肝细胞损伤诱发骨髓间充质干细胞间质化与促癌作用研究
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摘要
研究背景
近年来随着对肿瘤研究的深入,肿瘤微环境的重要性引起了各国政府和学术界的广泛关注。人们逐渐认识到肿瘤组织是由一群异质性的肿瘤细胞亚群所组成,不同的亚群在体内增生和形成肿瘤的能力具有显著差异,其中数量较少的肿瘤干细胞(cancer stemcell,CSC)才有成瘤及维持恶性显型的作用,而CSC又与肿瘤微环境之间存在着密切的联系。正常的微环境可以抑制肿瘤细胞的生长,而异常的微环境则可以促进肿瘤细胞的生长,这就意味着肿瘤的发生机制不仅仅是细胞恶变这一单方面因素,肿瘤微环境在参与调控肿瘤的发生发展中发挥着重要作用。尽管肿瘤微环境中的各种细胞在肿瘤发生发展过程中均起到一定作用,但肿瘤相关成纤维细胞(tumor associated fibroblasts, TAFs)在这一过程中的作用尤为重要,而骨髓来源间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)是TAFs的重要来源之一。肝纤维性病变是肝脏对各种原因所致肝损伤的“创伤愈合”反应,也是肝硬化发生的前奏和必经的中间环节,而肝硬化与肝癌的关系十分密切,但是它们之间的内在关系目前尚不明确,我们猜测肝纤维化很可能是肝癌潜在的诱导因素,但目前关于导致肝纤维化之前的诱发因素即肿瘤(肝癌)发生发展之前微环境变化对在肿瘤促进作用的研究还鲜有报道。
研究目的
本研究的目的在于探讨外源性致癌剂短时间作用对肝细胞基因表达水平的影响；探索损伤的肝细胞分泌因子是否导致微环境恶性转化以及转化后的微环境对肿瘤成瘤和迁移能力的影响。
研究方法
1.大鼠间充质干细胞的原代培养和鉴定
采用全骨髓贴壁法原代培养大鼠间充质干细胞,并检测其多向分化潜能以及细胞表面标记物。
2.外源性致癌物对BRL细胞的作用
1)MTT试验摸索黄曲霉毒素B1(aflatoxin B1,AFB1)对BRL细胞存活率的影响并绘制生长曲线,观察染毒后细胞增殖能力的变化；
2)染色体核型分析,检测AFB1染毒对BRL细胞染色体的影响；
3)MTT、单细胞凝胶电泳和Hoechst33258染色用来检测DEN对人胎肝干细胞的累积毒性效应；
4)实时定量PCR检测AFB1染毒对BRL细胞基因表达水平的影响
3.微环境的恶性改变以及其对肿瘤成瘤能力的影响
1)建立Transwell共培养模型：模拟损伤肝细胞对间充质干细胞的影响；
2)免疫组化检测诱导前后BMSCs α-SMA表达水平的变化；
3)软琼脂克隆试验、划痕试验以及细胞侵袭试验评估微环境改变对肿瘤细胞成瘤能力的影响。
研究结果
1.二乙基亚硝(DEN)对肝干细胞毒性效应
人胎肝干细胞经900、1350、1800μg/ml的DEN处理48h后,细胞存活率低于阴性对照组,差异有显著性(P<0.01)；人胎肝干细胞经过DEN处理24h后,出现染色体固缩,浓染等细胞凋亡表现；单细胞凝胶电泳实验发现,当DEN浓度为900和1350μg/ml时,具有致DNA断裂的作用,尾部DNA含量分别达到(18.44±4.99)%和(17.33-3.29)%,明显高于对照组(0.02±0.00)%,差异有显著性(P<0.01),但在浓度为1800和2250μg/ml时,彗星拖尾程度明显降低,差异有显著性(P<0.01)。
2. AFB1引起BRL细胞毒性效应
MTT显示AFB1剂量为3mmol/L时,BRL细胞存活率为43.59%显著低于对照组100%,差异具有显著性(P<0.05),计算IC50为2.96mmol/L。
3.成功培养大鼠间充质干细胞(BMSCs)
培养出的BMSCs细胞呈纺锤形或梭形的细胞,形似成纤维样细胞,检测其表面标记物CD29阳性,阳性率为92.5%：CD44阳性,阳性率为65%；CD45为阴性,其阳性率为3.5%,具有成骨、成脂性。
4.核型分析
AFB1染毒10h后.可观察到BRL细胞染色体出现断裂、裂隙和多倍体等改变。
5.定量PCR检测mRNA表达水平
检测了经AFB1染毒后的BRL细胞基因表达水平的变化,发现TGF-β、PDGF-A、PDGF-B、PDGF-C和PDGF-D表达量均有不同程度的上调。
6.损伤后的BRL细胞对BMSCs的影响
利用Transwell小室建立了AFB1染毒后的BRL细胞与BMSCs共培养模型,并且检测到BMSCs的a-SMA表达量上调。
7.恶性转化的间充质干细胞对HepG2细胞的影响
通过软琼脂克隆试验、划痕试验以及侵袭试验检测发现,诱导后BMSCs可显著增强HepG2的迁徙和侵袭能力。
研究结论
1.DEN对胎肝干细胞具有直接的毒性效应,引起DNA损伤,具有潜在的遗传毒性。
2.AFB1短期处理BRL细胞导致细胞TGF-β和PDGF mRNA表达水平升高,其分泌的细胞因子可以上调间充质干细胞α-SMA的表达。
3.BRL细胞非致瘤性损伤可改变微环境,从而增强肿瘤细胞的成瘤、迁徙和侵袭能力。
Background:
With the developing of cancer research, the tumor microenvironment has aroused widespread concern by governments and academia fields in recent years. People gradually realized that tumor is heterogeneity, and only a small part of cells which we called cancer stem cells (CSCs) are the source of tumor formation and development. Moreover, there was a close relationship between CSCs and the tumor microenvironment. It was found that normal