Calpain-2对阿霉素心肌毒性干预作用的研究及ragB-mGITRL DNA疫苗的研制
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摘要
心肌损伤可由病毒、细菌等感染,导致的心肌炎或化学药物的毒性作用等多种因素所介导,严重影响人类的健康。本论文涉及对阿霉素和牙龈卟啉菌两类心脏毒性诱因的十预性研究,旨在趋利避害,从源头上预防两类因素对心脏造成的病理性损伤。
阿霉素,是一种作用于DNA的葸环类抗生素,广泛应用于临床抗肿瘤的药物。阿霉素的应用带来多方面的副作用,包括恶心、呕吐和心律不齐等。其中当累积剂量达至(?)55mg/m2(?)(?),能够引起充血性心衰、扩张性心肌病甚至死亡,从而限制了其临床应用。迄今为止,阿霉素的急性和累积性心脏毒性的机制尚未完全清楚,更无预防和治疗阿霉素诱导心肌病行之有效的措施。
Calpain,即钙蛋白酶,属于钙离子依赖的半胱氨酸蛋白酶家族,广泛表达于哺乳动物以及多种其他生物。人类已鉴定的15种亚型的分布、结构和功能均有明显差异。其中“-calpain(calpain-1)和m-calpain(calpain-2)为普遍存在且研究深入。Calpain的激活参与机体多系统疾病,如糖尿病、阿尔滋海默氏病、动脉粥样硬化以及胃癌等。前期研究发现,calpastatin的过表达,加剧了阿霉素引起的急性心肌毒性,表明calpain与其密切相关,calpain-1或calpain-2是否参与了保护作用?其分子机制如何?尚待研究证实。
心血管系统的另一重要疾病动脉粥样硬化,心肌缺血性损伤是冠状动脉粥样硬化的不良后果,可有多种因素促其发生,其中,牙周炎就是一重要诱因。牙龈卟啉菌是牙周炎的主要致病菌,同时牙龈卟啉菌义参与并促进泡沬细胞的形成。鉴于此,研究牙龈卟啉菌疫苗以有效地预防牙周炎的发生,减少发生动脉粥样硬化的可能性。GITR-GITRL是新近发现的免疫应答发生过程中共刺激分子,若在使用牙龈卟啉菌疫苗时辅以GITRL,也许能增强抗原刺激作用,更加有效的清除入侵的病原微生物,对抗感染免疫起着推波助澜的作用。有待进一步研究证实。
目的
(1)我们前期研究发现在阿霉素刺激的原代心肌细胞和腹腔注射阿霉素引起calpastatin过表达小鼠急性心肌毒性模型的心脏中,降低了calpain的表达及其活性；因calpastatin在体内的过表达,加剧了小鼠的心功能障碍以及死亡率。以上研究表明,内源性calpain对阿霉素诱导的心肌毒性提供了保护作用,但哪些亚型的calpain参与保护作用,不甚清楚。本研究旨在探讨calpain-2在阿霉素诱导的心肌毒性中的作用及其可能的分子机制。
(2)为了进一步体内研究calpain-2在阿霉素诱导的心肌毒性中的作用,构建心肌特异性、可诱导性的calpain-2过表达转基因系统。在转基因小鼠的基础上,应用阿霉素诱导小鼠急性和慢性心肌毒性模型,研究calpain-2在此两种模型中所扮演的角色及其作用机制。
(3)已有研究表明,牙龈卟啉菌不仅与冠状动脉粥样硬化及其诱发的心肌缺血性损伤有关,而且其所导致的牙周炎还是众多疾病的诱因,如吸入性肺炎、慢性肾病、糖尿病等。因此,本课题通过研究制备牙龈卟啉菌外膜蛋白RagB的DNA疫苗,并辅以GITRL为佐剂,为预防牙龈卟啉菌感染性牙周炎,控制动脉粥样硬化等多种疾病的发生与发展奠定良好的基础。同时,探讨RagB-GITRL DNA疫苗对牙龈卟啉菌感染的保护作用及其分子机制,为DNA疫苗研究提供新的思路。
方法
(1)心肌特异性和可诱导性calpain-2过表达转基因小鼠的建立
PCR法获得5′端带有TetO和a-MHC启动子核酸序列和3′端带有hGH核酸序列的hcalpain-2片段,克隆至真核表达载体。质粒经显微注射技术导入小鼠受精卵,再将其植入受体小鼠子宫,发育成hcalpain-2转基因小鼠。选择hcalpain-2阳性的小鼠与tTA转基因小鼠交配,其后代进行基因型鉴定,酶谱法检测(?)hcalpain-2在心肌中的活性,western blot法评估hcalpain-2以及calpain1在转基因小鼠心脏或肺中的表达,并多普勒法检测小鼠的心脏功能。
(2)阿霉素诱导小鼠心肌毒性模型的建立及分析
将基因型鉴定后的转基因小鼠分成对照组和阿霉素处理组,每组中每种基因型小鼠至少四只,且阿霉素处理组中Tg-tTA/capn-2基因型小鼠至少6-8只。阿霉素诱导急性心肌毒性模型：12周龄的雄性四种基因型小鼠,按体重腹腔给予阿霉素；对照组小鼠腹腔给予无菌PBS。同时,另取Tg-capn-2和Tg-tTA/capn-2小鼠各六只,腹腔给予calpain(?)印制剂(CI—Ⅲ)。阿霉素诱导的慢性心肌毒性模型：8周龄雄性四种基因型小鼠,按体重腹腔给予阿霉素；对照组小鼠腹腔给予无菌PBS。将已构建好的模型小鼠进行心脏超声检查心功能的改变,并留取心脏标本用于后续试验。
(3)评估calpain-2对阿霉素诱导心肌毒性的保护作用机制
分离新生小鼠心肌细胞,进行以下处理：感染腺病毒Ad-calpain-2和Ad-HA,给予1μM的阿霉素,AMC底物法检测心肌细胞caspase-3活性、ELISA法检测DNA片段化水平和western blot法检测AKT磷酸化水、I平；感染腺病毒Ad-calpastatin和Ad-HA,给予1μM的阿霉素,AMC底物法检测心肌细胞caspase-3(?)活性、ELISA法检测DNA片段化水平和、western blot法检测AKT磷酸化水平
取出急性心肌毒性模型的小鼠心肌组织,进行以下处理：取-片心肌组织,应用AMC底物法检测caspase-3的活性；另取一片心肌组织,western blot法检测AKT的磷酸化水平。包埋阿霉素诱导的慢性心肌毒性模型的心脏组织,应用WGA染色法计量心肌细胞大小。(4)牙龈卟啉菌DNA疫苗的构建及体外表达
以ragB和mGITRL质粒为模板,PCR扩增ragB和mGITRL片段,克隆至载体pIRES。获得重组子pIRES-ragB和pIRES-ragB-mGITRL,用于后续试验。将成功构建的重组子转染COS-7细胞,western blot检测ragB和mGITRL在COS-7细胞中的表达情况。
(5)外膜蛋白ragB-mGITRL DNA疫苗在预防牙龈卟啉菌感染中的作用及其分子机制
