人类脐带间充质干细胞阻抑肝内胆管癌细胞生长的分子机制研究
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摘要
研究背景与目的
肝内胆管细胞癌([ntrahepatic cholangiocarcinoma, ICC),又被称为外周型胆管癌,或者胆管癌之肝内型,它是肝内胆管被覆上皮发生的,并起源于肝内二级分支以下胆管上皮的原发性肝癌的其中一种类型,据世界范围内统计,ICC约占原发性肝脏所发生恶性肿瘤的10%-15%,其发生率仅次于肝细胞癌(HCC),但近年来已经上升为肝内原发肿瘤导致死亡的第一位,目前认为,肝内胆管癌的发生与胆管结石、乙型病毒性肝炎、丙型病毒性肝炎感染以及其他胆管发生的炎症等因素密切相关。肝内胆管癌具有发生隐匿、临床症状不典型、恶性程度高、发展迅速、容易转移等特点,尽管目前在早期诊断和治疗方法上有了很大改进,但是仍不能改变其不良预后,因此发现新的、有效的抗肝内胆管癌的方法是当务之急。随着广大研究者对肿瘤分子机制不断的深入认识,现在已将恶性肿瘤定义为一种多因子、多步骤和多基因参与的全身性疾病,因此,在近10年中,肿瘤的生物学治疗以及基因治疗成为肿瘤治疗的研究热点和新希望。
间充质干细胞(MSCs)是干细胞的一种类型,因其能分化成间质组织而得名,MSCs是一类具有自我更新和多向分化潜能的细胞,在特定的诱导环境下,可进一步分化为骨、软骨、脂肪、肌腱、肌肉等组织,目前,MSCs已被广泛应用于细胞治疗及组织再生方面的研究。人脐带来源的间充质干细胞因较其他来源的干细胞具有更明显的优势,所以越来越受到人们的重视,研究表明,人脐带组织中分离得到的间充质干细胞既具有较强的增殖能力,又具有向其它类型细胞(神经元样细胞、少突胶质细胞、脂肪细胞及星形胶质细胞等)分化的能力,加之其来源广泛、易获得且具有较强的肿瘤趋化能力等特点,在细胞移植及基因治疗、组织工程等方面具有广阔的应用前景。因干细胞强的肿瘤趋化能力,很多研究者聚焦于其与肿瘤细胞之间的关系,
干细胞移植技术已应用于几种造血及非造血肿瘤的治疗,将干细胞应用于肿瘤治疗具有广泛的前景,有研究证实,某些实体肿瘤的生长可被干细胞所抑制,另有研究显示,这种抑制作用是通过干细胞自身分泌的可溶性活性因子实现的。
但是,目前尚没有关于间充质干细胞对肝内胆管癌细胞作用研究的相关报道,因此本研究对此作用及其分子机制进行了初步探讨。本研究通过Transwell共培养实验、MTT比色实验、DNA断裂实验等方法,检测脐带间充质干细胞对肝内胆管癌HCCC-9810细胞增殖和凋亡的影响,同时借助于Western Blot及免疫荧光染色方法,检测与肝内胆管癌发生发展密切相关通路中蛋白质表达水平的变化,进一步探讨脐带间充质干细胞作用于肝内胆管癌细胞生长可能的分子机制。
第一部分
人类脐带间充质干细胞对肝内胆管癌细胞生长的影响
研究目的
1、检测人类脐带间充质干细胞对肝内胆管癌HCCC-9810细胞生长的影响。
2、检测不同浓度人类脐带间充质干细胞条件培养基对肝内胆管癌HCCC-9810细胞生长的影响。
3、检测人类脐带间充干细胞条件培养基对肝内胆管癌HCCC-9810细胞生长的影响是否由诱导HCCC-9810细胞凋亡所介导。
4、检测人类脐带间充质干细胞及其条件培养基对裸鼠肝内胆管癌移植瘤生长的影响。
研究方法
1、采用transwell共培养系统共培养人类脐带间充质干细胞(HUVECs细胞作为对照)与HCCC-9810细胞,应用细胞计数法计数处理后的肿瘤细胞数。
2、配置不同浓度的干细胞条件培养基(10%、25%、50%、75%)处理HCCC-9810细胞,应用MTT细胞增殖实验及细胞克隆实验测定HCCC-9810细胞增殖率的变化。
3、应用Hoechst33258染色实验检测干细胞条件培养基诱导HCCC-9810细胞凋亡的作用。
4、采用皮下注射细胞悬液的方法,构建裸鼠移植瘤模型,测量注射不同细胞组间肿瘤发生率及体积大小,并采用脐带间充质干细胞条件培养基注射于已形成肿瘤部位的方法,测量肿瘤体积的变化。
5、采用SPSS16.0统计软件用于实验数据的统计学分析,数据的统计学意义通过独立组间学生t检验,采用(x±S)进行分析。
结果
1、脐带间充质干细胞对肝内胆管癌HCCC-9810细胞增殖的影响
HCCC-9810细胞与hUC-MSCs细胞共培养48h后,计数细胞为7.64×105个／m1,而与HUVECs细胞共培养48h后,细胞计数为11.98×105个/ml,由此可见,脐带间充质干细胞诱导的HCCC-9810肿瘤细胞抑制效应明显高于HUVECs对照组,与对照组相比,肿瘤细胞增殖率下降了34.4%,差异具明显的统计学意义(p<0.05)。为了进一步检测培养的脐带间充质干细胞微环境是否能抑制肿瘤细胞的生长,我们检测了干细胞条件培养基对HCCC-9810细胞增殖的影响,结果显示,HCCC-9810细胞经不同浓度间充质干细胞条件培养液处理后,细胞增殖抑制率呈现时间与剂量依赖性,HCCC-9810细胞经50%间充质干细胞条件培养基培养24小时后,增殖抑制率由6.21%增至49.86%,与对照组相比有显著差距。我们又采用了细胞克隆实验进一步证实这种抑制作用的存在,结果显示,与对照组相比,脐带间充质干细胞条件培养基处理组细胞克隆单位形成数明显低于对照组(p<0.01)。
2、脐带间充质干细胞条件培养液对HCCC-9810细胞凋亡的影响
为了证实干细胞条件培养基对HCCC-9810细胞的增殖抑制是否由凋亡介导,进一步采用DNA染色的方法观察核染色质形态。结果显示,HCCC-9810细胞经50%脐带间充质干细胞条件培养液处理48h后,经Hoechst33258染色呈现明显细胞凋亡,凋亡细胞比率明显高于对照组,与对照组相比,差异具明显的统计学意义(p<0.05VS.HUVEC对照组)。
3、脐带间充质干细胞抑制BALB/C裸鼠移植瘤的形成
结果显示,皮下注射不同细胞组50天后,注射HCCC.9810+MSC细胞组,移植瘤出现时间及肿瘤体积均明显低于对照组：观察至第35-40天,有7只裸鼠长出可视肿瘤(观察至第50天,平均肿瘤体积约为1.3cm3),此组中另外3只裸鼠始终无肿瘤发生。注射HCCC.9810细胞组,或者HCCC.9810+HUVEC细胞组中所有实验小鼠均形成了可视肿瘤(观察至第50天,平均肿瘤体积分别为2.6cn13,2.5cm3).实验的50-70天,分别给HCCC-9810实验组继续注射间充质干细胞或者HUVECs细胞的条件培养基,结果显示,注射间充质干细胞条件培养基组,原本已形成的肿瘤体积明显缩小,至实验结束,干细胞条件培养基处理组小鼠肿瘤的平均体积降至1.4cm3,而对照组肿瘤体积继续增长至3.5cm3,差异具显著的统计学意义。
结论
1、脐带间充质干细胞可抑制肝内胆管癌HCCC-9810细胞的生长。
2、经50%脐带间充质干细胞条件培养基处理肝内胆管癌HCCC-9810细胞24小时可达到细胞的半数抑制率。
3、脐带间充质干细胞可抑制BALB/C裸鼠移植瘤的形成,且其条件培养基可使已形成的肿瘤体积缩小。
4、脐带间充质干细胞对肝内胆管癌HCCC-9810细胞生长的抑制作用是通过诱导细胞凋亡所介导的。
第二部分
人类脐带间充质干细胞阻抑肝内胆管癌细胞生长的分子机制
研究目的
1、检测经脐带间充质干细胞条件培养基作用后,肝内胆管癌HCCC-9810细胞中PI3K/Akt信号转导通路主要蛋白的表达水平变化。
2、检测经脐带间充质干细胞条件培养基作用后,肝内胆管癌HCCC-9810细胞中Wnt/β-catenin信号转导通路主要蛋白的表达水平变化。
3、检测加入GSK-3β(?)抑制剂或者激活剂,或加入Akt激活剂,作为单一处理因素,或者联合脐带间充质干细胞条件培养基处理HCCC-9810细胞,所介导的肝内胆管癌细胞增殖及相关蛋白的变化。
研究方法
1、Western Blot方法检测肝内胆管癌HCCC-9810细胞经脐带间充质干细胞条件培养基处理前后p-PI3K、p-PDK1、p-Akt Akt\p-GSK-3β、GSK-3β、β-Catenin、 c-Myc、CyclinD1等蛋白的表达变化。
