人脑胶质瘤CD105表达和胶质瘤干细胞在血管生成+中的作用及机制初探_

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bFGF

basic fibroblast growth factor

? ???? BSA bovine serum albumin ?????? cDNA complementary DNA

??DNA

CSCs cancer stem cells ?? ?? DAB 3,3-diaminobenzidine tetrahydrochloride 3,3-?? ??? DMEM Dulbecco’s modified Eagle medium Dulbecco ?Eagle DMSO dimethyl sulfoxide

?? ?? DNA deoxyribonucleic acid

??????

dNTP deoxyribonucleoside triphosphate ????й?? E C

s endothelial cells ??? EDTA ethylene diaminetetraccetic acid ??? ?? EGF

epidermal growth factor

???? EP C

s endothelial progenitor cells ???? FCS

fetal calf serum

???? FIT C fluorescein isothiocyanate ?≒?? ?

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glioma-derived microvascular

endothelial cells

???? ?? ???

GFAP

glial fibrillary acidic protein

????? ?? h hour

HE hematoxylin eosin ? ???(??)

HEPES N-2-hydroxyethylpiperazine-N’-2- ethanesulfonic acid

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human umbilical vein endothelial cells

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MMP2 matrix metallopeptidase 2 ?? ???2 MMP9

matrix metallopeptidase 9

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mRNA messenger RNA RNA MVD microvessel density ?? Matrigel Matrigel ?? PBS phosphate buffered saline ???? ? PCR

polymerase chain reaction

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qPCR quantitative real-time P C

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RT-PCR

reverse transcription polymerase chain reaction

? ? ?? sec second 。

siRNA

small interfering RNA

???? TGF-? transforming growth factor-? ? ?? ?

TRITC tetraethyl rhodamine isothiocyanate ? ?? ?≠?? VEGF vascular endothelial growth factor

?? ???

VEGFR vascular endothelial growth factor receptor ?? ??? ?

vWF von Willebrand Factor ?? ? ?

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The Roles of CD105 Expression and Glioma Stem Cells in the Angiogenesis of Human Glioma and Their

Mechanisms

Abstract

Malignant glioma is the most common and aggressive primary brain tumor. Even after surgical excision, radio and chemotherapy, it shows a high percentage of recurrence and short survival time. Malignant glioma is highly vascularized tumors of human. Chemotherapy is often unsatisfactory because of the presence of blood-brain barrier. It is well known that angiogenesis is a key factor in the progression of malignant tumors. Many efforts have focused on new therapy approaches based on the inhibition of angiogenesis.

Anti-angiogenic therapy is efficient and specific, which avoids the blood-brain barrier and the side-effects of traditional therapy. Thus, it is expected to provide a new and more effective way for treatment of brain tumors. Anti-angiogenesis to treat cancer during the past 40 years has been made great progress. Several anti-angiogeneic drugs have been used for clinical trials, however, they showed limited improvement in overall survival.

Targeting tumor endothelial cells is now the main strategy for inhibition of angiogenesis. Therefore, the search for specific targets of tumor angiogenesis has been very important. Accumulating evidence has shown that C D105 (endoglin) specifically expressing on vascular endothelial cells participates in tumor angiogenesis. Thus, C D105 might be emerging as a promising target of anti-angiogenetic therapy of cancer. C D105 gene is located on human chromosome 9q34, encoding a dimer of the same type of membrane-bound glycoprotein, and CD105 has molecular weight of 180kD and locates on cytoplasm membrane. C D105 is a component of TGF-? receptor complex involved in cardiovascular development, body homeostasis and other physiological processes. C D105 has been identified as one of endothelial markers, and is correlated with tumor grade, stage, metastasis and prognosis. Currently, CD105 in glioma angiogenesis and its mechanism has not been fully elucidated.

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C ancer stem cells (C SC s), or tumor initiating cells, account for a very small cell population in a tumor and have self-renewal and differentiation capacities. Emerging evidence shows that CSCs may be directly or indirectly involved in the process of tumor angiogenesis. However, the possible interaction between GSCs and endothelia cells (ECs), and transdifferentiated EC phenotype of GSCs have not been clarified.