将构建并鉴定的重组子肌肉注射6周龄的雌性Balb/c小鼠,随机分成三组,分别注射pIRES-ragB、pIRES-ragB-mGITRL和pIRES。注射六周后,小鼠腹部皮下注射1×109的牙龈卟啉菌诱导溃疡模型(腹部皮下溃疡模型代替牙周炎模型),48小时后,测量溃疡面积。ELISA法检测血清中抗RagB抗体的滴度；分离脾脏细胞和骨髓细胞,ELISPOT法检测脾脏细胞和骨髓细胞中RagB特异性抗原形成细胞的数量；QRT-PCR法检测脾脏中细胞因子IL-21和IFN-y mRNA水平；FCM法检测脾脏中Tfh细胞和IFN-γ+T细胞的比例。
结果
(1)PCR扩增出5′端带入TetO和α-MHC启动子核酸序列和3′端带入hGH核酸序列的hcalpain-2片段,并构建至真核双表达载体。构建的质粒导入小鼠受精卵并植入受体小鼠的子宫,发育成带有hcalpain-2基因的小鼠。选择hcalpain-2阳性小鼠和tTA转基因小鼠交配获得四种基因型小鼠,即WT、Tg-capn-2、Tg-tTA和Tg-tTA/capn-2。Western blot证实hcalpain-2在心脏特异性并可诱导性表达,而对其他器官内源性calpain-1和calpain-2蛋白水平无影响。酶谱法结果显示,hcalpain-2在心肌中过表达提高calpain-2的活性。小鼠心功能检测表明,四种基因型小鼠的心脏收缩和舒张功能没有差异。
(2)建立阿霉素诱导的急性心肌毒性模型和慢性心肌毒性模型,心脏功能检测表明：在急性心肌毒性模型中,阿霉素处理组(WT、Tg-capn-2和Tg-tTA)与Sham组相比,明显降低心脏的收缩功能,而在calpain-2过表达的阿霉素处理组,心脏收缩功能明显改善,给予calpain抑制剂(CI-Ⅲ)后阻断了calpain-2过表达引起的心脏收缩功能的改善；在慢性心肌毒性模型中,阿霉素处理组(WT、Tg-capn-2和Tg-tTA)与Sham组相比,明显降低了心脏的收缩和舒张功能,而在calpain-2过表达的阿霉素处理组,两者都显著改善。
(3)在calpain-2过表达的心肌细胞中：①经阿霉素处理后,其caspase-3的活性与对照组相比显著降低,DNA片段化也明显改善,且AKT的磷酸化水平显著上调。然而,calpain-1在原代心肌细胞中的过表达升高了细胞caspase-3(?)舌性和增加了DNA片段化。②在calpain药理性抑制剂CⅠ-Ⅲ PD150606和生理性内源性抑制剂calpastatin的作用下,经阿霉素处理后,与未加抑制剂组相比心肌细胞的caspase-3(?)(?)性和DNA片段化都明显增高。③在calpastatin过表达的心肌细胞,经阿霉素处理后,与Ad-HA组相比AKT的磷酸化水平显著下调。④在阿霉素诱导的急性和慢性心肌毒性的心肌组织中,阿霉素提高了caspase-3的活性,而calpain-2的过表达则抵消了阿霉素引起的caspase-3活性的升高,同时增加了AKT的mRNA水平和蛋白磷酸化水平。同时calpain-2的过表达明显改善阿霉素引起的心肌肥大。
(4)PCR扩增出ragB和mGITRL片段,并将其克隆至真核表达载体pIRES上,获得重组子pIRES-ragB和pIRES-ragB-mGITRL。将重组子pIRES-ragB-mGITRL转入COS-7细胞,western blot结果显示,检测出RagB和mGITRL特异性条带。
(5) ELISA法检测结果显示,ragB免疫组产生较高滴度RagB特异性抗体(1：32000),而带有佐剂GITRL组,产生更高滴度的特异性抗体(1：64000)。ELISPOT分析结果表明,pIRES-ragB免疫的脾脏细胞和骨髓细胞产生了RagB特异性抗体形成细胞；而pIRES-ragB-mGITRL免疫组,RagB特异性抗体形成细胞增多三倍以上。同时,脾脏中细胞因子IL-21和IFN-γ mRNA水平在ragB组显著上调,GITRL进一步提高了两者的mRNA水平；Tfh和IFN-γ+T细胞在脾脏CD3+细胞中的比例因疫苗ragB的作用而显著上调,佐剂GITRL进一步上调两者的比例。牙龈卟啉菌感染引起小鼠腹部溃疡,疫苗的使用明显改善溃疡的形成,pIRES-ragB-mGITRL免疫组尤其明显。
结论
(1)成功构建了携带有TetO、α-MHC启动子和hGH核酸序列的hcalpain-2真核表达载体,并成功导入小鼠受精卵获得带有hcalpain-2基因的小鼠。继而与tTA转基因小鼠杂交,成功获得心肌特异性和可诱导性表达且各器官功能未受影响的hcalpain-2转基因小鼠。
(2)成功建立了阿霉素诱导的小鼠急性和慢性心肌毒性模型。calpain-2的过表达,发挥了对阿霉素诱导的心肌毒性的保护作用,有望成为预防和治疗阿霉素心肌毒性的靶点,从而推进阿霉素抗癌方面的应用。
(3)在体外,Calpain-2能增强原代心肌细胞AKT mRNA水平和蛋白磷酸化水平；在体内,同样能提高心肌组织的AKT mRNA水平和蛋白磷酸化水平。Calpain-2对阿霉素诱导的心肌毒性提供的保护作用可是通过AKT这条通路实现的。
(4)成功构建了真核表达载体pIRES-ragB和pIRES-ragB-mGITRL,并在表达细胞系COS-7细胞中有效表达。
(5)DNA疫苗免疫小鼠产：生了较高滴度的抗RagB抗体,并显示了对牙龈卟啉菌感染的保护作用；GITRL的联用进一步提高疫苗的保护效率,其作用可能是通过提高脾脏中Tfh细胞的比例,从而增强脾脏和骨髓中RagB特异性抗体形成细胞的数量,促进特异性抗体的产生；同时,GITRL还提高了脾脏中IFN-γ+T细胞的比例。总之,GITRL可能通过Tfh细胞和IFN-γ+T细胞两条途径,促进ragB疫苗的免疫效果和增强针对牙龈卟啉菌感染的保护作用。外膜蛋白ragB-mGITRL DNA疫苗有望用于预防牙龈卟啉菌的感染及其诱发的牙周炎,减少动脉粥样硬化、糖尿病等疾病的发生。GITRL则有望成为疫苗佐剂的重要候选者。
Myocardial injury was mediated by toxicity of chemical and myocarditis caused by infection of virus, bacteria and so forth. At same time, it seriously affected our health. In order to avoid disadvantages and prevent heart from pathological damage, this thesis involved the intervention study for two types of cardiac toxicity induced by doxorubicin and Porphyromonas gingivalis.
