Objective: To explore the application effect of optimized emergency nursing process in patients with hypertensive cerebral hemorrhage. Methods: A total of 74 patients with hypertensive cerebral hemorrhage admitted to the Emergency Department of Zhongda Hospital of Southeast University from March 2020 to January 2022 were selected as the study subjects.They were divided into an observation group and a control group based on a random number table method, with 37 patients in each group. The control group was given routine emergency nursing, while the observation group was given optimized emergency nursing process. The efficacy indicators of emergency care, the National Institutes of Health Stroke Scale（NIHSS） score, systolic blood pressure（SBP）, and diastolic blood pressure（DBP） were compared between the two groups. Results: The total time for triage and evaluation, emergency stay, electronic computed tomography examination, intravenous medication, and emergency treatment in the observation group was shorter than that in the control group, with statistically significant difference（P＜0.05）; After nursing,the NIHSS score in the two groups was lower than that before nursing, the NIHSS score in the observation group was lower than that in the control group, with statistically significant difference（P＜0.05）; After nursing, SBP and DBP in the observation group were lower than those in the control group, with statistically significant difference（P＜0.05）. Conclusion: The effect of optimized emergency nursing process for patients with hypertensive cerebral hemorrhage is significant. It can shorten emergency time,improve neurological function of patients, and decrease the levels of blood pressure.

Interleukin-6 (IL-6) overproduction has been considered to contribute to inflammatory damage of glomerular mesangial cells (GMCs) in human mesangial proliferative glomerulonephritis (MsPGN) and its rat model called Thy-1 nephritis (Thy-1N). However, the regulatory mechanisms of IL-6 expression in GMCs upon sublytic C5b-9 timulation remain poorly understood. We found that Krüppel-like factor 4 (KLF4) bound to the IL-6 promoter (−618 to −126 nt) and activated IL-6 gene transcription. Furthermore, lysine residue 224 of KLF4 was acetylated by p300/CBP-associated factor (PCAF), which was important for KLF4-mediated transactivation. Moreover, lysine residue 5 on histone H2B and lysine residue 9 on histone H3 at the IL-6 promoter were also acetylated by PCAF, which resulted in an increase in IL-6 transcription. Besides, NF-κB activation promoted IL-6 expression by elevating the expression of PCAF. Overall, these findings suggest that sublytic C5b-9-induced the expression of IL-6 involves KLF4-mediated transactivation, PCAF-mediated acetylation of KLF4 and histones, and NF-κB activation in GMCs.

孕产期抑郁症是围产期常见的并发症之一,严重影响母儿身心健康.目前,早期诊断孕产期抑郁症尤为困难,客观有效的生物学标志物对早发现早治疗孕产期抑郁症意义重大.近年来探索孕产期抑郁症早期诊断的生物标志物成为研究热点,主要在性激素、神经内分泌相关激素、免疫炎症分子、遗传及表观遗传学等方面.本文就近年来孕产期抑郁症生物学标志物的研究进展进行综述.

Rat Thy-1 nephritis (Thy-1N) is an experimental mesangial proliferative glomerulonephritis (MsPGN) for studying human MsPGN. Although sublytic C5b-9 complex formation on glomerular mesangial cells (GMCs) and renal MCP-1 and RANTES production in rats with Thy-1N have been proved, the role and mechanism of MCP-1 or RANTES synthesis in GMCs induced by sublytic C5b-9 are poorly elucidated. In this study, we first found the expression of transcription factor (KLF6), co-activator (KAT7) and chemokines (MCP-1 and RANTES) was all up-regulated both in renal tissue of Thy-1N rats (in vivo) and in sublytic C5b-9-induced GMCs (in vitro). Further in vitro experiments revealed that KLF6 bound to MCP-1 promoter (-297 to -123 nt) and RANTES promoter (-343 to -191 nt), leading to MCP-1 and RANTES gene transcription. Meanwhile, KAT7 also bound to the same region of MCP-1 and RANTES promoter in a KLF6-dependent manner, and KLF6 was acetylated by KAT7 at lysine residue 100, which finally promoted MCP-1 and RANTES expression. Moreover, our in vivo experiments discovered that knockdown of renal KAT7 or KLF6 gene obviously reduced MCP-1 and RANTES production, GMCs proliferation, ECM accumulation, and proteinuria secretion in Thy-1N rats. Collectively, our study indicates that sublytic C5b-9-induced MCP-1 and RANTES synthesis is associated with KAT7-mediated KLF6 acetylation and elevated KLF6 transcriptional activity, which might provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.

