重组大鼠肾上腺髓质素基因对骨骼肌缺血再灌注损伤保护作用研究

发布时间：2018-07-09 18:21

本文选题：重组肾上腺髓质素基因 + 骨骼肌　；参考：《湖南师范大学》2010年硕士论文

【摘要】： 目的：采用真核表达载体pVAX1,构建重组大鼠肾上腺髓质素(rrADM),直接注射法导入大鼠腓肠肌,通过构建大鼠骨骼肌缺血再灌注模型,探讨rrADM对骨骼肌缺血再灌注损伤是否具有保护作用。并比较rrADM、依达拉丰、维拉帕米、别嘌呤醇和罂粟碱在骨骼肌缺血再灌注损伤中保护作用的差异。方法： 1.SD大鼠1只,取大鼠肾上腺提取总RNA,用RT-PCR方法逆转录合成ADM基因的cDNA,以cDNA为模板扩增,将产物克隆至PMD-18T载体上,亚克隆至pVAX1真核表达载体上,获得rrADM质粒DNA,采用碱性裂解法大量提取质粒DNA,采用聚乙二醇分级沉淀法纯化质粒DNA。计算所获质粒DNA浓度。 2.SD大鼠18只,体重(203±18.4)g,按体重完全随机分为3组,每组6只。即正常对照组、空载体组、700μg·kgbw-1rrADM组。3组分别注射生理盐水1ml、pVAX1质粒DNA生理盐水溶液1ml、rrADM质粒DNA生理盐水溶液1ml,采用直接肌注法导入大鼠左后肢腓肠肌,质粒DNA注射剂量均为700μg·kgbw-1,每周一次,连续4周。4周后取注射部位肌肉,免疫组织化学法检测各组ADM的表达情况。 3.SD大鼠64只,体重(203±18.4)g,按体重完全随机分为8组,每组8只。即正常对照(C)组、缺血再灌注模型(IR)组、空载体(Ev)组、rrADM组、依达拉丰(Ed)组、维拉帕米(Vp)组、别嘌呤醇(Ap)组、罂粟碱(Pp)组。术前4周,Ev组、rrADM组分别腓肠肌注射pVAX1质粒DNA生理盐水溶液1ml、rrADM质粒DNA生理盐水溶液1ml,其它6组,分别腓肠肌注射等量的生理盐水。每周一次,连续4周,4周后用于骨骼肌缺血再灌注实验,缺血4小时,再灌注3小时。再灌注前15分钟,Ed组、Vp组、Ap组、Pp组分别腹腔注射Ed0.5mg/kg盐水溶液1ml、Vp 2mg/kg盐水溶液1ml、Ap 50mg/kg盐水溶液1ml、Pp5mg/kg盐水溶液1ml；其它4组分别腹腔注射等量的生理盐水。术毕,取大鼠腓肠肌组织并收集血清,检测血清肌酸激酶(CK)、乳酸脱氢酶(LDH)含量,检测肌组织丙二醛(MDA)、总超氧化物歧化酶(T-SOD)含量,并对肌组织进行病理形态学检查。结果： 1.所获基因序列与Genebank中的ADM基因序列比较,两者完全一致。对rrADM XbaⅠ和HindⅢ双酶切鉴定结果为阳性。纯化后的质粒DNA:OD260值为0.912,OD280值为0.498,OD260/OD280比值为1.831。所获质粒DNA浓度为4.560mg/ml。 2.注射部位的腓肠肌组织免疫组织化学检测显示：正常对照组、空载体组腓肠肌组织ADM表达水平低下,结果呈阴性；700μg·kgbw-1 rrADM组腓肠肌组织ADM表达水平高,结果呈阳性。 3.与IR组大鼠血浆CK、LDH及肌组织MDA含量比较,rrADM组、Ed组、Vp组、Ap组、Pp组水平均明显降低(P0.01)；与IR组大鼠肌组织T-SOD含量比较,rrADM组、Ed组、Vp组、Ap组、Pp组水平均明显升高(P0.01)。IR组与Ev组之间各指标差异均无统计学意义(P0.05)。rrADM组、Ed组、Vp组、Ap组、Pp组,5组与Ev组相比各指标差异均有统计学意义(P0.01)。rrADM组、Ed组、Vp组、Ap组、Pp组5组间各指标,除大鼠血浆LDH含量rrADM组与Ap组相比下降了18.20%(P0.05),其余各组间各指标差异均无统计学意义(P0.05)。病理学检查表明,IR组与Ev组注射部位骨骼肌镜下可见大面积肌组织结构紊乱,边界模糊不清,炎性细胞聚集、渗出较多,组织水肿严重。而rrADM组、Ed组、Vp组、Ap组、Pp组5组镜下偶见轻微的肌丝断裂或组织水肿,少量或未见炎性细胞聚集,肌组织结构正常,病理学改变明显好于IR组与Ev组。结论： 1.成功获得大鼠ADM基因,并成功的重组了大鼠肾上腺髓质素,大量提取和纯化的rrADM质粒DNA,符合动物实验的用药要求。 2.rrADM质粒DNA注射入大鼠腓肠肌后,使组织中ADM表达上调。表明质粒构建是成功的,质粒的导入和组织摄取情况较好,可用于下一阶段的实验。 3.rrADM能提高大鼠骨骼肌组织T-SOD活性,降低肌组织MDA含量和血浆CK、LDH含量,对大鼠骨骼肌缺血再灌注损伤有保护作用。依达拉丰、维拉帕米、别嘌呤醇和罂粟碱对大鼠骨骼肌缺血再灌注损伤有保护作用,且rrADM、依达拉丰、维拉帕米、别嘌呤醇和罂粟碱对大鼠骨骼肌缺血再灌注损伤的保护作用没有差异。
[Abstract]:Objective: to construct recombinant rat adrenomedullin (rrADM) by using eukaryotic expression vector pVAX1 and direct injection into the gastrocnemius muscle of rats. By constructing rat skeletal muscle ischemia reperfusion model, the protective effect of rrADM on skeletal muscle ischemia-reperfusion injury was investigated. And rrADM, Yida Laffont, Vera Pammy, allopurinol and poppy were compared. Protective effects of sodium millet on skeletal muscle ischemia-reperfusion injury.
Method:
1 rats of 1.SD were extracted from the rat adrenal gland to extract the total RNA. The cDNA of ADM gene was synthesized by RT-PCR method. The product was amplified by cDNA as a template. The product was cloned to PMD-18T vector. The subcloned to pVAX1 eukaryotic expression vector, rrADM plasmid DNA was obtained. The plasmid DNA was extracted by alkaline lysis method, and the plasmids were purified by polyethylene glycol classification precipitation method. DNA. was used to calculate the concentration of plasmid DNA.
18 2.SD rats, body weight (203 + 18.4) g, were divided into 3 groups according to the total weight of body weight, with 6 rats in each group. The normal control group, the empty body group, the 700 mu g / kgbw-1rrADM group were injected with the physiological saline 1ml, the pVAX1 plasmid DNA physiological saline solution 1ml, the rrADM plasmid DNA physiological saline solution 1ml, and the direct myocutaneous injection method was used to import the gastrocnemius muscle of the left hind limb of the rat and plasmid The injection volume was 700 g kgbw-1, once a week, 4 weeks after.4 weeks, the injection site muscles were taken, and the expression of ADM in each group was detected by immunohistochemistry.
