本文關鍵詞： 酶原位消化法 密度梯度離心法 肝星狀細胞 miR-34c ACSL1 Type I collage α-SMA　出處：《第二軍醫(yī)大學》2014年碩士論文　論文類型：學位論文

【摘要】：研究目的: 本課題組在前期研究中構建DMN大鼠肝纖維化模型，通過miRNA表達譜芯片檢測，發(fā)現(xiàn)： miR-34c可能在肝纖維化進程中發(fā)揮重要作用，且靶基因預測系統(tǒng)推測，ACSL1可能是miR-34c的作用靶點。因之，本實驗：將通過螢光素酶報告系統(tǒng)進一步驗證ACSL1是否為miR-34c的作用靶點；檢測miR-34c和ACSL1在不同狀態(tài)肝星狀細胞（Hepatic Stellate Cell，HSC）中的表達水平；同時在活化態(tài)HSC中下調(diào)miR-34c的基因表達水平，研究miR-34c調(diào)控靶基因ACSL1對HSC活化的影響作用。 研究方法： 第一部分：大鼠原代HSC的提取與鑒定 1、采用Friedman酶消化-密度梯度離心法：（1）對雄性SD大鼠行麻醉后，分離門靜脈進行插管，依次灌注D-Hank’s液、鏈酶蛋白酶消化液和膠原酶消化液；（2）將肝臟充分消化后過濾，離心，，用Nycodenz和DNase I液調(diào)整細胞懸液的密度，使密度達到1.040-1.055g/ml之間，鋪上無Ga的Gass液，提取星狀細胞；（3）細胞計數(shù)后，按比例接種于細胞皿中，放入CO2細胞培養(yǎng)箱中培養(yǎng)傳代。 2、在328nm紫外光的激發(fā)下顯微鏡觀察HSC。 3、細胞免疫熒光法檢測培養(yǎng)2天、14天HSC中Desmin和α-SMA的表達。 第二部分：miR-34c與ACSL1的靶向關系檢測及其在不同狀態(tài)HSC中表達水平的檢測 1、在課題組前期工作的基礎上，利用螢光素酶靶向報告系統(tǒng)驗證miR-34c和ACSL1是否存在靶向關系； 2、實時定量PCR檢測miR-34c和ACSL1在不同狀態(tài)的HSC中的表達水平。 第三部分：下調(diào)HSC中miR-34c表達對ACSL1表達及對HSC活化相關指標的影響 1、使用miR-34c inhibitor轉(zhuǎn)染活化態(tài)HSC（培養(yǎng)至第14天者），同時用空白組、NC組作為對照，并采用螢光轉(zhuǎn)染指標劑檢測轉(zhuǎn)染效率； 2、PCR檢測轉(zhuǎn)染后HSC中miR-34c和ACSL1的表達水平； 3、Western-blot技術檢測轉(zhuǎn)染后HSC中ACSL1蛋白表達水平； 4、利用實時定量PCR和Western-blot檢測轉(zhuǎn)染后HSC活化相關指標Type I collagen與α-SMAR mRNA及蛋白水平的表達。 研究結果： 1、本實驗采用酶原位消化法和密度梯度離心法，每只SD大鼠可獲得HSC約5-7×107； HSC在328nm紫外光的激發(fā)下發(fā)出黃綠色熒光，采用Desmin和α-SMA細胞免疫熒光法檢測靜止態(tài)和活化態(tài)HSC細胞（經(jīng)鑒定其細胞純度達到90%以上）。 2、經(jīng)螢光素酶靶向報告系統(tǒng)的驗證，證實ACSL1是miR-34c的靶基因；實時定量PCR檢測結果表明，miR-34c和ACSL1在不同狀態(tài)HSC中的表達水平差異有統(tǒng)計學意義(P0.01)。隨著HSC的活化，miR-34c的表達水平顯著增高；而ACSL1則受miR-34c的負性調(diào)控作用，其表達水平隨著HSC的活化明顯減少(P0.01)。 3、將miR-34c inhibitor轉(zhuǎn)染到14天HCS（活化態(tài)）中，熒光轉(zhuǎn)染指示劑檢測顯示轉(zhuǎn)染效率達到90%以上,14天HCS轉(zhuǎn)染miR-34c inhibitor后:（1）實時定量PCR檢測顯示miR-34c的表達量明顯低于空白組和對照組（P＜0.01）；且（2）ACSL1mRNA的表達量明顯高于空白組和對照組(P＜0.01）；（3）Western-blot技術檢測ACSL1蛋白水平，ACSL1的表達量明顯高于空白組和對照組；（4）分別利用實時定量PCR和Western-blot檢測α-SMAR和Type I collagen的表達水平：其mRNA和蛋白表達水平均明顯低于空白組和對照組（分別P＜0.01）。 結論： 經(jīng)酶原位消化法和密度梯度離心法可從SD大鼠肝臟成功提取HSC，經(jīng)鑒定：細胞純度較高，狀態(tài)良好，可為后續(xù)實驗提供所需的細胞。miR-34c隨著HSC的活化而表達增高，而其靶基因ACSL1受到miR-34c的負向調(diào)控，表達水平隨著HSC的活化而降低。下調(diào)活化態(tài)HSC中miR-34c的表達，能使ACSL1表達水平增高，同時也會對HSC活化指標α-SMA和Type I collagen的表達產(chǎn)生影響，即兩者表達量隨miR-34c表達的下調(diào)而顯著減少。因而，miR-34c可能是通過靶向調(diào)控ACSL1而影響HSC的活化狀態(tài)，進而參與肝纖維化進程。
[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.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575

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