Anti-VDAC3 recombinant antibody decreased human sperm motility and membrane integrity: A potential spermicide for contraception

Objective:To express recombinant protein that comprises an important fragment of human sperm specific voltage dependent anion channel 3 (VDAC3) protein as a potential molecule for generation of antibody, which can affect sperm function, aiming at spermicide development. Methods: The produce of VDAC3 recombinant protein encoded by cDNA sequence of human VDAC3 exon 5-8, based on experimental design of VDAC3 knock-out mice study. And after the purification of various human sperm VDAC3 recombinant proteins, epitope has been predicted in our recombinant protein determined by ElliPro program. Polyclonal antibody was produced for 14 wk. Then anti-VDAC3-exon 5-8 recombinant antiserum was inoculated to human sperm. After the process, antibody VDAC3 protein in human sperm was incubation with anti-VDAC3 recombinant antibody. Finally evaluation the effect of VDAC3 antiserum to human sperm motility and plasma membrane integrity was proceeded.Results: Human VDAC3 recombinant protein was successfully over-expressed in Escherichia coli and purified by affinity chromatography method. Purified human sperm VDAC3 recombinant protein could stimulate immune response in rabbit producing an antibody against VDAC3. Anti-VDAC3 recombinant antibody recognized VDAC3 antigen in human sperm could decrease human sperm motility and membrane integrity significantly.Conclusions:Anti-VDAC3 recombinant polyclonal antibody that we produced in rabbit by ourselves could decrease sperm motility and sperm membrane integrity. The authors suggest this polyclonal antibody could be used as a candidate agent for male contraception in the future. Furthermore, the authors intend to explore the effect of this antibody into sperm function aiming at male contraceptive vaccine development.

VDAC3与ABA受体蛋白相互作用的验证以及初步研究

酵母双杂交技术在拟南芥cDNA文库中筛选得到了与ABA受体蛋白RCAR1,RCAR3均有相互作用的蛋白质VDAC3,并已获得VDAC3基因的全长.VDAC3蛋白包含有3个结构域,分别为Porin3结构域,DUF3442结构域和SEG片段,现根据结构域分布,将VDAC3截短为包含DUF3442的VDAC3N以及包含SEG的VDAC3C两段.将VDAC3全长以及分别截短的169个氨基酸(VDAC3N)及105个氨基酸(VDAC3C)的片段进行酵母转化实验,与RCAR1或RCAR3共同转化后,VDAC3,VDAC3N的共转酵母能够在缺陷型培养基上生长,并且使X-Gal显色为蓝色,而VDAC3C则不能.这验证了全长的VDAC3与RCAR1,RCAR3的存在相互作用,并发现决定VDAC3蛋白与RCAR1,RCAR3是相互作用的结构域位于N端的DUF3442结构域.

电压依赖性阴离子通道VDAC3参与拟南芥先天免疫

线粒体外膜蛋白电压依赖性阴离子通道(VDAC)是动物细胞凋亡调控系统中的关键组分,但其在植物中的功能还不明确。拟南芥VDAC家族有4个成员,它们都能被病原菌诱导,VDAC1在由病原菌所引起的超敏性细胞死亡中发挥作用已有报道,但VDAC家族其他成员是否参与植物免疫反应还未见报道。本研究以拟南芥相关T-DNA插入突变体为材料,研究了VDAC3基因在植物抗病反应中的功能。病原相关分子模式flg22诱导的活性氧产生和胼胝质沉积在VDAC3突变体中都显著增强,表明VDAC3参与了病原相关分子模式触发的免疫反应。此外,效应因子激发的超敏性细胞死亡在vdac3突变体中也更明显,显示VDAC3也参与了效应子触发的免疫反应。以上结果说明,VDAC3在多个层面参与植物的抗病反应。

PAX3在缺氧状态下调控滋养细胞铁死亡的机制研究

目的探讨配对盒基因(PAX)3在缺氧状态下调控滋养细胞铁死亡的机制。方法该实验于2023年1月至12月在江南大学附属妇产医院优生优育医学研究所完成。分别在20%氧气的常氧状态下和2%氧气的缺氧状态下体外培养人绒毛膜滋养层细胞(HTR-8/SVneo),将HTR-8a/Svneo细胞按照不同的处理分为7组:常氧组、缺氧组、缺氧+NC质粒组、缺氧+PAX3(OE)组、缺氧+SLC40A1(OE)组、缺氧+VDAC3(OE)组、缺氧+PAX3(OE)+VDAC3(KD)组。运用质粒转染技术敲低电压依赖性阴离子通道亚型3(VDAC3)和过表达PAX3、SLC40A1、VDAC3来验证细胞中PAX3调控SLC40A1、VDAC3的表达。使用电子显微镜观察常氧状态、常氧+铁死亡诱导剂(Erastin)、缺氧状态下HTR-8/SVneo细胞线粒体情况。用活/死细胞双染色试剂盒检测细胞死亡率。采用免疫荧光细胞化学染色检测各组滋养细胞PAX3、SLC40A1、VDAC3的定位及表达。实时定量PCR法和Western blot法检测各组细胞PAX3、SLC40A1、VDAC3 mRNA和蛋白表达。使用独立样本t检验。结果(1)缺氧状态下的细胞线粒体结构与常氧状态+Erastin的线粒体结构相似,线粒体皱缩、体积减小,膜电子密度增高,内脊减少、模糊。(2)缺氧状态下,活/死细胞双染色试剂盒的结果显示细胞死亡率增加。免疫荧光结果显示细胞PAX3主要在胞核内表达,SLC40A1主要在胞浆内表达,VDAC3主要在线粒体中表达。(3)与常氧状态相比,缺氧处理组细胞死亡增多,HTR-8a/Svneo缺氧组中PAX3、SLC40A1 mRNA和蛋白表达量均降低;HTR-8a/Svneo缺氧组中VDAC3 mRNA和蛋白表达升高;敲低PAX3后,SLC40A1表达降低,细胞大量死亡;过表达PAX3后,SLC40A1表达增高,细胞存活率升高。结论缺氧会诱导滋养细胞铁死亡。PAX3通过调控滋养细胞铁死亡影响SLC40A1表达,但PAX3的敲除或过表达对VDAC3表达没有影响。