2、细胞免疫荧光染色方法检测肝内胆管癌HCCC-9810细胞经脐带间充质干细胞条件培养基处理前后p-Akt、p-GSK-3β、β-Catenin的表达变化。
3、分别加入GSK-3β特异性激活试剂SNP及特异性抑制试剂CHIR99021作为单一处理因素,或者与脐带间充质干细胞条件培养基联合作用后,观察肝内胆管癌HCCC-9810细胞增殖能力变化,并进一步检测β-Catenin的表达变化。
4、加入Akt特异性激活剂IGF-1预处理HCCC-9810细胞15min后,进一步观察脐带间充质干细胞条件培养基对肝内胆管癌HCCC-9810细胞增殖能力的影响。
结果
1、hUC-MSCs条件培养基可抑制肝内胆管癌HCCC-9810细胞中PI3K/Akt信号转导通路相关蛋白的表达
1) Western Blot实验结果显示,HCCC-9810细胞经50%hUC-MSCs条件培养基处理48小时后,与对照组相比,p-PI3K、p-PDK1、p-Akt、p-GSK-3β的表达明显下调,而总的Akt(T-Akt)与总的GSK-3β(T-GSK-3β)的表达无明显变化,而HUVEC条件培养基处理组上述蛋白表达与空白对照组相比无明显变化。统计显示,HUVEC条件培养基处理组与hUC-MSC条件培养基处理组相比,p-Akt与T-Akt的灰度比值分别为0.65±0.04及0.19±0.02(P<0.001),p-GSK-3β与T-GSK-3β的灰度比值分别为0.48±0.03及0.22±0.05(P<0.01),比值差异均具有明显的统计学意义。
2)细胞免疫荧光染色结果显示,与空白对照组相比,干细胞条件培养基处理组p-Akt、p-GSK-3β的相对荧光强度分别为37.3±0.8%、35.7±1.2%,空白对照组p-Ak、p-GSK-3β的蛋白荧光强度明显高于干细胞条件培养基处理组,两组间差异的统计学意义较显著(P<0.01, p-Akt; P<0.01, p-GSK-3β).
2、hUC-MSCs条件培养基可抑制肝内胆管癌HCCC-9810细胞中Wnnt信号转导通路相关蛋白的表达
1) Western Blot实验结果显示,肝内胆管癌HCCC-9810细胞经50%hUC-MSCs条件培养基处理48小时后,与空白对照组相比,β-catenin、c-Myc、cyclin-D1的表达明显下调,并且β-catenin的核富集量明显减少,而HUVEC条件培养基处理组β-catenin、c-Myc、cyclin-D1的蛋白表达情况几乎与空白对照组相似。
2)细胞荧光免疫染色结果显示,干细胞条件培养基处理组β-catenin在细胞核中荧光强度较空白对照组明显减弱。
3、hUC-MSCs条件培养基通过Akt通路调节GSK-3β的活性,从而下调β-catenin的表达,抑制Wnnt通路
经GSK-3β抑制剂(CHIR99021)处理后,48h观察点结果显示,CHIR99021与hUC-MSCs条件培养基联合处理组与hUC-MSCs条件培养基单独处理组相比,HCCC-9810细胞的抑制作用明显减弱p<0.001),并且CHIR99021与hUC-MSCs条件培养基联合处理组增加了β-catenin的蛋白表达水平。而经GSK-3β激活剂(SNP)处理后,48h观察点结果显示,SNP与hUC-MSCs条件培养基联合处理组与hUC-MSCs条件培养基单独处理组相比,HCCC-9810细胞的抑制作用明显增强(p<0.001),并且SNP与]hUC-MSCs条件培养基联合处理组下调了β-catenin的蛋白表达水平。加入Akt通路激活剂IGF-1预处理HCCC-9810细胞15min后,24h观察点结果显示,IGF-1与hUC-MSCs条件培养基联合处理组与hUC-MSCs条件培养基单独处理组相比,IGF-1与hUC-MSCs条件培养基联合处理组对HCCC-9810细胞的抑制作用明显下降(p<0.05)。
结论
1、hUC-MSCs条件培养基可通过抑制PI3K/Akt信号通路阻抑肝内胆管癌HCCC-9810细胞生长。
2、hUC-MSCs条件培养基亦可通过抑制Wnt/β-catenin信号通路阻抑肝内胆管癌HCCC-9810细胞生长。
3、hUC-MSCs条件培养基对肝内胆管癌细胞的抑制作用可能是通过以GSK-3β作为“桥梁蛋白”的Akt和Wnnt两条通路的串话实现的。
Background and Objective
Intrahepatic cholangiocarcinoma(ICC), also known as peripheral cholangiocarcinoma, is a malignancy whose pathogenesis involves abnormal biliary epithelial differentiation and is originated from the secondary bile duct epithelium branch. It is one kind of primary liver cancer. World widely, ICC accounted for about10%-15%of the primary liver malignant tumor, and it is the second common form of primary liver cancer next to that of hepatocellular carcinoma. Nowadays, it has been rising for primary tumor form among the liver cancer which leads to death. Presently, it is reported that the incidence of ICC is closely related with bile duct calculi, HBV/HCV infection and some other bile duct inflammation. ICC possess the following characteristics:hidden onset, atypical clinical symptoms, high malignant degree, rapid development, and easy to transfer. Despite advances in diagnosis and treatment, most patients present with advanced metastatic lesions that are not amenable to surgical excision or liver transplantation and the poor prognosis can not be changed. So, to find a new and effective method to apply to diagnose and treat ICC is our priority. With the development of molecular biology and the deep understanding to tumor molecular mechanism, researchers have been aware of that malignant tumor is one kind of systemic diseases involved by multiple factors and multiple genes. Therefore, in the recent10years, biological therapy and gene therapy have been become the hot spot in tumor treatment.
