本文選題：豬 + Hnrnpk��； 參考：《信陽師范學院》2015年碩士論文

【摘要】：核不均一核糖核蛋白k (heterogeneous nuclear ribonucleoprotein k, Hnrnpk)是一個多功能蛋白,參與DNA修復、轉錄和翻譯調控、RNA的剪切加工等生物學過程,能通過多種途徑對成肌細胞增殖與分化起重要調控作用,但其作用機制及信號通路尚不清楚。c-Src作為非受體蛋白酪氨酸激酶家族的一員,可以通過Rat/MEK/ERK和p38MAPK信號通路對骨骼肌肌生成過程起著重要的調節(jié)作用,但其在成肌細胞中的表達調控機制卻不清楚。以往研究表明Hnrnpk與c-Src存在多層次的互作并發(fā)揮重要生物學功能。本研究從豬Hnrnpk和c-Src基因的克隆及表達著手,以小鼠C2C12成肌細胞為模型,結合qRT-PCR、免疫印跡、細胞轉染、亞細胞定位、免疫熒光、過表達、RNA干涉、Co-IP、GST pull-down、酵母雙雜交、RIP等方法,深入探討Hnrnpk與c-Src的互作及其在C2C12成肌細胞增殖分化中的作用,這將為闡明骨骼肌發(fā)育和再生的復雜網絡調節(jié)機制奠定基礎,而且還可望為畜牧業(yè)提高動物的產肉量、肉品質及醫(yī)學上控制肌肉再生提供理論依據。本研究得到以下結果： 1. Hnrnpk基因的克隆及時空表達分析 克隆得到2056bp豬Hnrnpk基因的cDNA序列,包含1395bp的開放閱讀框,編碼464個氨基酸；Hnrnpk基因在梅山豬不同發(fā)育時期的背最長肌中均差異表達,胚胎期表達量最高,并且隨著生長發(fā)育呈下調表達。Hnrnpk基因在120日齡的大白豬肌肉和脂肪組織中相對高表達,腎、肺中次之,心、大腦、肝、脾和卵巢中的表達相對較低。而就四種不同類型肌肉組織而言,半腱肌和背最長肌中表達量最高,咬肌次之,股二頭肌中最低；Hnrnpk基因在C2C12細胞分化過程中表達下調,其直接調控的c-myc、c-Src、p21等基因表達趨勢與之相似。 2. c-Src基因的克隆及時空表達分析 克隆得到4126bp豬c-Src基因的cDNA序列,包含1629bp的開放閱讀框,編碼542個氨基酸；c-Src基因在大白豬和梅山豬肌肉中的表達模式相似,均在胚胎65日齡高表達,出生后3天表達量極顯著減少,而在21日齡又表達上調,此后隨著發(fā)育逐漸下調；兩品種相比,胚胎65日齡,c-Src基因在梅山豬中的表達量顯著高于大白豬(P0.05),而出生后3天、21天及6月齡大白豬中的表達量均極顯著高于梅山豬(P0.01); c-Src基因在豬不同組織中廣泛表達,在PT期,大腦中c-Src基因的表達量最高,肝臟中表達量最低。在3天,肝臟中c-Src基因的表達量最高,骨骼肌中表達量最低。在4月齡,肺中c-Src基因的表達量最高,骨骼肌中表達量最低；在C2C12細胞誘導分化過程中,c-Src基因隨著誘導分化表達先上調后逐漸下調。 3. Hnrnpk對C2C12細胞增殖分化的影響 超表達Hnrnpk基因后,成肌轉錄因子MyoG, Myf6的表達量下調,而MyoD卻上調,成熟骨骼肌相關分子標記物MLC2、MCK、TAK1下調,c-Src表達量上調,p21下調；siRNA干涉Hnrnpk后,成肌轉錄因子MyoG、Myf6和MyoD以及成熟骨骼肌相關分子標記物MLC2、 MCK表達量上調,c-Src表達量下調,p21上調。表明Hnrnpk可能通過上調c-Src和下調成肌轉錄因子MRFs家族的表達從而促進C2C12細胞增殖或抑制分化。 4. c-Src對C2C12細胞增殖分化的影響 超表達c-Src基因后,成肌轉錄因子MyoG、 Myf6、 MyoD的表達量下調,而且成熟骨骼肌相關分子標記物MLC2、 MCK、 TAK1表達量下調,p21表達量下調；PP2抑制劑處理C2C12細胞后,MyoG和MyoD分化標記基因、p21、凋亡基因Caspase3表達量均上調,p53和c-myc基因下調表達,而c-Src表達沒有變化。表明c-Src對C2C12細胞增殖分化發(fā)揮作用可能是通過直接或間接調節(jié)成肌轉錄因子MRFs家族和p21的表達來實現的。 5.Hnrnpk與c-Src的互作分析 通過ELM在線分析發(fā)現與Hnrnpk相互作用的眾多元件包括了c-Src蛋白的SH3結構域,進而采用Co-IP初步證實內源性的Hnrnpk和c-Src在分化的C2C12細胞中相互作用；此外,又用酵母雙雜交和GST pull-down方法對Hnrnpk與c-Src的互作進一步分析,酵母雙雜交結果表明Hnrnpk與c-Src的全長以及單獨的SH3結構域互作,與其他缺失體不互作,與c-Src全長的結合活性要強于單獨的SH3結構域；GST pull-down結果表明Hnrnpk能夠在體外與c-Src的全長及所有缺失體相互作用。RIP實驗結果表明分化的C2C12細胞中c-Src mRNA與Hnrnpk的結合能力要極顯著高于陰性對照IgG抗體(P0.01),而增殖的C2C12細胞中c-Src mRNA和Hnrnpk的結合能力與陰性對照IgG抗體相比差異不顯著(P0.05),表明Hnrnpk只在分化的C2C12細胞中與c-Src mRNA互作,這與鼠Hnrnpk在C2C12細胞中的亞細胞分布是一致的。
[Abstract]:The effects of Hnrnpk on the proliferation and differentiation of myoblasts were investigated by means of rat / MEK / ERK and p38MAPK signaling pathways .

