肝细胞生长因子促进肝星状细胞凋亡的机制研究

发布时间：2018-01-16 21:26

  本文关键词：肝细胞生长因子促进肝星状细胞凋亡的机制研究　出处：《广西医科大学》2012年博士论文　论文类型：学位论文

  更多相关文章： 骨髓间充质干细胞 全骨髓贴壁法 成骨分化 成脂分化 肝星状细胞 肿瘤坏死因子相关凋亡诱导配体 肝细胞生长因子 凋亡 死亡受体-5 骨髓间充质干细胞 肝星状细胞 肿瘤坏死因子相关凋亡诱导配体 肝细胞生长因子 死亡受体-

【摘要】：目的观察全骨髓贴壁法分离、培养大鼠骨髓间充质干细胞(BMSCs)的传代、鉴定及成骨分化、成脂分化的情况,为进一步研究BMSCs提供功能稳定的种子细胞。 方法采用全骨髓贴壁培养法对SD大鼠BMSCs进行分离、培养、纯化。在倒置相差显微镜下动态观察活体细胞形态学改变；MTT比色法测定OD值绘制生长曲线；用流式细胞仪鉴定BMSCs表面抗原；使用成骨诱导液及成脂诱导液分别诱导其向成骨细胞、成脂肪细胞分化。 结果(1)全骨髓贴壁法分离培养的BMSCs为贴壁生长的成纤维样细胞,传代后的细胞形态均一,呈漩涡状排列。(2)第3代BMSCs的生长曲线呈‘S’形,经历了三个生长时期：潜伏期、对数生长期和停滞期。(3)流式细胞仪检测BMSCs表面抗原结果示：CD29+99.45%、CD34+1.45%、CD44+99.52%、CD45+1.41%。(4)成骨、成脂细胞诱导分化后,分别使用茜素红及油红‘O’染色,其中矿化结节被染成橘红色、脂滴被染成红色。 结论全骨髓贴壁培养法分离、培养大鼠骨髓干细胞可获较高纯度的BMSCs,该类细胞具有向成骨细胞及脂肪细胞分化的潜能。 目的观察外源性肝细胞生长因子(HGF)在肿瘤坏死因子相关凋亡诱导配体(TRAIL)促进原代星状细胞(HSCs)凋亡中的作用及其可能机制。 方法复苏、传代SD大鼠原代HSCs,细胞增殖明显时用于实验。将实验分为以下4组：(1)HSCs空白对照组；(2)HGF组；(3)TRAIL组;(4)HGF+TRAIL组。各实验组细胞培养24h、48h。在倒置相差荧光显微镜下观察细胞形态。使用免疫细胞化学法了解alpha-平滑肌肌动蛋白(a-SMA)在HSCs胞浆中的表达情况以及MTT比色法检测外源性HGF及TRAIL分别对HSCs增殖的影响；流式细胞仪Annexin-V-FITC/PI双染法检测HSCs凋亡率以及流式细胞仪免疫荧光法检测HSCs表面DR5的荧光强度；使用Western blot法检测HSCs的DR5蛋白表达。 结果(1)免疫细胞化学法显示HSCs胞浆中a-SMA (+)。(2)MTT法结果显示：HGF及TRAIL分别在50-200ng/ml、0.5-1.5ug/ml各浓度下对HSCs增殖无影响,TRAIL在2ug/ml作用下对HSCs有抑制作用；24h、48hHSCs的OD值分别为：(0.22±0.02)%及(0.25±0.08)%,低于其它各组(P0.01)。(3)流式细胞仪检测各组HSCs的凋亡结果示：HGF+TRAIL组的24h、48h的凋亡率明显高于空白对照组、TRAIL组及HGF组(P0.01)。 (4)使用免疫荧光法检测HSCs表面DR5的荧光强度结果示：HGF+TRAIL组24h、48h的DR5荧光强度明显高于空白对照组、HGF组及TRAIL组(P0.01)。(5)Western blot法检测DR5蛋白的表达结果显示：HGF+TRAIL组中DR5蛋白明显高于空白对照组、HGF组及TRAIL组(P0.01)。 结论外源性HGF能促进TRAIL诱导的HSCs凋亡,可能与HGF能上调活化HSCs表面DR5蛋白表达有关。 目的观察慢病毒介导HGF-ShRNA转染骨髓间充质干细胞(BMSCs)及肝星状细胞(HSCs)后对BMSCs与HSCs共培养体系中HSCs凋亡的影响,探讨两种细胞来源的HGF在HSCs凋亡中的作用。为BMSCs移植治疗肝纤维化提供实验依据。 方法采用全骨髓贴壁培养法对SD大鼠BMSCs进行分离、培养、纯化,使用传代至第3-4代的细胞进行实验。大鼠原代HSCs复苏、传代。应用6孔培养板,在半透膜(transwell insert)上接种BMSCs(1.3×105cells/well),在6孔培养板上接种原代HSCs(1×105cells/well),建立上下双层细胞共培养体系,常规培养。将实验分为以下5组：(1)空白对照组：HSCs单独培养；(2)BMSCs+HSCs共培养组：BMSCs与HSCs共培养；(3)HSCs转染共培养组：使用慢病毒介导的HGF-ShRNA转染HSCs后与BMSCs共培养；(4)BMSCs转染共培养组：使用慢病毒介导的HGF-ShRNA转染BMSCs后与HSCs共培养；(5)TNF-a预处理组：TNF-a(100ng/ml)预处理BMSCs6h后与HSCs共培养；以上体系培养观察24h、48h、72h。在倒置相差显微镜下动态观察活体细胞形态学改变及转染后绿色荧光表达情况；Western blot检测转染组HGF蛋白的表达；酶联免疫吸附法(ELISA)检测共培养各组上清液中HGF及TRAIL的浓度以及转染后各组上清液HGF的浓度；流式细胞仪检测转染后荧光表达及Western blot法检测HGF蛋白的表达。Annexin Ⅴ-FITC/PI双染法检测HSCs凋亡率；荧光定量PCR、Western blot分别检测各组中DR5、Caspase-8mRNA及蛋白的表达。采用SPSS13.0统计软件进行数据分析。 结果(1)质粒测序结果与构建时序列结果一致。(2)转染后72h在导致相差荧光显微镜下观察两种细胞绿色荧光表达量约80%；BMSCs转染组及HSCs转染组中HGF蛋白下调分别为(60.2±2.5)%、(63.3±4.3)%；流式细胞仪检测BMSCs转染组绿色荧光表达阳性率为70%。(3)ELISA法检测转染组上清液中HGF浓度结果显示：BMSCs空白对照组及HSCs空白对照组中HGF的浓度分别高于BMSCs转染共培养组及HSCs转染共培养组(P0.01);(4)ELISA法检测共培养后各组上清液中HGF及TRAIL浓度结果提示：其中在TNF-a预处理组中HGF的浓度明显高于HSCs空白对照组、BMSCs+HSCs共培养组及BMSCs单独培养组(P0.01)；TRAIL在BMSCs组中的浓度明显高于空白对照组、BMSCs+HSCs共培养组及TNF-a预处理组(P0.01)。(5)流式细胞仪Annexin-Ⅴ-FITC/PI双染法检测结果示：TNF-a预处理组HSCs的凋亡率明显高于其它3组,且呈时间依赖性(P0.01);BMSCs转染共培养组中HSCs的凋亡率低于共培养组(P0.05)。(6)FQ-PCR、Western blotting检测共培养后各组HSCs的DR5、Caspase-8mRNA、蛋白表达,其中TNF-a预处理组中HSCs的DR5、Caspase-8mRNA及蛋白表达量明显高于HSCs空白对照组、BMSCs+HSCs共培养组、BMSCs转染共培养组及HSCs转染共培养组(P0.01)。BMSCs+HSCs共培养组中HSCs的DR5、Caspase-8mRNA及蛋白表达量明显高于BMSCs转染共培养组(P0.01)。 结论BMSCs来源的HGF通过上调HSCs中DR5及凋亡相关蛋白Caspase-8的表达促进HSCs的凋亡,而TNF-a预处理后能增强这一作用,HSCs自分泌的HGF在共培养体系中促进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.

