本文選題：重組腎上腺髓質(zhì)素基因 + 骨骼肌�。� 參考：《湖南師范大學(xué)》2010年碩士論文

【摘要】： 目的：采用真核表達(dá)載體pVAX1,構(gòu)建重組大鼠腎上腺髓質(zhì)素(rrADM),直接注射法導(dǎo)入大鼠腓腸肌,通過構(gòu)建大鼠骨骼肌缺血再灌注模型,探討rrADM對(duì)骨骼肌缺血再灌注損傷是否具有保護(hù)作用。并比較rrADM、依達(dá)拉豐、維拉帕米、別嘌呤醇和罌粟堿在骨骼肌缺血再灌注損傷中保護(hù)作用的差異。 方法： 1.SD大鼠1只,取大鼠腎上腺提取總RNA,用RT-PCR方法逆轉(zhuǎn)錄合成ADM基因的cDNA,以cDNA為模板擴(kuò)增,將產(chǎn)物克隆至PMD-18T載體上,亞克隆至pVAX1真核表達(dá)載體上,獲得rrADM質(zhì)粒DNA,采用堿性裂解法大量提取質(zhì)粒DNA,采用聚乙二醇分級(jí)沉淀法純化質(zhì)粒DNA。計(jì)算所獲質(zhì)粒DNA濃度。 2.SD大鼠18只,體重(203±18.4)g,按體重完全隨機(jī)分為3組,每組6只。即正常對(duì)照組、空載體組、700μg·kgbw-1rrADM組。3組分別注射生理鹽水1ml、pVAX1質(zhì)粒DNA生理鹽水溶液1ml、rrADM質(zhì)粒DNA生理鹽水溶液1ml,采用直接肌注法導(dǎo)入大鼠左后肢腓腸肌,質(zhì)粒DNA注射劑量均為700μg·kgbw-1,每周一次,連續(xù)4周。4周后取注射部位肌肉,免疫組織化學(xué)法檢測各組ADM的表達(dá)情況。 3.SD大鼠64只,體重(203±18.4)g,按體重完全隨機(jī)分為8組,每組8只。即正常對(duì)照(C)組、缺血再灌注模型(IR)組、空載體(Ev)組、rrADM組、依達(dá)拉豐(Ed)組、維拉帕米(Vp)組、別嘌呤醇(Ap)組、罌粟堿(Pp)組。術(shù)前4周,Ev組、rrADM組分別腓腸肌注射pVAX1質(zhì)粒DNA生理鹽水溶液1ml、rrADM質(zhì)粒DNA生理鹽水溶液1ml,其它6組,分別腓腸肌注射等量的生理鹽水。每周一次,連續(xù)4周,4周后用于骨骼肌缺血再灌注實(shí)驗(yàn),缺血4小時(shí),再灌注3小時(shí)。再灌注前15分鐘,Ed組、Vp組、Ap組、Pp組分別腹腔注射Ed0.5mg/kg鹽水溶液1ml、Vp 2mg/kg鹽水溶液1ml、Ap 50mg/kg鹽水溶液1ml、Pp5mg/kg鹽水溶液1ml；其它4組分別腹腔注射等量的生理鹽水。術(shù)畢,取大鼠腓腸肌組織并收集血清,檢測血清肌酸激酶(CK)、乳酸脫氫酶(LDH)含量,檢測肌組織丙二醛(MDA)、總超氧化物歧化酶(T-SOD)含量,并對(duì)肌組織進(jìn)行病理形態(tài)學(xué)檢查。 結(jié)果： 1.所獲基因序列與Genebank中的ADM基因序列比較,兩者完全一致。對(duì)rrADM XbaⅠ和HindⅢ雙酶切鑒定結(jié)果為陽性。純化后的質(zhì)粒DNA:OD260值為0.912,OD280值為0.498,OD260/OD280比值為1.831。所獲質(zhì)粒DNA濃度為4.560mg/ml。 2.注射部位的腓腸肌組織免疫組織化學(xué)檢測顯示：正常對(duì)照組、空載體組腓腸肌組織ADM表達(dá)水平低下,結(jié)果呈陰性；700μg·kgbw-1 rrADM組腓腸肌組織ADM表達(dá)水平高,結(jié)果呈陽性。 3.與IR組大鼠血漿CK、LDH及肌組織MDA含量比較,rrADM組、Ed組、Vp組、Ap組、Pp組水平均明顯降低(P0.01)；與IR組大鼠肌組織T-SOD含量比較,rrADM組、Ed組、Vp組、Ap組、Pp組水平均明顯升高(P0.01)。IR組與Ev組之間各指標(biāo)差異均無統(tǒng)計(jì)學(xué)意義(P0.05)。rrADM組、Ed組、Vp組、Ap組、Pp組,5組與Ev組相比各指標(biāo)差異均有統(tǒng)計(jì)學(xué)意義(P0.01)。rrADM組、Ed組、Vp組、Ap組、Pp組5組間各指標(biāo),除大鼠血漿LDH含量rrADM組與Ap組相比下降了18.20%(P0.05),其余各組間各指標(biāo)差異均無統(tǒng)計(jì)學(xué)意義(P0.05)。病理學(xué)檢查表明,IR組與Ev組注射部位骨骼肌鏡下可見大面積肌組織結(jié)構(gòu)紊亂,邊界模糊不清,炎性細(xì)胞聚集、滲出較多,組織水腫嚴(yán)重。而rrADM組、Ed組、Vp組、Ap組、Pp組5組鏡下偶見輕微的肌絲斷裂或組織水腫,少量或未見炎性細(xì)胞聚集,肌組織結(jié)構(gòu)正常,病理學(xué)改變明顯好于IR組與Ev組。 結(jié)論： 1.成功獲得大鼠ADM基因,并成功的重組了大鼠腎上腺髓質(zhì)素,大量提取和純化的rrADM質(zhì)粒DNA,符合動(dòng)物實(shí)驗(yàn)的用藥要求。 2.rrADM質(zhì)粒DNA注射入大鼠腓腸肌后,使組織中ADM表達(dá)上調(diào)。表明質(zhì)粒構(gòu)建是成功的,質(zhì)粒的導(dǎo)入和組織攝取情況較好,可用于下一階段的實(shí)驗(yàn)。 3.rrADM能提高大鼠骨骼肌組織T-SOD活性,降低肌組織MDA含量和血漿CK、LDH含量,對(duì)大鼠骨骼肌缺血再灌注損傷有保護(hù)作用。依達(dá)拉豐、維拉帕米、別嘌呤醇和罌粟堿對(duì)大鼠骨骼肌缺血再灌注損傷有保護(hù)作用,且rrADM、依達(dá)拉豐、維拉帕米、別嘌呤醇和罌粟堿對(duì)大鼠骨骼肌缺血再灌注損傷的保護(hù)作用沒有差異。
[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.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363

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