microenvironment can inhibit tumor development, and abnormal microenvironment can promote tumor development. This prompts that mutation might not be the unique factor in regulating the tumor formation, microenvironment might also be involved. Although a variety of cells in the tumor microenvironment play a certain role in the tumor development, the role of tumor associated fibroblasts (TAFs) might be more important. Bone marrow mesenchymal stem cells (BMSCs) are among the key sources of TAFs. Liver fibrosis is a wound healing response in liver, also a necessary intermediate of hepatocirrhosis. There is a close relationship between hepatic fibrosis and hepatocellular carcinoma, but the intrinsic relationship between them is unclear. We speculated that liver fibrosis might be a potential factor in hepatocellular carcinoma, but the role of the matrix before tumor had rarely been reported.
Objective:
We aimed to investigate the effect of gene expression in liver cells after short-time exposure to exogenous carcinogen, to explore the role of secreted factors of the damaged liver cells on malignant transformation of the microenvironment, and to clarify the influence of transformed microenvironment in tumorigenicity and tumor migration.
Methods:
1.Primary culture and appraisal the rat mesenchymal stem cells
We used the whole bone marrow adherent method to culture the rat bone marrow mesenchymal stem cells (BMSCs) which were identified by detecting its multi-directional differentiation potential and cell surface markers.
2.The roles of exogenous carcinogens on BRL cell
1) After exposed to Aflatoxin B1(AFB1), cells survival rate of BRL was
assessed by Methyl thiazolyl tetrazolium (MTT) assay. Through drawing growth curve,the differences in proliferation ability were evaluated before and after exposed to AFB1.
2) The chromosome damage of BRL cells exposed to AFB1was analysis by Karyotype.
3) MTT, SCGE and Hoechst33258stain were detect the cumulative cytotoxicity for DEN on human liver stem cells.
4) Real-time quantitative PCR was used to detect the levels of gene expressions change after the BRL cell exposed to AFB1.
3.The microenvironment variation and their effects on the ability of the tumor information.
1) To establish the Transwell co-cultured model and simulate the influence of damage liver cells on BMSCs.
2) Immunohistochemical was used to compare the level of α-SMA expression on BMSCs before and after induced.
3) Soft agar test, scratch test and invasion test were used to assess the effect of microenvironmental variation on tumor information.