Doxorubicin, as an anthracycline antibiotic intercalating into DNA, is widely used in the clinical treatment of tumor. Doxorubicin may bring some side effects such as nausea, vomiting and arrhythmia. Sometimes, it may cause congestive heart failure, dilated cardiomyopathy and even death when its cumulative dose reaches55mg/m2, for which its clinical application is limited. However, the mechanism of acute and cumulative doxorubicin cardiotoxicity has not yet been fully understood, nor is there any effective prevention or treatment of doxorubicin-induced cardiomyopathy.
Calpain which is widely expressed in mammalian and other biological varieties belongs to the family of calcium-dependent cysteine protease. There are already15subtypes of calpain respectively encoded by15genes have been identified by human, with distinct differences in distribution, composition and function of each subtype. In which the u-calpain (calpain-1) and m-calpain (calpain-2) are very common with intensive studies. The activation of calpain requires Ca2+. Its endogenous inhibitor is calpastatin which is the specific inhibitor of calpain. The activation of calpain is involved in many systematic diseases of the body, such as embryonic lethal, diabetes, Alzheimer's disease, myocardial infarction, heart ischemia-reperfusion injury, atherosclerosis and gastric cancer, etc. Our previous study showed that over-expression of calpastatin increased acute myocardial toxicity induced by doxorubicin, indicating calpain was closely related with acute myocardial toxicity induced by Adriamycin. Was calpain-1or calpain-2or both involved in the protective effect and in which way? How was the molecular mechanism? These are questions still need to be answered.
Atherosclerosis, another important disease of cardiovascular system, has multiple incentives in which paradentitis is a major one. Porphyromonas gingivalis is the main pathogenic bacteria of paradentitis and it also participates in and promotes the formation of foam cells. Considering this, will prophyromonas gingivalis vaccine which can effectively prevent the paradentitis also prevent atheroscelrosis? And with adjuvant GITRL, will it be more efficient in cleaning up the invading pathogenic microbes? Will it be a catalyst in anti-infectious immunity? There are more to be proved.
Purposes
(1) Our previous studies found out that in the model of injecting doxorubicin into the primary myocardial cells and abdominal cavity of mouse, causing acute myocardial toxicity, the expression and activity of calpain was lowered in its heart. In transgenic mouse with over expression of calpastatin, the injection of doxorubicin into abdominal cavity inducing acute myocardial toxicity exacerbated the mouse's cardiac dysfunction and increased mortality because the activity of calpain is inhibited. The above studies indicated that endogenous calpain provided a protection against myocardial toxicity induced by doxorubicin. However, the subtype of calpain that was involved in this protection is not so clear. Herein, our study was to explore the effect of calpain-2in the myocardial toxicity induced by doxorubicin and its possible molecular mechanism.
(2) In order to further study the effect of calpain-2in the myocardial toxicity induced by doxorubicin, a cardiac specific and inducible over-expression transgenic system of calpain was constructed. In transgenic mouse, we used doxorubicin to induce acute and chronic myocardial toxicity models and then studied the calpain-2's effect and mechanism in both models.
(3) Studies have shown that the occurrence and development of atherosclerosis are closely related with paradentitis of which the Porphyromonas gingivalis is the major pathogenic bacteria and involves in the occurrence of atherosclerosis. At the same time, paradentitis is also the incentive of multiple diseases like aspiration pneumonia, chronic kidney disease and diabetes, etc. Therefore, our study was to enhance the protection against Porphyromonas gingivalis'infection and reduce the incidence of paradentitis through preparing a DNA vaccine of outer membrane protein RagB of Porphyromonas gingivalis with adjuvant GITRL, thus to control the occurrence and development of multiple diseases such as atherosclerosis. Also, it was to further study the DNA vaccine's protective effect and molecular mechanism against the infection of Porphyromonas gingivalis to provide theoretic basis for DNA vaccine's future clinic application.
Methods
(1) Establishment of cardiac-specific and inducible calpain-2transgenic mice
Human calpain-2(hcalpain-2) nucleic acid sequence contained TetO and a-MHC promoter sequence at5'end and hGH sequence at3'end was obtained with PCR, and cloned into the eukaryotic expression vector. Recombinant plasmid was imported into mouse fertilized egg with microinjection, which implanted in womb of mice, and developed into hcalpain-2transgenic mice. Selecting hcalpain-2-positive mice were mated with tTA transgenic mice, and the offspring was genotyped with PCR. Hcalpain-2activity in the myocardium was detected with zymography. Western blot method was employed to assess hcalpain-2and calpain-1protein level in the heart or lung of transgenic mice. The heart function of mice was measured with Doppler method.
(2) Establishment and analysis of mice model with doxorubicin-induced cardiotoxicity
Transgenic mice identified with genotyping were divided into two groups, that is, control group and doxorubicin-treated group. At least four mice in each group and6-8mice in Tg-tTA/capn-2group with doxorubicin treatment. Doxorubicin-induced acute cardiotoxicity model:12-week old male mice were given doxorubicin by intraperitoneal injection at each genotype, and control group mice were given sterile PBS by intraperitoneal injection. At the same time, taking another six Tg-capn-2and Tg-tTA/capn-2mice respectively were treated with calpain inhibitor (CⅠ-Ⅲ) by intraperitoneal injection. Doxorubicin-induced chronic cardiotoxicity model:8-week old male mice were given doxorubicin by intraperitoneal injection according to the weight of mice; control group mice were given sterile PBS. Heart function of mice was measured with Doppler and hearts were harvested for subsequent experiments.
(3) To assess the protection mechanisms of calpain-2in doxorubicin-induced cardiotoxicity
Separating neonatal mouse cardiomyocytes were treated, as follows: Cardiomyocytes were infected with adenovirus calpain-2and HA respectively and given lμM doxorubicin. Caspase-3activity was detected with AMC substrate method in cardiomyocytes. ELISA method was employed to detect DNA fragmentation level and phosphorylated AKT was measured with western blot. Cardiomyocytes were infected with adenovirus calpastatin and HA respectively and given lμM doxorubicin. Caspase-3activity of cardiomyocytes was detected with AMC substrate method. ELISA method was employed to detect DNA fragmentation level and phosphorylated AKT was measured with western blot.
Myocardial tissue of mice in acute cardiotoxicity model was used for measuring caspase-3activity with AMC substrate method and detecting the phosphorylated AKT by western blot. The hearts of mice were embeded with wax, stained with WGA and measured the cell size with imageJ software in doxorubicin-induced chronic myocardial toxicity model.
