Objective To investigate the clinical meanings of lymphangiogenesis, lymph vessel invasion (LVI) and lymph node (LN) micrometastasis in gastric cancer. Methods The expression of D2-40 in 68 patients with gastric cancer of primary lesion and the expressions of CK20 and (or) CKpan in 791 lymph nodes from 51 cases which were detected by immunohistochemical staining were analyzed, as well as their clinicopathologic profiles. The relationship of lymph vessel density (LVD), LVI and LN micrometastasis with LN metastasis and other clinicopathologic parameters was analyzed respectively. Results Positive rate of LVI with HE (LVI-HE) and D2-40 (LVI-IM) staining was respectively 66.2%(45/68) and 76.5%(52/68), P=0.118. The positive rate of LVI-IM was related to deeper tumor invasion (P=0.044), later stage of TNM (P=0.003) and LN metastasis (P=0.000). Average LVD of 68 cases was (18.19±7.44)/HP. The increment of LVD was significantly associated with LVI-HE positive status （P=0.040）, LVI-IM positive status （P=0.001）, venous invasion （P=0.037）, later stage of TNM （P=0.020） and LN metastasis （P=0.001）. The survival rate of the group sharing ≥15/HP of LVD was significantly lower than that in the group sharing ≤14/HP of LVD in early period of follow-up （P=0.032）. The incidence of nodal involvement in 51 patients was increased from 74.5%(38/51) by HE staining to 88.2%(45/51) by CK (CK20 or CKpan) immunostaining. The detection rate of metastasized LN was increased from 32.0%（253/791） by HE staining to 41.5%（328/791） by CK immunostaining (Plt;0.001). The significant difference of LN micrometastasis detection rate between CK20 (8.7%) and CKpan (12.3%) was also identified (P=0.003). The increased number of LN micrometastasis was related to larger diameter of tumor (P=0.001), higher LVI-HE positive rate (P=0.040), deeper invasion of tumor (P=0.018) and later stage of TNM (P=0.012). Both LN stage and TNM stage were changed according to the detection of LN micrometastasis: Seven patients of N0 should be recognized as N1 (N0→N1)， 6 as N1→N2, 1 as N2→N3. Four patients of stage Ⅰb should be recognized as stage Ⅱ (Ⅰb→Ⅱ)， 4 as Ⅱ→Ⅲa， 3 as Ⅲa→Ⅲb， 1 as Ⅲb→Ⅳ. Conclusion Detection of D2-40 and CK in diagnosis of LVI and LN micrometastasis is better than HE staining. The combined detection of CK20 and CKpan may be much easier to find out the LN with micrometastasis. Later stage of TNM the tumor is, more frequently the LN micrometastasis happens. The relationships of LVI-IM, LVD and LN micrometastasis with LN metastasis in gastric cancer has been demonstrated. Patients with higher LVD share a lower survival rate in early period of follow-up.
摘要:目的: 探讨选择性内皮素A受体拮抗剂BQ123对人喉癌Hep2细胞裸鼠种植瘤的生长及血管形成的影响。 方法 :将实验动物裸鼠随机分为3组:BQ123［n =8,2mg/(kg·day)］、氟尿嘧啶组［n =8,2mg/(kg·day)］、生理盐水组(n =8),比较各组裸鼠成瘤体积、微血管密度(MVD)。 结果 :BQ123组肿瘤体积为(162±053)cm3,明显小于生理盐水组及氟尿嘧啶组,差异具有统计学意义;BQ123组的肿瘤组织中MVD高倍镜下为232,明显低于生理盐水组(586)及氟尿嘧啶组(395),差异具有统计学意义。 结论 :BQ123对人喉癌Hep2细胞在裸鼠体内有明显抑瘤作用,肿瘤的体积、肿瘤组织MVD显著低于对照组,表明BQ123可通过抑制肿瘤血管生成而显著抑制肿瘤生长。Abstract: Objective: To study the effects of endothelin A receptor blockade BQ123 on the implanted human laryngeal carcinoma angiogenesis of nude mouse. Methods : From March 2008 to July 2009, 24 Balb/c nude mice were randomly divided into three groups: BQ123 group ［〖WTBX〗n =8, BQ123 at 2mg/(kg·day)］, 5Fu group ［〖WTBX〗n =8, fluorouracil at 2mg/(kg·day)］ and the control group (〖WTBX〗n =8, normal saline). The carcinoma volume and microvascular density of each group were compared. Results : The tumor size of BQ123 group, which was (162±053)cm3 in average, was significant smaller than the tumor sizes of the other two group s. The average microvascular density score of the tumors in BQ123 group was 232 per hyper power len (HP), which was also significantly less than the average scores of control groups (586 and 395 respectively). Conclusion : Nude mouse experiments show that the carcinoma volume and microvascular density of BQ123 group are significantly lower than those of the control groups. BQ123 inhibits the growth of carcinoma by its inhibition of carcinoma angiogenesis.
