miRNA-34c靶向ACSL1影响肝星状细胞活化的研究

发布时间：2018-03-01 02:12

本文关键词： 酶原位消化法密度梯度离心法肝星状细胞 miR-34c ACSL1 Type I collage α-SMA　出处：《第二军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究目的: 本课题组在前期研究中构建DMN大鼠肝纤维化模型，通过miRNA表达谱芯片检测，发现： miR-34c可能在肝纤维化进程中发挥重要作用，且靶基因预测系统推测，ACSL1可能是miR-34c的作用靶点。因之，本实验：将通过萤光素酶报告系统进一步验证ACSL1是否为miR-34c的作用靶点；检测miR-34c和ACSL1在不同状态肝星状细胞（Hepatic Stellate Cell，HSC）中的表达水平；同时在活化态HSC中下调miR-34c的基因表达水平，研究miR-34c调控靶基因ACSL1对HSC活化的影响作用。研究方法：第一部分：大鼠原代HSC的提取与鉴定 1、采用Friedman酶消化-密度梯度离心法：（1）对雄性SD大鼠行麻醉后，分离门静脉进行插管，依次灌注D-Hank’s液、链酶蛋白酶消化液和胶原酶消化液；（2）将肝脏充分消化后过滤，离心，，用Nycodenz和DNase I液调整细胞悬液的密度，使密度达到1.040-1.055g/ml之间，铺上无Ga的Gass液，提取星状细胞；（3）细胞计数后，按比例接种于细胞皿中，放入CO2细胞培养箱中培养传代。 2、在328nm紫外光的激发下显微镜观察HSC。 3、细胞免疫荧光法检测培养2天、14天HSC中Desmin和α-SMA的表达。第二部分：miR-34c与ACSL1的靶向关系检测及其在不同状态HSC中表达水平的检测 1、在课题组前期工作的基础上，利用萤光素酶靶向报告系统验证miR-34c和ACSL1是否存在靶向关系； 2、实时定量PCR检测miR-34c和ACSL1在不同状态的HSC中的表达水平。第三部分：下调HSC中miR-34c表达对ACSL1表达及对HSC活化相关指标的影响 1、使用miR-34c inhibitor转染活化态HSC（培养至第14天者），同时用空白组、NC组作为对照，并采用萤光转染指标剂检测转染效率； 2、PCR检测转染后HSC中miR-34c和ACSL1的表达水平； 3、Western-blot技术检测转染后HSC中ACSL1蛋白表达水平； 4、利用实时定量PCR和Western-blot检测转染后HSC活化相关指标Type I collagen与α-SMAR mRNA及蛋白水平的表达。研究结果： 1、本实验采用酶原位消化法和密度梯度离心法，每只SD大鼠可获得HSC约5-7×107； HSC在328nm紫外光的激发下发出黄绿色荧光，采用Desmin和α-SMA细胞免疫荧光法检测静止态和活化态HSC细胞（经鉴定其细胞纯度达到90%以上）。 2、经萤光素酶靶向报告系统的验证，证实ACSL1是miR-34c的靶基因；实时定量PCR检测结果表明，miR-34c和ACSL1在不同状态HSC中的表达水平差异有统计学意义(P0.01)。随着HSC的活化，miR-34c的表达水平显著增高；而ACSL1则受miR-34c的负性调控作用，其表达水平随着HSC的活化明显减少(P0.01)。 3、将miR-34c inhibitor转染到14天HCS（活化态）中，荧光转染指示剂检测显示转染效率达到90%以上,14天HCS转染miR-34c inhibitor后:（1）实时定量PCR检测显示miR-34c的表达量明显低于空白组和对照组（P＜0.01）；且（2）ACSL1mRNA的表达量明显高于空白组和对照组(P＜0.01）；（3）Western-blot技术检测ACSL1蛋白水平，ACSL1的表达量明显高于空白组和对照组；（4）分别利用实时定量PCR和Western-blot检测α-SMAR和Type I collagen的表达水平：其mRNA和蛋白表达水平均明显低于空白组和对照组（分别P＜0.01）。结论：经酶原位消化法和密度梯度离心法可从SD大鼠肝脏成功提取HSC，经鉴定：细胞纯度较高，状态良好，可为后续实验提供所需的细胞。miR-34c随着HSC的活化而表达增高，而其靶基因ACSL1受到miR-34c的负向调控，表达水平随着HSC的活化而降低。下调活化态HSC中miR-34c的表达，能使ACSL1表达水平增高，同时也会对HSC活化指标α-SMA和Type I collagen的表达产生影响，即两者表达量随miR-34c表达的下调而显著减少。因而，miR-34c可能是通过靶向调控ACSL1而影响HSC的活化状态，进而参与肝纤维化进程。
[Abstract]:The purpose of the study is:
The research group to construct DMN rat model of hepatic fibrosis in the previous study, through miRNA microarray detection, found that miR-34c may play an important role in the progression of hepatic fibrosis, and target gene prediction system that ACSL1 may be a target of miR-34c. Therefore, this experiment: by luciferase reporter the system further verify whether ACSL1 is the target of miR-34c; detection of miR-34c and ACSL1 in the different states of hepatic stellate cells (Hepatic Stellate Cell, HSC) the expression level of miR-34c gene at the same time; down regulated in activated HSC expression level, study the role of miR-34c regulated target genes ACSL1 effects on the activation of HSC.
Research methods:
Part 1: extraction and identification of primary HSC in rats
1, using Friedman enzyme digestion density gradient centrifugation method: (1) in male SD rats after anesthesia, isolated portal vein intubation, followed by infusion of D-Hank 's fluid, pronase and collagenase digestion; (2) the liver will be fully digested after filtration, centrifugation, Nycodenz and DNase I adjust the liquid cell suspension density, the density is between 1.040-1.055g/ml, Gass with liquid Ga, extracted from stellate cells; (3) cell count, according to the proportion of cells inoculated into the CO2 cell culture dishes and cultured in the box.
2, microscopically observed HSC. under the excitation of 328nm ultraviolet light
3, cell immunofluorescence was used to detect the expression of Desmin and alpha -SMA in HSC for 2 days and 14 days.
The second part: detection of the target relationship between miR-34c and ACSL1 and the detection of the expression level in different states of HSC