目的 绒毛外滋养细胞焦亡是否参与子痫前期的发生发展值得探讨.文章旨在构建H2 O2诱导滋养细胞焦亡模型,探讨绒毛外滋养细胞焦亡是否参与子痫前期的发生发展,为探究子痫前期发病机制提供新的方向. 方法 人滋养细胞HTR-8/SVneo培养于1640+10％胎牛血清+1％抗生素中,培养12 h后,给予H2 O2(100、150、200、250μmol/L)处理细胞(2、4、6、12 h),对照为1640+10％胎牛血清+1％抗生素正常培养细胞.提取细胞总蛋白,Western blot检测焦亡相关分子蛋白水平的表达;RT-qPCR检测细胞中焦亡相关分子mRNA水平;倒置相差显微镜观察细胞形态学改变. 结果 当H2 O2为150μmol/L,处理人滋养细胞4 h时,焦亡相关分子NLRP3、caspase1、Cleaved caspase1、GSDMD及IL-1β 的蛋白水平明显高于对照,随着作用时间的延长及浓度的增加,蛋白质表达水平被抑制;当H2 O2为150μmol/L,作用2 h时,焦亡经典通路中上游"启动"信号关键分子NLRP3及IL-1β 的mRNA水平较对照明显升高(P<0.000);4 h时,焦亡经典通路中的关键分子GSDMD的mRNA水平及下游炎症因子IL-18的mRNA水平明显高于对照组(P<0.05).通过焦亡相关分子mRNA水平的逆向验证,H2 O2诱导滋养细胞焦亡模型最佳条件为150μmol/L和4 h,在该条件下,光镜下可明显看到细胞肿胀、碎裂及质膜气泡形成等细胞焦亡典型改变. 结论 体外成功建立了氧化应激反应诱导滋养细胞焦亡模型,模拟了氧化应激反应诱导滋养细胞发生焦亡损伤的病生理过程,为后续子痫前期发病机制的研究提供实验基础.

Mesangioproliferative glomerulonephritis (MsPGN) is characterized by the proliferation of glomerular mesangial cells (GMCs) and accumulation of extracellular matrix (ECM), followed by glomerulosclerosis and renal failure of patients. Although our previous studies have demonstrated that sublytic C5b‐9 complex formed on the GMC membrane could trigger GMC proliferation and ECM expansion of rat Thy‐1 nephritis (Thy‐1N) as an animal model of MsPGN, their mechanisms are still not fully elucidated. In the present studies, we found that the levels of response gene to complement 32 (RGC‐32), myeloid zinc finger 1 (MZF1), phosphorylated extracellular signal‐regulated kinase 5 (phosphorylated ERK5, p‐ERK5), F‐box only protein 28 (FBXO28) and TNF receptor‐associated factor 6 (TRAF6) were all markedly up‐regulated both in the renal tissues of rats with Thy‐1N (in vivo) and in the GMCs upon sublytic C5b‐9 stimulation (in vitro). Further in vitro experiments revealed that up‐regulated FBXO28 and TRAF6 could form protein complex binding to ERK5 and enhance ERK5 K63‐ubiquitination and subsequent phosphorylation. Subsequently, ERK5 activation contributed to MZF1 expression and MZF1‐dependent RGC‐32 up‐regulation, finally resulting in GMC proliferative response. Furthermore, the MZF1‐binding element within RGC‐32 promoter and the functions of FBXO28 domains were identified. Additionally, knockdown of renal FBXO28, TRAF6, ERK5, MZF1 and RGC‐32 genes respectively markedly reduced GMC proliferation and ECM production in Thy‐1N rats. Together, these findings indicate that sublytic C5b‐9 induces GMC proliferative changes in rat Thy‐1N through ERK5/MZF1/RGC‐32 axis activated by the FBXO28‐TRAF6 complex, which might provide a new insight into MsPGN pathogenesis.

