1.23KD rhALR表达、纯化、单克隆抗体制备及功能初步探索 2.rhALR对人肾小管上皮细胞缺氧复氧炎症反应干预及

发布时间：2018-06-30 08:35

本文选题：急性肾损伤 + 肝再生增强因子　；参考：《重庆医科大学》2015年博士论文

【摘要】：背景与目的肝再生增强因子(Augmenter of liver regeneration, ALR)是一种广泛存在于真核细胞的生长因子,不仅表达于肝脏,在肾脏也有表达。ALR含有两个亚型,分子量为15KD和23KD,分别存在于细胞浆和线粒体中。两种亚型含有共同的终止密码,不同的起始密码。23KD ALR存在于线粒体,参与细胞的能量代谢。目前对于ALR的生物学研究大多集中在15KD亚型上,但是对于23KD ALR的功能研究很少。本课题组前期研究发现15KD ALR在大鼠急性肾损伤后6h在髓质肾小管区开始升高,72h恢复正常水平；体外能够抑制肾小管上皮细胞凋亡及促进其增殖,提示15KD ALR在维持正常肾小管功能上发挥重要作用。由于急性肾损伤发生早期,常伴有线粒体功能紊乱,而且23KDALR与15KD ALR在序列上具有极高的相似性,因此我们推测23KDALR与15KD ALR是否存在同样抗凋亡、促增殖的作用。本研究中,我们拟通过生物工程学技术,构建、表达23KD rhALR,并制备单克隆抗体。为研究23KD ALR生物学功能提供实验基础,并初步比较与15KD ALR生物学功能差异。方法1.构建重组质粒pET28a-hALR:选取带有6-His标签的原核表达质粒pET28a,提取HepG2细胞总RNA,逆转录为cDNA,应用PCR方法,按照设计的引物扩增出含有BamH I/XhoI酶切位点的目的基因片段。通过双酶切目的片段和载体,在T4连接酶的作用下进行目的基因片段与载体连接。构建的重组质粒经过PCR、双酶切及基因测序方法鉴定。2.重组蛋白表达：选取最适时间点和最适IPTG浓度,诱导大肠杆菌BL21表达23KDrhALR蛋白,通过亲和层析技术获得纯化的目的蛋白。并对表达的蛋白进行Western blot检测。收集纯化的蛋白,将蛋白置于透析袋中,4℃进行透析。3.选取雌性Balb/c小鼠作为免疫动物。通过动物免疫、细胞融合、杂交瘤细胞筛选,获取能产生高滴度单抗的细胞株。将该细胞株注射入经降植烷预处理的小鼠腹腔,收集腹水得到相应单克隆抗体。应用ELISA、Western blot、快速定性试纸检测方法检测抗体的效价及Ig亚型。4.比较15KD 和 23KD rhALR对肝癌细胞增殖作用的影响。并在课题组前期研究的基础上,采用顺铂诱导HepG2凋亡,给予15KD和23KD rhALR后,流式细胞仪检测细胞凋亡情况。5.收集正常人和AKI患者的血液和尿液,应用间接ELISA方法检测其中23KD ALR的含量。结果1.成功构建pET28a-hALR重组质粒。将pET28a和pET28a-hALR同时进行PCR和双酶切鉴定。结果空质粒经PCR扩增和双酶切后未见目的条带,而重组质粒均有与预期大小相符的目的条带。重组质粒基因测序结果与pubme d数据库进行Blast比对,未发现目的片段基因有碱基缺失、插入和移位等改变。2.将重组质粒转化入BL21大肠杆菌中,以不同浓度IPTG和不同时间点诱导表达,用SDS-PAGE和Western blot检测蛋白表达情况。结果显示在IPTG浓度为0.6mM,诱导时间为5h时重组蛋白产量最高。3.收集诱导表达5h菌体,重悬于含溶菌酶的Binding buffer中,进行超声碎菌。收集上清进行亲和层析纯化目的蛋白。收集最后洗脱液,进行透析复性。4.通过免疫动物,获得致敏小鼠脾细胞,与预先用8-AG筛选好的Sp2/0细胞进行细胞融合获得杂交瘤细胞。杂交瘤细胞经过HAT、HT培养基筛选及有限稀释法培养后,获得特异性、稳定性杂交瘤细胞株2C11。