本文選題：缺血再灌注 + 腎上腺髓質(zhì)素�。� 參考：《湖南師范大學(xué)》2009年碩士論文

【摘要】： 目的: 構(gòu)建大鼠腎上腺髓質(zhì)素真核表達(dá)質(zhì)粒pVAX1-ADM,注射入大鼠腓腸肌,通過檢測肢體缺血再灌注后,注射部位腓腸肌組織中的丙二醛(MDA)、超氧化物歧化酶(SOD),血清中的肌酸激酶(CK)、乳酸脫氫酶(LDH)的水平,及組織形態(tài)學(xué)檢查來判斷重組質(zhì)粒對骨骼肌缺血再灌注損傷的保護(hù)作用。 方法: 1.取雄性SD大鼠腎上腺組織進(jìn)行總RNA的提取,RT-PCR方法得到ADM基因,克隆到T載體上,進(jìn)一步亞克隆到pVAX1真核表達(dá)載體上,得到重組質(zhì)粒pVAX1-ADM。 2.雄性SD大鼠32只,體重200±10g,按體重隨機(jī)分為4組,每組8只,即空載體組(pVAX1)、300μg重組質(zhì)粒組、500μg重組質(zhì)粒組和700μg重組質(zhì)粒組。四組腓腸肌注射含重組質(zhì)粒(pVAX1-ADM)的量分別為0μg、300μg、500μg和700μg,每周1次,共4周。取注射部位腓腸肌組織,RT-PCR和免疫組織學(xué)檢查,測定重組質(zhì)粒的轉(zhuǎn)錄水平和蛋白表達(dá)水平,獲取重組質(zhì)粒最適注射劑量。 3.雄性SD大鼠32只,體重200±10g,按體重隨機(jī)分為4組,每組8只,即正常對照組、缺血再灌注(IR)模型組、陽性藥物組(維拉帕米組)、重組質(zhì)粒組。術(shù)前,重組質(zhì)粒組腓腸肌注射重組質(zhì)粒700μg,其他三組注射與質(zhì)粒溶液等量的生理鹽水,每周1次,共4周。4周后進(jìn)行缺血再灌注實(shí)驗(yàn),缺血3h,再灌注2h。術(shù)中,維拉帕米組于再灌注前10分鐘,腹腔注射維拉帕米溶液2mg/kg,其他三組注射等量生理鹽水。術(shù)畢,取腓腸肌組織測定MDA的含量、SOD的活力,取血清測定CK、LDH的含量,及腓腸肌組織進(jìn)行組織學(xué)檢查。 結(jié)果: 1.獲得的大鼠腎上腺髓質(zhì)素基因的測序結(jié)果與GeneBank中序列進(jìn)行比對,完全一致。真核表達(dá)質(zhì)粒經(jīng)PCR、XbaⅠ和HindⅢ雙酶切鑒定顯示,重組質(zhì)粒為陽性。分光光度法對提取的重組質(zhì)粒進(jìn)行測定,結(jié)果為OD260=0.901,OD280=0.487,OD260/280=1.850,質(zhì)粒濃度為4.5mg/ml。 2.定量RT-PCR和免疫組織化學(xué)方法對注射重組質(zhì)粒的大鼠腓腸肌組織進(jìn)行測定,結(jié)果顯示,組織中的表達(dá)量隨重組質(zhì)粒注射量的增加而增加。 3.與IR模型組組相比,重組質(zhì)粒組腓腸肌組織中MDA的含量降低32.21%(p＜0.01),SOD的含量增加了61.69%(p＜0.05);血清CK的含量降低了50.69%(p＜0.05),LDH的含量降低了28.38%(p＜0.05)。 與維拉帕米組相比,重組質(zhì)粒組腓腸肌組織中MDA的含量增加了8.91%(p＞0.05),SOD的含量增加了23.71%(p＞0.05);血清中CK的含量增加了20.61%(p＞0.05),LDH的含量增加了36.17%,有統(tǒng)計(jì)學(xué)意義(p＜0.05)。 骨骼肌組織學(xué)檢查結(jié)果顯示重組質(zhì)粒組和維拉帕米組骨骼肌肌纖維少量斷裂和炎性細(xì)胞浸潤,骨骼肌水腫,間質(zhì)水腫明顯減輕。 結(jié)論: 1.目的基因ADM成功連接到真核表達(dá)載體中,提取的重組質(zhì)粒濃度為4.5mg/ml。 2.重組質(zhì)粒的注入可使大鼠腓腸肌ADM表達(dá)量提高,其中700μg重組質(zhì)粒組為最佳劑量組。 3.大鼠骨骼肌組織ADM表達(dá)量提高,可以減輕缺血再灌注過程中肌肉組織的損傷程度,對骨骼肌缺血再灌注損傷有保護(hù)作用。
[Abstract]:Purpose :