目的:检测早发型子痫前期患者血清中NLRP3、Caspase-1、IL-1β、IL-18的表达并探索其临床意义.方法:选取2016年1月至2018年1月在东南大学附属中大医院产科住院分娩的汉族单胎孕妇320例,子痫前期及正常妊娠孕妇各160例.比较纳入对象的临床资料,采用多因素Logistic回归方法分析子痫前期发病的高危因素.选取其中2017年7月至2017年12月间住院分娩汉族单胎孕妇30例,早发型子痫前期及正常妊娠孕妇各15例.采用ELISA法检测两组孕妇血清中NLRP3、Caspase-1、IL-1β、IL-18的表达情况.结果:1.子痫前期组与正常妊娠组孕妇及伴侣年龄、文化程度、孕前及分娩前的体重、体重指数(BMI)、高血压家族史有统计学差异(P<0.05).2.早发型子痫前期孕妇血清中NLRP3、Caspase-1、IL-1β、IL-18的表达水平较正常妊娠组均升高,其中IL-1β差异显著(P<0.05).结论:(1)低文化程度、孕前高体重指数及高血压家族史为子痫前期独立危险因素.(2)细胞焦亡与子痫前期的发病机制有重要联系,或可成为治疗子痫前期的新靶点.

Sublytic C5b-9 formation on glomerular mesangial cells in rat Thy-1 nephritis (Thy-1N), a model of human mesangioproliferative glomerulonephritis, is accompanied by the production of proinflammatory cytokines, but the relationship between sublytic C5b-9 and cytokine synthesis and the underlying mechanism remains unclear. To explore the problems mentioned above, in this study, we first examined the levels of proinflammatory ILs (e.g., IL-23 and IL-36a) as well as transcription factor (KLF4) and coactivator (PCAF) in the renal tissues of Thy-1N rats and in the glomerular mesangial cell line (HBZY-1) stimulated by sublytic C5b-9. Then, we further determined the role of KLF4 and PCAF in sublytic C5b-9-induced IL-23 and IL-36a production as well as the related mechanism. Our results showed that the levels of KLF4, PCAF, IL-23, and IL-36a were obviously elevated. Mechanistic investigation revealed that sublytic C5b-9 stimulation could increase IL-23 and IL-36a synthesis through KLF4 and PCAF upregulation, and KLF4 and PCAF could form a complex, binding to the IL-23 or IL-36a promoter in a KLF4-dependent manner, causing gene transcription. Importantly, KLF4 acetylation by PCAF contributed to sublytic C5b-9-induced IL-23 and IL-36a transcription. Besides, the KLF4 binding regions on IL-23 or IL-36a promoters and the KLF4 lysine site acetylated by PCAF were identified. Furthermore, silencing renal KLF4 or PCAF gene could significantly inhibit IL-23 or IL-36a secretion and tissue damage of Thy-1N rats. Collectively, these findings implicate that the KLF4/PCAF interaction and KLF4 acetylation by PCAF play a pivotal role in the sublytic C5b-9-mediated IL-23 and IL-36a production of Thy-1N rats.

目的:研究转录因子KLF4调控sublytic C5b-9刺激肾小球系膜细胞（glomerular mesangial cell,GMC）诱导促炎因子白介素-23（IL-23）生成的作用。方法:构建KLF4的短发夹状小干扰RNA（shKLF4）及过表达质粒（pIRES2-KLF4）。将pIRES2-KLF4或shKLF4转染GMC后再行sublytic C5b-9刺激,用qPCR、Western blot和ELISA法检查沉默或过表达KLF4基因后对GMC产生IL-23的影响。此外,构建IL-23基因近端启动子全长质粒,行荧光素酶报告基因实验测定沉默或过表达KLF4基因后对IL-23启动子活性的影响。结果:（1）Sublytic C5b-9刺激GMC后能明显促进IL-23的生成,而沉默KLF4基因后,由sublytic C5b-9诱导GMC产生的IL-23明显减少,但过表达KLF4后IL-23的水平则显著增加。（2）Sublytic C5b-9刺激GMC能显著上调IL-23的启动子活性,而沉默KLF4基因后由sublytic C5b-9诱导的IL-23启动子活性明显降低,但过表达KLF4后又能显著升高IL-23的启动子活性。结论:Sublytic C5b-9刺激诱导IL-23的生成可通过其刺激上调转录因子KLF4的表达而实现。