将该细胞株注射入经降植烷预处理小鼠腹腔内,小鼠产生腹水。腹水效价为10-5；抗体亚类重链为IgG1,轻链kappa链。间接ELISA和Western blot结果显示2C11分泌的单抗特异性良好。5.15KD rhALR能够促进肝癌细胞增殖,而23KD rhALR对肝癌细胞增殖无影响；15KD rhALR能够抑制顺铂引起的HepG2细胞凋亡,23KD rhALR对凋亡无影响。6.采用间接ELISA法检测正常人和AKI患者血液以及尿液,结果均为阴性。结论1.成功构建了23KD hALR原核表达质粒pET28a-hALR。2.成功诱导23KD rhALR表达,并通过亲和层析技术获得纯化的目的蛋白,为抗体制备提供了基础。3.通过单克隆抗体制备技术,获得稳定分泌23KD rhALR单克隆抗体细胞株2C11一株,为研究23KD ALR生物学功能提供了实验基础。4.外源性23KD rhALR不能促进肝癌细胞增殖,同时也不能抑制顺铂诱导的肝癌细胞凋亡。提示15KD ALR和23KD ALR存在功能差异；5.采用间接ELISA法检测正常人和急性肾损伤患者体液中23KDALR表达情况,均为阴性结果。背景与目的急性肾损伤是一种高发病率、高致死率的综合征。临床上多种因素均可引起急性肾损伤,包括感染、肾毒性药物、手术、失血等。虽然避免发病原因,可以较大程度上避免疾病的发生,但是目前对于急性肾损伤仍然缺乏相应的治疗手段,一旦疾病发生常常会导致无法换回的结果。肾脏缺血再灌注损伤是急性肾损伤最常见的原因之一,实验已经证实,肾脏发生缺血再灌注后,肾小管上皮细胞出现明显的凋亡、坏死；肾实质常伴有炎症细胞的明显浸润；实验动物在肾脏发生缺血再灌注损伤后往往也处于炎症反应状态。、因此缺血再灌注损伤中,炎症反应是疾病发生发展的中心环节。丝裂原活化蛋白激酶信号通路参与多种细胞生理、病理反应,也是参与炎症调节的重要信号通路。ERK,p38,JNK是该通路主要调控分子,它们与NF-κB一起组成调节机体炎症平衡的主要途径。肝再生增强因子是一种生长因子。前期研究己发现,15KD肝再生增强因子在缺血再灌注大鼠中表现出肾保护作用；在对体外经缺氧复氧处理后的大鼠肾小管上皮细胞也具有下调NF-κB表达,抑制炎症反应的作用。本研究为进一步探讨15KD ALR对人肾小管上皮细胞缺氧复氧反应及机制,我们拟通过对缺氧复氧模型细胞外源性加入15KD rhALR,并用其抗体阻断以及shRNA内源性干扰的方式观察细胞ALR表达、缺氧复氧后炎症因子的表达及ERK, p38, JNK信号通路和NF-κB核转位的变化情况,深入了解ALR在细胞缺氧复氧导致的炎症反应中的抗炎作用及机制。方法1.人肾小管上皮细胞(HK-2)为研究对象,以shRNA转染HK-2细胞72h作为内源性干扰ALR表达的实验方法。下一步实验将HK-2细胞随机分为A、B两组。A组：正常组,模型组,15KD rhALR处理组,15KD rhALR+15KD ALR抗体组；B组：正常组,模型组,shRNA/control组,shRNA/ALR组。所有细胞在缺氧复氧前,均经过无血清同步化处理24h,同步化后换为完全培养基；在前期研究的基础上,除正常组细胞外的其他细胞均进行缺氧6h,复氧12h处理。2.为研究外源性与内源性ALR的联系,将HK-2细胞分为正常组,15KD rhALR处理组(25μg/ml,50μg/ml)。外源性15KD rhALR处理24h后应用Real-time PCR 和 Western blot观察内源性ALR的变化。3.MTS方法检测转染shRNA后,HK-2细胞增殖率变化情况。4. ELISA和Real time PCR检测TNF-α、IL-6、MCP-1的蛋白和基因表达水平。5. Western blot检测pERK/ERK, pp38/p38, pJNK/JNK, NF-κB p65表达情况。6.