Mesenchymal stem cells: Molecular characteristics and clinical applications

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells with the capacity to differentiate into tissues of both mesenchymal and non-mesenchymal origin. MSCs can differentiate into osteoblastic, chondrogenic, and adipogenic lineages, although recent studies have demonstrated that MSCs are also able to differentiate into other lineages, including neuronal and cardiomyogenic lineages. Since their original isolation from the bone marrow, MSCs have been successfully harvested from many other tissues. Their ease of isolation and ex vivo expansion combined with their immunoprivileged nature has made these cells popular candidates for stem cell therapies. These cells have the potential to alter disease pathophysiology through many modalities including cytokine secretion, capacity to differentiate along various lineages, immune modulation and direct cell-cell interaction with diseased tissue. Here we first review basic features of MSC biology including MSC characteristics in culture, homing mechanisms, differentiation capabilities and immune modulation. We then highlight some in vivo and clinical evidence supporting the therapeutic roles of MSCs and their uses in orthopedic, autoimmune, and ischemic disorders.

The potential molecular mechanism of overexpression of uPA, IL-8, MMP-7 and MMP-9 induced by TRAIL in pancreatic cancer cell

BACKGROUND: TNF-related apoptosis-inducing ligand (TRAIL) is a death ligand of the TNF-superfamily that has been implicated in inducing apoptosis in some tumor cells. The purpose of this study was to find out if TRAIL could induce the expression of uPA, IL-8, MMP-7 and MMP-9. and to explore the corresponding potential signaling transduction pathway in pancreatic cancer cells. METHODS: Colo357wt, Panc89 and PancTu I cell lines were stimulated by TRAIL (100 ng/ml). Crystal violet cell vitality assay was used to check the sensitivity to TRAIL-induced apoptosis. Real-time RT-PCR tested the expression of uPA, IL-8, MMP-7 and MMP-9. RESULTS: TRAIL can stimulate the expression of uPA, IL-8, MMP-7 and MMP-9 in pancreatic cancer cell lines, especially in Colo357wt. The members of caspases, MEK1/2, PKC, and NF-kappa B are involved in TRAIL-induced expression of uPA, IL-8, MMP-7 and MMP-9. Furthermore, caspases play a different role in Colo357wt, Panc89 and PancTu I. CONCLUSIONS: TRAIL-treatment may result in the enhancement of invasion involving the signaling pathways of caspases, MEK1/2, PKC and NF-kappa B, in pancreatic cancer cells. It points to the necessity to carefully evaluate in vivo side effects of TRAIL.

Molecular pathogenesis and therapeutic strategies of human osteosarcoma

Osteosarcoma（OS）is a devastating illness with rapid rates of dissemination and a poor overall prognosis,despite aggressive standard-of-care surgical techniques and combination chemotherapy regimens.Identifying the molecular mechanisms involved in disease pathogenesis and progression may offer insight into new therapeutic targets.Defects in mesenchymal stem cell differentiation,abnormal expression of oncogenes and tumor suppressors,and dysregulation within various important signaling pathways have all been implicated in development of various disease phenotypes.As such,a variety of basic science and translational studies have shown promise in identifying novel markers and modulators of these disease-specific aberrancies.Born out of these and similar investigations,a variety of emerging therapies are now undergoing various phases of OS clinical testing.They broadly include angiogenesis inhibitors,drugs that act on the bone microenvironment,receptor tyrosine kinase inhibitors,immune system modulators,and other radio-or chemo-sensitizing agents.As new forms of drug delivery are being developed simultaneously,the possibility of targeting tumors locally while minimizing systemic toxicityis is seemingly more achievable now than ever.In this review,we not only summarize our current understanding of OS disease processes,but also shed light on the multitude of potential therapeutic strategies the scientific community can use to make long-term improvements in patient prognosis.