BACKGROUND/AIMS:The activation of complement system and the formation of C5b-9 complex have been confirmed in the glomeruli of patients with mesangioproliferative glomerulonephritis (MsPGN). However, the role and mechanism of C5b-9-induced injury in glomerular mesangial cell (GMC) are poorly understood. Rat Thy-1N is an animal model for studying MsPGN. It has been revealed that the attack of C5b-9 to the GMC in rat Thy-1N is sublytic, and sublytic C5b-9 can cause GMC apoptosis, but the underlying mechanism is not fully elucidated. To explore the role and regulatory mechanism of C5b-9 in MsPGN lesion, we used rat Thy-1N model and first detected the change of microRNA (miRNA) profiles both in Thy-1N rat renal tissues (in vivo) and in the cultured GMCs with sublytic C5b-9 stimulation (in vitro). Then we determined the effect of miR-3546, which increased both in vivo and in vitro, on GMC apoptosis upon sublytic C5b-9 as well as the involved mechanism.METHODS:Rat Thy-1N model was established and GMCs were treated with sublytic C5b-9. The rat renal cortex and the stimulated GMCs were obtained for miRNA microarray detection. Subsequently, the increased miRNAs were verified by real-time PCR. Meanwhile, to ascertain the ability of some miRNAs to upregulate cleaved caspase 3 and induce GMC apoptosis, the corresponding miRNA mimics were transfected into GMCs, followed by western blotting (WB) and flow cytometry mesurement. Thereafter, the miR-3546-targeted gene (SOX4) was predicted using bioinformatics approaches, and SOX4 expression in Thy-1N tissues and in the GMCs upon sublytic C5b-9 stimulation or miR-3546 mimic/inhibitor transfection were detected using real-time PCR and WB. To prove that miR-3546 can affect SOX4 gene transcription and SOX4 can regulate survivin expression, dual luciferase reporter assay, real-time PCR, WB and chromatin immunoprecipitation (ChIP) assays were performed. Furthermore, the role of miR-3546/SOX4/survivin axis in the GMC apoptosis induced by sublytic C5b-9 was examined using WB and flow cytometry.RESULTS:Compared with normal renal tissues and untreated GMCs, there were 43 and 62 upregulated miRNAs (> 2-fold) in Thy-1N tissues and sublytic C5b-9-stimulated GMCs respectively. A total of 17 miRNAs were increased both in vivo and in vitro, 11 of which were validated by real-time PCR. Among them, miR-3546 could markedly promote GMC apoptosis and inhibit SOX4 or survivin expression in response to sublytic C5b-9, and either SOX4 or survivin overexpression markedly rescued the GMC apoptosis mediated by miR-3546 mimic. Additionally, SOX4 overexpression could reverse the survivin suppression by miR-3546 mimic, and SOX4 could bind to survivin promoter (-1,278 to -853 nt) and activate survivin gene transcription.CONCLUSION:MiR-3546/ SOX4/survivin axis has a promoting role in the GMC apoptosis triggered by sublytic C5b-9, and our findings may provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.

目的:鉴定亚溶解型C5b-9刺激大鼠肾小球系膜细胞(GMC)诱导Kruppel样因子4(KLF4)与P300/CBP相关因子(PCAF)相互结合以及结合部位.方法:以亚溶解型C5b-9刺激GMC,应用免疫沉淀(IP)和免疫印迹(IB)法检测KLF4与PCAF相互结合情况.另构建KLF4、PCAF过表达和KLF4、PCAF不同结构域的质粒,将质粒转染GMC,应用IP和IB鉴定KLF4与PCAF的结合部位.结果与结论:亚溶解型C5b-9刺激GMC后能够诱导KLF4与PCAF相互结合,KLF4结合于PCAF转录结合结构域,而PCAF则结合在KLF4的转录激活结构域.

The apoptosis of glomerular mesangial cells (GMCs) in the early phase of rat Thy-1 nephritis (Thy-1N), a model of human mesangioproliferative glomerulonephritis (MsPGN), is primarily triggered by sublytic C5b-9. However, the mechanism of GMC apoptosis induced by sublytic C5b-9 remains unclear. In this study, we demonstrate that expressions of TNFR1-associated death domain–containing protein (TRADD) and IFN regulatory factor–1 (IRF-1) were simultaneously upregulated in the renal tissue of Thy-1N rats (in vivo) and in GMCs under sublytic C5b-9 stimulation (in vitro). In vitro, TRADD was confirmed to be a downstream gene of IRF-1, because IRF-1 could bind to TRADD gene promoter to promote its transcription, leading to caspase 8 activation and GMC apoptosis. Increased phosphorylation of p38 MAPK was verified to contribute to IRF-1 and TRADD production and caspase 8 activation, as well as to GMC apoptosis induced by sublytic C5b-9. Furthermore, phosphorylation of MEK kinase 2 (MEKK2) mediated p38 MAPK activation. More importantly, three sites (Ser153/164/239) of MEKK2 phosphorylation were identified and demonstrated to be necessary for p38 MAPK activation. In addition, silencing of renal MEKK2, IRF-1, and TRADD genes or inhibition of p38 MAPK activation in vivo had obvious inhibitory effects on GMC apoptosis, secondary proliferation, and urinary protein secretion in rats with Thy-1N. Collectively, these findings indicate that the cascade axis of MEKK2–p38 MAPK–IRF-1–TRADD–caspase 8 may play an important role in GMC apoptosis following exposure to sublytic C5b-9 in rat Thy-1N.