激光共聚焦观察NF-κB p65的核转位。结果1. shRNA/ALR干扰HK-2细胞内源性ALR表达,荧光显微镜下观察,可见转染效率大于80%. Real time PCR和Western blot结果显示与正常HK-2细胞比较,shRNA/ALR组ALR基因和蛋白水平明显下调(P0.05), shRNA/control组中ALR的表达无显著改变(P0.05)。2.外源性加入15KD rhALR和shRNA转染后,对HK-2细胞增殖无明显影响。3.与模型组相比,15KD rhALR处理组TNF-α、IL-6、MCP-1蛋白和基因表达水平明显降低(P0.05)。与15KD rhALR处理组比较,15KD rhALR+15KD ALR抗体组TNF-α、IL-6、MCP-1蛋白和基因表达水平明显升高(P0.05)。4.与模型组相比,15KD rhALR处理组ERK,p38,JNK磷酸化水平降低(P0.05)。与15KD rhALR处理组比较,15KD rhALR+15KD ALR抗体组ERK, p38, JNK磷酸化水平增加(P0.05)。NF-κB p65细胞核内表达情况,与模型组相比,15KD rhALR处理组细胞核中NF-κB p65表达水平降低(P0.05)。与15KD rhALR处理组相比,15KD rhALR +15KD ALR抗体组细胞核中NF-κB p65表达水平增加(P0.05)。5.与shRNA/control组比较,shRNA/ALR 组 TNF-α IL-6、MCP-1蛋白和基因表达水平明显降低(P0.05)。6.与shRNA/control组比较,shRNA/ALR组ERK, p38, JNK磷酸化水平明显下降(P0.05)。shRNA/ALR组细胞核中TNF-κB p65表达水平较shRNA/control组明显下降(P0.05)。应用激光共聚焦方法观察NF-κB p65核转位情况,其结果与Western b lot结果一致。7.与正常组比较,HK-2细胞经过缺氧复氧后,内源性ALR在蛋白和基因水平,表达均增加(P0.05)。外源性加入15KD rhALR(25μg/ml, 50μg/ml)处理细胞后,其内源性ALR的表达水平呈现随外源性ALR的浓度增加而减少的趋势(P0.05)。结论1.外源性15KD rhALR可一定程度上降低正常情况培养下HK-2细胞内源性ALR的表达。2.缺氧复氧能够导致HK-2细胞炎症反应；外源性加入15KD rhALR能够减轻炎症因子的产生。外源性15KD rhALR被其抗体阻断后,可部分抵消其抑制炎症的作用。3.外源性15KD rhALR降低MAPKs通路中ERK, p38, JNK的磷酸化,减少NF-KB核转位,从而减少下游炎症因子的产生。4. shRNA/ALR能够显著干扰内源性ALR的表达。干扰内源性ALR可减轻缺氧复氧导致的HK-2细胞炎症反应。5.干扰内源性ALR表达能够抑制MAPKs通路中ERK, p38, JNK的磷酸化, NF-κB核转位减少,减轻HK-2细胞炎症反应。
[Abstract]:BACKGROUND & OBJECTIVE : Augmenter of liver regeneration is a kind of growth factor widely present in eukaryotic cells , not only in the liver but also in the kidney . The two isoforms contain two subtypes , the molecular weight is 15KD and 23KD respectively . The two isoforms contain common termination codes and different starting codons .
