本文關(guān)鍵詞：肝細(xì)胞生長因子促進(jìn)肝星狀細(xì)胞凋亡的機(jī)制研究　出處：《廣西醫(yī)科大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 全骨髓貼壁法 成骨分化 成脂分化 肝星狀細(xì)胞 腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體 肝細(xì)胞生長因子 凋亡 死亡受體-5 骨髓間充質(zhì)干細(xì)胞 肝星狀細(xì)胞 腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體 肝細(xì)胞生長因子 死亡受體-

【摘要】：目的觀察全骨髓貼壁法分離、培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞(BMSCs)的傳代、鑒定及成骨分化、成脂分化的情況,為進(jìn)一步研究BMSCs提供功能穩(wěn)定的種子細(xì)胞。 方法采用全骨髓貼壁培養(yǎng)法對SD大鼠BMSCs進(jìn)行分離、培養(yǎng)、純化。在倒置相差顯微鏡下動態(tài)觀察活體細(xì)胞形態(tài)學(xué)改變；MTT比色法測定OD值繪制生長曲線；用流式細(xì)胞儀鑒定BMSCs表面抗原；使用成骨誘導(dǎo)液及成脂誘導(dǎo)液分別誘導(dǎo)其向成骨細(xì)胞、成脂肪細(xì)胞分化。 結(jié)果(1)全骨髓貼壁法分離培養(yǎng)的BMSCs為貼壁生長的成纖維樣細(xì)胞,傳代后的細(xì)胞形態(tài)均一,呈漩渦狀排列。(2)第3代BMSCs的生長曲線呈‘S’形,經(jīng)歷了三個(gè)生長時(shí)期：潛伏期、對數(shù)生長期和停滯期。(3)流式細(xì)胞儀檢測BMSCs表面抗原結(jié)果示：CD29+99.45%、CD34+1.45%、CD44+99.52%、CD45+1.41%。(4)成骨、成脂細(xì)胞誘導(dǎo)分化后,分別使用茜素紅及油紅‘O’染色,其中礦化結(jié)節(jié)被染成橘紅色、脂滴被染成紅色。 結(jié)論全骨髓貼壁培養(yǎng)法分離、培養(yǎng)大鼠骨髓干細(xì)胞可獲較高純度的BMSCs,該類細(xì)胞具有向成骨細(xì)胞及脂肪細(xì)胞分化的潛能。 目的觀察外源性肝細(xì)胞生長因子(HGF)在腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(TRAIL)促進(jìn)原代星狀細(xì)胞(HSCs)凋亡中的作用及其可能機(jī)制。 方法復(fù)蘇、傳代SD大鼠原代HSCs,細(xì)胞增殖明顯時(shí)用于實(shí)驗(yàn)。將實(shí)驗(yàn)分為以下4組：(1)HSCs空白對照組；(2)HGF組；(3)TRAIL組;(4)HGF+TRAIL組。各實(shí)驗(yàn)組細(xì)胞培養(yǎng)24h、48h。在倒置相差熒光顯微鏡下觀察細(xì)胞形態(tài)。使用免疫細(xì)胞化學(xué)法了解alpha-平滑肌肌動蛋白(a-SMA)在HSCs胞漿中的表達(dá)情況以及MTT比色法檢測外源性HGF及TRAIL分別對HSCs增殖的影響；流式細(xì)胞儀Annexin-V-FITC/PI雙染法檢測HSCs凋亡率以及流式細(xì)胞儀免疫熒光法檢測HSCs表面DR5的熒光強(qiáng)度；使用Western blot法檢測HSCs的DR5蛋白表達(dá)。 結(jié)果(1)免疫細(xì)胞化學(xué)法顯示HSCs胞漿中a-SMA (+)。(2)MTT法結(jié)果顯示：HGF及TRAIL分別在50-200ng/ml、0.5-1.5ug/ml各濃度下對HSCs增殖無影響,TRAIL在2ug/ml作用下對HSCs有抑制作用；24h、48hHSCs的OD值分別為：(0.22±0.02)%及(0.25±0.08)%,低于其它各組(P0.01)。(3)流式細(xì)胞儀檢測各組HSCs的凋亡結(jié)果示：HGF+TRAIL組的24h、48h的凋亡率明顯高于空白對照組、TRAIL組及HGF組(P0.01)。 (4)使用免疫熒光法檢測HSCs表面DR5的熒光強(qiáng)度結(jié)果示：HGF+TRAIL組24h、48h的DR5熒光強(qiáng)度明顯高于空白對照組、HGF組及TRAIL組(P0.01)。(5)Western blot法檢測DR5蛋白的表達(dá)結(jié)果顯示：HGF+TRAIL組中DR5蛋白明顯高于空白對照組、HGF組及TRAIL組(P0.01)。 結(jié)論外源性HGF能促進(jìn)TRAIL誘導(dǎo)的HSCs凋亡,可能與HGF能上調(diào)活化HSCs表面DR5蛋白表達(dá)有關(guān)。 目的觀察慢病毒介導(dǎo)HGF-ShRNA轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞(BMSCs)及肝星狀細(xì)胞(HSCs)后對BMSCs與HSCs共培養(yǎng)體系中HSCs凋亡的影響,探討兩種細(xì)胞來源的HGF在HSCs凋亡中的作用。為BMSCs移植治療肝纖維化提供實(shí)驗(yàn)依據(jù)。 方法采用全骨髓貼壁培養(yǎng)法對SD大鼠BMSCs進(jìn)行分離、培養(yǎng)、純化,使用傳代至第3-4代的細(xì)胞進(jìn)行實(shí)驗(yàn)。大鼠原代HSCs復(fù)蘇、傳代。應(yīng)用6孔培養(yǎng)板,在半透膜(transwell insert)上接種BMSCs(1.3×105cells/well),在6孔培養(yǎng)板上接種原代HSCs(1×105cells/well),建立上下雙層細(xì)胞共培養(yǎng)體系,常規(guī)培養(yǎng)。