芪苈强心胶囊对心肌梗死大鼠心脏IP3Rs/GRP75/VDAC1基因调控的机制研究

目的:探讨芪苈强心胶囊对心肌梗死大鼠线粒体Ca^(2+)转运相关基因的调控作用。方法:采用冠状动脉左前降支结扎术建立心肌梗死大鼠模型。术后随机将动物分到模型组、芪苈强心组和卡托普利组;同时设有假手术组作为对照。治疗四周后,通过心脏大体结构观察大鼠心脏梗死范围,HE染色观察大鼠心肌组织病理形态变化;实时荧光(Real⁃Time)PCR检测大鼠心脏梗死边缘区线粒体Ca^(2+)转运相关基因三磷酸肌醇受体2(IP3R2),葡萄糖调节蛋白75(GRP75),电压依赖性阴离子通道1(VDAC1),线粒体融合蛋白2(Mfn2),以及线粒体凋亡相关基因B淋巴细胞瘤⁃2(Bcl⁃2),Bcl⁃2相关X蛋白(Bax)mRNA表达变化;Western blot检测心肌组织Bcl⁃2,Bax蛋白表达变化;TUNEL染色检测心肌组织细胞凋亡率。结果:与假手术组相比,模型组大鼠心脏左室前壁呈现大面积梗死区域,心肌组织结构紊乱;线粒体Ca^(2+)转运相关基因IP3R2,GRP75,VDAC1,Mfn2 mRNA表达显著升高(P<0.05,P<0.01);线粒体凋亡相关分子Bcl⁃2 mRNA和蛋白表达均明显下降(P<0.01),Bax mRNA和蛋白表达均显著升高(P<0.01),心肌细胞凋亡率显著增加(P<0.01)。与模型组相比,芪苈强心组和卡托普利组心脏大体标本的梗死范围缩小,心肌纤维排列相对规整;线粒体Ca^(2+)转运相关基因IP3R2,GRP75,VDAC1,Mfn2 mRNA表达显著降低(P<0.01);线粒体凋亡相关分子Bcl⁃2 mRNA和蛋白表达有所提高(P<0.05,P<0.01),Bax mRNA和蛋白表达显著降低(P<0.05,P<0.01),心肌细胞凋亡率明显下降(P<0.01)。结论:芪苈强心胶囊能够改善心肌梗死大鼠心脏形态结构,其作用机制与调节线粒体Ca^(2+)转运复合体IP3R2/GRP75/VDAC1基因表达,进而抑制细胞凋亡有关。

Mechanism of Qiliqiangxin capsule on the regulation of IP3Rs/GRP75/VDAC1 gene in myocardial infarction rat heart

Objective:To investigate the regulatory effect of Qiliqiangxin Capsule on mitochondrial Ca^(2+)related genes in rats with myocardial infarction(MI).Methods:The rat model of MI was established by ligation of the left anterior descending coronary artery.After operation,the rats were randomly assigned to the model group,the Qiliqiangxin group and the captopril group;a sham-operated group was also available as a control.After four weeks of treatment,the extent of infarction in rats was observed by gross cardiac structure and the morphological changes of myocardial histopathology were observed by HE staining.Detection of mitochondrial Ca^(2+)transport-related genes such as inositol-1,4,5-trisphosphate receptor 2(IP3R2),glucose regulated protein 75(GRP75),voltage-dependent anion channel 1(VDAC1),and mitofusion 2(Mfn2)and mitochondrial apoptosis-related genes such as B-cell lymphoma-2(Bcl-2)and Bcl-2 related X protein(Bax)mRNA expression changes was measured by RT-PCR in the infarct margins of the heart;Western blot was used to detect changes in Bcl-2,Bax protein expression in myocardial tissue.The rate of apoptosis in cardiac myocardial tissue was detected by TUNEL staining.Results:Compared with the sham group,the anterior left ventricular wall of the model group showed a large area of infarction,and the structure of myocardial tissue was disordered.The mRNA expression level of mitochondrial Ca^(2+)transport-related genes such as IP3R2,GRP75,VDAC1,and Mfn2 were significantly increased(P<0.05,P<0.01);The mRNA and protein expression of Bcl-2,a molecule related to mitochondrial apoptosis,were significantly decreased(P<0.01),while the mRNA and protein expression of Bax were significantly increased(P<0.01);and apoptosis rate was significantly increased(P<0.01).Compared with the model group,the infarct size of cardiac gross specimens in the Qiliqiangxin group and the captopril group was reduced and myocardial fibers were relatively well ordered;The mRNA expression of mitochondrial Ca^(2+)transport-related genes such as IP3R2,GRP75,VDAC1,and Mfn2 were significantly reduced(P<0.01);the mRNA and protein expression of Bcl-2,a molecule related to mitochondrial apoptosis,were increased(P<0.05,P<0.01),and the mRNA and protein expression of Bax were significantly decreased(P<0.05,P<0.01).and apoptosis rate was significantly decreased(P<0.01).Conclusion:Qiliqiangxin Capsule can improve the morphological structure of the heart of rats with MI,and its mechanism is related to regulation of the gene expression of mitochondrial Ca^(2+)transport complex IP3R2/GRP75/VDAC1,thereby inhibiting apoptosis.