coli BL21 ( DE3 ) . The results showed that the expression of recombinant plasmid pET28a - hsupramolecule was determined by means of PCR , Western blot and rapid qualitative test . The recombinant plasmid was constructed by PCR , Western blot and rapid qualitative test . The expression of recombinant protein was detected by SDS - PAGE and Western blot . The results showed that the recombinant protein production was the highest when IPTG concentration was 0.6 mM and the induction time was 5 h .
The antibody subclass heavy chain was IgG1 , light chain kappa chain . Indirect ELISA and Western blot showed that 2C11 secreted monoclonal antibody was good .
Conclusion 1 . The recombinant plasmid pET28a - hADr2.2 has been successfully induced by the monoclonal antibody preparation , and the result is negative . Conclusion 1 . The recombinant plasmid pET28a - hADr2.2 has been successfully induced by indirect ELISA .
5 . The results of indirect ELISA for detecting the expression of 23KRUR in the body fluid of normal people and acute kidney injury are all negative . The background and purpose of acute renal injury are the syndrome of high incidence and high mortality . Although the cause of the disease is avoided , the disease can be avoided to a greater extent .
The renal parenchyma is often accompanied by the obvious infiltration of inflammatory cells ;
The activation of mitogen - activated protein kinase signal pathway is the central part of the development of the disease . The mitogen - activated protein kinase signaling pathway is a key regulatory molecule for the development of the disease .
In this study , we studied the anti - inflammatory effect and mechanism of 15KD alr on human renal tubular epithelial cells in vitro . In this study , we studied the anti - inflammatory effect and mechanism of 15KD alr on human renal tubular epithelial cells . Methods 1 . The cells of human renal tubular epithelial cells ( HK - 2 ) were randomly divided into two groups A and B .
group B : normal group , model group , shRNA / control group , shRNA / alr group . All the cells were treated with serum - free synchronized treatment for 24 h before hypoxia and oxygen - enriched and replaced with complete medium after synchronization .
On the basis of the previous study , the cells except the normal group were treated with hypoxia for 6 h and 12 h for 12 h . To study the relationship between exogenous and endogenous alr , HK - 2 cells were divided into normal group and 15KD rhalr treatment group ( 25渭g / ml , 50渭g / ml ) . After 24 h treatment with exogenous 15 KD rhADr , real - time PCR and Western blot were used to observe the changes of endogenous alr . 3 . The change of proliferation rate of HK - 2 cells was observed after the transfection of shRNA was detected by MTS . The levels of protein and gene expression of TNF - 伪 , IL - 6 , MCP - 1 were detected by ELISA and Real time PCR . The expression of pERK / ERK , pp38 / p38 , p38 , p38 , p38 , p38 , p38 , p38 , p38 and NF - 魏B was detected by Western blot , and the nuclear translocation of NF - 魏B was observed by laser co - focusing . The results showed that the expression of endogenous alr in HK - 2 cells was observed under the fluorescence microscope , and the transfection efficiency was more than 80 % . 3 . Compared with the model group , the expression of TNF - 伪 , IL - 6 , MCP - 1 and MCP - 1 were significantly lower than those in the model group ( P0.05 ) . Compared with the model group , the level of ERK , p38 , and the phosphorylation of the protein and protein in the nucleus of the 15KD rhalr + 15KD alr group were significantly decreased ( P0.05 ) .
3 . The exogenous 15 KD rhADr decreased the phosphorylation of ERK , p38 MAPK in the MAPKs pathway and reduced the NF - KB nuclear translocation , thus reducing the inflammatory response of HK - 2 cells induced by hypoxia and complex oxygen .
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R692.5

【相似文献】