1, on the basis of the earlier work of the group, the targeting relationship between miR-34c and ACSL1 was verified by the fluoro enzyme targeting report system.
2, real-time quantitative PCR was used to detect the expression level of miR-34c and ACSL1 in different states of HSC.
The third part: the effect of down regulation of miR-34c expression in HSC on ACSL1 expression and the related indexes of HSC activation
1, miR-34c inhibitor was used to transfect activated HSC (cultured to fourteenth days). At the same time, blank group and NC group were used as controls. The transfection efficiency was detected by fluorescent transfection indicator.
2, PCR was used to detect the expression level of miR-34c and ACSL1 in HSC after transfection.
3, Western-blot technique was used to detect the expression of ACSL1 protein in HSC after transfection.
4, real-time quantitative PCR and Western-blot were used to detect the expression of Type I collagen and the level of alpha -SMAR mRNA and protein after transfection of HSC.
The results of the study:
1, the enzymatic digestion and density gradient centrifugation, each SD rat could get about 5-7 HSC * 107 HSC; emit yellow green fluorescence under UV excitation at 328nm, detection of static state and active state of HSC cells by Desmin and -SMA (alpha cell immunofluorescence reached cell purity identification 90% above).
2, the luciferase targeting system validation report, confirmed that ACSL1 is the target gene miR-34c; real-time quantitative PCR detection results showed that there was significant difference in the expression level of miR-34c and ACSL1 in the different state of HSC (P0.01). With the activation of HSC, the expression level of miR-34c was significantly increased; negative role ACSL1 by miR-34c, the expression level with the activation of HSC was significantly reduced (P0.01).
3, the miR-34c inhibitor was transfected into HCS 14 days (activation state), fluorescence detection showed that the transfection efficiency of transfection indicator reached more than 90%, 14 days after the transfection of HCS miR-34c inhibitor: (1) the quantitative real-time PCR showed that miR-34c expression was significantly lower than that of the control group and the control group (P < 0.01); and (2) the expression of ACSL1mRNA was significantly higher than the control group and the control group (P < 0.01); (3) to detect the protein level of ACSL1 Western-blot, the expression of ACSL1 was significantly higher than the control group and control group; (4) the expression level of PCR and real-time quantitative Western-blot detection of -SMAR Type and I collagen alpha were used: the expression level of the mRNA and the protein was significantly lower than that of the control group and the control group (P < 0.01).
Conclusion:
In situ by enzyme digestion and density gradient centrifugation can be successfully extracted HSC from SD rat liver cells were identified: high purity, good condition, can provide the required.MiR-34c cells for subsequent experiments with the activation of HSC and the expression of negative regulation and its target gene ACSL1 by miR-34c, the expression level decreased with the activation of HSC. Expression of activated miR-34c in HSC state, the expression of ACSL1 was increased, but also influence the expression of -SMA and Type I index collagen on activation of HSC, which is the expression with the down-regulation of miR-34c expression decreased significantly. Therefore, miR-34c may be activated by targeting ACSL1 the effect of HSC, and is involved in the progression of hepatic fibrosis.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575

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