(4) Construction and expression in vitro of Porphyromonas gingivalis DNA vaccine
RagB and mGITRL nucleic acid sequences were acquired with PCR from plasmid contained RagB and mGITRL respectively and cloned into the vector pIRES. Getting recombinant, that is, pIRES-ragB and pIRES-ragB-mGITRL. The constructed recombinant was transfected into COS-7cells, and then western blot was used to detect ragB and mGITRL expression.
(5) The role and molecular mechanism of the outer membrane protein ragB DNA vaccine and adjuvant mGITRL against Porphyromonas gingivalis infection
Six-week female Balb/c mice were immunized with recombinant by intramuscular injection in the three groups, that is, pIRES-ragB, pIRES-ragB-mGITRL, and pIRES. Mice immunized for six weeks, were infected with1×109Porphyromonas gingivalis by subcutaneous injection to induce ulcer model. After48hours, measuring the ulcer area. The titer of anti-RagB antibody in serum was detected with ELISA. ELISPOT assay was employed to measure the number of specific antigen-forming cells in the spleen cells and bone marrow cells. IL-21and IFN-γ mRNA level in spleen were assessed by QRT-PCR and Tfh cells and IFN-γ+T cells ratio in spleen were detected with FCM.
Results
(1) Acquiring hcalpain-2fragment with TetO and a-MHC promoter at5'end and hGH nucleic acid sequence at3'ends, and have been cloned into eukaryotic expression vector. The constructed plasmid was introduced into mouse fertilized eggs, which was implanted in the uterus of the recipient mice, and developed into hcalpain-2transgenic mice. At last, we acquired four genotypes of mice, that is, WT, Tg-capn-2, Tg-tTA and Tg-tTA/capn-2. Western blot analysis showed hcalpain-2was inducible and cardiac-specific expression and had no effect on endogenous calpain-1and calpain-2protein levels in other organs. Zymography results indicated that hcalpain-2over-expression in the myocardium increased the activity of calpain-2. Detection of cardiac function showed no difference in systolic and diastolic function among four genotypes of mice.
(2) Establishment of doxorubicin-induced acute and chronic cardiotoxicity model, and measurement result of cardiac function showed that:In the acute myocardial toxicity model, doxorubicin-treated group (WT, Tg-capn-2and Tg-tTA groups) mice were significantly reduced the systolic function of heart compared with sham group. However, owing to calpain-2over-expression, systolic function of mice was improved significantly. But given the calpain inhibitor (CⅠ-Ⅲ), the protection of calpain-2was canceled immediately. In the chronic myocardial toxicity model, doxorubicin treatment group (WT, Tg-capn-2and Tg-tTA groups) also were obviously reduced systolic and diastolic function of mice compared with sham group, but the heart function of mice was improved significantly attributing to calpain-2over-expression.
(3) At the calpain-2over-expression of myocardial cells treated with doxorubicin, caspase-3activity was significantly decreased comparing with control group; DNA fragmentation was also significantly improved; the level of phosphorylated AKT was significantly up-regulated. However, calpain-1over-expression in primary cardiomyocytes increased caspase-3activity and DNA fragmentation. At the effect of calpain physiological inhibitor (CⅠ-Ⅲ and PD150606) and endogenous inhibitor (calpastatin), caspase-3activity and DNA fragmentation of cardiomyocytes treated with doxorubicin were significantly increased comparing with sham group. Calpastatin over-expression in myocardial cells downregulated the AKT phosphorylation comparing with control group after treating with doxorubicin. In the heart tissue of doxorubicin-induced acute and chronic cardiotoxicity model, the caspase-3activity was increased comparing with sham group and canceled owing to calpain-2over-expression. At the same time, over-expression of calpain-2increased AKT mRNA and phosphorylated protein levels. Also, calpain-2over-expression significantly improved doxorubicin-induced myocardial hypertrophy.
(4) PCR amplified ragB, and mGITRL fragment, and cloned into the eukaryotic expression vector pIRES. Acquired recombinants pIRES-ragB and pIRES-ragB-mGITRL. Recombinant pIRES-ragB-mGITRL was transfected into COS-7cells, and western blot results displayed two specific bands, RagB and mGITRL.
(5) Results of ELISA assay showed that mice immunized with ragB DNA vaccine produced high titers of RagB specific antibody (1:32000), while given adjuvant GITRL, produced higher titer specific antibody (1:64000). ELISPOT analysis results showed that RagB specific antigen-forming cells were produced in the spleen cells and bone marrow cells of mice immunized with ragB DNA vaccine, with the effect of adjuvant GITRL, RagB specific antigen-forming cells were increased by more than three times. IL-21and of IFN-y mRNA in the spleen and protein in serum of mice immunized with ragB DNA vaccine were significantly raised, while given GITRL further increasing the levels. The proportion of Tfh and IFN-γ+T-cells in CD3+cells of spleen significantly up-regulated due to the application of ragB DNA vaccine, and the adjuvant GITRL further raised the amplification of two cells. The application of DNA vaccine significantly decreased lesion area caused by Porphyromonas gingivalis via abdominal infection, and the participation of adjuvant, further narrowing the lesion size.
Conclusions
(1) Eukaryotic expression vector contained hcalpain-2sequence with TetO, a-MHC promoter and hGH nucleic acid sequence was successfully constructed and imported into mouse fertilized eggs, which produced a transgenic mice carried hcalpain-2gene. And then mating with tTA transgenic mice produced an offspring with calpain-2cardiac-specific and inducible expression. Hcalpain-2over-expression in heart unaffected function of various organs in transgenic mice.
(2) Doxorubicin-induced mice acute and chronic cardiotoxicity model were successfully constructed. Calpain-2over-expression providing protection against doxorobicin induced cardiac toxicity is expected to become a target for the prevention and treatment of doxorubicin induced cardiotoxicity, thereby enhancing doxorubicin anticancer applications.
(3) Calpain-2improved AKT mRNA and protein phosphorylation levels in primary myocardial cells in vitro and myocardial tissue in vivo. Calpain-2provided protection against doxorubicin induced cardiac toxicity via AKT pathway.
(4) The successful construction of eukaryotic expression vector, pIRES-ragB mGITRL and pIRES-ragB-mGITRL, which successfully expressed in COS-7cell line.
(5) Mice immunized with DNA vaccine produced anti-RagB antibody and provided a protective effect for Porphyromonas gingivalis infection, while the application of adjuvant GITRL further improved the protection efficiency of the vaccine. The adjuvant GITRL increased the quantity of the RagB specific antibody-forming cells in the spleen and bone marrow by raising the proportion of Tfh cells in spleen, which should further improved the production of specific antibodies. Meanwhile, the GITRL increased the ratio of IFN-γ+T cells in spleen. In summary, GITRL played a important role in enhancing protection function of ragB DNA vaccine against Porphyromonas gingivalis infection via two ways: The Tfh cells and the IFN-γ+T cells. Outer membrane protein ragB DNA vaccine was hopefully to prevent Porphyromonas gingivalis infection as well as the occurrence of periodontitis, thereby reducing the occurrence and development of atherosclerosis, diabetes and other diseases. The GITRL was expected to be the major candidate for vaccine adjuvants.