64 3.SD rats, body weight (203 + 18.4) g, were divided into 8 groups according to the total weight of body weight, that is, the normal control (C) group, the ischemic reperfusion model (IR) group, the Ev group, the rrADM group, the group of Yida Laffont (Ed), the Vera Pammy (Vp) group, the allopurinol (Ap) group, the papaverine (Pp) group. The gastrocnemius was injected into the gastrocnemius muscle by injecting the plasmids respectively. Saline solution 1ml, rrADM plasmid DNA physiological saline solution 1ml, the other 6 groups, respectively, the gastrocnemius injection of the same amount of physiological saline. Once a week, 4 weeks, 4 weeks after the skeletal muscle ischemia reperfusion experiment, 4 hours of ischemia, reperfusion for 3 hours, Ed, Vp, Ap, Pp group, Pp group, Ed0.5mg/kg saline solution 1ml, Vp 2mg /kg saline solution 1ml, Ap 50mg/kg saline solution 1ml, Pp5mg/kg saline solution 1ml; the other 4 groups were intraperitoneally injected with equal amount of physiological saline. After the operation, the rat gastrocnemius muscle tissue was collected and serum was collected, serum creatine kinase (CK), lactate dehydrogenase (LDH) content was detected, the content of malondialdehyde (MDA), total superoxide dismutase (T-SOD) content in muscle tissue was detected, and the content of the total superoxide dismutase (T-SOD) was detected and the content of the total superoxide dismutase (T-SOD) was detected. The muscle tissue was examined by pathomorphology.
Result:
The 1. gene sequences were compared with the ADM gene sequences in Genebank. The results of both rrADM Xba I and Hind III double enzyme digestion were positive. The DNA:OD260 value of the purified plasmid was 0.912, the OD280 value was 0.498, the OD260/OD280 ratio was 1.831. and the concentration of DNA was 4.560mg/ ml..
2. the immunohistochemical staining of the gastrocnemius tissue in the injection site showed that the ADM expression level of the gastrocnemius muscle tissue in the normal control group was low and the result was negative, and the ADM expression level of the gastrocnemius muscle in the 700 g / kgbw-1 rrADM group was high and the result was positive.
3. and IR group CK, LDH and muscle tissue MDA content, rrADM group, Ed group, Vp group, Ap group, and Pp group level decreased significantly (P0.01), and IR group muscle tissue T-SOD content. In group Vp, group Ap and group Pp, the difference between the 5 groups was statistically significant (P0.01).RrADM, Ed, Vp, Ap, and Pp groups of the 5 groups. Except for the LDH content of the rat plasma, the rrADM group decreased by 18.20%, and there was no statistical difference between the other groups. Under the skeletal muscle microscope, the tissue structure disorder of large area muscle, the boundary blurred, the inflammatory cell aggregation, the exudation more, the tissue edema were serious. In group rrADM, group Ed, group Vp, group Ap, and group Pp, the 5 groups showed slight muscle filament breakage or tissue edema, little or no inflammatory cell aggregation, normal muscle structure and pathological changes obviously better than that in group Pp. Group IR and group Ev.
Conclusion:
1. the rat ADM gene was successfully obtained, and the rrADM plasmid DNA, which was successfully extracted from rat adrenomedullin and extracted and purified, was in line with the requirements of animal experiments.
After injection of 2.rrADM plasmid DNA into the gastrocnemius muscle, the expression of ADM in the tissue was up-regulated, indicating that the plasmid construction was successful. The plasmid introduction and tissue uptake were better, which could be used in the next stage of the experiment.
3.rrADM can improve the T-SOD activity of skeletal muscle tissue in rats, reduce the MDA content of muscle tissue and the plasma CK, LDH content, and protect the skeletal muscle ischemia reperfusion injury in rats. IDA Laffont, Vera Pammy, allopurinol and papaverine have protective effect on skeletal muscle ischemia reperfusion injury in rats, and rrADM, Yida Laffont, Vera Pammy, allopurine The protective effects of alcohol and papaverine on skeletal muscle ischemia-reperfusion injury in rats were not different.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

【参考文献】