小檗碱抗肿瘤作用与Wnt/-βcaten in信号转导关系

目的证明小檗碱抗肿瘤作用机制可能与信号转导过程的调控有关。方法采用细胞增殖抑制和Hoechst 33258染色凋亡实验比较小檗碱和黄连总碱对人结肠癌HCT116和SW480细胞的作用。利用Tcf-4报告基因研究小檗碱对肿瘤细胞的信号转导影响。结果小檗碱在5—40mg·L^-1浓度范围内呈浓度依赖性和时间依赖性抑制人结肠癌HCT116和SW480细胞的增殖；小檗碱（20mg·L^-1）处理72h后的HCT116和SW480细胞出现明显凋亡；相当于小檗碱浓度的黄连总碱有类似于小檗碱的作用。20—40mg·L^-1小檗碱和黄连总碱均能明显抑制β—catenin／Tcf介导的转录活性。结论黄连总碱的抗肿瘤作用可能与其主要成分小檗碱有关；其抗肿瘤作用机制至少与抑制Wnt／β-catenin信号通路有关。

shRNA表达载体构建方法的优化

目的探讨shRNA表达载体的构建方法 ,以加速RNA干扰研究的进程。方法对shRNA表达载体的构建过程进行分析和监测 ,并加以优化。结果发现shRNA表达载体构建的退火过程容易产生障碍 ,经优化退火缓冲液的NaCl含量后 ,能明显提高退火效率及shRNA表达载体构建的成功率。结论shRNA表达载体构建的退火过程需加以关注 ,退火缓冲液中NaCl含量应提高至 2 0

钙结合蛋白S100A2对Wnt/β-catenin信号途径活性的上调作用

目的研究钙结合蛋白S100A2对Wnt/β-catenin信号途径活性的影响,并探讨其可能的机制。方法原核诱导表达GST-hS100A2,经纯化后加入骨肉瘤细胞株MG63和人结肠癌细胞株HCT116的培养液中,Western blot检测细胞中β-catenin含量的变化;荧光素酶活性分析法检测S100A2对HEK293细胞中β-catenin/TCF4活性的影响;以表达GSK-3β、DVL、Axin的相应质粒分别转染HEK293细胞,GST-Pulldown/Western blot实验检测S100A2与这些蛋白质和β-catenin之间的相互作用。结果S100A2使MG63和HCT116细胞中β-catenin含量增加、β-catenin/TCF4活性增强;S100A2分别与β-catenin和GSK-3β之间存在相互作用,而与DVL和Axin之间则未发现相互作用。结论S100A2可以上调Wnt/β-catenin信号途径的活性,其机制可能涉及S100A2与β-catenin和GSK-3β之间的相互作用。

RNA干扰抑制结肠癌细胞血管内皮生长因子表达

目的:应用RNA干扰技术抑制结肠癌血管内皮生长因子(VEGF)表达。方法:将VEGF基因作为RNA干扰的靶区,通过E-RNAi网上提供的服务,设计两个特异的RNA干扰序列,将其装入含U6启动子的载体上,构建成抗VEGF基因的小发夹样RNA(shRNA)表达载体,再转染人结肠癌细胞HT29,通过RT-PCR、Northern blotting、免疫荧光和Western blotting,观察VEGF表达受抑的程度。结果:成功构建了两种抗VEGF基因的shRNA表达载体,RT-PCR、Northern blotting、免疫荧光和Western blotting,均发现其能明显抑制HT29细胞VEGF基因的表达,抑制率分别达42%、88%、73%和82%。结论:针对VEGF基因的shRNA表达载体能够明显抑制结肠癌细胞VEGF基因的表达。

凋亡抑制因子survivin基因的克隆及在真核细胞中的表达

目的 :克隆Survivin编码序列并在真核细胞中表达。方法 :RT -PCR扩增Survivin编码序列 ,建立与绿色荧光蛋白(GFP)共表达的融合重组质粒 ,转染哺乳动物细胞 ,利用GFP绿色荧光和WesternBlot检测Survivin蛋白的表达。结果 :RT -PCR扩增出 4 2 1bp的Survivin编码序列 ,构建融合重组质粒pEGFP -C1-Survivin ,利用GFP荧光和WesternBlot证实Sur vivin蛋白的表达。结论 :成功克隆Survivin基因并在真核细胞中表达。