In this study, human glioma specimens were used to study the C D105 protein expression and its relationship with tumor grading and patient survival. Furthermore, fresh glioma specimens were used for endothelial cells and culture of GSCs. In addition, human ECs (HUVE-12 cells) were used for the study of CD105 function.

The main results and conclusions are as follows:

1. CD105 protein was found expressed in glioma tissues. Microvessel density (CD105- MVD) was statistically different between WHO grades of gliomas, i.e., the higher the grades, the higher the CD105-MVD. More importantly, high CD105-MVD was correlated with poor prognosis of patients. High and low C D105-MVD values in the patients with survival rate were significantly correlated (Log Rank test: 24.27; P <0.001). Thus, CD105 can be a marker for classification of glioma grade to predict prognosis of patients.

2. Glioma derived microvascular endothelial cells were isolated from primary human glioma, and were strongly positive for CD105, CD31, CD34, F?-R, but without expression of GFAP and ?-SMA, the marker for the smooth muscle and pericytes. Culture in Matrigel showed spontaneous tubular formation of endothelial cells.

3. Tumorspheres were obtained from primary glioma cells, suggesting their features of cancer glioma stem cells (GSC s). Tumorspheres were found to grow into EC s during co-culture and to transdifferentiate into EC phenotype in the presence of endothelial medium.

4. Silencing C D105 gene expression showed inhibition of angiogenesis in vitro. By flow cytometry, real-time quantitative PC R and western blot evaluation of lentiviral interference effects. Virus-positive cell rate was 94% measured by flow cytometry. Real-time PC R and western blot showed that C D105mRNA and protein levels in the CD105-silenced cells - shCD105 were significantly lower than shCon and Mock group. AS compared with Mock, the proliferation of shC D105 cells in the presence of VEGF was decreased. Transwell experiment showed significantly reduction in the number of migrating

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cells (P <0.01), and scratch test revealed inhibited migration (P <0.01). Tubular formation in Matrigel was also significantly reduced (P <0.01). Real-time PCR showed that CD105 gene silencing could reduce the expression of VEGFR2 mRNA and MMP2 mRNA.

In summary, our results suggest that CD105 expression with high CD105-MVD values is predictive for poor prognosis of glioma patients, and C D105 protein is a promoting regulator for glioma angiogenesis. Moreover, we found that glioma stem cells could interact with endothelia cells and present EC phenotype to participate in the angiogenesis of glioma.

Key words: angiogenesis; CD105 (Endoglin); glioma; tumor vascular endothelial cells;

cancer stem cells; microvessel density; prognosis.

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?????CD105?? ??? ?? ??? ??

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??? ? ? ? ????????? ? ?? ?,?? ?? ? ?? ? н? ? ?? ?儈?? ??亴 ? ?????? ?о??? ? ? ???? ????? ?? ? ? ??? ?? ????? ???? ? 儈????а? ??? ?? 儈 ?? ?н ???? ? ? ???, ? ?? ? ??????? , ??????? а? ? ?? ??? ???? ?40 伎 ? ?? ?? ?? ? ?ウ ??? ?? ??????? ?? ??? ??? ? ???фн ?? ? ?на??? ??? ???? ??? ????? ??? ?? ? ?? ? ??? ? ? ? ??? ?? ?? ? ?? VEGF??? ??? ??? ? ? ??? FGF-2?? ??? ? ???? ?? ??? ??????? ?? ? ??? ?????? ?? ?? ??

? ??? ? ?? ??? ????? ??????? ?? ????? ???? ??? ????????ウ ?? ? ? ?CD105(Endoglin) ?????? ???? 儈???о????? ? ? ? ??? ??????? ???CD105?? ??????9q34? ????? а? ?????? ???? ??180kD? ???????CD105 TGF?￡? ? ? ? о? ?? ?? ?っ ?????? CD105??? ??ウ???? ?TGF-￡ ? ?? о?? ????? ?л?? TGF-￡? ?? ??CD105 ??? ??? ???????ф ?? ?? ??? 亴 ? ? ? ??? ????? ???? CD105 ?????? ?? ? ? ?

* ?仈 ??、 ?(No.30700284)? ?? ??ウ ? ?973? ?亩?(No.2010CB529403)? ?

