Objective To investigate the effects of lymphangiogenesis on cancer growth and metastasis.Methods Human lymphatic endothelial cells(HLyECs)were isolated and purified from human lymph node by magnetic beads coated with antibody against human VEGFR3.Human breast cancer cell line(MDA-MB-435)or human osteosarcoma cell line(MG-63)was inoculated alone or co-inoculated with HLyECs subcutaneously into nude mice.The weight of tumor and lung surface metastasis were detected;peri-tumor lymphatic vessels were shown by Evans blue,intra-tumor lymphatic vessels were shown by immunohistochemistry for human and mouse PDPN.Conditioned medium of tumor on proliferation of HLyECs was evaluated by MTT assay.Results Compared with inoculating tumor cells alone,the growth and metastasis of MDA-MB-435 were promoted by co-inoculating HLyECs.The peritumoral and intra-tumoral lymphatic vessel density of MDA-MB-435 were increased by co-inoculating HLyECs.Both hPDPN-and mPDPN-positive lymphatic vessels were found.But co-inoculating HLyECs had no effect on lymphatic vessels density of MG-63.Neither intra-nor peri-tumor lymphatic vessels were found.The proliferation of HLyECs was increased by conditioned medium of MDA-MB-435 but not by MG-63.Conclusion Growth and lymphangiogenesis of breast cancer can be enhanced by co-inoculating lymphatic endothelial cells.

Objective To investigate the effect of IH764-3 on the leukocyte-mediated hypoxia-reoxygention injury of human brain microvascular endothelial cell (HBM VEC). Methods MTT assay was used to detect the survival of HBMVEC; gelatin zymography was used to check the activity of MMPs. The level of reactive oxygen species (ROS) in leukocyte was determined via commercially available kit, and the indirect enzyme-linked immunosorbent assay (ELISA) was used to quantify the contents of TNF-α, IL-1α, IL-2 and INF-γ in leukocyte culture medium. Results Survival of HBMVEC was impaired by hypoxia-reoxygenation, which was aggravated by supernatant of activated leukocytes but was attenuated by IH764-3. Leukocytes produced high level of MMP-9, ROS and cytokines (TNF-α, IL-1α, IL-2, IFN-γ) after hypoxia-reoxygenation, the process was inhibited by IH 764-3. Furthermore, IH764-3 could effectively reverse hypoxia-reoxygenation injury of HBMVEC with supernatant of activated leukocytes. Conclusion IH764-3 can protect HBMVEC from leukocyte-involved hypoxia-reoxygenation injury by attenuating the activation of leukocytes and inhibiting the pathogenic effects of leukocytes products.

Objective To investigate the effect of IH764-3 on the leukocyte-mediated hypoxia-reoxygention injury of human brain microvascular endothelial cell (HBMVEC). Methods MTT assay was used to detect the survival of HBMVEC; gelatin zymography was used to check the activity of MMPs. The level of reactive oxygen species (ROS) in leukocyte was determined via commercially available kit,and the indirect enzyme-linked immunosorbent assay (ELISA) was used to quantify the contents of TNF-?,IL-1?,IL-2 and INF-? in leukocyte culture medium.Results Survival of HBMVEC was impaired by hypoxia-reoxygenation,which was aggravated by supernatant of activated leukocytes but was attenuated by IH764-3. Leukocytes produced high level of MMP-9,ROS and cytokines (TNF-?,IL-1?,IL-2,IFN-?) after hypoxia-reoxygenation,the process was inhibited by IH 764-3. Furthermore,IH764-3 could effectively reverse hypoxia-reoxygenation injury of HBMVEC with supernatant of activated leukocytes. Conclusion IH764-3 can protect HBMVEC from leukocyte-involved hypoxia-reoxygenation injury by attenuating the activation of leukocytes and inhibiting the pathogenic effects of leukocytes products.

Objective To discuss the ultrastructural pathological characteristics and dynamic changes of brain vessel after subarachnoid hemorrhage(SAH),and the mechanism of these changes in delayed cerebral vasospasm.Methods SAH model was made by infusing blood twice into the cistern magna of Japanese rabbits.The animals were divided randomly into SAH group,saline group,puncture group and blank group,at 1 h,3 d,5 d,7 d and 10 d after the first infusion the animals were perfused and basilar artery was harvested.Ultrastructural changes were observed under light microscope and electron microscope.Results Under the light microscope,the vessel wall became thick,the vessel cavity became narrow,the endothelia cells became swollen,vacuoles could be found in the chromatin,inner elastic membrane became reductus and broke.Under the electron microscope,the close connection between the endothelial cells disappeared,the membrane of the cells fell off,and the mitochondria became swollen,vacuoles could be seen,the chromatin became concentrated,heterochromatin could be seen,smooth muscle became deformed,chromatin became uneven, myofilament had derangement and fragmentation and dissolved,vacuolus could be seen in the kytoplasm,mitochondrion became swollen.The structural change of basilar artery under the light microscope got similar to that under the electron microscope;slight change was observed right after 1 h of SAH,significant change was observed at 3 d,and most obvious change was observed between 5 d and 7 d.Conclusion Ultrastructural changes were observed in the basilar artery after SAH,and significant dynamic changes were observed in the progress.The damage of endothelia cells may be the important factors which cause delayed cerebral vasospasm.

