肾上腺髓质素对骨骼肌缺血再灌注损伤的保护作用研究

发布时间：2018-04-22 07:05

本文选题：缺血再灌注 + 肾上腺髓质素　；参考：《湖南师范大学》2009年硕士论文

【摘要】： 目的: 构建大鼠肾上腺髓质素真核表达质粒pVAX1-ADM,注射入大鼠腓肠肌,通过检测肢体缺血再灌注后,注射部位腓肠肌组织中的丙二醛(MDA)、超氧化物歧化酶(SOD),血清中的肌酸激酶(CK)、乳酸脱氢酶(LDH)的水平,及组织形态学检查来判断重组质粒对骨骼肌缺血再灌注损伤的保护作用。方法: 1.取雄性SD大鼠肾上腺组织进行总RNA的提取,RT-PCR方法得到ADM基因,克隆到T载体上,进一步亚克隆到pVAX1真核表达载体上,得到重组质粒pVAX1-ADM。 2.雄性SD大鼠32只,体重200±10g,按体重随机分为4组,每组8只,即空载体组(pVAX1)、300μg重组质粒组、500μg重组质粒组和700μg重组质粒组。四组腓肠肌注射含重组质粒(pVAX1-ADM)的量分别为0μg、300μg、500μg和700μg,每周1次,共4周。取注射部位腓肠肌组织,RT-PCR和免疫组织学检查,测定重组质粒的转录水平和蛋白表达水平,获取重组质粒最适注射剂量。 3.雄性SD大鼠32只,体重200±10g,按体重随机分为4组,每组8只,即正常对照组、缺血再灌注(IR)模型组、阳性药物组(维拉帕米组)、重组质粒组。术前,重组质粒组腓肠肌注射重组质粒700μg,其他三组注射与质粒溶液等量的生理盐水,每周1次,共4周。4周后进行缺血再灌注实验,缺血3h,再灌注2h。术中,维拉帕米组于再灌注前10分钟,腹腔注射维拉帕米溶液2mg/kg,其他三组注射等量生理盐水。术毕,取腓肠肌组织测定MDA的含量、SOD的活力,取血清测定CK、LDH的含量,及腓肠肌组织进行组织学检查。结果: 1.获得的大鼠肾上腺髓质素基因的测序结果与GeneBank中序列进行比对,完全一致。真核表达质粒经PCR、XbaⅠ和HindⅢ双酶切鉴定显示,重组质粒为阳性。分光光度法对提取的重组质粒进行测定,结果为OD260=0.901,OD280=0.487,OD260/280=1.850,质粒浓度为4.5mg/ml。 2.定量RT-PCR和免疫组织化学方法对注射重组质粒的大鼠腓肠肌组织进行测定,结果显示,组织中的表达量随重组质粒注射量的增加而增加。 3.与IR模型组组相比,重组质粒组腓肠肌组织中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)。与维拉帕米组相比,重组质粒组腓肠肌组织中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%,有统计学意义(p＜0.05)。骨骼肌组织学检查结果显示重组质粒组和维拉帕米组骨骼肌肌纤维少量断裂和炎性细胞浸润,骨骼肌水肿,间质水肿明显减轻。结论: 1.目的基因ADM成功连接到真核表达载体中,提取的重组质粒浓度为4.5mg/ml。 2.重组质粒的注入可使大鼠腓肠肌ADM表达量提高,其中700μg重组质粒组为最佳剂量组。 3.大鼠骨骼肌组织ADM表达量提高,可以减轻缺血再灌注过程中肌肉组织的损伤程度,对骨骼肌缺血再灌注损伤有保护作用。
[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 卤 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 卤 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 .

【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

【引证文献】