發(fā)布時間：2018-06-30 08:35

  本文選題：急性腎損傷 + 肝再生增強因子��； 參考：《重慶醫(yī)科大學》2015年博士論文

【摘要】：背景與目的肝再生增強因子(Augmenter of liver regeneration, ALR)是一種廣泛存在于真核細胞的生長因子,不僅表達于肝臟,在腎臟也有表達。ALR含有兩個亞型,分子量為15KD和23KD,分別存在于細胞漿和線粒體中。兩種亞型含有共同的終止密碼,不同的起始密碼。23KD ALR存在于線粒體,參與細胞的能量代謝。目前對于ALR的生物學研究大多集中在15KD亞型上,但是對于23KD ALR的功能研究很少。本課題組前期研究發(fā)現(xiàn)15KD ALR在大鼠急性腎損傷后6h在髓質(zhì)腎小管區(qū)開始升高,72h恢復(fù)正常水平；體外能夠抑制腎小管上皮細胞凋亡及促進其增殖,提示15KD ALR在維持正常腎小管功能上發(fā)揮重要作用。由于急性腎損傷發(fā)生早期,常伴有線粒體功能紊亂,而且23KDALR與15KD ALR在序列上具有極高的相似性,因此我們推測23KDALR與15KD ALR是否存在同樣抗凋亡、促增殖的作用。本研究中,我們擬通過生物工程學技術(shù),構(gòu)建、表達23KD rhALR,并制備單克隆抗體。為研究23KD ALR生物學功能提供實驗基礎(chǔ),并初步比較與15KD ALR生物學功能差異。方法1.構(gòu)建重組質(zhì)粒pET28a-hALR:選取帶有6-His標簽的原核表達質(zhì)粒pET28a,提取HepG2細胞總RNA,逆轉(zhuǎn)錄為cDNA,應(yīng)用PCR方法,按照設(shè)計的引物擴增出含有BamH I/XhoI酶切位點的目的基因片段。通過雙酶切目的片段和載體,在T4連接酶的作用下進行目的基因片段與載體連接。構(gòu)建的重組質(zhì)粒經(jīng)過PCR、雙酶切及基因測序方法鑒定。2.重組蛋白表達：選取最適時間點和最適IPTG濃度,誘導(dǎo)大腸桿菌BL21表達23KDrhALR蛋白,通過親和層析技術(shù)獲得純化的目的蛋白。并對表達的蛋白進行Western blot檢測。收集純化的蛋白,將蛋白置于透析袋中,4℃進行透析。3.選取雌性Balb/c小鼠作為免疫動物。通過動物免疫、細胞融合、雜交瘤細胞篩選,獲取能產(chǎn)生高滴度單抗的細胞株。將該細胞株注射入經(jīng)降植烷預(yù)處理的小鼠腹腔,收集腹水得到相應(yīng)單克隆抗體。應(yīng)用ELISA、Western blot、快速定性試紙檢測方法檢測抗體的效價及Ig亞型。4.比較15KD 和 23KD rhALR對肝癌細胞增殖作用的影響。并在課題組前期研究的基礎(chǔ)上,采用順鉑誘導(dǎo)HepG2凋亡,給予15KD和23KD rhALR后,流式細胞儀檢測細胞凋亡情況。5.收集正常人和AKI患者的血液和尿液,應(yīng)用間接ELISA方法檢測其中23KD ALR的含量。結(jié)果1.成功構(gòu)建pET28a-hALR重組質(zhì)粒。將pET28a和pET28a-hALR同時進行PCR和雙酶切鑒定。結(jié)果空質(zhì)粒經(jīng)PCR擴增和雙酶切后未見目的條帶,而重組質(zhì)粒均有與預(yù)期大小相符的目的條帶。重組質(zhì)�；驕y序結(jié)果與pubme d數(shù)據(jù)庫進行Blast比對,未發(fā)現(xiàn)目的片段基因有堿基缺失、插入和移位等改變。2.將重組質(zhì)粒轉(zhuǎn)化入BL21大腸桿菌中,以不同濃度IPTG和不同時間點誘導(dǎo)表達,用SDS-PAGE和Western blot檢測蛋白表達情況。結(jié)果顯示在IPTG濃度為0.6mM,誘導(dǎo)時間為5h時重組蛋白產(chǎn)量最高。3.收集誘導(dǎo)表達5h菌體,重懸于含溶菌酶的Binding buffer中,進行超聲碎菌。收集上清進行親和層析純化目的蛋白。收集最后洗脫液,進行透析復(fù)性。4.通過免疫動物,獲得致敏小鼠脾細胞,與預(yù)先用8-AG篩選好的Sp2/0細胞進行細胞融合獲得雜交瘤細胞。雜交瘤細胞經(jīng)過HAT、HT培養(yǎng)基篩選及有限稀釋法培養(yǎng)后,獲得特異性、穩(wěn)定性雜交瘤細胞株2C11。