Mesenchymal stem cells (MSCs) is one kind of stem cells, possessing a multiple-differentiation potential which permits these cells to differentiate into a variety of mesodermal cell lineages, including bone, cartilage, fat, muscle and tendon. Therefore, MSCs have been widely used in studies of cell therapy and tissue regeneration. In contrast to hMSCs from other resources, human umbilical cord mesenchymal stem cells(hUC-MSCs), as a new source of seed cells, get more and more attention due to their availability, low immunogenicity, as well as strong tropism for tumors. It is reported that hUC-MSCs not only have strong proliferation ability, but also have the ability to differentiate to some other cells (such as neurons, astrocytes, fat cells and oligodendrocytes, etc). Because of its strong tropism for tumors, many researchers have been focus on its relationship with the tumor cells. Nowadays, stem cell transplantation technology has been applied in several kinds of hematologic and non-hematologic malignancies. Previous studies have shown that the development and growth of some human solid malignancies can be inhibited by mesenchymal stem cells. And other studies have demonstrated that human mesenchymal stem cells may inhibit tumor cell phenotypes by secreting certain soluble factors.
Because the mechanism of human mesenchymal stem cells effects on human intrahepatic cholangiocarcinoma has not been reported, in the current study, we sought to shed light on this phenomenon. Co-culture systems, MTT assays and DNA fragmentation assays were used to determine the intrahepatic cholangiocarcinoma HCCC-9810cells proliferation and apoptosis. Western Blot and immunofluorescence staining methods were used to determine the expression level of proteins which were closely related to several pathways in intrahepatic cholangiocarcinoma to identify the possible molecular mechanism.
Part I
Effect of human umbilical cord mesenchymal stem cells on intrahepatic cholangiocarcinoma cells
Objective
1. To detect the effect of hUC-MSCs on human intrahepatic cholangiocarcinoma HCCC-9810cells.
2. To detect the effect of various concentrations of conditioned media from hUC-MSCs (10%,25%,50%and75%) on human intrahepatic cholangiocarcinoma HCCC-9810cells.
3. To evaluate whether the effect of conditioned media from hUC-MSCs on HCCC-9810cells was mediated by apoptosis.
4. To detect the effect of hUC-MSCs on tumor formation in BALB/c Nude Mice.
Methods
1. Co-culture systems were established by using transwell6-well plates to detect cell proliferation.
2. HCCC-9810cells were treated with various concentrations of conditioned media from hUC-MSCs or HUVECs(10%、25%、50%and75%) to detect cell proliferation using MTT and colony-forming assay.
3. Hoechst33258assay were used to determine cell apoptosis.
4. BALA/c nude mice transplantation was established to detect tumor formation.
5. SPSS16.0software was used for all statistical analysis. Statistical significance was assessed by comparing mean values (±SD) using the Student's t-test for independent groups.
Results
1. Effect of human umbilical cord mesenchymal stem cells on cholangiocarcinoma cells
The numbers of HCCC-9810cells co-cultured with hMSCs and HUVECs for48h were7.64X105个/ml and11.98X105个/ml, respectively. The hMSC-induced inhibitory effects on tumor cell proliferation were significantly greater than in controls. Compared to that of the control, the cell proliferation rate decreased34.4%(P<0.05). In order to determine whether the cultured hUC-MSCs microenvironment could inhibit tumor cell growth, we tested the effect of conditioned media from hUC-MSCs on HCCC-9810cells. HCCC-9810cells treated with various concentrations hUC-MSC-conditioned media resulted in dose-dependent and time-dependent inhibition of cell proliferation. The proliferation inhibition rate increased from6.21%to49.86%when HCCC-9810cells were cultured with50%hUC-MSC-conditioned media for24h. Treatment of HCCC-9810cells with50%hUC-MSCs conditioned media led to a significant reduction in the number of colony-forming units relative to control cells. The mean numbers of colony-forming units in the hUC-MSC media treatment group, and in the HUVEC media control group were14.7and35.7, respectively (P<0.01).
2. The hUC-MSCs conditioned media treatment resulted in an induction of apoptosis in HCCC-9810cells
To further evaluate whether the inhibitory effect was mediated by inducing cell apoptosis, DNA staining assay was used. Treatment of HCCC-9810cells with50%conditioned media for48h resulted in apoptosis. We counted the apoptotic cells depending on the presence of cell rounding, detachment, and nuclear fragmentation. The means±SEM for the percentage of apoptotic cells in the treatment group and the control groups were (48.1±2.98)%,(9.3±3.05)%, and (9.6±3.51)%, respectively. Differences between the treatment group and control groups were statistically significant (P<0.05).
3. Tumor formation was inhibited by hUC-MSCs in BALB/c nude mice
Results showed that on the50th day after injection, the mice injected with HCCC-9810cells and hUC-MSCs had a lower tumor incidence than the control groups, and the mean volume of tumors of the mice injected with tumor cells and MSCs was dramatically lower than that of control groups:7mice developed detectable tumors on day35-40(average tumor volume=1.3cm3on day50), and3mice had not developed any tumors when they were killed on day50. By contrast, mice injected with HCCC-9810only, or a mixture of HCCC-9810and HUVEV, formed detectable tumors (average tumor volumes were2.6cm3and2.5cm3, respectively on day50). From day50to day70, mice in the three subgroups derived from Group4were injected with conditioned media from hUC-MSCs or HUVECs in the tumor sites, or received no treatment. The mean tumor volume in the hUC-MSCs group was significantly smaller compared with that of the control. The average tumor volume decreased to1.4cm3in the MSCs group, whereas it continued to increase to3.5cm3in the HUVEC control group by day70. Taken together, these observations suggested that hUC-MSCs may inhibit tumor growth in animals.
Conclusion
1、Human umbilical cord mesenchymal stem cells could inhibit the growth of HCCC-9810cells.
2、When HCCC-9810cells were cultured with50%hUC-MSCs-conditioned media for24h, the half inhibitory rate could be up to.