目的:构建大鼠生长阻滞及DNA损伤诱导基因45(growth arrest and DNA-damage-inducible gene 45,Gadd45)β/γ启动子(全长和截短)荧光素酶报告质粒,并观察在大鼠肾小球系膜细胞(glomerular messangial cells,GMCs)中过表达激活转录因子3(activating transcription factor 3,ATF3)后分别对大鼠Gadd45β/γ基因启动活性的影响.同时筛选其可能的ATF3结合位点.方法:采用PCR技术,将扩增出的大鼠Gadd45β基因启动子全长(-1 105 ～ +236 nt)和Gadd45γ基因启动子全长(-913～+72nt)分别插入到荧光素酶报告基因载体pGL3-basic中,获得Gadd45β/γ基因启动子全长荧光素酶报告质粒(pGL3-Gadd45β/γ-FL),再将pGL3-Gadd45β/γ-FL分别与课题组前期构建的大鼠野生型ATF3过表达质粒(pIRES2/ATF3)共转染GMCs,检测其荧光素酶活性,以确定ATF3对Gadd45β/γ基因的启动作用.另用生物信息学软件预测Gadd45β/γ基因启动子上ATF3潜在的结合位点,并据此构建4个Gadd45β和3个Gadd45γ基因启动子截短片段的荧光素酶报告质粒.将Gadd45β/γ基因启动子全长和各截短片段的荧光素酶报告质粒与pIRES2/ATF3共转染GMCs,再行荧光素酶活性测定,以筛选ATF3的结合位点.结果:菌液PCR及核酸测序证实,大鼠Gadd45β/-γ基因启动子(全长和截短)的荧光素酶报告质粒均构建成功.将pGL3-Gadd45β/γ-FL分别和pIRES2/ATF3共转染GMCs发现,Gadd45β/γ基因启动子活性均显著增加.而将Gadd45β启动子全长及4个截短质粒分别与pIRES2/ATF3共转染GMCs后显示,pGL3-Gadd45β-4的启动活性显著低于pGL3-Gadd45β-FL、pGL3-Gadd45β-1、pGL3-Gadd45β-2和pGL3-Gadd45β-3.提示ATF3可能结合在Gadd45β基因启动子的(-146～+23 nt)区域.同样,将Gadd45γ启动子全长及3个截短质粒分别与pIRES2/ATF3共转染GMCs后显示,pGL3-Gadd45γ-2、pGL3-Gadd45γ-3的启动活性显著低于pGL3-Gadd45γ-FL和pGL3-Gadd45γ-1.提示ATF3可能结合在Gadd45γ基因启动子的(-456～-61 nt)区域,且这段区域可能包含1个以上ATF3结合位点.结论:成功构建了大鼠Gadd45β/γ基因启动子全长及各截短片段荧光素酶报告质粒,并初步确定了ATF3在Gadd45β/γ基因启动子上的结合区域.

The apoptosis of glomerular mesangial cells (GMCs) is considered to be an important contributor to the initiation and development of rat Thy-1 nephritis (Thy-1N) and is accompanied by sublytic C5b-9 deposition. However, the mechanism by which sublytic C5b-9 triggers GMC apoptosis has not been elucidated. In this study, functional and histological examinations were performed on GMCs treated with sublytic C5b-9 (in vitro) and renal tissues of Thy-1N rats (in vivo). The in vitro studies found that sublytic C5b-9 could trigger GMC apoptosis through upregulating Egr-1, ATF3, and Gadd45 expression. Egr-1-mediated post-transcriptional modulation of ATF3, Egr-1/ATF3-enhanced Gadd45 promoter activity, and p300-mediated ATF3 acetylation were all involved in GMC apoptosis. More importantly, the effective binding elements for Egr-1 and ATF3 to Gadd45β/γ promoters and the ATF3 acetylation site were identified. In vivo, silencing renal p300, Egr-1, ATF3, and Gadd45β/γ significantly decreased GMC apoptosis, secondary GMC proliferation, and urinary protein secretion in Thy-1N rats. Together, these findings implicate that sublytic C5b-9-induced activation of Egr-1/p300-ATF3/Gadd45 axis plays a critical role in GMC apoptosis in Thy-1N rats.

