肝刺激因子通過抑制肝臟星狀細(xì)胞活化減輕肝臟纖維化的研究
發(fā)布時(shí)間：2018-07-31 14:21
【摘要】：背景和目的肝臟受到損傷后,肝實(shí)質(zhì)細(xì)胞發(fā)生壞死或凋亡,誘發(fā)炎癥反應(yīng),分泌多種細(xì)胞因子,進(jìn)而刺激靜息狀態(tài)的肝星狀細(xì)胞(hepatic Stellate Cell,HSC)發(fā)生活化,進(jìn)一步轉(zhuǎn)分化為肌成纖維細(xì)胞(myofibroblast,MFB)。肌成纖維細(xì)胞可以分泌大量包含膠原在內(nèi)的細(xì)胞外基質(zhì),導(dǎo)致肝臟纖維化。由此可見,HSC活化是肝纖維化發(fā)生與發(fā)展中的核心事件。鑒于HSC在肝臟纖維化發(fā)生發(fā)展過程中的重要作用,研究其活化的分子機(jī)制將有助于逆轉(zhuǎn)或治療肝臟纖維化。肝刺激因子(hepatic Stimulator Substance,HSS)是一種能刺激肝細(xì)胞增殖的生物活性物質(zhì)。近期報(bào)道顯示,HSS可通過保護(hù)線粒體膜孔道,維持細(xì)胞內(nèi)鈣離子穩(wěn)態(tài)抑制細(xì)胞凋亡,發(fā)揮肝細(xì)胞保護(hù)作用,因而被認(rèn)為是肝細(xì)胞內(nèi)重要的存活因子(survival factor)。HSS與肝纖維化的發(fā)生發(fā)展也有一定關(guān)系。在小鼠肝纖維化模型中,過表達(dá)HSS可顯著地減輕纖維化癥狀,而敲減HSS可明顯加重小鼠CCl4和膽總管結(jié)扎誘導(dǎo)的肝纖維化(本實(shí)驗(yàn)室未發(fā)表資料),但HSS在肝臟纖維化發(fā)病與演化過程中的機(jī)制依然沒有清楚地闡明。鑒于有報(bào)道稱,HSS在肝實(shí)質(zhì)細(xì)胞和HSC內(nèi)均存在表達(dá),我們不禁要問,HSS能否通過抑制肝星狀細(xì)胞活化來(lái)達(dá)到減輕抑制肝臟纖維化的目的?為此,本實(shí)驗(yàn)培養(yǎng)HSC,通過調(diào)節(jié)其細(xì)胞內(nèi)HSS表達(dá)水平,研究HSS對(duì)HSC活化的影響,并探討其對(duì)肝纖維化產(chǎn)生的影響。本實(shí)驗(yàn)旨在從全新視角揭示HSS與HSC活化的關(guān)系,為預(yù)防及治療肝纖維化提供理論基礎(chǔ)。方法1、細(xì)胞模型的建立:選擇并培養(yǎng)具有活化表型的肝星狀細(xì)胞(LX-2)細(xì)胞系,并通過穩(wěn)定轉(zhuǎn)染的方法構(gòu)建HSS高表達(dá)(HSS-Tx)和HSS敲減(HSS-sh RNA)的LX-2細(xì)胞系。2、HSC活化分子標(biāo)記物的鑒定:通過western blot的方法檢測(cè)HSS-Tx和HSSsh RNA細(xì)胞中HSC活化分子標(biāo)記物a-SMA和Ⅲ型膠原的表達(dá),以研究HSS與HSC活化之間的關(guān)系。3、活化型HSC細(xì)胞特征的檢測(cè):HSC活化特征包括增殖加快、遷移增強(qiáng)和凋亡減少。通過流式細(xì)胞儀分析細(xì)胞周期、MTS實(shí)驗(yàn)測(cè)定細(xì)胞活力和免疫熒光檢測(cè)Ki67的陽(yáng)性細(xì)胞數(shù),研究HSS-Tx和HSS-sh RNA細(xì)胞增殖能力的變化;通過Transwell和Xcelligence實(shí)驗(yàn)檢測(cè)兩組細(xì)胞遷移能力的改變;并檢測(cè)caspase-3/7活性以分析兩組細(xì)胞的凋亡情況。4、HSS影響HSC活化的分子通路的探究:分別利用磷酸化MAPK抗體芯片和磷酸化酪氨酸激酶抗體芯片檢測(cè)兩組細(xì)胞差異活化的關(guān)鍵蛋白質(zhì),以研究HSS影響HSC活化的分子通路。5、HSS影響HSC細(xì)胞遷移的機(jī)制研究:分別利用高內(nèi)涵細(xì)胞成像分析技術(shù)和激光共聚焦掃描顯微鏡檢測(cè)細(xì)胞微絲的分布,纖維狀-肌動(dòng)蛋白(F-actin)和球狀-肌動(dòng)蛋白(G-actin)的含量,以分析兩組細(xì)胞微絲的變化及其與遷移的關(guān)系。胞漿鈣離子與微絲裝配及細(xì)胞遷移關(guān)系密切,利用鈣離子示蹤劑顯示胞漿內(nèi)鈣離子含量,反映鈣離子變化和細(xì)胞運(yùn)動(dòng)的關(guān)系。6、HSS影響HSC線粒體動(dòng)態(tài)性及線粒體鈣離子的研究:HSS定位于HSC的線粒體,為探討HSS是否通過影響線粒體功能進(jìn)而改變HSC細(xì)胞遷移能力,首先利用活細(xì)胞染料標(biāo)記兩組細(xì)胞內(nèi)的線粒體,并用激光共聚焦掃描顯微鏡和配套軟件分析細(xì)胞的線粒體形態(tài),通過ATP含量測(cè)定及Seahorse實(shí)驗(yàn)檢測(cè)線粒體功能的變化,并使用線粒體鈣離子示蹤劑測(cè)定兩組細(xì)胞線粒體鈣離子的分布情況。7、回復(fù)實(shí)驗(yàn):為進(jìn)一步確定上述實(shí)驗(yàn)獲得的實(shí)驗(yàn)結(jié)果,利用RNAi的方法抑制HSS-Tx細(xì)胞內(nèi)HSS的表達(dá),重復(fù)5和6的實(shí)驗(yàn),即測(cè)定細(xì)胞內(nèi)微絲的含量及分布,線粒體形態(tài)的變化等。結(jié)果1、Western blot結(jié)果顯示,HSS轉(zhuǎn)染的LX-2細(xì)胞(HSS-Tx)中HSS表達(dá)明顯高于vector細(xì)胞,而且HSS-sh RNA轉(zhuǎn)染的LX-2細(xì)胞(HSS-sh RNA)中HSS表達(dá)顯著低于vector細(xì)胞,且在傳代過程中能維持HSS高表達(dá)或低表達(dá)狀態(tài),說明細(xì)胞模型構(gòu)建成功,可用于后續(xù)實(shí)驗(yàn)。2、Western blot結(jié)果顯示,HSS-Tx細(xì)胞中a-SMA(P0.05)和III型膠原(P0.01)的表達(dá)明顯低于HSS-sh RNA細(xì)胞,說明HSS可抑制HSC的活化。3、MTS法測(cè)定細(xì)胞活力結(jié)果顯示,HSS-Tx組細(xì)胞活力在傳代后36 h開始弱于HSS-sh RNA細(xì)胞(P0.01),而且這種狀況一直持續(xù)至72 h(P0.05)。流式細(xì)胞儀結(jié)果顯示,HSS-Tx組G0/G1期細(xì)胞比例明顯多于HSS-sh RNA細(xì)胞。與此類似,其Ki67陽(yáng)性細(xì)胞數(shù)也明顯少于HSS-sh RNA(P0.05)。Transwell實(shí)驗(yàn)結(jié)果顯示,在相同時(shí)間內(nèi)穿過小孔的HSS-Tx細(xì)胞數(shù)目明顯少于HSS-sh RNA細(xì)胞,同樣Xcelligence實(shí)驗(yàn)結(jié)果顯示在接種后1 h至25 h,HSS-Tx細(xì)胞的遷移能力均明顯弱于sh RNA細(xì)胞(P0.0001),以上結(jié)果提示轉(zhuǎn)染HSS基因可以抑制LX-2細(xì)胞的增殖及遷移。4、磷酸化MAPK通路抗體芯片結(jié)果顯示,高表達(dá)HSS與敲減HSS兩組細(xì)胞中,包括ERK,P38和AKT等在內(nèi)的18種信號(hào)分子的磷酸化水平未見明顯差異,而71個(gè)磷酸化酪氨酸激酶抗體芯片結(jié)果顯示HSS-sh RNA細(xì)胞中Blk,Fyn,TNK1,FAK和TXK等與微絲裝配相關(guān)激酶的磷酸化水平明顯高于HSS-Tx組,提示HSS可能通過抑制上述激酶的活性,從而限制微絲裝配。5、利用熒光標(biāo)記的鬼筆環(huán)肽來(lái)標(biāo)記HSS-Tx和HSS-sh RNA細(xì)胞內(nèi)F-actin,通過高內(nèi)涵細(xì)胞成像系統(tǒng)和激光共聚焦掃描顯微鏡顯示F-actin含量和分布。