將該細胞株注射入經(jīng)降植烷預(yù)處理小鼠腹腔內(nèi),小鼠產(chǎn)生腹水。腹水效價為10-5；抗體亞類重鏈為IgG1,輕鏈kappa鏈。間接ELISA和Western blot結(jié)果顯示2C11分泌的單抗特異性良好。5.15KD rhALR能夠促進肝癌細胞增殖,而23KD rhALR對肝癌細胞增殖無影響；15KD rhALR能夠抑制順鉑引起的HepG2細胞凋亡,23KD rhALR對凋亡無影響。6.采用間接ELISA法檢測正常人和AKI患者血液以及尿液,結(jié)果均為陰性。結(jié)論1.成功構(gòu)建了23KD hALR原核表達質(zhì)粒pET28a-hALR。2.成功誘導(dǎo)23KD rhALR表達,并通過親和層析技術(shù)獲得純化的目的蛋白,為抗體制備提供了基礎(chǔ)。3.通過單克隆抗體制備技術(shù),獲得穩(wěn)定分泌23KD rhALR單克隆抗體細胞株2C11一株,為研究23KD ALR生物學功能提供了實驗基礎(chǔ)。4.外源性23KD rhALR不能促進肝癌細胞增殖,同時也不能抑制順鉑誘導(dǎo)的肝癌細胞凋亡。提示15KD ALR和23KD ALR存在功能差異；5.采用間接ELISA法檢測正常人和急性腎損傷患者體液中23KDALR表達情況,均為陰性結(jié)果。背景與目的急性腎損傷是一種高發(fā)病率、高致死率的綜合征。臨床上多種因素均可引起急性腎損傷,包括感染、腎毒性藥物、手術(shù)、失血等。雖然避免發(fā)病原因,可以較大程度上避免疾病的發(fā)生,但是目前對于急性腎損傷仍然缺乏相應(yīng)的治療手段,一旦疾病發(fā)生常常會導(dǎo)致無法換回的結(jié)果。腎臟缺血再灌注損傷是急性腎損傷最常見的原因之一,實驗已經(jīng)證實,腎臟發(fā)生缺血再灌注后,腎小管上皮細胞出現(xiàn)明顯的凋亡、壞死；腎實質(zhì)常伴有炎癥細胞的明顯浸潤；實驗動物在腎臟發(fā)生缺血再灌注損傷后往往也處于炎癥反應(yīng)狀態(tài)。、因此缺血再灌注損傷中,炎癥反應(yīng)是疾病發(fā)生發(fā)展的中心環(huán)節(jié)。絲裂原活化蛋白激酶信號通路參與多種細胞生理、病理反應(yīng),也是參與炎癥調(diào)節(jié)的重要信號通路。ERK,p38,JNK是該通路主要調(diào)控分子,它們與NF-κB一起組成調(diào)節(jié)機體炎癥平衡的主要途徑。肝再生增強因子是一種生長因子。前期研究己發(fā)現(xiàn),15KD肝再生增強因子在缺血再灌注大鼠中表現(xiàn)出腎保護作用；在對體外經(jīng)缺氧復(fù)氧處理后的大鼠腎小管上皮細胞也具有下調(diào)NF-κB表達,抑制炎癥反應(yīng)的作用。本研究為進一步探討15KD ALR對人腎小管上皮細胞缺氧復(fù)氧反應(yīng)及機制,我們擬通過對缺氧復(fù)氧模型細胞外源性加入15KD rhALR,并用其抗體阻斷以及shRNA內(nèi)源性干擾的方式觀察細胞ALR表達、缺氧復(fù)氧后炎癥因子的表達及ERK, p38, JNK信號通路和NF-κB核轉(zhuǎn)位的變化情況,深入了解ALR在細胞缺氧復(fù)氧導(dǎo)致的炎癥反應(yīng)中的抗炎作用及機制。方法1.人腎小管上皮細胞(HK-2)為研究對象,以shRNA轉(zhuǎn)染HK-2細胞72h作為內(nèi)源性干擾ALR表達的實驗方法。下一步實驗將HK-2細胞隨機分為A、B兩組。A組：正常組,模型組,15KD rhALR處理組,15KD rhALR+15KD ALR抗體組；B組：正常組,模型組,shRNA/control組,shRNA/ALR組。所有細胞在缺氧復(fù)氧前,均經(jīng)過無血清同步化處理24h,同步化后換為完全培養(yǎng)基；在前期研究的基礎(chǔ)上,除正常組細胞外的其他細胞均進行缺氧6h,復(fù)氧12h處理。2.為研究外源性與內(nèi)源性ALR的聯(lián)系,將HK-2細胞分為正常組,15KD rhALR處理組(25μg/ml,50μg/ml)。外源性15KD rhALR處理24h后應(yīng)用Real-time PCR 和 Western blot觀察內(nèi)源性ALR的變化。3.MTS方法檢測轉(zhuǎn)染shRNA后,HK-2細胞增殖率變化情況。