and normal skin. One of the genes P311 was found to be noticeable. It showed that the increase of [1-2].P311 protein in the early hypertrophic scar tissue of the burned patients is not one of any kind. Known protein family, few studies on its function,.Pan, etc. found that P311 gene can induce TGF- beta 1 non dependent myofibroblast phenotype change [3].Fujitani and so on that adenovirus mediated P311 gene regulation can promote the lateral axonal regeneration of the facial nerve injury in rats to further explore the potential division of the P311 gene in hypertrophic scars. In the submechanism, we screened the adult liver cDNA library by using the yeast two hybrid system to fuse the P311 of the Gal4 DNA binding domain as the bait protein, and obtained the candidate protein HT036[5]. that interacted with the P311.
The HT036 gene is a new function unknown gene. Its sequence was first submitted to the gene pool by Xu and so on in 2001 to encode a cell protein. In order to explore the possible mechanism of HT036 gene in the formation of hypertrophic scar, we first detected the difference in the expression of the gene in hypertrophic scar and the normal skin of the androgyny, and then observed the HT036 gene in the fibroblast. Finally, the protein was expressed in prokaryotic expression system to prepare for future research.
Research contents and methods
First, the expression of HT036 gene is different between hypertrophic scar and normal skin.
The samples were cleaned by PBS, the total tissue RNA was extracted, and the expression of HT036mRNA was detected by RT-PCR kit. In order to ensure the linear range of RT-PCR, 30 cycles were selected. Beta -actin was used as internal control, and the expression of Quantity One was analyzed with software Quantity One.
Two, the role of HT036 and P311 in fibroblast transdifferentiation.
The human HT036 encoding gene was obtained by PCR, and the eukaryotic expression vector pEGFP-N2 was cloned through EcoR I and SaL I double enzyme digestion. The human embryo lung fibroblast cell line (MRC-5) was bought from American ATCC, using 10% calf serum, 100U/ml penicillin G, 100ug/ml chain mycin DMEM culture. 48 The total protein of cell was extracted by Western blot and the expression of GAPDH was detected by Western and Quantity One. The same method was used to transfect 293 cells, collect 72 hours culture supernatant, and detect the fibrosis related index TGF- beta 1, MMP-2, MMP-9. with ELISA.
Three, prokaryotic expression, identification and induction kinetics of HT036 protein.
The prokaryotic expression vector pET30a (+) -HT036 was constructed according to the above method. After enzyme digestion and sequencing identification, the recombinant vector was transformed into Escherichia coli DE3 (BL21) pLysS strain. The protein expression was induced by IPTG, and the specific His antibody was identified, and the optimal induction time and induction concentration of IPTG were analyzed.
Research results
First, the expression of HT036 gene is different between hypertrophic scar and normal skin.
The expression of HT036 in 3 patients decreased in hypertrophic scar tissue, and decreased by 70%, 75% and 46%. respectively compared with normal skin.
Two, the role of HT036 and P311 in fibroblast transdifferentiation.
HT036 inhibited the expression of alpha -SMA in MRC-5 cells, while P311 induced its expression. The relative expression of alpha -SMA in the blank group, HT036 group, P311 group and co transfected group decreased the TGF- beta 1, MMP-2, MMP-9 secretion of 0.13,0.03,0.18,0.05.HT036, respectively. Compared with the P311 group, the decrease was 46.4%, 33% and 46.4% respectively.
Three, prokaryotic expression, identification and induction kinetics of HT036 protein.
The prokaryotic expression vector pET30a (+) -HT036 was successfully constructed, and the successful expression of.SDS-PAGE in Escherichia coli showed a strong protein band at the molecular weight of 29KDa, and the 20% table.Western blot of the total protein detected a single anti His positive band. The best induction time was 4h after adding IPTG, and there was no obvious expression between the IPTG induced concentration of IPTG. Difference.
research conclusion
1. The expression of HT036 in hypertrophic scar tissue is reduced.
Two, HT036 can inhibit the transdifferentiation of fibroblasts.
Three, HT036 protein was successfully expressed.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R644;R346

【参考文献】