ObjectiveTo find the role of oncogene cmet and suppressor gene p53 in the process of tumor angiogenesis and their clinical significance. MethodsBy immunohistochemical method and computer image analysis technique, microvessel count and cmet, p53 protein expression were quantitatively determined in 80 cases of breast carcinoma and 20 cases of breast fibroadenoma. ResultsThe high microvessel count and the positive expression of cmet, p53 were significantly correlated with histologic grade, lymph node metastasis and the stage of the tumor (P＜0.01). The high microvessel count was significantly correlated with the positive expression of cmet and p53 (P＜0.01).ConclusionBoth oncogene cmet and suppressor gene p53 modulate tumor angiogenesis of breast carcinoma.
【Abstract】Objective To understand the features of lymphatic vessel, and to summarize the foundation and mechanism of the promotion and inhibition of tumor lymphangiogenesis recorded on the current studies of animal experiments and clinical researches. Methods The related literatures of the structural features of lymphatic vessel, lymphatic endothelial molecular markers, the origin of lymphatic tumors, the molecular mechanisms and regulatory factors were reviewed, and the relationship between tumor lymphangiogenesis and lymphatic metastasis, the treatment targeting at the formation of the anti-tumor lymphatic vessel and its existing problems were also analyzed. Results Hyperplasia of lymphatic vessels occurred during the process of tumor formation and progression. The structural features of the lymphatic vessels in the tumor were conducive to tumor lymphatic metastasis. In recent years, methods of anti-lymphangiogenesis and inhibition of tumor lymphatic metastasis had achieved considerable success in animal experiments. However, there were still a lot of problems need to be solved. Conclusion Tumor lymphangiogenesis has a significantly positive correlation between tumor lymphatic metastasis and patients’ prognosis, which may indicate that treatment against the formation of tumor lymphatic vessel maybe effective.
Objective To investigate the effect of M2-like macrophage/microglia-derived mitochondria transplantation in treatment of mouse spinal cord injury (SCI). Methods BV2 cells were classified into M1 (LPS treatment), M2 (IL-4 treatment), and M0 (no treatment) groups. After receiving M1 and M2 polarization, BV2 cells received microscopic observation, immunofluorescence staining [Arginase-1 (Arg-1)] and flow cytometry [inducible nitric oxide synthase (iNOS), Arg-1] to determine the result of polarization. MitoSox Red and 2, 7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) stainings were used to evaluate mitochondrial function difference. Mitochondria was isolated from M2-like BV2 cells through differential velocity centrifugation for following transplantation. Then Western blot was used to measure the expression levels of the relevant complexes (complexes Ⅱ, Ⅲ, Ⅳ, and Ⅴ) in the oxidative phosphorylation (OXPHOS), and compared with M2-like BV2 cells to evaluate whether the mitochondria were obtained. Thirty-six female C57BL/6 mice were randomly divided into 3 groups (n=12). Mice from sham group were only received the T10 laminectomy. After the T10 spinal cord injury (SCI) model was prepared in the SCI group and mitochondria transplantation (MT) group, mitochondrial storage solution and mitochondria (100 μg) derived from M2-like BV2 cells were injected into the injured segment, respectively. After operation, the Basso Mouse Scale (BMS) score was performed to evaluate the motor function recovery. And immunofluorescence staining, lycopersicon esculentum agglutinin (LEA)-FITC staining, and ELISA [vascular endothelial growth factor A (VEGFA)] were also performed. Results After polarization induction, BV2 cells in M1 and M2 groups showed specific morphological changes of M1-like and M2-like macrophages, respectively. Immunofluorescence staining showed