The protective effect of recombinant plasmid on ischemia - reperfusion injury of skeletal muscle was determined by detecting the level of malondialdehyde ( MDA ) , superoxide dismutase ( SOD ) , creatine kinase ( CK ) , lactate dehydrogenase ( LDH ) in muscle tissue of rat adrenal medulla after ischemia - reperfusion .


Method :


1 . The total RNA was extracted from the adrenal tissues of male SD rats . The ADM gene was obtained by RT - PCR and cloned into the T vector , and then subcloned into the eukaryotic expression vector of vector pAX1 to obtain the recombinant plasmid pAX1 - ADM .


2 . Thirty - two male SD rats weighing 200 鹵 10g were randomly divided into four groups according to their weight : 8 rats in each group , i.e . empty vector group , 300 渭g recombinant plasmid group , 500 渭g recombinant plasmid group and 700 渭g recombinant plasmid group .


3 . Thirty - two male SD rats weighing 200 鹵 10g were randomly divided into four groups according to their weight : 8 rats in each group , i.e . normal control group , ischemia / reperfusion ( IR ) model group , positive drug group ( verapamil group ) and recombinant plasmid group .


Results :


1 . The sequencing result of the obtained rat adrenomedullin gene was identical to that in GeneBank . The eukaryotic expression plasmid was identified by PCR , Xba I and HindIII . The recombinant plasmid was determined by spectrophotometry . The results were as follows : OD260 = 0.901 , OD280 = 0.487 , OD260 / 280 = 1.850 , and the plasmid concentration was 4.5mg / ml .


2 . Quantitative RT - PCR and immunohistochemistry method were used to determine the muscle tissue of rats injected with recombinant plasmid . The results showed that the expression of recombinant plasmid increased with the increase of the injection amount of recombinant plasmid .


3 . Compared with IR model group , the content of MDA decreased by 32.21 % ( p & lt ; 0.01 ) , the content of SOD increased by 61.69 % ( p & lt ; 0.05 ) , the content of serum CK decreased by 50.69 % ( p & lt ; 0.05 ) , and the content of LDH decreased by 28.38 % ( p & lt ; 0.05 ) .


Compared with verapamil group , the content of MDA increased 8.91 % ( p & gt ; 0.05 ) , the content of SOD increased 23.71 % ( p & gt ; 0.05 ) , the content of CK in serum increased 20.61 % ( p & gt ; 0.05 ) , and the content of LDH increased by 36.17 % ( p & lt ; 0.05 ) .


The histological examination of skeletal muscle showed that the recombinant plasmid group and verapamil group had a small number of broken and inflammatory cell infiltration , skeletal muscle edema and interstitial edema .


Conclusion :


1 . The target gene ADM was successfully ligated into the eukaryotic expression vector , and the concentration of the extracted recombinant plasmid was 4.5mg / ml .


2 . The injection of recombinant plasmid could increase the expression of ADM in the rat . Of these , 700 渭g recombinant plasmid group was the best dose group .


3 . The expression of ADM in skeletal muscle tissue of rats is increased , and the injury degree of muscle tissue during ischemia - reperfusion can be reduced , and the injury of skeletal muscle ischemia - reperfusion injury is protected .

【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R363

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 楊鋒;劉志坤;何葵;;腎上腺髓質(zhì)素真核表達(dá)載體對皮瓣缺血再灌注損傷的影響[J];中國組織工程研究與臨床康復(fù);2011年28期

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本文編號：1786109