3、hUC-MSCs could inhibit the tumor formation in BALB/c Nude mice, and the formed tumors shrank in size after injection of conditioned media from hUC-MSCs.
4、The inhibitory effect of hUC-MSCs on HCCC-9810cells was mediated by inducing HCCC-9810cells apoptosis.
Part II
The molecular mechanism of human umbilical cord mesenchymal stem cells suppress cholangiocarcinoma cells
Objective
1. To examine the effects of conditioned media from hUC-MSCs on the PI3K/Akt signaling pathway.
2. To investigate the effects of conditioned media from hUC-MSCs on the Wnt/β-catenin signaling pathway.
3. To detect the changes of HCCC-9810cell proliferation ability after activation or inhibitation Akt or Wnt signaling pathway.
Methods
1. Western Blot was used to observe the change of p-PI3K、p-PDK1、p-Akt、Akt、 p-GSK-3β、GSK-3β、β-Catenin、c-Myc. Cyclin D1in HCCC-9810cells after treatment with hUC-MSCs conditioned media.
2. Immunofluorescence was used to detect the change of p-Akt、p-GSK-3β、β-Catenin in HCCC-9810cells after treatment with hUC-MSCs conditioned media.
3. MTT assay was used to detect the changes of cell proliferation ability after activation or inhibition GSK-3β in HCCC-9810cells.
4. MTT assay was used to detect the changes of cell proliferation ability with or without IGF-1preincubation for15min. Results
1. Inhibition of Akt Signaling Using Conditioned Media from hUC-MSC Cultures in tumor cells
(1) Immunoblot analysis showed that, compared with control groups, treatment of HCCC-9810cells with hUC-MSC conditioned media resulted in reduced phosphorylation of PI3K、PDK1、Akt and GSK-3P, while the total Akt and GSK-3β level did not change. However, HUVEC conditioned media failed to down-regulate the expression of these proteins. In the HUVEC control group and MSCs group, the ratios of phospho-to total Akt were0.65±0.04and0.19±0.02, respectively, while the ratios of phospho-to total Gsk-3β was0.48±0.03and0.22±0.05, respectively. There were substantial differences in the phospho-to total Akt and Gsk-3β ratios between the treated group and control group (P<0.001;P<0.01, versus controls, respectively).
(2) The expression of phospho-Akt(Ser473) and phospho-GSK-3β (Ser9) were examined through indirect immunofluorescence staining. Compared to the controls, the relative luciferase activities for p-Akt and p-GSK-3β were37.3±0.8%and35.7±1.2%, respectively. In contrast to the control, the immunofluorescence staining of phospho-Akt (Ser473) and phospho-GSK-3β (ser9) in the treatment group was significantly weaker than that of control(P<0.01, p-Akt;P<0.01, p-GSK-3p)
2. Down-regulation of Wnt signaling in tumor cells by conditioned media from hUC-MSC cultures
(1) Immunoblot results showed that treatment of HCCC-9810cells with hUC-MSC conditioned media resulted in the down-regulation of β-catenin、c-Myc and cyclin-D1. However, conditioned media from HUVEC failed to down-regulate the expression of these proteins.
(2) Immunofluorescence cytology showed that treatment of HCCC-9810cells with conditioned media from hUC-MSC resulted in a decrease in β-catenin nuclear assembly.
3. The hUC-MSC conditioned media decreased β-catenin expression and inhibited Wnt signaling by regulating GSK-3β activity by the Akt signaling pathway
The GSK-3β inhibitors CHIR99021significantly rescued HCCC-9810cells from the inhibitory effects of hUC-MSCs conditioned media (P<0.001), whereas the GSK-3β activator SNP significantly increased the inhibitory effects of hUC-MSCs conditioned media (P<0.001). Treatment with CHIR99021increased β-catenin protein levels in the conditioned media-treated group, while SNP treatment resulted in decreased β-catenin protein levels. We further treated with hUC-MSC conditioned media for24h, with or without IGF-1(200ng/mL) preincubation for15min. The results showed that exposure to IGF-1significantly blocked the inhibitory effects of hUC-MSC conditioned media on HCCC-9810cells (P<0.05).
Conclusion
1、Human umbilical cord mesenchymal stem cells could inhibit the growth of cholangiocarcinoma cells via PI3K/Akt signaling pathway
2、Human umbilical cord mesenchymal stem cells could inhibit the growth of cholangiocarcinoma cells via Wnt/β-catenin signaling pathway.