將實(shí)驗(yàn)分為以下5組：(1)空白對照組：HSCs單獨(dú)培養(yǎng)；(2)BMSCs+HSCs共培養(yǎng)組：BMSCs與HSCs共培養(yǎng)；(3)HSCs轉(zhuǎn)染共培養(yǎng)組：使用慢病毒介導(dǎo)的HGF-ShRNA轉(zhuǎn)染HSCs后與BMSCs共培養(yǎng)；(4)BMSCs轉(zhuǎn)染共培養(yǎng)組：使用慢病毒介導(dǎo)的HGF-ShRNA轉(zhuǎn)染BMSCs后與HSCs共培養(yǎng)；(5)TNF-a預(yù)處理組：TNF-a(100ng/ml)預(yù)處理BMSCs6h后與HSCs共培養(yǎng)；以上體系培養(yǎng)觀察24h、48h、72h。在倒置相差顯微鏡下動態(tài)觀察活體細(xì)胞形態(tài)學(xué)改變及轉(zhuǎn)染后綠色熒光表達(dá)情況；Western blot檢測轉(zhuǎn)染組HGF蛋白的表達(dá)；酶聯(lián)免疫吸附法(ELISA)檢測共培養(yǎng)各組上清液中HGF及TRAIL的濃度以及轉(zhuǎn)染后各組上清液HGF的濃度；流式細(xì)胞儀檢測轉(zhuǎn)染后熒光表達(dá)及Western blot法檢測HGF蛋白的表達(dá)。Annexin Ⅴ-FITC/PI雙染法檢測HSCs凋亡率；熒光定量PCR、Western blot分別檢測各組中DR5、Caspase-8mRNA及蛋白的表達(dá)。采用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)分析。 結(jié)果(1)質(zhì)粒測序結(jié)果與構(gòu)建時(shí)序列結(jié)果一致。(2)轉(zhuǎn)染后72h在導(dǎo)致相差熒光顯微鏡下觀察兩種細(xì)胞綠色熒光表達(dá)量約80%；BMSCs轉(zhuǎn)染組及HSCs轉(zhuǎn)染組中HGF蛋白下調(diào)分別為(60.2±2.5)%、(63.3±4.3)%；流式細(xì)胞儀檢測BMSCs轉(zhuǎn)染組綠色熒光表達(dá)陽性率為70%。(3)ELISA法檢測轉(zhuǎn)染組上清液中HGF濃度結(jié)果顯示：BMSCs空白對照組及HSCs空白對照組中HGF的濃度分別高于BMSCs轉(zhuǎn)染共培養(yǎng)組及HSCs轉(zhuǎn)染共培養(yǎng)組(P0.01);(4)ELISA法檢測共培養(yǎng)后各組上清液中HGF及TRAIL濃度結(jié)果提示：其中在TNF-a預(yù)處理組中HGF的濃度明顯高于HSCs空白對照組、BMSCs+HSCs共培養(yǎng)組及BMSCs單獨(dú)培養(yǎng)組(P0.01)；TRAIL在BMSCs組中的濃度明顯高于空白對照組、BMSCs+HSCs共培養(yǎng)組及TNF-a預(yù)處理組(P0.01)。(5)流式細(xì)胞儀Annexin-Ⅴ-FITC/PI雙染法檢測結(jié)果示：TNF-a預(yù)處理組HSCs的凋亡率明顯高于其它3組,且呈時(shí)間依賴性(P0.01);BMSCs轉(zhuǎn)染共培養(yǎng)組中HSCs的凋亡率低于共培養(yǎng)組(P0.05)。(6)FQ-PCR、Western blotting檢測共培養(yǎng)后各組HSCs的DR5、Caspase-8mRNA、蛋白表達(dá),其中TNF-a預(yù)處理組中HSCs的DR5、Caspase-8mRNA及蛋白表達(dá)量明顯高于HSCs空白對照組、BMSCs+HSCs共培養(yǎng)組、BMSCs轉(zhuǎn)染共培養(yǎng)組及HSCs轉(zhuǎn)染共培養(yǎng)組(P0.01)。BMSCs+HSCs共培養(yǎng)組中HSCs的DR5、Caspase-8mRNA及蛋白表達(dá)量明顯高于BMSCs轉(zhuǎn)染共培養(yǎng)組(P0.01)。 結(jié)論BMSCs來源的HGF通過上調(diào)HSCs中DR5及凋亡相關(guān)蛋白Caspase-8的表達(dá)促進(jìn)HSCs的凋亡,而TNF-a預(yù)處理后能增強(qiáng)這一作用,HSCs自分泌的HGF在共培養(yǎng)體系中促進(jìn)其凋亡中的作用甚少。
[Abstract]:Objective To observe the passage, identification, osteogenic differentiation and adipogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) separated by whole bone marrow adherent method, and provide a functional stable seed cell for further study of BMSCs.
Using the method of whole bone marrow adherent culture method was separated on BMSCs SD rats were cultured, purified. Under the inverted microscope observation in vivo dynamic changes in cell morphology; MTT colorimetric determination of OD growth curve; flow cytometry was used to identify BMSCs surface antigen; use of osteogenic and adipogenic induced by liquid liquid respectively. To induce osteoblasts, adipocytes.
