本文關(guān)鍵詞： 法醫(yī)病理學(xué) 差異顯示技術(shù) 錨定引物 隨機(jī)引物　出處：《山西醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:運(yùn)用差異顯示技術(shù)結(jié)合銀染初步篩選大鼠皮膚挫傷后皮膚、肌肉的基因表達(dá)差異,旨在尋找一種或幾種與損傷相關(guān)的敏感基因,為法醫(yī)學(xué)病理學(xué)實(shí)踐中推斷時(shí)間提供科學(xué)、有效的參考依據(jù)。 方法:實(shí)驗(yàn)分為正常對(duì)照組(n=6)與實(shí)驗(yàn)損傷組(n=6)。實(shí)驗(yàn)損傷組:用戊巴比妥鈉腹腔注射麻醉大鼠,常規(guī)手術(shù)消毒后,用250克重力錘在150cm高度自由落下,造成右后肢股四頭肌處皮膚、肌肉挫傷,于挫傷后4h將大鼠脫頸處死,于挫傷處取皮膚肌肉組織。正常對(duì)照組:將大鼠脫頸處死,取相應(yīng)皮膚肌肉組織。提取對(duì)照組與損傷組的總RNA,經(jīng)質(zhì)量評(píng)價(jià)后,無降解的RNA進(jìn)行反轉(zhuǎn)錄-聚合酶鏈?zhǔn)椒磻?yīng)。以4種錨定引物和3種隨機(jī)引物,共3×4=12種組合的非特異性引物進(jìn)行擴(kuò)增,對(duì)擴(kuò)增后的產(chǎn)物進(jìn)行1.5%的瓊脂糖凝膠電泳,初步篩選差異條帶。待確定正常組與損傷組有差異條帶之后,將擴(kuò)增產(chǎn)物進(jìn)行8%的聚丙烯酰胺凝膠電泳,電壓400V、時(shí)間3h。對(duì)聚丙烯酰胺凝膠進(jìn)行固定、漂洗、染色、顯色、終止、漂洗。用無菌刀片切下差異條帶,進(jìn)行2次擴(kuò)增。經(jīng)過TIANquick Maxi Purification Kit純化之后的擴(kuò)增產(chǎn)物與pMD18-T Vector連接并轉(zhuǎn)染到JM109感受態(tài)細(xì)胞中,之后在SOC培養(yǎng)基、LB瓊脂平板培養(yǎng)基(Amp+)中進(jìn)行培養(yǎng)。挑選白色單菌落接種到含有Amp+LB液體培養(yǎng)基過夜培養(yǎng),送菌液進(jìn)行測(cè)序,測(cè)序結(jié)果提交GenBank進(jìn)行對(duì)比。 結(jié)果:(1)實(shí)驗(yàn)組與對(duì)照組皮膚肌肉總RNA的質(zhì)量符合實(shí)驗(yàn)要求,其A260/A280值可達(dá)1.913。(2)PCR擴(kuò)增產(chǎn)物進(jìn)行1.5%的瓊脂糖凝膠電泳后,實(shí)驗(yàn)組皮膚與肌肉與對(duì)照組比較出現(xiàn)差異條帶。(3)PCR擴(kuò)增產(chǎn)物進(jìn)行8%的聚丙烯酰胺凝膠電泳,銀染后實(shí)驗(yàn)組皮膚與肌肉出現(xiàn)差異條帶,差異條帶大約在150-600bp之間,總計(jì)22條差異條帶。(4)2次擴(kuò)增產(chǎn)物與pMD18-T Vector連接并轉(zhuǎn)染到JM109感受態(tài)細(xì)胞中,LB瓊脂平板培養(yǎng)基(Amp+)中進(jìn)行培養(yǎng)。出現(xiàn)白色菌落與藍(lán)色菌落。(5)菌液進(jìn)行測(cè)序,測(cè)序結(jié)果提交GenBank進(jìn)行對(duì)比,發(fā)現(xiàn)5個(gè)差異表達(dá)片段與GenBank的大鼠肌鈣蛋白、核酸結(jié)合蛋白、細(xì)胞色素c氧化酶、Rattus norvegicus myxovirus (influenza virus) resistance 2、Mouse DNA sequence from clone RP23-403G13有同源性,其余的差異條帶沒有同源性。 結(jié)論:大鼠皮膚損傷過程中有多種基因參與表達(dá)。肌鈣蛋白與細(xì)胞色素c氧化酶在損傷后,它們的mRNA表達(dá)均增加,但不清楚對(duì)損傷過程是一種保護(hù)作用或是損害作用,以及與損傷時(shí)間是否存在一定數(shù)量關(guān)系還有待進(jìn)一步研究。
[Abstract]:Objective: to screen the difference of gene expression in the muscle of rat skin contusion by differential display technique combined with silver staining in order to search for one or more sensitive genes related to injury. To provide a scientific and effective reference for the practice of forensic pathology. Methods: the experiment was divided into normal control group and experimental injury group. Experimental injury group: rats were anesthetized with pentobarbital sodium intraperitoneally. After routine operation, 250 g gravity hammer was used to drop freely at 150 cm height. The skin and muscle contusion of the quadriceps femoris of the right hind limb were caused, the rats were killed at 4 hours after contusion, and the skin muscle tissue was taken from the contusion. The skin muscle tissue was taken. The total RNAs of the control group and the injured group were extracted. After the quality evaluation, the non-degradable RNA was reverse transcription-polymerase chain reaction. Four kinds of anchoring primers and three kinds of random primers were used. A total of 12 combinations of nonspecific primers were amplified, the amplified products were amplified by 1.5% agarose gel electrophoresis, and the differential bands were preliminarily screened. Polyacrylamide gel electrophoresis of 8%, voltage 400V, time 3 hours. The polyacrylamide gel was fixed, rinsed, dyed, colored, stopped, rinsed. After purification by TIANquick Maxi Purification Kit, the product was ligated with pMD18-T Vector and transfected into JM109 receptive cells. After that, the culture was carried out in the SOC medium, LB Agar plate medium, and the white colony was inoculated into the liquid medium containing Amp LB for overnight culture, and the bacterial solution was sequenced. The sequencing results were submitted to GenBank for comparison. Results 1) the quality of total RNA in skin and muscle of the experimental group and the control group met the requirements of the experiment, and the A260 / A280 value of the A260 / A280 value could reach 1.913.02 / 2 PCR amplification product after 1.5% agarose gel electrophoresis. Compared with the control group, the skin and muscle of the experimental group showed different bands of polyacrylamide gel electrophoresis of 8%, and the difference bands of the skin and muscle of the experimental group were about 150-600 BP after silver staining. A total of 22 differentially amplified products were linked to pMD18-T Vector and transfected into the LB-LB Agar plate medium for culture. The white colony and blue colony were sequenced. The sequencing results were submitted to GenBank for comparison. It was found that the five differentially expressed fragments were homologous to GenBank rat troponin, nucleic acid binding protein and cytochrome c oxidase (Rattus norvegicus myxovirus influenza virus) resistance 2 mouse DNA sequence from clone RP23-403G13, but the other differentially expressed bands had no homology. Conclusion: there are many genes involved in the expression of troponin and cytochrome c oxidase in the process of skin injury in rats. The expression of mRNA is increased after injury, but it is not clear whether the expression of troponin or cytochrome c oxidase is a protective or damaging effect on the process of injury. And whether there is a certain amount of relationship with the time of injury remains to be further studied.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：D919

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