阿霉素,是一种作用于DNA的葸环类抗生素,广泛应用于临床抗肿瘤的药物。阿霉素的应用带来多方面的副作用,包括恶心、呕吐和心律不齐等。其中当累积剂量达至(?)55mg/m2(?)(?),能够引起充血性心衰、扩张性心肌病甚至死亡,从而限制了其临床应用。迄今为止,阿霉素的急性和累积性心脏毒性的机制尚未完全清楚,更无预防和治疗阿霉素诱导心肌病行之有效的措施。
Calpain,即钙蛋白酶,属于钙离子依赖的半胱氨酸蛋白酶家族,广泛表达于哺乳动物以及多种其他生物。人类已鉴定的15种亚型的分布、结构和功能均有明显差异。其中“-calpain(calpain-1)和m-calpain(calpain-2)为普遍存在且研究深入。Calpain的激活参与机体多系统疾病,如糖尿病、阿尔滋海默氏病、动脉粥样硬化以及胃癌等。前期研究发现,calpastatin的过表达,加剧了阿霉素引起的急性心肌毒性,表明calpain与其密切相关,calpain-1或calpain-2是否参与了保护作用?其分子机制如何?尚待研究证实。
心血管系统的另一重要疾病动脉粥样硬化,心肌缺血性损伤是冠状动脉粥样硬化的不良后果,可有多种因素促其发生,其中,牙周炎就是一重要诱因。牙龈卟啉菌是牙周炎的主要致病菌,同时牙龈卟啉菌义参与并促进泡沬细胞的形成。鉴于此,研究牙龈卟啉菌疫苗以有效地预防牙周炎的发生,减少发生动脉粥样硬化的可能性。GITR-GITRL是新近发现的免疫应答发生过程中共刺激分子,若在使用牙龈卟啉菌疫苗时辅以GITRL,也许能增强抗原刺激作用,更加有效的清除入侵的病原微生物,对抗感染免疫起着推波助澜的作用。有待进一步研究证实。
目的
(1)我们前期研究发现在阿霉素刺激的原代心肌细胞和腹腔注射阿霉素引起calpastatin过表达小鼠急性心肌毒性模型的心脏中,降低了calpain的表达及其活性；因calpastatin在体内的过表达,加剧了小鼠的心功能障碍以及死亡率。以上研究表明,内源性calpain对阿霉素诱导的心肌毒性提供了保护作用,但哪些亚型的calpain参与保护作用,不甚清楚。本研究旨在探讨calpain-2在阿霉素诱导的心肌毒性中的作用及其可能的分子机制。
(2)为了进一步体内研究calpain-2在阿霉素诱导的心肌毒性中的作用,构建心肌特异性、可诱导性的calpain-2过表达转基因系统。在转基因小鼠的基础上,应用阿霉素诱导小鼠急性和慢性心肌毒性模型,研究calpain-2在此两种模型中所扮演的角色及其作用机制。
(3)已有研究表明,牙龈卟啉菌不仅与冠状动脉粥样硬化及其诱发的心肌缺血性损伤有关,而且其所导致的牙周炎还是众多疾病的诱因,如吸入性肺炎、慢性肾病、糖尿病等。因此,本课题通过研究制备牙龈卟啉菌外膜蛋白RagB的DNA疫苗,并辅以GITRL为佐剂,为预防牙龈卟啉菌感染性牙周炎,控制动脉粥样硬化等多种疾病的发生与发展奠定良好的基础。同时,探讨RagB-GITRL DNA疫苗对牙龈卟啉菌感染的保护作用及其分子机制,为DNA疫苗研究提供新的思路。
方法
(1)心肌特异性和可诱导性calpain-2过表达转基因小鼠的建立
PCR法获得5′端带有TetO和a-MHC启动子核酸序列和3′端带有hGH核酸序列的hcalpain-2片段,克隆至真核表达载体。质粒经显微注射技术导入小鼠受精卵,再将其植入受体小鼠子宫,发育成hcalpain-2转基因小鼠。选择hcalpain-2阳性的小鼠与tTA转基因小鼠交配,其后代进行基因型鉴定,酶谱法检测(?)hcalpain-2在心肌中的活性,western blot法评估hcalpain-2以及calpain1在转基因小鼠心脏或肺中的表达,并多普勒法检测小鼠的心脏功能。
(2)阿霉素诱导小鼠心肌毒性模型的建立及分析
将基因型鉴定后的转基因小鼠分成对照组和阿霉素处理组,每组中每种基因型小鼠至少四只,且阿霉素处理组中Tg-tTA/capn-2基因型小鼠至少6-8只。阿霉素诱导急性心肌毒性模型：12周龄的雄性四种基因型小鼠,按体重腹腔给予阿霉素；对照组小鼠腹腔给予无菌PBS。同时,另取Tg-capn-2和Tg-tTA/capn-2小鼠各六只,腹腔给予calpain(?)印制剂(CI—Ⅲ)。阿霉素诱导的慢性心肌毒性模型：8周龄雄性四种基因型小鼠,按体重腹腔给予阿霉素；对照组小鼠腹腔给予无菌PBS。将已构建好的模型小鼠进行心脏超声检查心功能的改变,并留取心脏标本用于后续试验。
(3)评估calpain-2对阿霉素诱导心肌毒性的保护作用机制
分离新生小鼠心肌细胞,进行以下处理：感染腺病毒Ad-calpain-2和Ad-HA,给予1μM的阿霉素,AMC底物法检测心肌细胞caspase-3活性、ELISA法检测DNA片段化水平和western blot法检测AKT磷酸化水、I平；感染腺病毒Ad-calpastatin和Ad-HA,给予1μM的阿霉素,AMC底物法检测心肌细胞caspase-3(?)活性、ELISA法检测DNA片段化水平和、western blot法检测AKT磷酸化水平
取出急性心肌毒性模型的小鼠心肌组织,进行以下处理：取-片心肌组织,应用AMC底物法检测caspase-3的活性；另取一片心肌组织,western blot法检测AKT的磷酸化水平。包埋阿霉素诱导的慢性心肌毒性模型的心脏组织,应用WGA染色法计量心肌细胞大小。(4)牙龈卟啉菌DNA疫苗的构建及体外表达
以ragB和mGITRL质粒为模板,PCR扩增ragB和mGITRL片段,克隆至载体pIRES。获得重组子pIRES-ragB和pIRES-ragB-mGITRL,用于后续试验。将成功构建的重组子转染COS-7细胞,western blot检测ragB和mGITRL在COS-7细胞中的表达情况。
(5)外膜蛋白ragB-mGITRL DNA疫苗在预防牙龈卟啉菌感染中的作用及其分子机制
将构建并鉴定的重组子肌肉注射6周龄的雌性Balb/c小鼠,随机分成三组,分别注射pIRES-ragB、pIRES-ragB-mGITRL和pIRES。注射六周后,小鼠腹部皮下注射1×109的牙龈卟啉菌诱导溃疡模型(腹部皮下溃疡模型代替牙周炎模型),48小时后,测量溃疡面积。ELISA法检测血清中抗RagB抗体的滴度；分离脾脏细胞和骨髓细胞,ELISPOT法检测脾脏细胞和骨髓细胞中RagB特异性抗原形成细胞的数量；QRT-PCR法检测脾脏中细胞因子IL-21和IFN-y mRNA水平；FCM法检测脾脏中Tfh细胞和IFN-γ+T细胞的比例。