抑制Dicer基因对shRNA功能发挥的影响

本文将Dicer基因的RNA酶III结构域作为靶区,设计并构建了两个抗Dicer基因的小发夹样RNA(shRNA)表达载体,将其转染2215、结肠癌TC细胞和基因组中整合有绿色荧光蛋白基因(GFP)的HepG2A9细胞,通过RT-PCR评价RNA干扰抑制Dicer基因表达的效率;当HepG2A9细胞Dicer基因表达被上述RNA干扰抑制时,再转染抗GFP的shRNA表达载体,通过RT-PCR和荧光显微镜观察GFP表达水平。结果显示,在不同细胞系中,这两个抗Dicer基因shRNA表达载体,均能明显抑制Dicer基因的表达;当Dicer基因受抑时,后续转染抗GFP的shRNA表达载体不能有效抑制GFP的表达。结果表明,抗Dicer基因shRNA表达载体,能够明显抑制Dicer基因的表达;shRNA表达载体的功能发挥需要Dicer酶的直接参与。

Improved method increases sensitivity for circulating hepatocellular carcinoma cells

AIM:To improve an asialoglycoprotein receptor(ASGPR)-based enrichment method for detection of circulating tumor cells(CTCs)of hepatocellular carcinoma(HCC).METHODS:Peripheral blood samples were collected from healthy subjects,patients with HCC or various other cancers,and patients with hepatic lesions or hepatitis.CTCs were enriched from whole blood by extracting CD45-expressing leukocytes with monoclonal antibody coated-beads following density gradient centrifugation.The remaining cells were cytocentrifuged on polylysine-coated slides.Isolated cells were treated by triple immunofluorescence staining with CD45antibody and a combination of antibodies against ASGPR and carbamoyl phosphate synthetase 1(CPS1),used as liver-specific markers,and costained with DAPI.The cell slide was imaged and stained tumor cells that met preset criteria were counted.Recovery,sensitivity and specificity of the detection methods were determined and compared by spiking experiments with various types of cultured human tumor cell lines.Expression of ASGPR and CPS1 in cultured tumor cells and tumor tissue specimens was analyzed by flow cytometry and triple immunofluorescence staining,respectively.RESULTS:CD45 depletion of leukocytes resulted in a significantly greater recovery of multiple amounts of spiked HCC cells than the ASGPR+selection(P s<0.05).The expression rates of either ASGPR or CPS1were different in various liver cancer cell lines,ranging between 18%and 99%for ASGPR and between 9%and 98%for CPS1.In both human HCC tissues and liver cancer cell lines,there were a few HCC cells that did not stain positive for ASGPR or CPS1.The mixture of monoclonal antibodies against ASGPR and CPS1identified more HCC cells than either antibody alone.However,these antibodies did not detect any tumor cells in blood samples spiked with the human breastcancer cell line MCF-7 and the human renal cancer cell line A498.ASGPR+or/and CPS1+CTCs were detected in 29/32(91%)patients with HCC,but not in patients with any other kind of cancer or any of the other test subjects.Furthermore,the improved method detected a higher CTC count in all patients examined than did the previous method(P=0.001),and consistently achieved 12%-21%higher sensitivity of CTC detection in all seven HCC patients with more than 40 CTCs.CONCLUSION:Negative depletion enrichment combined with identification using a mixture of antibodies against ASGPR and CPS1 improves sensitivity and specificity for detecting circulating HCC cells.