Results:
1. The toxicity effect of DEN in fetal hepatic stem cells
The survival rate of fetal hepatic stem cell was significantly lower than that of control group (P<0.05) at the dose of900,1350and1800μg/ml after48h treatment. When human hepatic stem cells were exposed to DEN for24h, cellular apoptosis occurred with karyopyknosis and deep staining. Break in DNA strands was found at the concentrations of900and1350μg/ml of DEN; the content of DNA in tail [(18.44±4.99)%, (17.33±3.29)%, respectively] was higher than the control [(0.015±0.004)%](P<0.01), while the comet length was decreased at the higher concentrations of DEN (1800and2250μg/ml)
2. The toxicity effect of AFB1in BRL cells
The survival rate of BRL cells was43.59% at the dose of3mmol/L AFB1by MTT assay. It was significantly lower than the control (P<0.05). The value of IC50was2.96mmol/L
3. Successfully cultured rat BMSCs
The cells showed spindle-shaped or spindle-shaped, and the cell shape was similarly to fibroblast cells. Detection of surface markers on BMSCs showed that positive rate of CD29, CD44, CD45was92.5%,65%,3.5%respectively. In the conditioned medium, BMSCs could differentiate into osteoblast or fat cells.
4. Karyotype analysis
Chromosomal abnormalities including polyploidy, gap, and breakage were observed in BRL after exposure to AFB1for1Oh.
5. Real-time quantitative PCR detected the mRNA levels
The expressions of TGF-β, PDGF-A, PDGF-B, PDGF-C and PDGF-D mRNAs were increased in varied degrees in AFB1-exposed BRL compared with untreated BRL cells.
6. The effect of damaged BRL in BMSCs
We co-cultured AFB1-exposed BRL and BMSCs by using Tranwell chamber, and detected upregulation of a-SMA expression in BMSCs.
7. To investigate the effect of malignant transformed BMSCs on HepG2cell line
Tumorigenicity, tumor migration and invasion abilities of HepG2were enhanced in malignant transformed BMSCs by using soft-agar cloning assay, scratch test and invasive test.
Conclusions
1. DEN could induce directly DNA damage of human hepatic stem cells in vitro, so it might have some potential genetic toxicity;
2. Pretreatment with AFB1increased the TGF-β and PDGF mRNA levels of BRL cells, and the cytokines of secreted by injured BRL cells up-regulated the expression of α-SMA in BMSCs.
3. Precancerous injuries of BRL cells could cause the microenvironment change, which can enhance the ability of tumor formation, migration and invasion.
近年来随着对肿瘤研究的深入,肿瘤微环境的重要性引起了各国政府和学术界的广泛关注。人们逐渐认识到肿瘤组织是由一群异质性的肿瘤细胞亚群所组成,不同的亚群在体内增生和形成肿瘤的能力具有显著差异,其中数量较少的肿瘤干细胞(cancer stemcell,CSC)才有成瘤及维持恶性显型的作用,而CSC又与肿瘤微环境之间存在着密切的联系。正常的微环境可以抑制肿瘤细胞的生长,而异常的微环境则可以促进肿瘤细胞的生长,这就意味着肿瘤的发生机制不仅仅是细胞恶变这一单方面因素,肿瘤微环境在参与调控肿瘤的发生发展中发挥着重要作用。尽管肿瘤微环境中的各种细胞在肿瘤发生发展过程中均起到一定作用,但肿瘤相关成纤维细胞(tumor associated fibroblasts, TAFs)在这一过程中的作用尤为重要,而骨髓来源间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)是TAFs的重要来源之一。肝纤维性病变是肝脏对各种原因所致肝损伤的“创伤愈合”反应,也是肝硬化发生的前奏和必经的中间环节,而肝硬化与肝癌的关系十分密切,但是它们之间的内在关系目前尚不明确,我们猜测肝纤维化很可能是肝癌潜在的诱导因素,但目前关于导致肝纤维化之前的诱发因素即肿瘤(肝癌)发生发展之前微环境变化对在肿瘤促进作用的研究还鲜有报道。