結(jié)果顯示,HSS-Tx組細(xì)胞內(nèi)微絲數(shù)目明顯少于HSS-sh RNA組(P0.01),而且分布更為不規(guī)則。利用同樣的方法標(biāo)記細(xì)胞內(nèi)G-actin,結(jié)果顯示HSS-Tx組細(xì)胞內(nèi)G-actin的含量顯著高于HSS-sh RNA組,提示HSS抑制HSC細(xì)胞微絲的裝配,減少應(yīng)力纖維的生成。細(xì)胞免疫熒光實(shí)驗(yàn)顯示HSS-Tx組細(xì)胞內(nèi)胞漿內(nèi)鈣離子的含量明顯少于HSS-sh RNA組(P0.001)。6、利用Mito-tracker特異性標(biāo)記活細(xì)胞線粒體,通過激光共聚焦掃描顯微鏡成像和相關(guān)軟件分析顯示,與HSS-sh RNA細(xì)胞相比,HSS-Tx細(xì)胞內(nèi)線粒體形態(tài)變得更短而且更圓(P0.0001),而且Western blot結(jié)果顯示,與線粒體融合相關(guān)的MFN-2表達(dá)降低(P0.01),說明HSS抑制了HSC細(xì)胞線粒體融合。測(cè)定細(xì)胞內(nèi)線粒體氧代謝(采用Seahorse儀器),結(jié)果顯示,HSS-Tx細(xì)胞的基礎(chǔ)代謝能力下降,ATP含量明顯低于HSS-sh RNA細(xì)胞(P0.0001)。線粒體融合受到抑制,不僅使線粒體功能受到破壞,而且也影響到線粒體鈣離子穩(wěn)態(tài),進(jìn)而影響整個(gè)胞漿鈣離子穩(wěn)態(tài)。利用線粒體鈣離子示蹤劑顯示HSS高/低表達(dá)細(xì)胞內(nèi)線粒體內(nèi)鈣離子的變化,結(jié)果顯示,HSS-Tx細(xì)胞線粒體內(nèi)的鈣離子濃度明顯低于HSS-sh RNA細(xì)胞(P0.0001)。7、Western blot結(jié)果顯示,HSS-Tx細(xì)胞轉(zhuǎn)染HSS si RNA pool后,HSS表達(dá)明顯下調(diào)。轉(zhuǎn)染HSS si RNA pool 3 d后,激光共聚焦掃描顯微鏡成像結(jié)果顯示微絲分布開始變得規(guī)則,微絲含量明顯增加,這些改變持續(xù)至轉(zhuǎn)染7 d,而且更為明顯。同樣轉(zhuǎn)染3 d后,線粒體開始變長(zhǎng),轉(zhuǎn)染7 d時(shí),線粒體形態(tài)改變最為明顯;與之對(duì)應(yīng),細(xì)胞內(nèi)ATP含量增加。結(jié)論1.HSS可以抑制HSCs活化。2.HSS破壞HSCs的線粒體動(dòng)態(tài)性,使ATP合成減少,線粒體內(nèi)鈣離子含量下降,進(jìn)而降低胞漿內(nèi)鈣離子濃度,抑制微絲裝配,使HSCs活化與遷移能力下降。
[Abstract]:After the injury of the background and objective liver, the necrosis or apoptosis of liver parenchyma cells, induced inflammatory reaction, secreting a variety of cytokines, and then stimulating the resting state of hepatic Stellate Cell (HSC) to live, and further convert into myofibroblast (myofibroblast, MFB). Myofibroblast can secrete a large number of bags. The extracellular matrix, including collagen, leads to liver fibrosis. This shows that HSC activation is a core event in the development and development of liver fibrosis. In view of the important role of HSC in the development of liver fibrosis, the study of its activation molecular mechanism will help to reverse or treat liver fibrosis. Hepatic Stimulator Substance, HSS) is a bioactive substance that stimulates the proliferation of hepatocytes. Recently, it has been reported that HSS can protect the mitochondrial membrane pore, maintain intracellular calcium ion homeostasis and inhibit cell apoptosis and play a protective role in liver cells. Therefore, it is considered to be an important survival factor (survival factor).HSS in liver cells and the occurrence of liver fibrosis. Development also has some relationship. In the mouse liver fibrosis model, overexpression of HSS can significantly reduce the symptoms of fibrosis, while knockout HSS can obviously aggravate the liver fibrosis induced by CCl4 and choledochal ligation in mice. However, the mechanism of HSS in the pathogenesis and evolution of liver fibrosis is still not clearly elucidated. It is reported that HSS is expressed in both liver parenchyma cells and HSC. We can not help asking whether HSS can reduce hepatic fibrosis by inhibiting the activation of hepatic stellate cells. To this end, this experiment is to cultivate HSC and to study the effect of HSS on the activation of HSC by regulating the level of HSS expression in the cells, and to explore its effect on liver fibrosis. This experiment aims to reveal the relationship between HSS and HSC activation from a new perspective, and to provide a theoretical basis for the prevention and treatment of liver fibrosis. Method 1, the establishment of cell model: selecting and cultivating the activated phenotype of hepatic stellate cells (LX-2) cell lines, and constructing HSS high expression (HSS-Tx) and HSS knockout (HSS-sh RNA) LX through