结果
(1)PCR扩增出5′端带入TetO和α-MHC启动子核酸序列和3′端带入hGH核酸序列的hcalpain-2片段,并构建至真核双表达载体。构建的质粒导入小鼠受精卵并植入受体小鼠的子宫,发育成带有hcalpain-2基因的小鼠。选择hcalpain-2阳性小鼠和tTA转基因小鼠交配获得四种基因型小鼠,即WT、Tg-capn-2、Tg-tTA和Tg-tTA/capn-2。Western blot证实hcalpain-2在心脏特异性并可诱导性表达,而对其他器官内源性calpain-1和calpain-2蛋白水平无影响。酶谱法结果显示,hcalpain-2在心肌中过表达提高calpain-2的活性。小鼠心功能检测表明,四种基因型小鼠的心脏收缩和舒张功能没有差异。
(2)建立阿霉素诱导的急性心肌毒性模型和慢性心肌毒性模型,心脏功能检测表明：在急性心肌毒性模型中,阿霉素处理组(WT、Tg-capn-2和Tg-tTA)与Sham组相比,明显降低心脏的收缩功能,而在calpain-2过表达的阿霉素处理组,心脏收缩功能明显改善,给予calpain抑制剂(CI-Ⅲ)后阻断了calpain-2过表达引起的心脏收缩功能的改善；在慢性心肌毒性模型中,阿霉素处理组(WT、Tg-capn-2和Tg-tTA)与Sham组相比,明显降低了心脏的收缩和舒张功能,而在calpain-2过表达的阿霉素处理组,两者都显著改善。
(3)在calpain-2过表达的心肌细胞中：①经阿霉素处理后,其caspase-3的活性与对照组相比显著降低,DNA片段化也明显改善,且AKT的磷酸化水平显著上调。然而,calpain-1在原代心肌细胞中的过表达升高了细胞caspase-3(?)舌性和增加了DNA片段化。②在calpain药理性抑制剂CⅠ-Ⅲ PD150606和生理性内源性抑制剂calpastatin的作用下,经阿霉素处理后,与未加抑制剂组相比心肌细胞的caspase-3(?)(?)性和DNA片段化都明显增高。③在calpastatin过表达的心肌细胞,经阿霉素处理后,与Ad-HA组相比AKT的磷酸化水平显著下调。④在阿霉素诱导的急性和慢性心肌毒性的心肌组织中,阿霉素提高了caspase-3的活性,而calpain-2的过表达则抵消了阿霉素引起的caspase-3活性的升高,同时增加了AKT的mRNA水平和蛋白磷酸化水平。同时calpain-2的过表达明显改善阿霉素引起的心肌肥大。
(4)PCR扩增出ragB和mGITRL片段,并将其克隆至真核表达载体pIRES上,获得重组子pIRES-ragB和pIRES-ragB-mGITRL。将重组子pIRES-ragB-mGITRL转入COS-7细胞,western blot结果显示,检测出RagB和mGITRL特异性条带。
(5) ELISA法检测结果显示,ragB免疫组产生较高滴度RagB特异性抗体(1：32000),而带有佐剂GITRL组,产生更高滴度的特异性抗体(1：64000)。ELISPOT分析结果表明,pIRES-ragB免疫的脾脏细胞和骨髓细胞产生了RagB特异性抗体形成细胞；而pIRES-ragB-mGITRL免疫组,RagB特异性抗体形成细胞增多三倍以上。同时,脾脏中细胞因子IL-21和IFN-γ mRNA水平在ragB组显著上调,GITRL进一步提高了两者的mRNA水平；Tfh和IFN-γ+T细胞在脾脏CD3+细胞中的比例因疫苗ragB的作用而显著上调,佐剂GITRL进一步上调两者的比例。牙龈卟啉菌感染引起小鼠腹部溃疡,疫苗的使用明显改善溃疡的形成,pIRES-ragB-mGITRL免疫组尤其明显。
结论
(1)成功构建了携带有TetO、α-MHC启动子和hGH核酸序列的hcalpain-2真核表达载体,并成功导入小鼠受精卵获得带有hcalpain-2基因的小鼠。继而与tTA转基因小鼠杂交,成功获得心肌特异性和可诱导性表达且各器官功能未受影响的hcalpain-2转基因小鼠。
(2)成功建立了阿霉素诱导的小鼠急性和慢性心肌毒性模型。calpain-2的过表达,发挥了对阿霉素诱导的心肌毒性的保护作用,有望成为预防和治疗阿霉素心肌毒性的靶点,从而推进阿霉素抗癌方面的应用。
(3)在体外,Calpain-2能增强原代心肌细胞AKT mRNA水平和蛋白磷酸化水平；在体内,同样能提高心肌组织的AKT mRNA水平和蛋白磷酸化水平。Calpain-2对阿霉素诱导的心肌毒性提供的保护作用可是通过AKT这条通路实现的。
(4)成功构建了真核表达载体pIRES-ragB和pIRES-ragB-mGITRL,并在表达细胞系COS-7细胞中有效表达。
(5)DNA疫苗免疫小鼠产：生了较高滴度的抗RagB抗体,并显示了对牙龈卟啉菌感染的保护作用；GITRL的联用进一步提高疫苗的保护效率,其作用可能是通过提高脾脏中Tfh细胞的比例,从而增强脾脏和骨髓中RagB特异性抗体形成细胞的数量,促进特异性抗体的产生；同时,GITRL还提高了脾脏中IFN-γ+T细胞的比例。总之,GITRL可能通过Tfh细胞和IFN-γ+T细胞两条途径,促进ragB疫苗的免疫效果和增强针对牙龈卟啉菌感染的保护作用。外膜蛋白ragB-mGITRL DNA疫苗有望用于预防牙龈卟啉菌的感染及其诱发的牙周炎,减少动脉粥样硬化、糖尿病等疾病的发生。GITRL则有望成为疫苗佐剂的重要候选者。
Myocardial injury was mediated by toxicity of chemical and myocarditis caused by infection of virus, bacteria and so forth. At same time, it seriously affected our health. In order to avoid disadvantages and prevent heart from pathological damage, this thesis involved the intervention study for two types of cardiac toxicity induced by doxorubicin and Porphyromonas gingivalis.