3、Suppression of cholangiocarcinoma cell growth by human umbilical cord mesenchymal stem cells:A possible role of Wnt and Akt signaling
肝内胆管细胞癌([ntrahepatic cholangiocarcinoma, ICC),又被称为外周型胆管癌,或者胆管癌之肝内型,它是肝内胆管被覆上皮发生的,并起源于肝内二级分支以下胆管上皮的原发性肝癌的其中一种类型,据世界范围内统计,ICC约占原发性肝脏所发生恶性肿瘤的10%-15%,其发生率仅次于肝细胞癌(HCC),但近年来已经上升为肝内原发肿瘤导致死亡的第一位,目前认为,肝内胆管癌的发生与胆管结石、乙型病毒性肝炎、丙型病毒性肝炎感染以及其他胆管发生的炎症等因素密切相关。肝内胆管癌具有发生隐匿、临床症状不典型、恶性程度高、发展迅速、容易转移等特点,尽管目前在早期诊断和治疗方法上有了很大改进,但是仍不能改变其不良预后,因此发现新的、有效的抗肝内胆管癌的方法是当务之急。随着广大研究者对肿瘤分子机制不断的深入认识,现在已将恶性肿瘤定义为一种多因子、多步骤和多基因参与的全身性疾病,因此,在近10年中,肿瘤的生物学治疗以及基因治疗成为肿瘤治疗的研究热点和新希望。
间充质干细胞(MSCs)是干细胞的一种类型,因其能分化成间质组织而得名,MSCs是一类具有自我更新和多向分化潜能的细胞,在特定的诱导环境下,可进一步分化为骨、软骨、脂肪、肌腱、肌肉等组织,目前,MSCs已被广泛应用于细胞治疗及组织再生方面的研究。人脐带来源的间充质干细胞因较其他来源的干细胞具有更明显的优势,所以越来越受到人们的重视,研究表明,人脐带组织中分离得到的间充质干细胞既具有较强的增殖能力,又具有向其它类型细胞(神经元样细胞、少突胶质细胞、脂肪细胞及星形胶质细胞等)分化的能力,加之其来源广泛、易获得且具有较强的肿瘤趋化能力等特点,在细胞移植及基因治疗、组织工程等方面具有广阔的应用前景。因干细胞强的肿瘤趋化能力,很多研究者聚焦于其与肿瘤细胞之间的关系,
干细胞移植技术已应用于几种造血及非造血肿瘤的治疗,将干细胞应用于肿瘤治疗具有广泛的前景,有研究证实,某些实体肿瘤的生长可被干细胞所抑制,另有研究显示,这种抑制作用是通过干细胞自身分泌的可溶性活性因子实现的。
但是,目前尚没有关于间充质干细胞对肝内胆管癌细胞作用研究的相关报道,因此本研究对此作用及其分子机制进行了初步探讨。本研究通过Transwell共培养实验、MTT比色实验、DNA断裂实验等方法,检测脐带间充质干细胞对肝内胆管癌HCCC-9810细胞增殖和凋亡的影响,同时借助于Western Blot及免疫荧光染色方法,检测与肝内胆管癌发生发展密切相关通路中蛋白质表达水平的变化,进一步探讨脐带间充质干细胞作用于肝内胆管癌细胞生长可能的分子机制。
第一部分
人类脐带间充质干细胞对肝内胆管癌细胞生长的影响
研究目的
1、检测人类脐带间充质干细胞对肝内胆管癌HCCC-9810细胞生长的影响。
2、检测不同浓度人类脐带间充质干细胞条件培养基对肝内胆管癌HCCC-9810细胞生长的影响。
3、检测人类脐带间充干细胞条件培养基对肝内胆管癌HCCC-9810细胞生长的影响是否由诱导HCCC-9810细胞凋亡所介导。
4、检测人类脐带间充质干细胞及其条件培养基对裸鼠肝内胆管癌移植瘤生长的影响。
研究方法
1、采用transwell共培养系统共培养人类脐带间充质干细胞(HUVECs细胞作为对照)与HCCC-9810细胞,应用细胞计数法计数处理后的肿瘤细胞数。
2、配置不同浓度的干细胞条件培养基(10%、25%、50%、75%)处理HCCC-9810细胞,应用MTT细胞增殖实验及细胞克隆实验测定HCCC-9810细胞增殖率的变化。
3、应用Hoechst33258染色实验检测干细胞条件培养基诱导HCCC-9810细胞凋亡的作用。
4、采用皮下注射细胞悬液的方法,构建裸鼠移植瘤模型,测量注射不同细胞组间肿瘤发生率及体积大小,并采用脐带间充质干细胞条件培养基注射于已形成肿瘤部位的方法,测量肿瘤体积的变化。
5、采用SPSS16.0统计软件用于实验数据的统计学分析,数据的统计学意义通过独立组间学生t检验,采用(x±S)进行分析。
结果
1、脐带间充质干细胞对肝内胆管癌HCCC-9810细胞增殖的影响
HCCC-9810细胞与hUC-MSCs细胞共培养48h后,计数细胞为7.64×105个／m1,而与HUVECs细胞共培养48h后,细胞计数为11.98×105个/ml,由此可见,脐带间充质干细胞诱导的HCCC-9810肿瘤细胞抑制效应明显高于HUVECs对照组,与对照组相比,肿瘤细胞增殖率下降了34.4%,差异具明显的统计学意义(p<0.05)。为了进一步检测培养的脐带间充质干细胞微环境是否能抑制肿瘤细胞的生长,我们检测了干细胞条件培养基对HCCC-9810细胞增殖的影响,结果显示,HCCC-9810细胞经不同浓度间充质干细胞条件培养液处理后,细胞增殖抑制率呈现时间与剂量依赖性,HCCC-9810细胞经50%间充质干细胞条件培养基培养24小时后,增殖抑制率由6.21%增至49.86%,与对照组相比有显著差距。我们又采用了细胞克隆实验进一步证实这种抑制作用的存在,结果显示,与对照组相比,脐带间充质干细胞条件培养基处理组细胞克隆单位形成数明显低于对照组(p<0.01)。
2、脐带间充质干细胞条件培养液对HCCC-9810细胞凋亡的影响
为了证实干细胞条件培养基对HCCC-9810细胞的增殖抑制是否由凋亡介导,进一步采用DNA染色的方法观察核染色质形态。结果显示,HCCC-9810细胞经50%脐带间充质干细胞条件培养液处理48h后,经Hoechst33258染色呈现明显细胞凋亡,凋亡细胞比率明显高于对照组,与对照组相比,差异具明显的统计学意义(p<0.05VS.HUVEC对照组)。
3、脐带间充质干细胞抑制BALB/C裸鼠移植瘤的形成
结果显示,皮下注射不同细胞组50天后,注射HCCC.9810+MSC细胞组,移植瘤出现时间及肿瘤体积均明显低于对照组：观察至第35-40天,有7只裸鼠长出可视肿瘤(观察至第50天,平均肿瘤体积约为1.3cm3),此组中另外3只裸鼠始终无肿瘤发生。注射HCCC.9810细胞组,或者HCCC.9810+HUVEC细胞组中所有实验小鼠均形成了可视肿瘤(观察至第50天,平均肿瘤体积分别为2.6cn13,2.5cm3).实验的50-70天,分别给HCCC-9810实验组继续注射间充质干细胞或者HUVECs细胞的条件培养基,结果显示,注射间充质干细胞条件培养基组,原本已形成的肿瘤体积明显缩小,至实验结束,干细胞条件培养基处理组小鼠肿瘤的平均体积降至1.4cm3,而对照组肿瘤体积继续增长至3.5cm3,差异具显著的统计学意义。
结论
1、脐带间充质干细胞可抑制肝内胆管癌HCCC-9810细胞的生长。
2、经50%脐带间充质干细胞条件培养基处理肝内胆管癌HCCC-9810细胞24小时可达到细胞的半数抑制率。
3、脐带间充质干细胞可抑制BALB/C裸鼠移植瘤的形成,且其条件培养基可使已形成的肿瘤体积缩小。
4、脐带间充质干细胞对肝内胆管癌HCCC-9810细胞生长的抑制作用是通过诱导细胞凋亡所介导的。
第二部分
人类脐带间充质干细胞阻抑肝内胆管癌细胞生长的分子机制
研究目的
1、检测经脐带间充质干细胞条件培养基作用后,肝内胆管癌HCCC-9810细胞中PI3K/Akt信号转导通路主要蛋白的表达水平变化。
2、检测经脐带间充质干细胞条件培养基作用后,肝内胆管癌HCCC-9810细胞中Wnt/β-catenin信号转导通路主要蛋白的表达水平变化。
3、检测加入GSK-3β(?)抑制剂或者激活剂,或加入Akt激活剂,作为单一处理因素,或者联合脐带间充质干细胞条件培养基处理HCCC-9810细胞,所介导的肝内胆管癌细胞增殖及相关蛋白的变化。
研究方法
1、Western Blot方法检测肝内胆管癌HCCC-9810细胞经脐带间充质干细胞条件培养基处理前后p-PI3K、p-PDK1、p-Akt Akt\p-GSK-3β、GSK-3β、β-Catenin、 c-Myc、CyclinD1等蛋白的表达变化。
2、细胞免疫荧光染色方法检测肝内胆管癌HCCC-9810细胞经脐带间充质干细胞条件培养基处理前后p-Akt、p-GSK-3β、β-Catenin的表达变化。
3、分别加入GSK-3β特异性激活试剂SNP及特异性抑制试剂CHIR99021作为单一处理因素,或者与脐带间充质干细胞条件培养基联合作用后,观察肝内胆管癌HCCC-9810细胞增殖能力变化,并进一步检测β-Catenin的表达变化。
4、加入Akt特异性激活剂IGF-1预处理HCCC-9810细胞15min后,进一步观察脐带间充质干细胞条件培养基对肝内胆管癌HCCC-9810细胞增殖能力的影响。
结果
1、hUC-MSCs条件培养基可抑制肝内胆管癌HCCC-9810细胞中PI3K/Akt信号转导通路相关蛋白的表达
1) Western Blot实验结果显示,HCCC-9810细胞经50%hUC-MSCs条件培养基处理48小时后,与对照组相比,p-PI3K、p-PDK1、p-Akt、p-GSK-3β的表达明显下调,而总的Akt(T-Akt)与总的GSK-3β(T-GSK-3β)的表达无明显变化,而HUVEC条件培养基处理组上述蛋白表达与空白对照组相比无明显变化。统计显示,HUVEC条件培养基处理组与hUC-MSC条件培养基处理组相比,p-Akt与T-Akt的灰度比值分别为0.65±0.04及0.19±0.02(P<0.001),p-GSK-3β与T-GSK-3β的灰度比值分别为0.48±0.03及0.22±0.05(P<0.01),比值差异均具有明显的统计学意义。
2)细胞免疫荧光染色结果显示,与空白对照组相比,干细胞条件培养基处理组p-Akt、p-GSK-3β的相对荧光强度分别为37.3±0.8%、35.7±1.2%,空白对照组p-Ak、p-GSK-3β的蛋白荧光强度明显高于干细胞条件培养基处理组,两组间差异的统计学意义较显著(P<0.01, p-Akt; P<0.01, p-GSK-3β).