Risk factors for intrahepatic cholangiocarcinoma:A case-control study in China

AIM: To carry out a hospital-based case-control study to investigate risk factors for intrahepatic cholangiocarcinoma (ICC) in China. METHODS: A total of 312 ICC cases and 438 matched controls were included in the study. The presence of diabetes mellitus,hypertention,hepatolithiasis,primary sclerosing cholangitis,liver fluke infection (Clonorchis sinensis ),was investigated through clinical records. Blood from all participants was tested for hepatitis B surface antigen (HBsAg) and anti-HCV antibodies. Odds ratios (OR) and 95% confi dence intervals (95% CI) were estimated using conditional logistic regression. RESULTS: Compared with controls,ICC patients had a higher prevalence of HBsAg seropositivity (48.4% vs 9.6%,P < 0.000),and hepatolithiasis (5.4% vs 1.1%,P = 0.001). By multivariate analysis,the signif icant risk factors for development of ICC were HBsAg seropositivity (adjusted OR,8.876,95% CI,5.973-13.192),and hepatolithiasis (adjusted OR,5.765,95% CI,1.972-16.851). The prevalence of anti-HCV seropositivity,diabetes mellitus,hypertention,cigarette smoking,and alcohol consumption were not significantly different between cases and controls. CONCLUSION: These findings suggest that HBV infection and hepatolithiasis are strong risk factors for development of ICC in China.

Growth factor-and cytokine-driven pathways governing liver stemness and differentiation

Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/pro-genitor cell expansion and differentiation, and the rel-evance of these signals in liver development, regeneration and carcinogenesis. Tyrosine kinase receptor signaling, in particular, c-Met, epidermal growth factor receptors or fibroblast growth factor receptors, contribute to prolifera-tion, survival and differentiation of liver stem/progenitor cells. Different evidence suggests a dual role for the trans-forming growth factor (TGF)-β signaling pathway in liver stemness and differentiation. On the one hand, TGF-βmediates progression of differentiation from a progenitor stage, but on the other hand, it contributes to the expan-sion of liver stem cells. Hedgehog family ligands are nec-essary to promote hepatoblast proliferation but need to be shut off to permit subsequent hepatoblast differentiation. In the same line, the Wnt family and β-catenin/T-cell fac-tor pathway is clearly involved in the maintenance of liver stemness phenotype, and its repression is necessary for liver differentiation during development. Collectively, data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates to contribute, under adequate conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role. From a clinical perspective, this is a fundamental issue for liver stem/progenitor cell-based therapies.

BMP signaling in mesenchymal stem cell differentiation and bone formation

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and have diverse functions during development and organogenesis. BMPs play a major role in skeletal development and bone formation, and disruptions in BMP signaling cause a variety of skeletal and extraskeletal anomalies. Several knockout models have provided insight into the mechanisms responsible for these phenotypes. Proper bone formation requires the differentiation of osteoblasts from mesenchymal stem cell (MSC) precursors, a process mediated in part by BMP signaling. Multiple BMPs, including BMP2, BMP6, BMP7 and BMP9, promote osteoblastic differentiation of MSCs both in vitro and in vivo. BMP9 is one of the most osteogenic BMPs, yet it is a poorly characterized member of the BMP family. Several studies demonstrate that the mechanisms controlling BMP9-mediated osteogenesis differ from other osteogenic BMPs, but little is known about these specific mechanisms. Several pathways critical to BMP9-mediated osteogenesis are also important in the differentiation of other cell lineages, including adipocytes and chondrocytes. BMP9 has also demonstrated translational promise in spinal fusion and bone fracture repair. This review will summarize our current knowledge of BMP-mediated osteogenesis, with a focus on BMP9, by presenting recently completed work which may help us to further elucidate these pathways.

RNA interference-mediated gene silencing of vascular endothelial growth factor in colon cancer cells

AIM： TO inhibit the expression of vascular endothelial growth factor （VEGF） in colon cancer cell line by RNA interference （RNAi）.METHODS： Followed the service of E-RNAi, we designed and constructed two kinds of shRNA expression vectors aiming at the VEGF gene, then transfected them into colon cancer HT29 cells by lipofectamineTM 2000. The level of VEGF mRNA was investigated by RT-PCR and Northern blotting. The protein expression of VEGF was observed by immunofluoresence staining and Western blotting.RESULTS： We got two kinds of VEGF specific shRNA expression vectors which could efficiently inhibit the expression of VEGF in HT29 cells. RT-PCR, Northern blotting, immunofluoresence staining and Western blotting showed that inhibition rate for VEGF expression was up to 42%, 89%, 73% and 82%, respectively.CONCLUSION： The expression of VEGF can be inhibited by RNA interference in HT29 cells.