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???? ??(cancer stem cells, C SC s) ?? ??(tumor initiating cells) ????? ???? ?? ? ???CSCs ??? ? ? ? ? ?????? ? ? ? ?? ? о????? ? ???? ??? ???????? ??? ? ???????? ?

傼 ???? ??? ??ウ???????CD105????о?? ??? 亴 ? ??? 勌???? ? ? ?????? ?? ???(glioma derived microvascular endothelial cells?GDMECs) ??? ??(glioma stem cells,GSCs)? ? ?? ?? ? ? ? ?HUVE-12??? ?ウCD105 ?????? ?? ??

? ???? ?? л?

1. C D105?? н ??????? ?CD105??? ?? (CD105-MVD)о???WHO ? ??? (????)о儈? (???) ?? ? (P<0.001)?儈??CD105-MVD о ??? ? ?? (Log Rank?傼?24.27?P <0.001)? ?????? ? 亴????? ?亴 ? ??а?

2. ?? ????? ?? ??? ??? ???? ??????CD105,CD31,CD34,F?-Rag? ?? ???????GFAP ━?? ????￠-SMA? ; Matrigelй? ???? ? ? ?

3. ? ??? ??? ???? ? ?GSCsо ??? ?GSCs ? ? ? ??? ?? ? ? GSCs ??? ? ?

4.?? ?? RNA?? ? ? ???CD105 ? ??? ? ???? ?? ??? ? ?PCR ? ???? ?? ??? ?? ???? ?? ??? 94%? ? ?PCR? ? ?? ?shCD105??CD105mRNA ??≤ ???↓ ?? Mock??shCD105оMock?????? VEGF ? ?Transwell 傼???? ? (P<0.01)? ? 傼??? (P<0.01)?Matrigel? ? ? (P<0.01)? ? ?PCR?? CD105 ?唈 VEGFR2 mRNA ≤ ???? л?MMP2????

к? 傼? ? ?CD105о????? ? ?? ???亴 ? ??а?CD105 ??? ↓ ? ?? ? ??? ???

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??? ?ウ俆?? ? ???? ??о ??? ? ??? ? ? ???? ??????? ? ??? ? ? ?а? ??

??φ??? (angiogenesis)?CD105(Endoglin)????????? ?????? ??? ?? (microvessel density)?亴 (prognosis)

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?????CD105?? ??? ?? ??? ??

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????? ??? ?? ???[1], ? ????? ?? ???????? ???? ??а[2-7]????? ??? (anti-angiogenesis)???? н? ? ??? ???? ? 儈????а???? ????? ??????? ? о ? ? ????? ? (neovascularization) ??? ??? ?? ?(vasculogenesis)? ?????? ????? ??????? ? (angiogenic switch) ???╄? ? ??? ?? ??? ???? ? 儈? ???а,?? ?? ? ?? ?? ?????????? ?ウ? ?? ???? ? ? ?? ? ?? ???? ? ????? ? ??? ? ? ?ウ ??? ???儈??CD105[8]? ? ? ? ??↓ ??? ?н?? ???CD105??ウ ??CD105 ???? ?????? ???? CD105 ????? ???? ? н??? ???а??ウCD105 ?????? ?? ? ?? ??? ?? ? ??????? ??? ??

??? ? ??? ?? ????? (VEGF) ? ?????? (FGF) ???? ?(Ang) ?? ?? ￡(TGF-￡) ? ?? ??? ??? ? ??オ????≤ ??? ??? ? ???? ? ?CD105 о? ?? ￡(TGF-￡) ?? ? [9, 10]?

CD105 ?? ??? ???? ?儈?? ????? ?ウ?? ? ??[11-17]?CD105??????9q34? ????? а? ?????? ???? ??180kD? ????????ウ? ?CD105 ?оTGF-￡ ? ? ? ? ???? ? ? ??? ?CD105 TGF ￡??? ??? ??TGF-￡??? ? ?? ??? ?????? ? ?CD34 F?R? ??CD105 ? ??? ????? ????

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?ウ?? ??? ??? ?й???? ? ??? ? ? siRNA ? ??????CD105????о? 亴 ? ???? ? ?CD105 ??? ? ?? ? ? ???? ?? ???? ??? ?? ?? ??а???CD105 ?????? ?? ?? ??