2、hUC-MSCs条件培养基可抑制肝内胆管癌HCCC-9810细胞中Wnnt信号转导通路相关蛋白的表达
1) Western Blot实验结果显示,肝内胆管癌HCCC-9810细胞经50%hUC-MSCs条件培养基处理48小时后,与空白对照组相比,β-catenin、c-Myc、cyclin-D1的表达明显下调,并且β-catenin的核富集量明显减少,而HUVEC条件培养基处理组β-catenin、c-Myc、cyclin-D1的蛋白表达情况几乎与空白对照组相似。
2)细胞荧光免疫染色结果显示,干细胞条件培养基处理组β-catenin在细胞核中荧光强度较空白对照组明显减弱。
3、hUC-MSCs条件培养基通过Akt通路调节GSK-3β的活性,从而下调β-catenin的表达,抑制Wnnt通路
经GSK-3β抑制剂(CHIR99021)处理后,48h观察点结果显示,CHIR99021与hUC-MSCs条件培养基联合处理组与hUC-MSCs条件培养基单独处理组相比,HCCC-9810细胞的抑制作用明显减弱p<0.001),并且CHIR99021与hUC-MSCs条件培养基联合处理组增加了β-catenin的蛋白表达水平。而经GSK-3β激活剂(SNP)处理后,48h观察点结果显示,SNP与hUC-MSCs条件培养基联合处理组与hUC-MSCs条件培养基单独处理组相比,HCCC-9810细胞的抑制作用明显增强(p<0.001),并且SNP与]hUC-MSCs条件培养基联合处理组下调了β-catenin的蛋白表达水平。加入Akt通路激活剂IGF-1预处理HCCC-9810细胞15min后,24h观察点结果显示,IGF-1与hUC-MSCs条件培养基联合处理组与hUC-MSCs条件培养基单独处理组相比,IGF-1与hUC-MSCs条件培养基联合处理组对HCCC-9810细胞的抑制作用明显下降(p<0.05)。
结论
1、hUC-MSCs条件培养基可通过抑制PI3K/Akt信号通路阻抑肝内胆管癌HCCC-9810细胞生长。
2、hUC-MSCs条件培养基亦可通过抑制Wnt/β-catenin信号通路阻抑肝内胆管癌HCCC-9810细胞生长。
3、hUC-MSCs条件培养基对肝内胆管癌细胞的抑制作用可能是通过以GSK-3β作为“桥梁蛋白”的Akt和Wnnt两条通路的串话实现的。
Background and Objective
Intrahepatic cholangiocarcinoma(ICC), also known as peripheral cholangiocarcinoma, is a malignancy whose pathogenesis involves abnormal biliary epithelial differentiation and is originated from the secondary bile duct epithelium branch. It is one kind of primary liver cancer. World widely, ICC accounted for about10%-15%of the primary liver malignant tumor, and it is the second common form of primary liver cancer next to that of hepatocellular carcinoma. Nowadays, it has been rising for primary tumor form among the liver cancer which leads to death. Presently, it is reported that the incidence of ICC is closely related with bile duct calculi, HBV/HCV infection and some other bile duct inflammation. ICC possess the following characteristics:hidden onset, atypical clinical symptoms, high malignant degree, rapid development, and easy to transfer. Despite advances in diagnosis and treatment, most patients present with advanced metastatic lesions that are not amenable to surgical excision or liver transplantation and the poor prognosis can not be changed. So, to find a new and effective method to apply to diagnose and treat ICC is our priority. With the development of molecular biology and the deep understanding to tumor molecular mechanism, researchers have been aware of that malignant tumor is one kind of systemic diseases involved by multiple factors and multiple genes. Therefore, in the recent10years, biological therapy and gene therapy have been become the hot spot in tumor treatment.
Mesenchymal stem cells (MSCs) is one kind of stem cells, possessing a multiple-differentiation potential which permits these cells to differentiate into a variety of mesodermal cell lineages, including bone, cartilage, fat, muscle and tendon. Therefore, MSCs have been widely used in studies of cell therapy and tissue regeneration. In contrast to hMSCs from other resources, human umbilical cord mesenchymal stem cells(hUC-MSCs), as a new source of seed cells, get more and more attention due to their availability, low immunogenicity, as well as strong tropism for tumors. It is reported that hUC-MSCs not only have strong proliferation ability, but also have the ability to differentiate to some other cells (such as neurons, astrocytes, fat cells and oligodendrocytes, etc). Because of its strong tropism for tumors, many researchers have been focus on its relationship with the tumor cells. Nowadays, stem cell transplantation technology has been applied in several kinds of hematologic and non-hematologic malignancies. Previous studies have shown that the development and growth of some human solid malignancies can be inhibited by mesenchymal stem cells. And other studies have demonstrated that human mesenchymal stem cells may inhibit tumor cell phenotypes by secreting certain soluble factors.
Because the mechanism of human mesenchymal stem cells effects on human intrahepatic cholangiocarcinoma has not been reported, in the current study, we sought to shed light on this phenomenon. Co-culture systems, MTT assays and DNA fragmentation assays were used to determine the intrahepatic cholangiocarcinoma HCCC-9810cells proliferation and apoptosis. Western Blot and immunofluorescence staining methods were used to determine the expression level of proteins which were closely related to several pathways in intrahepatic cholangiocarcinoma to identify the possible molecular mechanism.