Results (1) the method of whole bone marrow adherent cultured BMSCs fibroblast like cells adherent growth, cell morphology after passage, a spiral arrangement. (2) the growth curve of the third generation of BMSCs is "S" shape, has experienced three growth stages: incubation period, logarithmic growth phase and stop demurrage. (3) detection of BMSCs surface antigen shows the results of flow cytometry: CD29+99.45%, CD34+1.45%, CD44+99.52%, CD45+1.41%. (4) osteogenic, adipogenic differentiation, respectively using alizarin red and oil red "O" staining, the mineralized nodules turned orange, lipid droplets were dyed red.
Conclusion the whole bone marrow adherent culture and isolation of rat bone marrow stem cells can get higher purity BMSCs, which has potential to differentiate into osteoblasts and adipocytes.
Objective To observe the effect and possible mechanism of exogenous hepatocyte growth factor (HGF) on the apoptosis of primary stellate cells (HSCs) induced by TNF related apoptosis inducing ligand (TRAIL).
Method of recovery, passaging SD rat HSCs cell proliferation was used in the experiment. The experiment was divided into the following 4 groups: (1) HSCs control group; (2) HGF group; (3) TRAIL group; (4) HGF+TRAIL group. The experimental group cell culture 24h, 48h. under the inverted fluorescence under the microscope. The cell morphology was observed using immunocytochemical method to understand alpha- smooth muscle actin (a-SMA) in the cytoplasmic HSCs expression and MTT assay of exogenous HGF and TRAIL on proliferation of HSCs; the fluorescence intensity of flow cytometry to detect the apoptosis of HSCs Annexin-V-FITC/PI double staining method and flow cytometry fluorescence detection of HSCs surface DR5; use Western blot method to detect the expression of HSCs DR5 protein.
Results (1) immunocytochemical method showed that a-SMA cytoplasmic HSCs (+). (2) the results of MTT showed that HGF and TRAIL respectively in 50-200ng/ml, 0.5-1.5ug/ml concentration had no effect on the proliferation of HSCs, the inhibitory effect of TRAIL on HSCs in the presence of 2ug/ml; 24h, 48hHSCs: 0.22 (OD value respectively. + 0.02)% and (0.25 + 0.08)%, lower than that of other groups (P0.01). (3) the apoptosis of HSCs was detected by flow cytometry showed that HGF+TRAIL group 24h, the apoptosis rate of 48h was significantly higher than the control group, TRAIL group and HGF group (P0.01).
(4) the results showed the fluorescence intensity using immunofluorescence detection of HSCs surface DR5: group HGF+TRAIL 24h DR5, the fluorescence intensity of 48h was significantly higher than the control group, HGF group and TRAIL group (P0.01). (5) the expression of DR5 protein was detected by blot Western HGF+TRAIL in DR5 group showed significantly higher than white eggs the blank control group, HGF group and TRAIL group (P0.01).
