本文選題：HT036 + α-SMA��； 參考：《第三軍醫(yī)大學(xué)》2007年碩士論文

【摘要】： 深度燒傷常常導(dǎo)致患者發(fā)生增生性瘢痕,臨床表現(xiàn)為色紅、突出和質(zhì)硬。往往給患者身心帶來(lái)嚴(yán)重傷害。增生性瘢痕的發(fā)病機(jī)制已成為燒傷修復(fù)領(lǐng)域研究的重點(diǎn)。 我們?cè)谇捌谘芯恐羞x用含4096個(gè)人類基因的表達(dá)譜芯片對(duì)5例增生性瘢痕患者及自身正常皮膚進(jìn)行差異表達(dá)基因的篩選,發(fā)現(xiàn)其中一個(gè)基因P311引人注目,它在燒傷病人早期增生性瘢痕組織中表達(dá)顯著增高[1-2]。P311蛋白不屬于任何一種已知蛋白家族,對(duì)其功能少有研究。Pan等發(fā)現(xiàn)P311基因可以誘導(dǎo)TGF-β1非依賴的肌成纖維細(xì)胞表型樣改變[3]。Fujitani等證實(shí)腺病毒介導(dǎo)的P311基因上調(diào)可以促進(jìn)大鼠面神經(jīng)損傷側(cè)軸突再生[4]。為進(jìn)一步探討P311基因在增生性瘢痕中的潛在分子機(jī)制,我們利用酵母雙雜交系統(tǒng),以融合Gal4 DNA結(jié)合域的P311為誘餌蛋白,篩選了成人肝cDNA文庫(kù),獲得了與P311相互作用的候選蛋白HT036[5]。 HT036基因做為一功能未知新基因,其序列由Xu等在2001年首次提交基因庫(kù),編碼一胞內(nèi)蛋白。為探討HT036基因在增生性瘢痕形成中的可能機(jī)制,我們首先檢測(cè)了該基因在增生性瘢痕和同體正常皮膚中的表達(dá)差異,隨后觀察了HT036基因在成纖維細(xì)胞轉(zhuǎn)分化中的作用,最后在原核表達(dá)系統(tǒng)中表達(dá)該蛋白,為后續(xù)研究做準(zhǔn)備。 研究?jī)?nèi)容和方法 一、HT036基因在增生性瘢痕和同體正常皮膚中表達(dá)差異。 樣本經(jīng)PBS清洗,提取組織總RNA,通過(guò)RT-PCR試劑盒檢測(cè)HT036mRNA表達(dá)量,為保證RT-PCR在線性范圍,擴(kuò)增選取30個(gè)循環(huán)。采用β-actin為內(nèi)對(duì)照,并用軟件Quantity One分析表達(dá)量。 二、HT036和P311在成纖維細(xì)胞轉(zhuǎn)分化中的作用研究。 通過(guò)PCR獲得人HT036編碼基因,經(jīng)EcoRⅠ和SaLⅠ雙酶切,克隆至真核表達(dá)載體pEGFP-N2,經(jīng)酶切和測(cè)序鑒定正確。人胚肺成纖維細(xì)胞株(MRC-5)購(gòu)自美國(guó)ATCC,采用含10%小牛血清、100U/ml青霉素G、100ug/ml鏈霉素DMEM培養(yǎng)。采用LipofectamineTM2000轉(zhuǎn)染MRC-5細(xì)胞,48小時(shí)提取細(xì)胞總蛋白,通過(guò)Western blot檢測(cè)α-SMA和內(nèi)參GAPDH,并用軟件Quantity One分析表達(dá)量。采用同樣的方法轉(zhuǎn)染293細(xì)胞,收集72小時(shí)培養(yǎng)上清,用ELISA檢測(cè)纖維化相關(guān)指標(biāo)TGF-β1,MMP-2,MMP-9。 三、HT036蛋白原核表達(dá)、鑒定和誘導(dǎo)動(dòng)力學(xué)研究 我們按前述方法構(gòu)建原核表達(dá)載體pET30a(+)-HT036,經(jīng)酶切和測(cè)序鑒定正確后,重組載體轉(zhuǎn)化至大腸桿菌DE3(BL21)pLysS菌株。通過(guò)IPTG誘導(dǎo)蛋白表達(dá),并用特異性His抗體鑒定,并且對(duì)IPTG最佳誘導(dǎo)時(shí)間和誘導(dǎo)濃度進(jìn)行分析。 研究結(jié)果 一、HT036基因在增生性瘢痕和同體正常皮膚中表達(dá)差異。 3例患者的HT036表達(dá)在增生性瘢痕組織中都降低,與同體正常皮膚相比,分別降低70%,75%和46%。 二、HT036和P311在成纖維細(xì)胞轉(zhuǎn)分化中的作用研究。 HT036抑制MRC-5細(xì)胞α-SMA表達(dá),而P311誘導(dǎo)其表達(dá)。α-SMA相對(duì)表達(dá)量在空白組、HT036組、P311組和共轉(zhuǎn)染組分別為0.13,0.03,0.18,0.05。HT036降低293細(xì)胞TGF-β1,MMP-2,MMP-9的分泌。與P311組相比,分別降低46.4%,33%和29%。 三、HT036蛋白原核表達(dá)、鑒定和誘導(dǎo)動(dòng)力學(xué)研究 成功構(gòu)建了原核表達(dá)載體pET30a(+)-HT036,并在大腸桿菌中成功表達(dá)。SDS-PAGE分析顯示在29KDa分子量處出現(xiàn)強(qiáng)的蛋白帶,達(dá)總蛋白的20%表左右。Western blot檢測(cè)出單一的抗His陽(yáng)性條帶。最佳誘導(dǎo)時(shí)間為加入IPTG后4h,各IPTG誘導(dǎo)濃度間表達(dá)量無(wú)明顯差別。 研究結(jié)論 一、增生性瘢痕組織中HT036表達(dá)降低。 二、HT036能抑制成纖維細(xì)胞轉(zhuǎn)分化。 三、成功表達(dá)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R644;R346

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本文編號(hào)：2004641