the stable transfection method. Identification of -2 cell line.2, HSC activation molecular markers: the expression of HSC activated molecular markers a-SMA and type III collagen in HSS-Tx and HSSsh RNA cells was detected by Western blot method to investigate the relationship between HSS and HSC activation. The cell cycle was analyzed by flow cytometry. The cell viability and immunofluorescence of Ki67 positive cells were measured by MTS test. The proliferation ability of HSS-Tx and HSS-sh RNA cells was studied. The changes in the cell migration ability of the two groups were detected by Transwell and Xcelligence tests, and the activity of caspase-3/7 was detected to analyze the apoptosis of the two groups of cells. .4, HSS affecting the molecular pathway of HSC activation: using phosphorylated MAPK antibody chip and phosphorylated tyrosine kinase antibody chip to detect the key protein of two groups of cell differentiation activation respectively, in order to study the mechanism of HSS affecting the HSC activation molecular pathway.5, HSS affecting the migration of HSC cells: high intension cell imaging analysis respectively The distribution of cell microfilaments, fibrous actin (F-actin) and spherical actin (G-actin) were detected by technique and laser confocal scanning microscope to analyze the changes of cell microfilaments and their relationship with migration in two groups. Cytoplasmic calcium ions were closely related to microfilament assembly and cell migration, and intracellular calcium tracers were used to display the cytoplasm. Calcium ion content, reflecting the relationship of calcium ion change and cell movement.6, HSS affects the dynamics of HSC mitochondria and the study of mitochondrial calcium ion: HSS is located in the mitochondria of HSC, to explore whether HSS can change the migration ability of HSC cells by influencing mitochondrial function and then change the mitochondria in two groups of cells by using living cell dyes. The morphology of mitochondria in cells was analyzed by laser confocal scanning microscope (confocal scanning microscope) and the software was used to detect the changes of mitochondrial function through ATP content determination and Seahorse test. The distribution of calcium ion in the mitochondria of two groups of cells was measured by mitochondrial calcium tracer.7, and the experimental results were further determined to determine the experimental results obtained by the above experiments. The expression of HSS in HSS-Tx cells was inhibited by RNAi, and 5 and 6 experiments were repeated, that was to determine the content and distribution of intracellular microfilament and the change of mitochondria morphology. Results 1, Western blot showed that the HSS expression in LX-2 cells (HSS-Tx) transfected by HSS was significantly higher than that of vector cells. The expression was significantly lower than that of vector cells, and the high expression or low expression of HSS could be maintained during the passage process, indicating that the cell model was successfully constructed and can be used in the follow-up experimental.2. The results of Western blot showed that the expression of a-SMA (P0.05) and III type collagen (P0.01) in HSS-Tx cells was significantly lower than that of HSS-sh RNA cells. The results of cell viability showed that the cell vitality of HSS-Tx group began to be weaker than HSS-sh RNA cells (P0.01) at 36 h after passage, and this condition continued to 72 h (P0.05). The result of flow cytometry showed that the proportion of G0/G1 stage in HSS-Tx group was more than HSS-sh RNA cells. 