研究目的
本研究的目的在于探讨外源性致癌剂短时间作用对肝细胞基因表达水平的影响；探索损伤的肝细胞分泌因子是否导致微环境恶性转化以及转化后的微环境对肿瘤成瘤和迁移能力的影响。
研究方法
1.大鼠间充质干细胞的原代培养和鉴定
采用全骨髓贴壁法原代培养大鼠间充质干细胞,并检测其多向分化潜能以及细胞表面标记物。
2.外源性致癌物对BRL细胞的作用
1)MTT试验摸索黄曲霉毒素B1(aflatoxin B1,AFB1)对BRL细胞存活率的影响并绘制生长曲线,观察染毒后细胞增殖能力的变化；
2)染色体核型分析,检测AFB1染毒对BRL细胞染色体的影响；
3)MTT、单细胞凝胶电泳和Hoechst33258染色用来检测DEN对人胎肝干细胞的累积毒性效应；
4)实时定量PCR检测AFB1染毒对BRL细胞基因表达水平的影响
3.微环境的恶性改变以及其对肿瘤成瘤能力的影响
1)建立Transwell共培养模型：模拟损伤肝细胞对间充质干细胞的影响；
2)免疫组化检测诱导前后BMSCs α-SMA表达水平的变化；
3)软琼脂克隆试验、划痕试验以及细胞侵袭试验评估微环境改变对肿瘤细胞成瘤能力的影响。
研究结果
1.二乙基亚硝(DEN)对肝干细胞毒性效应
人胎肝干细胞经900、1350、1800μg/ml的DEN处理48h后,细胞存活率低于阴性对照组,差异有显著性(P<0.01)；人胎肝干细胞经过DEN处理24h后,出现染色体固缩,浓染等细胞凋亡表现；单细胞凝胶电泳实验发现,当DEN浓度为900和1350μg/ml时,具有致DNA断裂的作用,尾部DNA含量分别达到(18.44±4.99)%和(17.33-3.29)%,明显高于对照组(0.02±0.00)%,差异有显著性(P<0.01),但在浓度为1800和2250μg/ml时,彗星拖尾程度明显降低,差异有显著性(P<0.01)。
2. AFB1引起BRL细胞毒性效应
MTT显示AFB1剂量为3mmol/L时,BRL细胞存活率为43.59%显著低于对照组100%,差异具有显著性(P<0.05),计算IC50为2.96mmol/L。
3.成功培养大鼠间充质干细胞(BMSCs)
培养出的BMSCs细胞呈纺锤形或梭形的细胞,形似成纤维样细胞,检测其表面标记物CD29阳性,阳性率为92.5%：CD44阳性,阳性率为65%；CD45为阴性,其阳性率为3.5%,具有成骨、成脂性。
4.核型分析
AFB1染毒10h后.可观察到BRL细胞染色体出现断裂、裂隙和多倍体等改变。
5.定量PCR检测mRNA表达水平
检测了经AFB1染毒后的BRL细胞基因表达水平的变化,发现TGF-β、PDGF-A、PDGF-B、PDGF-C和PDGF-D表达量均有不同程度的上调。
6.损伤后的BRL细胞对BMSCs的影响
利用Transwell小室建立了AFB1染毒后的BRL细胞与BMSCs共培养模型,并且检测到BMSCs的a-SMA表达量上调。
7.恶性转化的间充质干细胞对HepG2细胞的影响
通过软琼脂克隆试验、划痕试验以及侵袭试验检测发现,诱导后BMSCs可显著增强HepG2的迁徙和侵袭能力。
研究结论
1.DEN对胎肝干细胞具有直接的毒性效应,引起DNA损伤,具有潜在的遗传毒性。
2.AFB1短期处理BRL细胞导致细胞TGF-β和PDGF mRNA表达水平升高,其分泌的细胞因子可以上调间充质干细胞α-SMA的表达。
3.BRL细胞非致瘤性损伤可改变微环境,从而增强肿瘤细胞的成瘤、迁徙和侵袭能力。
Background:
With the developing of cancer research, the tumor microenvironment has aroused widespread concern by governments and academia fields in recent years. People gradually realized that tumor is heterogeneity, and only a small part of cells which we called cancer stem cells (CSCs) are the source of tumor formation and development. Moreover, there was a close relationship between CSCs and the tumor microenvironment. It was found that normal microenvironment can inhibit tumor development, and abnormal microenvironment can promote tumor development. This prompts that mutation might not be the unique factor in regulating the tumor formation, microenvironment might also be involved. Although a variety of cells in the tumor microenvironment play a certain role in the tumor development, the role of tumor associated fibroblasts (TAFs) might be more important. Bone marrow mesenchymal stem cells (BMSCs) are among the key sources of TAFs. Liver fibrosis is a wound healing response in liver, also a necessary intermediate of hepatocirrhosis. There is a close relationship between hepatic fibrosis and hepatocellular carcinoma, but the intrinsic relationship between them is unclear. We speculated that liver fibrosis might be a potential factor in hepatocellular carcinoma, but the role of the matrix before tumor had rarely been reported.