Doxorubicin, as an anthracycline antibiotic intercalating into DNA, is widely used in the clinical treatment of tumor. Doxorubicin may bring some side effects such as nausea, vomiting and arrhythmia. Sometimes, it may cause congestive heart failure, dilated cardiomyopathy and even death when its cumulative dose reaches55mg/m2, for which its clinical application is limited. However, the mechanism of acute and cumulative doxorubicin cardiotoxicity has not yet been fully understood, nor is there any effective prevention or treatment of doxorubicin-induced cardiomyopathy.
Calpain which is widely expressed in mammalian and other biological varieties belongs to the family of calcium-dependent cysteine protease. There are already15subtypes of calpain respectively encoded by15genes have been identified by human, with distinct differences in distribution, composition and function of each subtype. In which the u-calpain (calpain-1) and m-calpain (calpain-2) are very common with intensive studies. The activation of calpain requires Ca2+. Its endogenous inhibitor is calpastatin which is the specific inhibitor of calpain. The activation of calpain is involved in many systematic diseases of the body, such as embryonic lethal, diabetes, Alzheimer's disease, myocardial infarction, heart ischemia-reperfusion injury, atherosclerosis and gastric cancer, etc. Our previous study showed that over-expression of calpastatin increased acute myocardial toxicity induced by doxorubicin, indicating calpain was closely related with acute myocardial toxicity induced by Adriamycin. Was calpain-1or calpain-2or both involved in the protective effect and in which way? How was the molecular mechanism? These are questions still need to be answered.
Atherosclerosis, another important disease of cardiovascular system, has multiple incentives in which paradentitis is a major one. Porphyromonas gingivalis is the main pathogenic bacteria of paradentitis and it also participates in and promotes the formation of foam cells. Considering this, will prophyromonas gingivalis vaccine which can effectively prevent the paradentitis also prevent atheroscelrosis? And with adjuvant GITRL, will it be more efficient in cleaning up the invading pathogenic microbes? Will it be a catalyst in anti-infectious immunity? There are more to be proved.
Purposes
(1) Our previous studies found out that in the model of injecting doxorubicin into the primary myocardial cells and abdominal cavity of mouse, causing acute myocardial toxicity, the expression and activity of calpain was lowered in its heart. In transgenic mouse with over expression of calpastatin, the injection of doxorubicin into abdominal cavity inducing acute myocardial toxicity exacerbated the mouse's cardiac dysfunction and increased mortality because the activity of calpain is inhibited. The above studies indicated that endogenous calpain provided a protection against myocardial toxicity induced by doxorubicin. However, the subtype of calpain that was involved in this protection is not so clear. Herein, our study was to explore the effect of calpain-2in the myocardial toxicity induced by doxorubicin and its possible molecular mechanism.
(2) In order to further study the effect of calpain-2in the myocardial toxicity induced by doxorubicin, a cardiac specific and inducible over-expression transgenic system of calpain was constructed. In transgenic mouse, we used doxorubicin to induce acute and chronic myocardial toxicity models and then studied the calpain-2's effect and mechanism in both models.
(3) Studies have shown that the occurrence and development of atherosclerosis are closely related with paradentitis of which the Porphyromonas gingivalis is the major pathogenic bacteria and involves in the occurrence of atherosclerosis. At the same time, paradentitis is also the incentive of multiple diseases like aspiration pneumonia, chronic kidney disease and diabetes, etc. Therefore, our study was to enhance the protection against Porphyromonas gingivalis'infection and reduce the incidence of paradentitis through preparing a DNA vaccine of outer membrane protein RagB of Porphyromonas gingivalis with adjuvant GITRL, thus to control the occurrence and development of multiple diseases such as atherosclerosis. Also, it was to further study the DNA vaccine's protective effect and molecular mechanism against the infection of Porphyromonas gingivalis to provide theoretic basis for DNA vaccine's future clinic application.
Methods
(1) Establishment of cardiac-specific and inducible calpain-2transgenic mice
Human calpain-2(hcalpain-2) nucleic acid sequence contained TetO and a-MHC promoter sequence at5'end and hGH sequence at3'end was obtained with PCR, and cloned into the eukaryotic expression vector. Recombinant plasmid was imported into mouse fertilized egg with microinjection, which implanted in womb of mice, and developed into hcalpain-2transgenic mice. Selecting hcalpain-2-positive mice were mated with tTA transgenic mice, and the offspring was genotyped with PCR. Hcalpain-2activity in the myocardium was detected with zymography. Western blot method was employed to assess hcalpain-2and calpain-1protein level in the heart or lung of transgenic mice. The heart function of mice was measured with Doppler method.
(2) Establishment and analysis of mice model with doxorubicin-induced cardiotoxicity
Transgenic mice identified with genotyping were divided into two groups, that is, control group and doxorubicin-treated group. At least four mice in each group and6-8mice in Tg-tTA/capn-2group with doxorubicin treatment. Doxorubicin-induced acute cardiotoxicity model:12-week old male mice were given doxorubicin by intraperitoneal injection at each genotype, and control group mice were given sterile PBS by intraperitoneal injection. At the same time, taking another six Tg-capn-2and Tg-tTA/capn-2mice respectively were treated with calpain inhibitor (CⅠ-Ⅲ) by intraperitoneal injection. Doxorubicin-induced chronic cardiotoxicity model:8-week old male mice were given doxorubicin by intraperitoneal injection according to the weight of mice; control group mice were given sterile PBS. Heart function of mice was measured with Doppler and hearts were harvested for subsequent experiments.
(3) To assess the protection mechanisms of calpain-2in doxorubicin-induced cardiotoxicity
Separating neonatal mouse cardiomyocytes were treated, as follows: Cardiomyocytes were infected with adenovirus calpain-2and HA respectively and given lμM doxorubicin. Caspase-3activity was detected with AMC substrate method in cardiomyocytes. ELISA method was employed to detect DNA fragmentation level and phosphorylated AKT was measured with western blot. Cardiomyocytes were infected with adenovirus calpastatin and HA respectively and given lμM doxorubicin. Caspase-3activity of cardiomyocytes was detected with AMC substrate method. ELISA method was employed to detect DNA fragmentation level and phosphorylated AKT was measured with western blot.
Myocardial tissue of mice in acute cardiotoxicity model was used for measuring caspase-3activity with AMC substrate method and detecting the phosphorylated AKT by western blot. The hearts of mice were embeded with wax, stained with WGA and measured the cell size with imageJ software in doxorubicin-induced chronic myocardial toxicity model.
(4) Construction and expression in vitro of Porphyromonas gingivalis DNA vaccine
RagB and mGITRL nucleic acid sequences were acquired with PCR from plasmid contained RagB and mGITRL respectively and cloned into the vector pIRES. Getting recombinant, that is, pIRES-ragB and pIRES-ragB-mGITRL. The constructed recombinant was transfected into COS-7cells, and then western blot was used to detect ragB and mGITRL expression.