05) the results of.Transwell experiment showed that the number of HSS-Tx cells passing through the pores in the same time was significantly less than that of HSS-sh RNA cells. The same results showed that the migration ability of HSS-Tx cells was significantly weaker than sh RNA cells (P0.0001) at 1 h to 25 h after the inoculation. The results suggested that the transfection of the HSS gene could inhibit the proliferation of the cells. And migrating.4, the phosphorylated MAPK pathway antibody chip results showed that the phosphorylation level of 18 signal molecules, including ERK, P38 and AKT, was not significantly different in the high expression HSS and the knockout HSS two groups, while the 71 phosphorylated tyrosine kinase antibody chips showed Blk, Fyn, TNK1, etc. and microfilament in HSS-sh RNA cells. The level of phosphorylation of the kinases is obviously higher than that of the HSS-Tx group, suggesting that HSS may inhibit the assembly of.5 by inhibiting the activity of the above kinase, and using the fluorescent labeled phimic cyclic peptide to mark the F-actin in HSS-Tx and HSS-sh RNA cells. The content and distribution of F-actin are displayed by the high intension cell imaging system and the laser confocal scanning microscope. The results showed that the number of intracellular microfilaments in the HSS-Tx group was significantly less than that of the HSS-sh RNA group (P0.01), and the distribution was more irregular. The same method was used to mark the intracellular G-actin. The results showed that the content of G-actin in the HSS-Tx group was significantly higher than that of the HSS-sh RNA group, suggesting that HSS inhibits the assembly of HSC fine cell microfilaments and reduces the formation of stress fibers. Cell immunity is reduced. The intracellular calcium content in HSS-Tx group was obviously less than that of group HSS-sh RNA (P0.001).6, and the mitochondria of living cells were marked by Mito-tracker specificity. The morphology of mitochondria in HSS-Tx cells became shorter than that of HSS-sh RNA cells. More circular (P0.0001), and Western blot results showed that the expression of MFN-2 associated with mitochondrial fusion decreased (P0.01), indicating that HSS inhibited mitochondrial fusion of HSC cells. Intracellular mitochondrial mitochondrial oxygen metabolism (Seahorse instrument) was measured. The results showed that the basal metabolic ability of HSS-Tx cells decreased and ATP content was significantly lower than HSS-sh RNA cells. Mitochondrial fusion is inhibited, which not only destroys mitochondrial function, but also affects the homeostasis of mitochondrial calcium ion, and then affects the whole plasma calcium homeostasis. The mitochondrial calcium ion changes in HSS high / low expression cells are displayed by the mitochondrial calcium tracer. The results show that the calcium ion concentration in the mitochondria of HSS-Tx cells Significantly lower than HSS-sh RNA cell (P0.0001).7, Western blot results showed that HSS-Tx cells transfected HSS Si RNA pool, HSS expression decreased obviously. Laser confocal scanning microscopy showed that microfilament distribution began to become regular, microfilament content increased significantly, these changes continued to transfection of 7, and more After transfection of 3 D, mitochondria began to grow, and the mitochondrial morphologic changes were most obvious when transfected to 7 d, and the content of ATP in the cells increased. Conclusion 1.HSS inhibited the mitochondrial dynamics of HSCs activated.2.HSS, reduced the synthesis of ATP, decreased the content of calcium ions in the mitochondria, and then reduced the intracellular calcium concentration. Inhibition of microfilament assembly reduces HSCs activation and migration ability.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575.2