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【参考文献】

相关期刊论文 前10条

1 Gerald Minuk;;肝星状细胞中细胞凋亡和存活的信号调控(英文)[J];中南大学学报(医学版);2007年05期

2 高杰;赵勇华;康鹏;梁明;李树臣;;TRAIL诱导大鼠肝星状细胞系HSC-T6凋亡及机制研究[J];临床肝胆病杂志;2010年03期

3 董薇;肝细胞生长因子及其临床应用研究进展[J];微生物学免疫学进展;2001年03期

4 杨丽;张荣华;谢厚杰;蔡宇;黄丰;;建立大鼠骨髓间充质干细胞稳定分离培养体系与鉴定[J];中国组织工程研究与临床康复;2009年06期

5 胡昆鹏;林楠;林继宗;邓美海;汤照峰;项鹏;许瑞云;;人骨髓间质干细胞对肝星状细胞的体外调控[J];中国组织工程研究与临床康复;2009年27期

6 宫恩年;商丰元;郭子宽;楼晓;;肝细胞生长因子基因修饰的骨髓间充质干细胞移植治疗兔股骨头坏死(英文)[J];中国组织工程研究与临床康复;2009年28期

7 苏思标;姜海行;王东旭;覃山羽;梁梓宇;;骨髓间充质干细胞调控肝星状细胞RhoA、P27的表达[J];世界华人消化杂志;2009年32期

8 陈国忠;姜海行;陆正峰;肖健;梁梓宇;覃山羽;;骨髓间充质干细胞共培养对肝星状细胞增殖、凋亡和RohA表达的调控[J];世界华人消化杂志;2010年16期

9 赵东长,陈蕊,余伟华,雷俊霞,彭延文,刘宇,张秀明,李树浓,项鹏;骨髓间质干细胞抑制肝星状细胞增殖与活化的体外研究[J];中国病理生理杂志;2005年06期

10 梁忆波;崔琳;于建宪;纪萍;;组织芯片检测结直肠癌中HGF/c-met的表达及其与肿瘤血管生成的关系[J];中国普外基础与临床杂志;2010年07期

，

本文编号：1434897