4. ELISA和Real time PCR檢測TNF-α、IL-6、MCP-1的蛋白和基因表達水平。5. Western blot檢測pERK/ERK, pp38/p38, pJNK/JNK, NF-κB p65表達情況。6.激光共聚焦觀察NF-κB p65的核轉(zhuǎn)位。結(jié)果1. shRNA/ALR干擾HK-2細胞內(nèi)源性ALR表達,熒光顯微鏡下觀察,可見轉(zhuǎn)染效率大于80%. Real time PCR和Western blot結(jié)果顯示與正常HK-2細胞比較,shRNA/ALR組ALR基因和蛋白水平明顯下調(diào)(P0.05), shRNA/control組中ALR的表達無顯著改變(P0.05)。2.外源性加入15KD rhALR和shRNA轉(zhuǎn)染后,對HK-2細胞增殖無明顯影響。3.與模型組相比,15KD rhALR處理組TNF-α、IL-6、MCP-1蛋白和基因表達水平明顯降低(P0.05)。 與15KD rhALR處理組比較,15KD rhALR+15KD ALR抗體組TNF-α、IL-6、MCP-1蛋白和基因表達水平明顯升高(P0.05)。4.與模型組相比,15KD rhALR處理組ERK,p38,JNK磷酸化水平降低(P0.05)。與15KD rhALR處理組比較,15KD rhALR+15KD ALR抗體組ERK, p38, JNK磷酸化水平增加(P0.05)。NF-κB p65細胞核內(nèi)表達情況,與模型組相比,15KD rhALR處理組細胞核中NF-κB p65表達水平降低(P0.05)。與15KD rhALR處理組相比,15KD rhALR +15KD ALR抗體組細胞核中NF-κB p65表達水平增加(P0.05)。5.與shRNA/control組比較,shRNA/ALR 組 TNF-α IL-6、MCP-1蛋白和基因表達水平明顯降低(P0.05)。6.與shRNA/control組比較,shRNA/ALR組ERK, p38, JNK磷酸化水平明顯下降(P0.05)。shRNA/ALR組細胞核中TNF-κB p65表達水平較shRNA/control組明顯下降(P0.05)。應(yīng)用激光共聚焦方法觀察NF-κB p65核轉(zhuǎn)位情況,其結(jié)果與Western b lot結(jié)果一致。7.與正常組比較,HK-2細胞經(jīng)過缺氧復(fù)氧后,內(nèi)源性ALR在蛋白和基因水平,表達均增加(P0.05)。外源性加入15KD rhALR(25μg/ml, 50μg/ml)處理細胞后,其內(nèi)源性ALR的表達水平呈現(xiàn)隨外源性ALR的濃度增加而減少的趨勢(P0.05)。結(jié)論1.外源性15KD rhALR可一定程度上降低正常情況培養(yǎng)下HK-2細胞內(nèi)源性ALR的表達。2.缺氧復(fù)氧能夠?qū)е翲K-2細胞炎癥反應(yīng)；外源性加入15KD rhALR能夠減輕炎癥因子的產(chǎn)生。外源性15KD rhALR被其抗體阻斷后,可部分抵消其抑制炎癥的作用。3.外源性15KD rhALR降低MAPKs通路中ERK, p38, JNK的磷酸化,減少NF-KB核轉(zhuǎn)位,從而減少下游炎癥因子的產(chǎn)生。4. shRNA/ALR能夠顯著干擾內(nèi)源性ALR的表達。干擾內(nèi)源性ALR可減輕缺氧復(fù)氧導(dǎo)致的HK-2細胞炎癥反應(yīng)。5.干擾內(nèi)源性ALR表達能夠抑制MAPKs通路中ERK, p38, JNK的磷酸化, NF-κB核轉(zhuǎn)位減少,減輕HK-2細胞炎癥反應(yīng)。
[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 - 偽 , 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 - 偽 , 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 .
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R692.5

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