Part I
Effect of human umbilical cord mesenchymal stem cells on intrahepatic cholangiocarcinoma cells
Objective
1. To detect the effect of hUC-MSCs on human intrahepatic cholangiocarcinoma HCCC-9810cells.
2. To detect the effect of various concentrations of conditioned media from hUC-MSCs (10%,25%,50%and75%) on human intrahepatic cholangiocarcinoma HCCC-9810cells.
3. To evaluate whether the effect of conditioned media from hUC-MSCs on HCCC-9810cells was mediated by apoptosis.
4. To detect the effect of hUC-MSCs on tumor formation in BALB/c Nude Mice.
Methods
1. Co-culture systems were established by using transwell6-well plates to detect cell proliferation.
2. HCCC-9810cells were treated with various concentrations of conditioned media from hUC-MSCs or HUVECs(10%、25%、50%and75%) to detect cell proliferation using MTT and colony-forming assay.
3. Hoechst33258assay were used to determine cell apoptosis.
4. BALA/c nude mice transplantation was established to detect tumor formation.
5. SPSS16.0software was used for all statistical analysis. Statistical significance was assessed by comparing mean values (±SD) using the Student's t-test for independent groups.
Results
1. Effect of human umbilical cord mesenchymal stem cells on cholangiocarcinoma cells
The numbers of HCCC-9810cells co-cultured with hMSCs and HUVECs for48h were7.64X105个/ml and11.98X105个/ml, respectively. The hMSC-induced inhibitory effects on tumor cell proliferation were significantly greater than in controls. Compared to that of the control, the cell proliferation rate decreased34.4%(P<0.05). In order to determine whether the cultured hUC-MSCs microenvironment could inhibit tumor cell growth, we tested the effect of conditioned media from hUC-MSCs on HCCC-9810cells. HCCC-9810cells treated with various concentrations hUC-MSC-conditioned media resulted in dose-dependent and time-dependent inhibition of cell proliferation. The proliferation inhibition rate increased from6.21%to49.86%when HCCC-9810cells were cultured with50%hUC-MSC-conditioned media for24h. Treatment of HCCC-9810cells with50%hUC-MSCs conditioned media led to a significant reduction in the number of colony-forming units relative to control cells. The mean numbers of colony-forming units in the hUC-MSC media treatment group, and in the HUVEC media control group were14.7and35.7, respectively (P<0.01).
2. The hUC-MSCs conditioned media treatment resulted in an induction of apoptosis in HCCC-9810cells
To further evaluate whether the inhibitory effect was mediated by inducing cell apoptosis, DNA staining assay was used. Treatment of HCCC-9810cells with50%conditioned media for48h resulted in apoptosis. We counted the apoptotic cells depending on the presence of cell rounding, detachment, and nuclear fragmentation. The means±SEM for the percentage of apoptotic cells in the treatment group and the control groups were (48.1±2.98)%,(9.3±3.05)%, and (9.6±3.51)%, respectively. Differences between the treatment group and control groups were statistically significant (P<0.05).
3. Tumor formation was inhibited by hUC-MSCs in BALB/c nude mice
Results showed that on the50th day after injection, the mice injected with HCCC-9810cells and hUC-MSCs had a lower tumor incidence than the control groups, and the mean volume of tumors of the mice injected with tumor cells and MSCs was dramatically lower than that of control groups:7mice developed detectable tumors on day35-40(average tumor volume=1.3cm3on day50), and3mice had not developed any tumors when they were killed on day50. By contrast, mice injected with HCCC-9810only, or a mixture of HCCC-9810and HUVEV, formed detectable tumors (average tumor volumes were2.6cm3and2.5cm3, respectively on day50). From day50to day70, mice in the three subgroups derived from Group4were injected with conditioned media from hUC-MSCs or HUVECs in the tumor sites, or received no treatment. The mean tumor volume in the hUC-MSCs group was significantly smaller compared with that of the control. The average tumor volume decreased to1.4cm3in the MSCs group, whereas it continued to increase to3.5cm3in the HUVEC control group by day70. Taken together, these observations suggested that hUC-MSCs may inhibit tumor growth in animals.
Conclusion
1、Human umbilical cord mesenchymal stem cells could inhibit the growth of HCCC-9810cells.
2、When HCCC-9810cells were cultured with50%hUC-MSCs-conditioned media for24h, the half inhibitory rate could be up to.
3、hUC-MSCs could inhibit the tumor formation in BALB/c Nude mice, and the formed tumors shrank in size after injection of conditioned media from hUC-MSCs.
4、The inhibitory effect of hUC-MSCs on HCCC-9810cells was mediated by inducing HCCC-9810cells apoptosis.
Part II
The molecular mechanism of human umbilical cord mesenchymal stem cells suppress cholangiocarcinoma cells
Objective
1. To examine the effects of conditioned media from hUC-MSCs on the PI3K/Akt signaling pathway.
2. To investigate the effects of conditioned media from hUC-MSCs on the Wnt/β-catenin signaling pathway.
3. To detect the changes of HCCC-9810cell proliferation ability after activation or inhibitation Akt or Wnt signaling pathway.
Methods
1. Western Blot was used to observe the change of p-PI3K、p-PDK1、p-Akt、Akt、 p-GSK-3β、GSK-3β、β-Catenin、c-Myc. Cyclin D1in HCCC-9810cells after treatment with hUC-MSCs conditioned media.
2. Immunofluorescence was used to detect the change of p-Akt、p-GSK-3β、β-Catenin in HCCC-9810cells after treatment with hUC-MSCs conditioned media.
3. MTT assay was used to detect the changes of cell proliferation ability after activation or inhibition GSK-3β in HCCC-9810cells.
4. MTT assay was used to detect the changes of cell proliferation ability with or without IGF-1preincubation for15min. Results
1. Inhibition of Akt Signaling Using Conditioned Media from hUC-MSC Cultures in tumor cells
(1) Immunoblot analysis showed that, compared with control groups, treatment of HCCC-9810cells with hUC-MSC conditioned media resulted in reduced phosphorylation of PI3K、PDK1、Akt and GSK-3P, while the total Akt and GSK-3β level did not change. However, HUVEC conditioned media failed to down-regulate the expression of these proteins. In the HUVEC control group and MSCs group, the ratios of phospho-to total Akt were0.65±0.04and0.19±0.02, respectively, while the ratios of phospho-to total Gsk-3β was0.48±0.03and0.22±0.05, respectively. There were substantial differences in the phospho-to total Akt and Gsk-3β ratios between the treated group and control group (P<0.001;P<0.01, versus controls, respectively).