Clinicopathologic characteristics of intrahepatic cholangiocarcinoma in patients with positive serum a-fetoprotein

AIM:To explore clinicopathologic characteristics of intrahepatic cholangiocarcinoma (ICC) in patients with positive serum a-fetoprotein (AFP). METHODS:One hundred and thirty one patients who underwent surgical dissection for pathologically confirmed ICC were divided into a positive AFP (> 20 ng/mL) group (n = 32) and a negative AFP group (n = 99), whose clinicopathologic features were analyzed and compared. RESULTS:The positive rate of HBsAg and liver cirrhosis of the positive AFP group was higher than that of the negative AFP group, while the positive rate of CA19-9 (> 37 U/mL) and the lymph node metastasis rate was lower. CONCLUSION:ICC patients with positive AFP share many clinicopathologic similarities with hepatocellular carcinoma.

Dual effect of WISP-1 in diverse pathological processes

Wnt-1 inducible signaling pathway-1（WISP-1）, also known as CCN-4, belongs to the connective tissue growth factor（CTGF） family. WISP-1 is primarily expressed in embryonic stem cells and is involved in adult organ development. WISP-1 participates in many cellular processes, including proliferation, differentiation, apoptosis and adhesion. In addition, WISP-1 plays an important role in diverse pathophysiological processes, such as embryonic development, inflammation, injury repairs and cancers. Recent studies showed that WISP-1 was highly correlated with tumor progression and malignant transformation, whereas it played an oncogenic role in colorectal cancer,cholangiocarcinoma, hepatocellular carcinoma and breast cancer. However, interestingly, WISP-1 exerts a tumorsuppressing role in lung and prostate cancers. WISP-1 promotes cell proliferation, adhesion, motility, invasion,metastasis and epithelial-to-mesenchymal transition via particular signaling pathways. In this review, we discussed the structure, expression profile, functions, clinical significance and potential mechanisms of WISP-1 in cancer and non-neoplastic diseases.

RNA Interference-Mediated Gene Silencing of Vascular Endothelial Growth Factor

OBJECTIVE To inhibit the expression of the vascular endothelial growth factor (VEGF) by RNA interference, and to observe the effect in different cells line. METHODS Using the services of E-RNAi, we designed and constructed two kinds of shRNAs expression vectors which were aimed at the VEGF gene. These vectors were then transfected into HEK293, colon cancer HT29, Hela and HepG2 cells by LipofectamineTM 2000. The level of VEGF mRNA was determined by RT-PCR and Northern blotting and the VEGF expression was examined by immunofluoresence staining. RESULTS The two kinds of VEGF specific shRNAs expression vectors were found to efficiently inhibit the expression of VEGF in HEK293 and HT29 cells by RT-PCR analysis, with inhibition rates of 72% and 42%, respectively; but the inhibition rates were reduced to 28% in Hela cells and 13% in HepG2 cells. Northern blotting showed that the inhibition rates of VEGF mRNA expression were 88% and 89% in HEK293 and HT29 cells, respectively. The inhibition rate of VEGF protein expression in HT29 cells was 73% based on immunofluoresence staining. CONCLUSION The expression of VEGF was inhibited by RNA interference, but differed with various cells lines, showing that RNA interference was cell-line dependent.

New genes emerging for colorectal cancer predisposition

Colorectal cancer(CRC)is one of the most frequent neoplasms and an important cause of mortality in the developed world.This cancer is caused by both genetic and environmental factors although 35%of the variation in CRC susceptibility involves inherited genetic differences.Mendelian syndromes account for about5%of the total burden of CRC,with Lynch syndrome and familial adenomatous polyposis the most common forms.Excluding hereditary forms,there is an important fraction of CRC cases that present familial aggregation for the disease with an unknown germline genetic cause.CRC can be also considered as a complex disease taking into account the common diseasecommom variant hypothesis with a polygenic model of inheritance where the genetic components of common complex diseases correspond mostly to variants of low/moderate effect.So far,30 common,low-penetrance susceptibility variants have been identified for CRC.Recently,new sequencing technologies including exomeand whole-genome sequencing have permitted to add a new approach to facilitate the identification of new genes responsible for human disease predisposition.By using whole-genome sequencing,germline mutations in the POLE and POLD1 genes have been found to be responsible for a new form of CRC genetic predisposition called polymerase proofreading-associated polyposis.