本文編號(hào)：2155862
[Abstract]:After the injury of the background and objective liver, the necrosis or apoptosis of liver parenchyma cells, induced inflammatory reaction, secreting a variety of cytokines, and then stimulating the resting state of hepatic Stellate Cell (HSC) to live, and further convert into myofibroblast (myofibroblast, MFB). Myofibroblast can secrete a large number of bags. The extracellular matrix, including collagen, leads to liver fibrosis. This shows that HSC activation is a core event in the development and development of liver fibrosis. In view of the important role of HSC in the development of liver fibrosis, the study of its activation molecular mechanism will help to reverse or treat liver fibrosis. Hepatic Stimulator Substance, HSS) is a bioactive substance that stimulates the proliferation of hepatocytes. Recently, it has been reported that HSS can protect the mitochondrial membrane pore, maintain intracellular calcium ion homeostasis and inhibit cell apoptosis and play a protective role in liver cells. Therefore, it is considered to be an important survival factor (survival factor).HSS in liver cells and the occurrence of liver fibrosis. Development also has some relationship. In the mouse liver fibrosis model, overexpression of HSS can significantly reduce the symptoms of fibrosis, while knockout HSS can obviously aggravate the liver fibrosis induced by CCl4 and choledochal ligation in mice. However, the mechanism of HSS in the pathogenesis and evolution of liver fibrosis is still not clearly elucidated. It is reported that HSS is expressed in both liver parenchyma cells and HSC. We can not help asking whether HSS can reduce hepatic fibrosis by inhibiting the activation of hepatic stellate cells. To this end, this experiment is to cultivate HSC and to study the effect of HSS on the activation of HSC by regulating the level of HSS expression in the cells, and to explore its effect on liver fibrosis. This experiment aims to reveal the relationship between HSS and HSC activation from a new perspective, and to provide a theoretical basis for the prevention and treatment of liver fibrosis. Method 1, the establishment of cell model: selecting and cultivating the activated phenotype of hepatic stellate cells (LX-2) cell lines, and constructing HSS high expression (HSS-Tx) and HSS knockout (HSS-sh RNA) LX through the stable transfection method. Identification of -2 cell line.2, HSC activation molecular markers: the expression of HSC activated molecular markers a-SMA and type III collagen in HSS-Tx and HSSsh RNA cells was detected by Western blot method to investigate the relationship between HSS and HSC activation. The cell cycle was analyzed by flow cytometry. The cell viability and immunofluorescence of Ki67 positive cells were measured by MTS test. The proliferation ability of HSS-Tx and HSS-sh RNA cells was studied. The changes in the cell migration ability of the two groups were detected by Transwell and Xcelligence tests, and the activity of caspase-3/7 was detected to analyze the apoptosis of the two groups of cells. .4, HSS affecting the molecular pathway of HSC activation: using phosphorylated MAPK antibody chip and phosphorylated tyrosine kinase antibody chip to detect the key