(2) The expression of phospho-Akt(Ser473) and phospho-GSK-3β (Ser9) were examined through indirect immunofluorescence staining. Compared to the controls, the relative luciferase activities for p-Akt and p-GSK-3β were37.3±0.8%and35.7±1.2%, respectively. In contrast to the control, the immunofluorescence staining of phospho-Akt (Ser473) and phospho-GSK-3β (ser9) in the treatment group was significantly weaker than that of control(P<0.01, p-Akt;P<0.01, p-GSK-3p)
2. Down-regulation of Wnt signaling in tumor cells by conditioned media from hUC-MSC cultures
(1) Immunoblot results showed that treatment of HCCC-9810cells with hUC-MSC conditioned media resulted in the down-regulation of β-catenin、c-Myc and cyclin-D1. However, conditioned media from HUVEC failed to down-regulate the expression of these proteins.
(2) Immunofluorescence cytology showed that treatment of HCCC-9810cells with conditioned media from hUC-MSC resulted in a decrease in β-catenin nuclear assembly.
3. The hUC-MSC conditioned media decreased β-catenin expression and inhibited Wnt signaling by regulating GSK-3β activity by the Akt signaling pathway
The GSK-3β inhibitors CHIR99021significantly rescued HCCC-9810cells from the inhibitory effects of hUC-MSCs conditioned media (P<0.001), whereas the GSK-3β activator SNP significantly increased the inhibitory effects of hUC-MSCs conditioned media (P<0.001). Treatment with CHIR99021increased β-catenin protein levels in the conditioned media-treated group, while SNP treatment resulted in decreased β-catenin protein levels. We further treated with hUC-MSC conditioned media for24h, with or without IGF-1(200ng/mL) preincubation for15min. The results showed that exposure to IGF-1significantly blocked the inhibitory effects of hUC-MSC conditioned media on HCCC-9810cells (P<0.05).
Conclusion
1、Human umbilical cord mesenchymal stem cells could inhibit the growth of cholangiocarcinoma cells via PI3K/Akt signaling pathway
2、Human umbilical cord mesenchymal stem cells could inhibit the growth of cholangiocarcinoma cells via Wnt/β-catenin signaling pathway.
3、Suppression of cholangiocarcinoma cell growth by human umbilical cord mesenchymal stem cells:A possible role of Wnt and Akt signaling
引文
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2. Palmer WC, Patel T. Are common factors involved in the pathogenesis of primary liver cancers?A meta-analysis of risk factors for intrahepatic cholangiocarcinoma. J Hepatol,2012,57(1):69-76.
3. Malhi H, Gores GJ. Cholangiocarcinoma:modem advances in understanding a deadly old disease. J Hepatol,2006,45(6):856-867.
4. Konstadoulakis MM, Roayaie S, Gomatos IP, et a 1. Fifteen-year, single-center experience with the surgical management of intrahepatic cholangiocarcinoma:operative results and long-term outcome. Surgery,2008,143(3):366-374.
5. Endo I, Gonen M, Yopp AC, et al. Intrahepatic cholangiocareinoma:rising frequency, improved survival, and determinants of outcome after resection. Ann Surg,2008,248(1):84-96.
6. Blechacz B, Gores GJ. Cholangiocarcinoma:advances in pathogenesis, diagnosis, and treatment. Hepatology,2008,48(1):308-321.
7.Nathan H, Pawlik TM, Wolfgang CL, et al. Trends in survival after surgery for cholangiocarcinoma:a 30-year population-based SEER database analysis. J Gastrointest Surg,2007,11(11):1488-1496, discussion 1496-1497.
8. Nakanuma Y, Harada K, lshikawa A, et al. Anatomic and molecular pathology of intrahepatic cholangiocarcinoma. J Hepatobiliary Pancreat Surg,2003,10(4):265-281.
9. Ribas A, Butterfield LH, Glaspy JA, et al. Current developments in cancer vaccines and cellular immunotherapy. J Clin Oncol,2003,21(12):2415-2432.
10. Llovet JM. Updated treatment approach to hepatocellular carcinoma. J Gastroenterol,2005, 40(3):225-235.
11. Jiang Y, Jahaqirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature,2002,418(6893):41-49.
12. Tocci A, Forte L. Mesenchymal stem cell:use and perspectives. Hematol J,2003,4(2):92-96.
13. Stagg J, Lejeune L, Paquin A, et al. Marrow stromal cells for interleukin-2 delivery in cancer immunotherapy. Hum Gene Ther,2004,15(6):597-608.
14. Nakamura K, Ito Y, Kawano Y, et al. Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther,2004,11(14):1155-1164.
15. Maestroni GJ, Hertens E, Galli P. Factor(s) from nonmacrophage bone marrow stromal cells inhibit Lewis lung carcinoma and B16 melanoma growth in mice. Cell Mol Life Sci,1999,55:663-667.
16. Khakoo AY, Pati S, Anderson SA, et al. Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi's sarcoma. J Exp Med,2006,203(5):1235-1247.
17. Karnoub AE, Dash AB, Vo AP, et al. Mesenchymal stem cells within tumor stroma promote breast cancer metastasis. Nature,2007,449(7162):557-563.
18. Paget S. The distribution of secondary growths in cancer of the breast. Cancer Metastasis Rev,1989, 8(2):98-101.
19. Park CC, Bissell MJ, Barcellos-Hoff MH. The influence of the microenvironment on the malignant phenotype. Mol Med Today,2000,6(8):324-329.
20. Bexell D, Gunnarsson S, Tormin A, et al. Bone marrow multipotent mesenchymal stroma cells act as pericyte-like migratory vehicles in experimental gliomas. Mol Ther,2009,17(1):183-190.
21. Honczarenko M, Le Y, Swierkowski M, et al. Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells,2006,24(4):1030-1041.
22. Wu Y, Chen L, Scott PG, et al. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells,2007,25(10):2648-2659.
23. Galmiche MC, Koteliansky VE, Briere J, et al. Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway. Blood, 1993,82(1):66-76.
24. Ball SG, Shuttleworth AC, Kielty CM. Direct cell contact influences bone marrow mesenchymal stem cell fate. Int J Biochem Cell Biol,2004,36(4):714-727.
25. Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res,2004,94(5):678-685.
26. Annabi B, Naud E, Lee YT, et al. Vascular progenitors derived from murine bone marrow stromal cells are regulated by fibroblast growth factor and are avidly recruited by vascularizing tumors. J Cell Biochem, 2004,91(6):1146-1158.
27. Makinoshima H, Dezawa M. Pancreatic cancer cells activate CCL5 expression in mesenchymal stromal cells through the insulin-like growth factor-1 pathway. FEBS Lett,2009,583(22):3697-3703.
28. Xu WT, Bian ZY, Fan QM, et al. Human mesenchymal stem cells (hMSCs) target osteosarcoma and promote its growth and pulmonary metastasis. Cancer Lett,2009,281(1):32-41.
29. Ramasamy R,Lam EW, Soeiro I, et al. Mesenchymal stem cells inhibit proliferation and apoptosis of tumor cells:impact on in vivo tumor growth. Leukemia,2007,21(2):304-310.
30. Glennie S, Soeiro I, Dyson PJ, et al. Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells. Blood,2005,105(7):2821-2827.
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