Objective To design a new experimental facility to induce rat diffuse axonal injury model. Methods Rat diffuse axonal injury was induced by a new experimental facility,which was developed to let the rat head spin 90 degree at the moment to cause shearing injury.Vital sign and behavior of rat were measured simultaneously.The rats were sacrificed at 2 h,6 h,12 h,24 h,36 h,72 h and 10 d after injury,respectively,and the brain tissues were removed to prepare paraffin section.Then silver staining and HE staining were conducted to investigate changes of axonal fibers. Results There were unconsciousness,respiratory rhythm disturbance and hyporeflexia of pupil light reflex immediately after injury,and reactiveness decrease and activity retardation still existed even after resuming consciousness.At anatomical scene,subarachnoid hemorrhage or cerebroventricular haemorrhage were widespread.At an early stage,there were swelling,collapse,and axonal retraction ball formation at cortico-medulla junction,callosum,brainstem,and cerebellar white matter under microscope.But at the later stage,gitter cell proliferation and nest-like aggregation were major pathophysiological changes at focal brain tissue. Conclusion The new experimental facility is suit able to be used to induce rat diffuse axonal injury,since it is convenient,controlable,and precise.

Objective: To establish an in vitro model of brain blood barrier (BBB) using cultured mouse brain microvascular endothelial cells (BMVEC). Methods: Mouse BMVEC were seeded on micro pore membrane of gelatin coated cell culture insert and cultured to confluence. The establishment of BBB was preliminary judged by a 4 h water leaking test. The tight junctions between BMVEC were demonstrated by scanning and transmission electron microscope. The transendothelial electrical resistance(TEER) over BMVEC was measured. The permeability of Horseradish peroxidase (HRP) through the BBB was analyzed and the effect of RMP 7 on permeability of the BBB was investigated. Results: The 4 h water leaking test became positive when BMVEC were cultured to confluence. By scanning and transmission electron microscope, the tight junctions were demonstrated on confluent BMVEC. The TEER over BMVEC monolayer increased 3.2 and 7.68 times and the permeability rates for HRP were 13.4% and 6.7% respectively, as compared with sub confluent BMVEC and human umbilical vein endothelial cell monolayer(HUVEC). The HRP permeability rate in the model of BBB increased 2.7 times after treatment with RMP 7. Conclusion: The established in vitro model of BBB has basic characteristics of BBB in vivo , and is suitable for central nervous system (CNS) drug research over BBB.

Objective To investigate the effect of mitochondrial fission on the function of pancreatic β cells.Methods INS-1 stable cell lines allowing inducible expression of either wild-type dynamin-related protein 1 (Drp-1 WT)or its dominant-negative mutant(Drp-1 K38A)were used.The effect of mitochondrial fission on the function of pancreatic β cells were investigated under different concentrations of glucose.Results There were increased mitochondrial fission and disintegration of the mitochondrial reticulum into multiple punctiform organelles in Drp-1 WT cells induced with doxycycline under high glucose condition.Insulin secretion(P<0.01),mitochondrial membrane potential(P<0.05),and ATP content(P<0.05)were decreased and cytochrome C expression was increased after the expression of Drp-1 WT under high glucose condition while these changes were markedly mild in Drp-1 K38A expression cells.Conclusion The increased mitochondrial fission inhibits pancreatic β cell function.