protein of two groups of cell differentiation activation respectively, in order to study the mechanism of HSS affecting the HSC activation molecular pathway.5, HSS affecting the migration of HSC cells: high intension cell imaging analysis respectively The distribution of cell microfilaments, fibrous actin (F-actin) and spherical actin (G-actin) were detected by technique and laser confocal scanning microscope to analyze the changes of cell microfilaments and their relationship with migration in two groups. Cytoplasmic calcium ions were closely related to microfilament assembly and cell migration, and intracellular calcium tracers were used to display the cytoplasm. Calcium ion content, reflecting the relationship of calcium ion change and cell movement.6, HSS affects the dynamics of HSC mitochondria and the study of mitochondrial calcium ion: HSS is located in the mitochondria of HSC, to explore whether HSS can change the migration ability of HSC cells by influencing mitochondrial function and then change the mitochondria in two groups of cells by using living cell dyes. The morphology of mitochondria in cells was analyzed by laser confocal scanning microscope (confocal scanning microscope) and the software was used to detect the changes of mitochondrial function through ATP content determination and Seahorse test. The distribution of calcium ion in the mitochondria of two groups of cells was measured by mitochondrial calcium tracer.7, and the experimental results were further determined to determine the experimental results obtained by the above experiments. The expression of HSS in HSS-Tx cells was inhibited by RNAi, and 5 and 6 experiments were repeated, that was to determine the content and distribution of intracellular microfilament and the change of mitochondria morphology. Results 1, Western blot showed that the HSS expression in LX-2 cells (HSS-Tx) transfected by HSS was significantly higher than that of vector cells. The expression was significantly lower than that of vector cells, and the high expression or low expression of HSS could be maintained during the passage process, indicating that the cell model was successfully constructed and can be used in the follow-up experimental.2. The results of Western blot showed that the expression of a-SMA (P0.05) and III type collagen (P0.01) in HSS-Tx cells was significantly lower than that of HSS-sh RNA cells. The results of cell viability showed that the cell vitality of HSS-Tx group began to be weaker than HSS-sh RNA cells (P0.01) at 36 h after passage, and this condition continued to 72 h (P0.05). The result of flow cytometry showed that the proportion of G0/G1 stage in HSS-Tx group was more than HSS-sh RNA cells. 05) the results of.Transwell experiment showed that the number of HSS-Tx cells passing through the pores in the same time was significantly less than that of HSS-sh RNA cells. The same results showed that the migration ability of HSS-Tx cells was significantly weaker than sh RNA cells (P0.0001) at 1 h to 25 h after the inoculation. The results suggested that the transfection of the HSS gene could inhibit the proliferation of the cells. And migrating.4, the phosphorylated MAPK pathway antibody chip results showed that the phosphorylation level of 18 signal molecules, including ERK, P38 and AKT, was not significantly different in the high expression HSS and the knockout HSS two groups, while the 71 phosphorylated tyrosine kinase antibody chips showed Blk, Fyn, TNK1, etc. and microfilament in HSS-sh RNA cells. The level of phosphorylation of the