| PurposeFrom the epidemiology invest, cancer patiens account for approximately 10%～20%of brain metastases cases, and in Chinese, the latest brain metastases cases is about 17000 per year. In all kinds of brain metastases cases, patients with lung cancer account for approximately 30%of brain metastases cases. More specially, small cell lung cancer (SCLC) has the propensity to progress aggressively and early metastasize to brain. Brain metastases are a source of significant morbidity and mortality in patients with SCLC. So, to investe the mechanism of how SCLC cell metastases to brain is very significance to SCLC's prevention and cure.However, cancer metastases are complected patho-stepes. Cancer cells flowed in blood get to distant organ, which firstly extravasate from blood vessels to parenchyma. Thus, the vascellum'permeability effectes cancer cells metastses. A key event of brain metastases is that SCLC cells migrate across the blood-brain barrier (BBB), which separates blood from the brain parenchyma. We had observed that the activation of Rho/ROCK signaling in human brain micrivascular endothelial cells (HBMECs) was required for SCLC cells transendothelial migration, and the increase of brain endothelial cell permeability(FEBS Letters,2006,580:4252-4260).Recently, the relationship between vascular endothelial growth factor(VEGF) and cancer was focused by people. VEGF has been shown to increase vascular permeability, and exerted a pro-angiogennic influence in a variety of human tumours. Placental growth factor (PLGF) is one of vascular endothelial growth factor (VEGF) family, plays biological functions in many pathological conditions. Found in research of tumor, PLGF affected the proliferation of tumor mainly through the promotion of new tumor blood vessels. PLGF is known to specifically bind with Flt-1, then Play a role in angiogenesis in coordination with VEGF. In addition, PLGF can recruit the hematopoietic stem cells and mononuclear cells which in the circulation to the primary tumor, and promotes the proliferation of tumor blood vessels. Recent studies show that,PLGF has dual roles at the occurrence of tumor.On the one hand,it can act directly on tumor cells and affect the migration of tumor cells. On the other hand,it can act on HBMEC and affect its function.We further found that PLGF bound with Flt-1 which is on HBMEC and then changed the integrity of tight junction between HBMECs.The purpose of this study is at the basis of preliminary studies through adoptting animal experiments to further verify whether PLGF participated the process of SCLC migration through blood-brain barrier into brain.Methods1,Identification of PLGF expression in different lung cancers by RT-qPCR.2,Comparion the invading through HBMEC monlayer among six lung cancer cell lines.3,Appling RNAi technology, use Adenovirus as a vector to construct siPLGF expression system.(1) Construction of recombinant adeno virus①Construction of si-PLGF shuttle.②Analysis of recombinant shuttle plasmid:shuttle(si PLGF).③Cotransformed linearized shuttle and adenoviral backbone.④Construction of recombinant adeno virus. ⑤Identification the recombinant adenovirus.(2) Packing virus①AD293 cell culture.②Primary virus harvested.③Second time virus harvested.④Third time virus harvested.⑤Purified adeno virus.4,Infected SCLC and identificated functions(1) Infected NCI-H250,NCI-H209,NCI-H446.(2) Real-time PCR identificated functions(3) Transmigration through HBMEC monlayer.5,Experiments in vivo(1) Inoculated different lung cancer cells into cardiac ventricle and then Analysis their tumorigenicity in vivo.①Treatment before the cells inoculated.②inoculated six kinds of lung cancer cells into cardiac ventricle of Nude mice.③Observation of brain and lung tumor cases through HE staining.(2) Inoculated infected SCLC into one internal carotid artery of Nude mice.①Infected the small cell lung cancer cells.②Inoculate SCLC into one internal cartid artery of Nude mice.③Observation of brain and lung tumor cases through HE staining.Results1. PLGF was expressed in different lung cancer cells.PLGF mRNA expression levels were different in lung cancer cells. SCLC cells came from brain metastases (NCI-H 250) expressed PLGF highly; SCLC cell came from bone marrow metastases (NCI-H 209) expressed PLGF Slightly lower than the former; SCLC cell came from plenral fluid of lung tumours (NCI-H 446) expressed lower-level PLGF; Non-small cell lung cancer cells expressed low-levels PLGF.2. Comparion the invading through HBMEC monlayer among six kinds of lung cancer cell lines. SCLC invaded through HBMEC monlayer more than the other cell lines.3. Construction of Ad-shuttle-si PLGF expression system and gained virus particles.(1) Successed construction of si-PLGF and construction of recombinant asdeno virus.(2) Gained purified high titer virus particle,1.5×1011 viruses.4. Ad-shuttle-si PLGF infected SCLC and migration experiments in vitro.(1) After virus infected, about 80%SCLC cells could be transfected.(2) Ad-si-PLGF SCLC cells expressed less PLGF than other two congtrol group.(3) PLGF silenced SCLC cells transmigrated through HBMEC monlayer less than other teo control groups.5. Experiments in vivo(1) The tumorigenicity were significant different in vivo, inoculating six kinds Of lung cancer cells via Ventricular.NCI-H446 and NCI-H460 tumor cells of the best, NCI-H292 and A549 tumor cells better, and NCI-H 209 and NCI-H 250 cells were less tumorigenicity. The six lung cancer cells in the lung tumor were visible, but did not found a case of brain metastases.(2) Inoculate infected SCLC into nude mice via internal carotid artery. Established small-cell lung cancer cell animal model of brain metastases. Detected metastatic brain tumors of nude mice which inoculated infected SCLC,the migration of different SCLC cell lines was distinct. NCI-H209 had better migration.It was not detected metastatic brain tumors in the nude mice which inoculated PLGF silenced SCLC.ConclusionsPLGF participated the process of SCLC migration through blood-brain barrier into brain. |
PDF Full Text Request