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1.Folkman, J. Tumor angiogenesis: therapeutic implications. N Engl J Med, 1971,

285(21):1182-6

2.Ferrara, N.,Kerbel, R.S. Angiogenesis as a therapeutic target. Nature, 2005,

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3.Loges, S., Roncal, C.,C armeliet, P. Development of targeted angiogenic medicine.

Journal of Thrombosis and Haemostasis, 2009, 7(1):21-33

4.Hanahan, D.,Weinberg, R.A. Hallmarks of cancer: the next generation. C ell, 2011,

144(5):646-74

5.Ellis, L.M.,Hicklin, D.J. VEGF-targeted therapy: mechanisms of anti-tumour activity.

Nature Reviews Cancer, 2008, 8(8):579-91

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S.,Gasparini, G. Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst, 1992, 84(24):1875-87

7.Holleb, A.I.,Folkman, J. Tumor angiogenesis. CA Cancer J Clin, 1972, 22(4):226-9

8.Minhajat, R., Mori, D., Yamasaki, F., Sugita, Y., Satoh, T.,Tokunaga, O. Endoglin

(CD105) expression in angiogenesis of colon cancer: analysis using tissue microarrays and comparison with other endothelial markers. Virchows Archiv, 2005, 448(2):127-134

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superfamily coreceptors in cancer. Biochimica Et Biophysica Acta-Molecular Basis of Disease, 2009, 1792(10):954-973

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vascular cell physiology. Journal of Cellular Biochemistry, 2007, 102(6):1375-88

11.Dallas, N.A., Samuel, S., Xia, L., Fan, F., Gray, M.J., Lim, S.J.,Ellis, L.M. Endoglin

(C D105): A marker of tumor vasculature and potential target for therapy. C linical

Cancer Research, 2008, 14(7):1931-1937

12.Fonsatti, E., Nicolay, H.J., Altomonte, M., C ovre, A.,Maio, M. Targeting cancer

vasculature via endoglin/C D105: a novel antibody-based diagnostic and therapeutic

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strategy in solid tumours. Cardiovascular Research, 2010, 86(1):12-9

13.Fonsatti, E.,Maio, M. Highlights on endoglin (C D105): from basic findings towards

clinical applications in human cancer. J Transl Med, 2004, 2(1):18

14.Maio, M., Altomonte, M.,Fonsatti, E. Is it the primetime for endoglin (CD105) in the

clinical setting? Cardiovascular Research, 2006, 69(4):781-783

15.van Meeteren, L.A., Goumans, M.J.,Ten Dijke, P. TGF-beta Receptor Signaling

Pathways in Angiogenesis; Emerging Targets for Anti-Angiogenesis Therapy. C urr Pharm Biotechnol, 2011

16.Seon, B.K., Haba, A., Matsuno, F., Takahashi, N., Tsujie, M., She, X., Harada, N.,

Uneda, S., Tsujie, T., Toi, H., Tsai, H.,Haruta, Y. Endoglin-targeted cancer therapy.

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Syro, L.V., Kovacs, K.,Lloyd, R.V. Endoglin (C D105): a review of its role in angiogenesis and tumor diagnosis, progression and therapy. Anticancer Research, 2011, 31(6):2283-90

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?? (MVD)? ?100 л ??? ?3??? ? ?н ???400 ? ?? (? ? ? )? ??? ?MVD ?н? ??? ?? ???? ?? ? ?1? ????????>8???????н???CD105??? ???? ??CD105-MVD?

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?? ? ±? (x±s)?????? ?? ? ? ? ????? T?傼(Independent-sample T test)??? ??? ? ?? ? (One-way ANOVA)??к?? ?SPSS 13.0?????? ??P<0.05?? ? ??P<0.01? ? ??? ??

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CD105? ????????? ?? ?????к, ???? ? ??, ???????????CD105??? ???? ??, ??? ? ???? ??????? ? ????? ??? ????? CD105 ? ???н ? ???? CD105 ??? на, ↓ ??? ?? ???? ??? ,儈? ???? ??? ( 1-1)?

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