Objective To evaluate the reproducibility of ADC measurements at 1.5 vs 3.0 T and at 1.5 T of different scanners in liver,spleen and pancreas of healthy volunteers.Methods Abdominal DWI were performed on 33 healthy volunteers by using GE 1.5 T,Siemens 1.5 T and Philips 3.0 T MR scanners.The mean ADC values of liver,spleen,pancreatic head,body,and tail were calculated.The ADC data were analyzed by using paired-sample t tests.Results The mean ADC of liver at GE 1.5 T,Siemens 1.5T and Philips 3.0 T were (1.56 ±0.10) ×10-3,(1.67 ±0.15) ×10-3 and(1.35 ±0.12) ×10-3 mm2/s,spleen were (0.96±0.10) × 10 3,(0.98 ±0.11) ×10-3and(0.81 ±0.14) × 10-3 mm2/s,pancreatic head were (2.09 ± 0.27) × 10-3,(2.20 ± 0.21) × 10-3 and (2.05 ± 0.27) × 10-3 mm2/s,pancreatic body were (2.03 ± 0.27) × 10-3,(2.09 ± 0.30) × 10-3 and (1.76 ± 0.25) × 10-3 mm2/s,pancreatic tail were (1.88 ± 0.28) × 10-3,(1.88 ± 0.27) × 10-3 and (1.56 ± 0.27) × 10-3 mm2/s,respectively.From the aspect of different field strength MR scanners,there were significant differences in mean ADC of liver (t =11.073,P ＜0.01 in GE 1.5 T vs Philips 3.0 T; t =12.795,P ＜0.01 in Siemens 1.5 T vs Philips 3.0 T),spleen (t =4.143,P ＜ 0.01 in GE 1.5 T vs Philips 3.0 T; t =5.376,P ＜ 0.01 in Siemens 1.5 T vs Philips 3.0 T),pancreatic body (t =4.677,P ＜ 0.01 in GE 1.5 T vs Philips 3.0 T; t =5.174,P ＜0.01 in Siemens 1.5 T vs Philips 3.0 T) and tail (t =5.356,P ＜0.01 in GE 1.5 T vs Philips 3.0 T; t =4.648,P ＜0.01 in Siemens 1.5 T vs Philips 3.0 T),but there were no significant differences in mean ADC of pancreatic head (t =0.340,P ＞ 0.05 in GE 1.5 T vs Philips 3.0 T; t =1.349,P ＞ 0.05 in Siemens 1.5 T vs Philips3.0 T).From the aspect of different 1.5 T MR scanners,there were significant differences in mean ADC of liver (t =-4.563,P ＜ 0.01),but there were no significant differences in mean ADC of spleen (t =-0.732,P ＞ 0.05),pancreatic head (t =-0.879,P ＞ 0.05),body (t =-1.020,P ＞0.05) and tail (t =0.054,P ＞ 0.05).Conclusion Between 1.5 T and 3.0 T MR scanners,there were significant differences in mean ADC of liver,spleen,pancreatic body and tail,but there were no significant differences in mean ADC of pancreatic head.At different 1.5 T MR scanners,there were significant differences in mean ADC of liver,but there were no significant differences in mean ADC of spleen,pancreatic head,body and tail.

BACKGROUND:Bone marrow mesenchyme stem cells are important non-hematopoietic stem cells in the bone marrow, which can stimulate angiogenesis. While, Slit can also stimulate angiogenesis, as many studies have proved. OBJECTIVE:To review the biological functions, clinical application and effects of bone marrow mesenchymal stem cells and Slit2 on promoting angiogenesis. METHODS:A computer-based online research of CNKI and PubMed databases was performed to col ect articles published between 1980 and 2014 with the keywords“MSCs”and“Slit2”in Chinese and English. There were 436 articles after the initial survey. Final y, 65 articles were included according inclusion and exclusion criteria. RESULTS AND CONCLUSION:Both bone marrow mesenchymal stem cells and Slit2 play an important role in promoting angiogenesis, but the relevance of bone marrow mesenchymal stem cells and Slit2 is stil controversial. If assuming that bone marrow mesenchymal stem cells secrete Slit2, more researches should be done to reveal whether bone marrow mesenchymal stem cells promoting angiogenesis is relevant to Slit2 and through which signaling pathway Slit2/Robo functions to adjust bone marrow mesenchymal stem cells thus to promote angiogenesis. If relevant, the transplantation of the Slit2 and bone marrow mesenchymal stem cells wil be a promising treatment of cerebral infarction and other central nervous injuries.

OBJECTIVE:To develop a method for simultaneous determination of irinotecan(CPT-11)and its active metabo-lite 7-ethyl-10-hydroxycamptothecin(SN-38)in human plasma.METHODS:After precipitated by acetonitrile and acidified with hy-drochloric acid,using camptothecin as internal standard,the plasma sample was determined by HPLC-FLD. The determination was performed on Waters Luna C18column with mobile phase consisted of 0.05 mol/L sodium dihydrogen phosphate-acetonitrile(70:30, V/V,adjusted pH to 4.0 by phosphoric acid)at flow rate of 1 mL/min. The excitation wavelength was set at 380 nm;the emis-sion wavelengths of CPT-11 and SN-38 were set at 480 nm and 535 nm,respectively. The column temperature was 25 ℃ and the sample size was 20 μL. RESULTS:The linear ranges were 200-1 000 ng/mL for CPT-11(r=0.999 4,n=5)and 5-45 ng/mL for SN-38(r=0.999 2,n=5). RSDs of inter-day and intra-day were 1.68%-5.57%. The relative recoveries of CPT-11 and SN-38 were 90.12%-106.93%(RSD<8%,n=5)and 92.07%-102.56%(RSD<6%,n=5);the extraction recoveries of CPT-11 and SN-38 were 72.23%-86.56%(RSD<6%,n=5)and 71.98%-83.44%(RSD<7%,n=5),respectively. The plasma concentra-tions of CPT-11 and SN-38 in 5 patients with colon cancer were 431.13-617.19,13.97-31.89 ng/mL(1 h after intravenous drip-ping)and 398.14-584.43,11.61-29.94 ng/mL(2 h after intravenous dripping). CONCLUSIONS:The method is simple,rapid, sensitive,reproducible and suitable for the determination of plasma concentration and pharmacokinetic study of CPT-11 and its metabolite SN-38.