(5) The role and molecular mechanism of the outer membrane protein ragB DNA vaccine and adjuvant mGITRL against Porphyromonas gingivalis infection
Six-week female Balb/c mice were immunized with recombinant by intramuscular injection in the three groups, that is, pIRES-ragB, pIRES-ragB-mGITRL, and pIRES. Mice immunized for six weeks, were infected with1×109Porphyromonas gingivalis by subcutaneous injection to induce ulcer model. After48hours, measuring the ulcer area. The titer of anti-RagB antibody in serum was detected with ELISA. ELISPOT assay was employed to measure the number of specific antigen-forming cells in the spleen cells and bone marrow cells. IL-21and IFN-γ mRNA level in spleen were assessed by QRT-PCR and Tfh cells and IFN-γ+T cells ratio in spleen were detected with FCM.
Results
(1) Acquiring hcalpain-2fragment with TetO and a-MHC promoter at5'end and hGH nucleic acid sequence at3'ends, and have been cloned into eukaryotic expression vector. The constructed plasmid was introduced into mouse fertilized eggs, which was implanted in the uterus of the recipient mice, and developed into hcalpain-2transgenic mice. At last, we acquired four genotypes of mice, that is, WT, Tg-capn-2, Tg-tTA and Tg-tTA/capn-2. Western blot analysis showed hcalpain-2was inducible and cardiac-specific expression and had no effect on endogenous calpain-1and calpain-2protein levels in other organs. Zymography results indicated that hcalpain-2over-expression in the myocardium increased the activity of calpain-2. Detection of cardiac function showed no difference in systolic and diastolic function among four genotypes of mice.
(2) Establishment of doxorubicin-induced acute and chronic cardiotoxicity model, and measurement result of cardiac function showed that:In the acute myocardial toxicity model, doxorubicin-treated group (WT, Tg-capn-2and Tg-tTA groups) mice were significantly reduced the systolic function of heart compared with sham group. However, owing to calpain-2over-expression, systolic function of mice was improved significantly. But given the calpain inhibitor (CⅠ-Ⅲ), the protection of calpain-2was canceled immediately. In the chronic myocardial toxicity model, doxorubicin treatment group (WT, Tg-capn-2and Tg-tTA groups) also were obviously reduced systolic and diastolic function of mice compared with sham group, but the heart function of mice was improved significantly attributing to calpain-2over-expression.
(3) At the calpain-2over-expression of myocardial cells treated with doxorubicin, caspase-3activity was significantly decreased comparing with control group; DNA fragmentation was also significantly improved; the level of phosphorylated AKT was significantly up-regulated. However, calpain-1over-expression in primary cardiomyocytes increased caspase-3activity and DNA fragmentation. At the effect of calpain physiological inhibitor (CⅠ-Ⅲ and PD150606) and endogenous inhibitor (calpastatin), caspase-3activity and DNA fragmentation of cardiomyocytes treated with doxorubicin were significantly increased comparing with sham group. Calpastatin over-expression in myocardial cells downregulated the AKT phosphorylation comparing with control group after treating with doxorubicin. In the heart tissue of doxorubicin-induced acute and chronic cardiotoxicity model, the caspase-3activity was increased comparing with sham group and canceled owing to calpain-2over-expression. At the same time, over-expression of calpain-2increased AKT mRNA and phosphorylated protein levels. Also, calpain-2over-expression significantly improved doxorubicin-induced myocardial hypertrophy.
(4) PCR amplified ragB, and mGITRL fragment, and cloned into the eukaryotic expression vector pIRES. Acquired recombinants pIRES-ragB and pIRES-ragB-mGITRL. Recombinant pIRES-ragB-mGITRL was transfected into COS-7cells, and western blot results displayed two specific bands, RagB and mGITRL.
(5) Results of ELISA assay showed that mice immunized with ragB DNA vaccine produced high titers of RagB specific antibody (1:32000), while given adjuvant GITRL, produced higher titer specific antibody (1:64000). ELISPOT analysis results showed that RagB specific antigen-forming cells were produced in the spleen cells and bone marrow cells of mice immunized with ragB DNA vaccine, with the effect of adjuvant GITRL, RagB specific antigen-forming cells were increased by more than three times. IL-21and of IFN-y mRNA in the spleen and protein in serum of mice immunized with ragB DNA vaccine were significantly raised, while given GITRL further increasing the levels. The proportion of Tfh and IFN-γ+T-cells in CD3+cells of spleen significantly up-regulated due to the application of ragB DNA vaccine, and the adjuvant GITRL further raised the amplification of two cells. The application of DNA vaccine significantly decreased lesion area caused by Porphyromonas gingivalis via abdominal infection, and the participation of adjuvant, further narrowing the lesion size.
Conclusions
(1) Eukaryotic expression vector contained hcalpain-2sequence with TetO, a-MHC promoter and hGH nucleic acid sequence was successfully constructed and imported into mouse fertilized eggs, which produced a transgenic mice carried hcalpain-2gene. And then mating with tTA transgenic mice produced an offspring with calpain-2cardiac-specific and inducible expression. Hcalpain-2over-expression in heart unaffected function of various organs in transgenic mice.
(2) Doxorubicin-induced mice acute and chronic cardiotoxicity model were successfully constructed. Calpain-2over-expression providing protection against doxorobicin induced cardiac toxicity is expected to become a target for the prevention and treatment of doxorubicin induced cardiotoxicity, thereby enhancing doxorubicin anticancer applications.
(3) Calpain-2improved AKT mRNA and protein phosphorylation levels in primary myocardial cells in vitro and myocardial tissue in vivo. Calpain-2provided protection against doxorubicin induced cardiac toxicity via AKT pathway.
(4) The successful construction of eukaryotic expression vector, pIRES-ragB mGITRL and pIRES-ragB-mGITRL, which successfully expressed in COS-7cell line.
(5) Mice immunized with DNA vaccine produced anti-RagB antibody and provided a protective effect for Porphyromonas gingivalis infection, while the application of adjuvant GITRL further improved the protection efficiency of the vaccine. The adjuvant GITRL increased the quantity of the RagB specific antibody-forming cells in the spleen and bone marrow by raising the proportion of Tfh cells in spleen, which should further improved the production of specific antibodies. Meanwhile, the GITRL increased the ratio of IFN-γ+T cells in spleen. In summary, GITRL played a important role in enhancing protection function of ragB DNA vaccine against Porphyromonas gingivalis infection via two ways: The Tfh cells and the IFN-γ+T cells. Outer membrane protein ragB DNA vaccine was hopefully to prevent Porphyromonas gingivalis infection as well as the occurrence of periodontitis, thereby reducing the occurrence and development of atherosclerosis, diabetes and other diseases. The GITRL was expected to be the major candidate for vaccine adjuvants.
引文
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