kinases is obviously higher than that of the HSS-Tx group, suggesting that HSS may inhibit the assembly of.5 by inhibiting the activity of the above kinase, and using the fluorescent labeled phimic cyclic peptide to mark the F-actin in HSS-Tx and HSS-sh RNA cells. The content and distribution of F-actin are displayed by the high intension cell imaging system and the laser confocal scanning microscope. The results showed that the number of intracellular microfilaments in the HSS-Tx group was significantly less than that of the HSS-sh RNA group (P0.01), and the distribution was more irregular. The same method was used to mark the intracellular G-actin. The results showed that the content of G-actin in the HSS-Tx group was significantly higher than that of the HSS-sh RNA group, suggesting that HSS inhibits the assembly of HSC fine cell microfilaments and reduces the formation of stress fibers. Cell immunity is reduced. The intracellular calcium content in HSS-Tx group was obviously less than that of group HSS-sh RNA (P0.001).6, and the mitochondria of living cells were marked by Mito-tracker specificity. The morphology of mitochondria in HSS-Tx cells became shorter than that of HSS-sh RNA cells. More circular (P0.0001), and Western blot results showed that the expression of MFN-2 associated with mitochondrial fusion decreased (P0.01), indicating that HSS inhibited mitochondrial fusion of HSC cells. Intracellular mitochondrial mitochondrial oxygen metabolism (Seahorse instrument) was measured. The results showed that the basal metabolic ability of HSS-Tx cells decreased and ATP content was significantly lower than HSS-sh RNA cells. Mitochondrial fusion is inhibited, which not only destroys mitochondrial function, but also affects the homeostasis of mitochondrial calcium ion, and then affects the whole plasma calcium homeostasis. The mitochondrial calcium ion changes in HSS high / low expression cells are displayed by the mitochondrial calcium tracer. The results show that the calcium ion concentration in the mitochondria of HSS-Tx cells Significantly lower than HSS-sh RNA cell (P0.0001).7, Western blot results showed that HSS-Tx cells transfected HSS Si RNA pool, HSS expression decreased obviously. Laser confocal scanning microscopy showed that microfilament distribution began to become regular, microfilament content increased significantly, these changes continued to transfection of 7, and more After transfection of 3 D, mitochondria began to grow, and the mitochondrial morphologic changes were most obvious when transfected to 7 d, and the content of ATP in the cells increased. Conclusion 1.HSS inhibited the mitochondrial dynamics of HSCs activated.2.HSS, reduced the synthesis of ATP, decreased the content of calcium ions in the mitochondria, and then reduced the intracellular calcium concentration. Inhibition of microfilament assembly reduces HSCs activation and migration ability.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
1 ;Effects of augmentation of liver regeneration recombinant plasmid on rat hepatic fibrosis[J];World Journal of Gastroenterology;2005年16期
2 楊曉明,王愛民,周平,王清明,魏漢東,吳祖澤,賀福初;Protective effect of recombinant human augmenter of liver regeneration on CCl_4-induced hepatitis in mice[J];Chinese Medical Journal;1998年07期
3 楊曉明,謝玲,吳祖澤,賀福初;肝部分切除后肝再生增強(qiáng)因子信使核糖核酸(mRNA)表達(dá)增強(qiáng)[J];生理學(xué)報(bào);1997年05期
4 楊曉明,謝玲,邱兆華,宮鋒,吳祖澤,賀福初;肝再生增強(qiáng)因子的cDNA克隆、表達(dá)及表達(dá)產(chǎn)物的生物活性研究[J];生物化學(xué)雜志;1997年02期
，本文編號(hào)：2155862
本文鏈接：http://sikaile.net/yixuelunwen/xiaohjib/2155862.html
最近更新
教材專著