Objective:
We aimed to investigate the effect of gene expression in liver cells after short-time exposure to exogenous carcinogen, to explore the role of secreted factors of the damaged liver cells on malignant transformation of the microenvironment, and to clarify the influence of transformed microenvironment in tumorigenicity and tumor migration.
Methods:
1.Primary culture and appraisal the rat mesenchymal stem cells
We used the whole bone marrow adherent method to culture the rat bone marrow mesenchymal stem cells (BMSCs) which were identified by detecting its multi-directional differentiation potential and cell surface markers.
2.The roles of exogenous carcinogens on BRL cell
1) After exposed to Aflatoxin B1(AFB1), cells survival rate of BRL was
assessed by Methyl thiazolyl tetrazolium (MTT) assay. Through drawing growth curve,the differences in proliferation ability were evaluated before and after exposed to AFB1.
2) The chromosome damage of BRL cells exposed to AFB1was analysis by Karyotype.
3) MTT, SCGE and Hoechst33258stain were detect the cumulative cytotoxicity for DEN on human liver stem cells.
4) Real-time quantitative PCR was used to detect the levels of gene expressions change after the BRL cell exposed to AFB1.
3.The microenvironment variation and their effects on the ability of the tumor information.
1) To establish the Transwell co-cultured model and simulate the influence of damage liver cells on BMSCs.
2) Immunohistochemical was used to compare the level of α-SMA expression on BMSCs before and after induced.
3) Soft agar test, scratch test and invasion test were used to assess the effect of microenvironmental variation on tumor information.
Results:
1. The toxicity effect of DEN in fetal hepatic stem cells
The survival rate of fetal hepatic stem cell was significantly lower than that of control group (P<0.05) at the dose of900,1350and1800μg/ml after48h treatment. When human hepatic stem cells were exposed to DEN for24h, cellular apoptosis occurred with karyopyknosis and deep staining. Break in DNA strands was found at the concentrations of900and1350μg/ml of DEN; the content of DNA in tail [(18.44±4.99)%, (17.33±3.29)%, respectively] was higher than the control [(0.015±0.004)%](P<0.01), while the comet length was decreased at the higher concentrations of DEN (1800and2250μg/ml)
2. The toxicity effect of AFB1in BRL cells
The survival rate of BRL cells was43.59% at the dose of3mmol/L AFB1by MTT assay. It was significantly lower than the control (P<0.05). The value of IC50was2.96mmol/L
3. Successfully cultured rat BMSCs
The cells showed spindle-shaped or spindle-shaped, and the cell shape was similarly to fibroblast cells. Detection of surface markers on BMSCs showed that positive rate of CD29, CD44, CD45was92.5%,65%,3.5%respectively. In the conditioned medium, BMSCs could differentiate into osteoblast or fat cells.
4. Karyotype analysis
Chromosomal abnormalities including polyploidy, gap, and breakage were observed in BRL after exposure to AFB1for1Oh.
5. Real-time quantitative PCR detected the mRNA levels
The expressions of TGF-β, PDGF-A, PDGF-B, PDGF-C and PDGF-D mRNAs were increased in varied degrees in AFB1-exposed BRL compared with untreated BRL cells.
6. The effect of damaged BRL in BMSCs
We co-cultured AFB1-exposed BRL and BMSCs by using Tranwell chamber, and detected upregulation of a-SMA expression in BMSCs.
7. To investigate the effect of malignant transformed BMSCs on HepG2cell line
Tumorigenicity, tumor migration and invasion abilities of HepG2were enhanced in malignant transformed BMSCs by using soft-agar cloning assay, scratch test and invasive test.
Conclusions
1. DEN could induce directly DNA damage of human hepatic stem cells in vitro, so it might have some potential genetic toxicity;
2. Pretreatment with AFB1increased the TGF-β and PDGF mRNA levels of BRL cells, and the cytokines of secreted by injured BRL cells up-regulated the expression of α-SMA in BMSCs.
3. Precancerous injuries of BRL cells could cause the microenvironment change, which can enhance the ability of tumor formation, migration and invasion.
引文
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