目的:构建大鼠补体应答基因32(response gene to complement,RGC32)启动子(全长和截短)荧光素酶报告质粒,并观察人胚肾细胞(HEK293)过表达髓锌指基因1(myeloid zinc finger gene1,MZF1)对大鼠RGC32基因启动活性的影响.同时,筛选其可能的MZF1结合位点.方法:将RGC32基因启动子全长(-686～-1 nt)插入到荧光素酶报告基因载体pGL3-basic中,获得RGC32基因启动子全长荧光素酶报告质粒(pGL3-RGC32-FL)后,再将pGL3-RGC32-FL与本课题组前期构建的大鼠野生型MZF1表达质粒(pIRES2-EGFP-MZF1)共转染HEK293细胞,检测其荧光素酶活性,以确定MZF1对RGC32基因的启动作用.另用生物信息学软件预测RGC32基因启动子上转录因子MZF1潜在的结合位点,并据此构建3个RGC32基因启动子截短的荧光素酶报告质粒(即pGL3-RGC32-1 、pGL3-RGC32-2和pGL3-RGC32-3).将上述RGC32基因启动子全长和各截短的荧光素酶报告质粒与MZF1过表达质粒共转染HEK293细胞,再行荧光素酶活性测定,以筛选MZF1的结合位点.结果:菌液PCR及核酸测序证实,大鼠RGC32基因启动子(全长和各截短)的荧光素酶报告质粒均构建成功.将pGL3-RGC32-FL和pIRES2-EGFP-MZF1共转染HEK293细胞发现,RGC32基因启动子活性显著增加.而将pGL3-RGC32-FL、pGL3-RGC32-1、pGL3-RGC32-2和pGL3-RGC32-3分别与pIRES2-EGFP-MZF1共转染HEK293细胞后显示,pGL3-RGC32-3的启动活性显著低于pGL3-RGC32-FL、pGL3-RGC32-1和pGL3-RGC32-2.提示MZF1可能结合在RGC32基因启动子的-286～-86 nt区域.结论:成功构建了大鼠RGC32基因启动子全长及截短荧光素酶报告质粒,证实过表达MZF1可促进RGC32基因的启动,并初步筛查出转录因子MZF1在RGC32基因启动子上可能的结合区域.

目的:构建大鼠趋化因子CCL5基因启动子(全长和截短)荧光素酶报告质粒,检测大鼠肾小球系膜细胞(glomerular mesangial cell,GMC)中过表达Kruppel样转录因子6(Kruppel-like factor 6,KLF6)对CCL5基因启动子活性的影响.同时,筛选KLF6与CCL5基因启动子区的结合位点.方法:采用PCR技术,将扩增出的大鼠CCL5基因启动子全长序列(-1744nt ～-14nt)插入荧光素酶报告基因载体pGL3-basic中,获得CCL5基因启动子全长荧光素酶报告质粒(pGL3-CCL5-FL).然后,将pGL3-CCL5-FL与大鼠野生型KLF6表达质粒(pIRES2/KLF6)共转染GMC,测定其荧光素酶活性.另用生物信息学软件预测CCL5基因启动子上KLF6潜在的结合位点,并据此构建出4个CCL5基因启动子截短的荧光素酶报告质粒(即pGL3-CCL5-1～4).将上述CCL5基因启动子全长和各截短的荧光素酶报告质粒分别与KLF6过表达质粒共转染GMC,再行荧光素酶活性的测定,初筛KLF6可能的结合部位.结果:菌液PCR以及核酸测序结果证实,上述所有启动子荧光素酶报告质粒均构建成功.pGL3-CCL5-FL和pIRES2/KLF6共转染GMC结果显示,CCL5基因启动子的活性显著增强.pGL3-CCL5-FL、pGL3-CCL5-1～4分别与pIRES2/KLF6共转染GMC后发现,pGL3-CCL5-4的启动活性显著降低.提示KLF6可能结合在CCL5基因启动子的-343nt ～-191nt区域.结论:本实验成功构建了大鼠CCL5基因启动子全长及截短荧光素酶报告质粒,并初步筛查出KLF6在CCL5基因启动子上可能的结合部位在-343nt ～-191nt区域.