Salvianolic acid B increases recombinant adeno-associated virus transduction through redirecting trafficking pathways and stabilizing perinuclear accumulations

OBJECTIVE To screen small molecule compounds from traditional Chinese medicine that can enhance recombinant adeno-associated virus(rAAV) transduction.METHODS Recombinant adeno-associated virus(rAAV) has been established as a powerful tool for in vivo gene transfer and achieved much promise in gene therapy applications.However,widespread clinical use has been limited by transduction efficiency.In the current study,we screened a panel of small molecule compound from traditional Chinese medicine focused on AAV intracellular trafficking process and found salvianolic acid B can significantly enhance rAAV2 transduction.RESULTS Salvianolic acid B caused a dose-depen.dent increase in rAAV2 transduction regardless of vector dose,genome architecture,and over a broad range of cell line from various cell type and species(HEK293,HeLa,HepG2,Huh-7,CHO-K1,LO-2).Salvianolic acid B treatment redirected rAAV2 particles toward large vesicles positive for late endosomal(Rab7) and lysosomal(LAMP1) markers.Furthermore,salvianolic acid B acted to increase accumulation of viral particles at the perinuclear region.CONCLUSION In summary,our results suggest that salvi.anolic acid B redirects rAAV2 toward more productive trafficking pathways and stabilizes perinuclear accumulations of vectors,facilitating productive nuclear trafficking.

Why some tumours trigger neovascularisation and others don't:the story thus far

Background:Angiogenesis is not essential for tumours to develop and expand,as cancer can also grow in a nonangiogenic fashion,but why this type of growth occurs is unknown.Surprisingly,our data from mRNA transcription profiling did not show any differences in the classical angiogenic pathways,but differences were observed in mitochondrial metabolic pathways,suggesting a key role for metabolic reprogramming.We then validated these results with mRNA profiling by investigating differential protein expression via immunohistochemistry in angiogenic and non-angiogenic non-small cell lung cancers(NSCLCs).Methods:Immunohistochemical staining for 35 angiogenesis- and hypoxia-related biomarkers were performed on a collection of 194 angiogenic and 73 non-angiogenic NSCLCs arranged on tissue microarrays.Sequencing of P53 was performed with frozen tissue samples of NSCLC.Results:The non-angiogenic tumours were distinguished from the angiogenic ones by having higher levels of proteins associated with ephrin pathways,mitochondria,cell biogenesis,and hypoxia-inducible factor 1(HIF1) regulation by oxygen and transcription of HIF-controlled genes but lower levels of proteins involved in the stroma,cell-cell signaling and adhesion,integrins,and Delta-Notch and epidermal growth factor(EGF)-related signaling.However,proteins classically associated with angiogenesis were present in both types of tumours at very comparable levels.Cytoplasmic expression of P53 was strongly associated with non-angiogenic tumours.A pilot investigation showed that P53 mutations were observed in 32.0%of angiogenic cases but in 71.4%of non-angiogenic tumours.Conclusions:Our observations thus far indicate that both angiogenic and non-angiogenic tumours experience hypoxia/HIF and vascular endothelial growth factor(VEGF) pathway protein expression in a comparable fashion.However,angiogenesis does not ensue in the non-angiogenic tumours.Surprisingly,metabolic reprogramming seems to distinguish these two types of neoplastic growth.On the basis of these results,we raise the hypothesis that in some,but not in all cases,initial tissue remodeling and/or inflammation could be one of the secondary steps necessary to trigger angiogenesis.In the non-angiogenic tumours,in which neovascularisation fails to occur,HIF pathway activation could be the driving force toward metabolic reprogramming.