Cloning and Expression Analysis of 1 . Hnrnpk Gene

The cDNA sequence of the 2056bp porcine Hnrnpk gene was cloned , and the cDNA sequence was 1395bp open reading frame , and 464 amino acids were encoded .
The expression of Hnrnpk gene in muscle and adipose tissue of 120 - day - old pig muscle and adipose tissue was the highest , and the expression of Hnrnpk gene in muscle and adipose tissue was relatively low .
The expression of Hnrnpk gene was down regulated in C2C12 cell differentiation , and its direct regulation of c - myc , c - myc , p21 and other genes were similar .

Cloning and Expression Analysis of the 2 . c - src Gene

the cDNA sequence of the 4126 bp porcine c - src gene was cloned , and a 1629 bp open reading frame was obtained , and 542 amino acids were encoded ;
the expression pattern of c - src gene in muscle of pig and meishan pig was similar , which was expressed at the age of 65 days of embryo , and the expression level was significantly decreased at the third day after birth , while the expression rate was up - regulated at the age of 21 , and then gradually decreased with development .
Compared with the two breeds , the expression of c - src gene was significantly higher in the pigs than that in Mishan ( P0.05 ) , while the expression of c - src gene was the highest in the three days after birth . The c - src gene expression was the highest in the liver and the lowest in skeletal muscle .
During the induction and differentiation of C2C12 cells , the c - src gene was down - regulated with the increase of induction differentiation .

3 . Effect of Hnrnpk on proliferation and differentiation of C2C12 cells

After overexpression of Hnrnpk gene , myogenic transcription factor MyoG , Myf6 expression was down - regulated , while MyoD was up - regulated , and the mature skeletal muscle - related molecular markers MLC2 , MCK , TAK1 were down - regulated , and the expression of c - src was up - regulated and p21 down - regulated ;
After interfering with Hnrnpk by siRNA , myogenic transcription factors , MyoG , Myf6 and MyoD , and mature skeletal muscle - related molecular markers MLC2 , MCK expression were up - regulated , and the expression of c - src was down - regulated , and p21 was up - regulated . It was suggested that Hnrnpk might promote the proliferation of C2C12 cells or inhibit differentiation by up - regulating the expression of c - src and down - regulating the MRFs family .

4 . Effects of c - src on the proliferation and differentiation of C2C12 cells

The expression of myogenic transcription factor MyoG , Myf6 , MyoD was down regulated after overexpression of c - src gene , and the expression of related molecular markers MLC2 , MCK , TAK1 of mature skeletal muscle was down regulated , and the expression level of p21 was down regulated .
After treatment of C2C12 cells , the expression of MyoG and MyoD differentiation marker gene , p21 and apoptosis gene Caspase3 were up - regulated , and the expression of p53 and c - myc was downregulated .

5 . Interaction between Hnrnpk and c - src

Many elements interacting with Hnrnpk were found by ELM on - line analysis , including the SH3 domain of c - src protein , and then Co - IP was used to demonstrate the interaction of endogenous Hnrnpk and c - src in differentiated C2C12 cells .
In addition , the interaction of Hnrnpk and c - src was further analyzed by yeast two - hybrid and GST pull - down methods . The results showed that Hnrnpk was the full - length of c - src and that of the isolated SH3 domains , which did not interact with other deletions , and the binding activity of the full length of c - src was stronger than that of the single SH3 domain ;
The results of RIP showed that Hnrnpk was significantly higher than that of negative control IgG antibody ( P0.01 ) , and the binding ability of c - src mRNA and Hnrnpk in C2C12 cells was significantly higher than that of negative control IgG antibody ( P0.05 ) .

【學位授予單位】：信陽師范學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S828

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本文編號：1739028