Conclusion exogenous HGF can promote the apoptosis of HSCs induced by TRAIL, which may be related to the ability of HGF to increase the expression of DR5 protein on the surface of HSCs.
Objective To observe the effect of lentivirus mediated HGF-ShRNA transfection of bone marrow mesenchymal stem cells (BMSCs) and hepatic stellate cells (HSCs) on BMSCs HSCs were co cultured with the apoptotic effect of HSCs system, discusses two kinds of sources of HGF in HSCs cell apoptosis. To provide the experimental basis for BMSCs transplantation in the treatment of liver fibrosis.
Using the method of whole bone marrow adherent culture method was separated on BMSCs SD rats were cultured, purified, experiments were performed using the passage to the 3-4 generation of cells. Primary rat HSCs recovery, passaged. Application of 6 Hole culture plate (Transwell insert) on the membrane with BMSCs (1.3 * 105cells/well), in the 6 hole the culture plate inoculation of primary HSCs (1 * 105cells/well), conventional culture establish the upper and lower double cell co culture system. The experiment was divided into the following 5 groups: (1) control group: HSCs culture alone; (2) co culture group BMSCs+HSCs: BMSCs co cultured with HSCs; (3) co culture group HSCs transfection: using lentivirus mediated HGF-ShRNA transfection of HSCs after co cultured with BMSCs; (4) the group co cultured with BMSCs transfection using lentivirus mediated HGF-ShRNA transfection of BMSCs after co cultured with HSCs; (5) TNF-a group: TNF-a (100ng/ml) after pretreatment of BMSCs6h co cultured with HSCs or above; training system A observation of 24h, 48h, 72h. dynamic under the inverted microscope to observe the expression of green fluorescence in vivo cell morphological changes and expression of Western after transfection; blot transfection group HGF protein; enzyme linked immunosorbent assay (ELISA) of HGF and TRAIL in the supernatant after transfection, the supernatant concentration and the concentration of HGF in co culture detection; the expression of.Annexin V -FITC/PI flow cytometry after transfection and Western blot fluorescence expression of HGF protein was detected by double staining of apoptosis rate was determined by HSCs method; fluorescence quantitative PCR, Western and blot were detected in DR5, Caspase-8mRNA and protein expression. The data were analyzed by SPSS13.0 statistical software.
Results (1) the construction sequence and plasmid sequencing results. (2) 72h after transfection resulted in the difference of the two kinds of cells were observed under fluorescence microscope about 80% green fluorescent expression; BMSCs transfection group and HSCs transfection group HGF protein expression respectively (60.2 + 2.5)% and (63.3 + 4.3)%; flow cytometry BMSCs transfection group expression of green fluorescence positive rate was 70%. (3) concentration results of HGF ELISA assay in the supernatant of transfected group showed that the concentration of BMSCs in blank control group and HSCs control group were higher than those in HGF transfected with BMSCs co cultured group and transfected with HSCs co culture group (P0.01); (4) ELISA detection of co culture suggests that HGF and TRAIL concentration results in each supernatant was: the concentration of TNF-a in pretreatment group, HGF was significantly higher than that in HSCs control group, BMSCs+HSCs group and BMSCs were cultured in single culture group (P0.01); the concentration of TRAIL in BMSCs group was significantly higher than that of blank The control group, BMSCs+HSCs co cultured group and TNF-a pretreatment group (P0.01). (5) flow cytometry Annexin- V -FITC/PI double staining showed that the apoptosis of TNF-a HSCs pretreatment group was significantly higher than that of the other 3 groups, with a time dependent (P0.01); BMSCs transfection group HSCs co cultured apoptosis the rate is lower than the co culture group (P0.05). (6) FQ-PCR, after HSCs DR5, Western blotting were cultured to detect Caspase-8mRNA protein expression, HSCs TNF-a pretreatment group, DR5, Caspase-8mRNA and protein expression of HSCs was significantly higher than that in blank control group, group of co cultured BMSCs +HSCs, transfection of BMSCs co culture group transfection of HSCs and co culture group (P0.01 group) in HSCs DR5 were cultured for.BMSCs+HSCs, Caspase-8mRNA and protein expression was significantly higher than that of BMSCs by co culture group (P0.01).
Conclusion HGF from BMSCs promotes HSCs apoptosis by upregulated the expression of DR5 and apoptosis related protein Caspase-8 in HSCs, while TNF-a pretreatment can enhance this effect. HSCs's autocrine HGF has little effect on promoting apoptosis in co culture system.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R363

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