that the positive expression of M2-like macrophages marker (Arg-1) was significantly higher in M2 group than in M0 group and M1 group (P<0.05). Flow cytometry showed that the expression of M1-like macrophage marker (iNOS) was significantly higher in M1 group than in M0 group and M2 group (P<0.05), and the expression of Arg-1 was significantly higher in M2 group than in M0 group and M1 group (P<0.05). MitoSox Red and DCFH-DA stainings showed that the fluorescence intensity of the M2 group was significantly lower than that of the M1 group (P<0.05), and there was no significant difference with the M0 group (P>0.05). The M2-like BV2 cells-derived mitochondria was identified through Western blot assay. Animal experiments showed that the BMS scores of MT group at 21 and 28 days after operation were significantly higher than those of SCI group (P<0.05). At 14 days after operation, the number of iNOS-positive cells in MT group was significantly lower than that in SCI group (P<0.05), but still higher than that in sham group (P<0.05); the number of LEA-positive cells and the expression of VEGFA in MT group were significantly more than those in the other two groups (P<0.05). Conclusion M2-like macrophage/microglia-derived mitochondria transplantation can promote angiogenesis and inhibit inflammatory M1-like macrophage/microglia polarization after mouse SCI to improve function recovery.
Objective To investigate the effects of the MKN-45 gastric cancer cell exosomes carrying microRNA-552 (miR-552) on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVEC). Methods ① The MKN-45 cells were divided into MKN-45 blank control group (no transfection), MKN-45 miR-552 inhibitor group [transfection of plasmid inhibiting mir-552 expression (mir-552 inhibitor plasmid)], and MKN-45 negative control group [transfection of negative control plasmid (empty plasmid)], the exosomes were extracted, purified, and identified. Western blotting was used to detect the protein expression of exosomal markers [CD63, CD9, and tumor susceptibility gene 101 (TSG101)]. ② The HUVEC cells were divided into HUVEC control group (added PBS), HUVEC-exosome group (co-cultured with exosomes of MKN-45 cell), HUVEC-negative control exosome group (co-cultured with exosomes of MKN-45 cell transfected with negative control plasmid), and HUVEC-miR-552 inhibitor exosome group (co-cultured with exosomes of MKN-45 cell transfected with miR-552 inhibitor plasmid), exosomes tracing experiment was used to detect whether exosomes entered HUVEC cells. Real-time fluorescent quantitative PCR method was used to detect the expression of miR-552, the MTT method was used to detect the proliferation of HUVEC cells, the Transwell chamber method was used to detect the migration of HUVEC cells, the angiogenesis test was used to detect the angiogenesis ability. Results This study successfully extracted exosomes from MKN-45 gastric cancer cells. Observed by transmission electron microscope, the exosomes were all round or elliptical, with a diameter of 100–150 nm, and the exosomal vesicle structure could be seen. Western blotting detection showed that the surface markers of exosomes (CD63, CD9, and TSG101 protein) were expressed in exosomes. The results of the tracing experiment showed that exosomes derived from MKN-45 cells were successfully internalized by HUVEC cells. After MKN-45 cells were transfected with miR-552 inhibitor plasmid, compared with the MKN-45 blank control group and MKN-45 negative control group, the relative expression level of miR-552 in the exosomes decreased (P<0.05). Compared with the HUVEC control group, the cell proliferation rate at 24, 48 and 74 h increased, as well as number of migration, tubule formation nodes, and relative expression level of miR-552 in the HUVEC-exosomes group increased (P<0.05). Compared with the HUVEC-negative control exosome group, the cell proliferation rate at 24, 48 and 74 h decreased, as well as the number of migration, tubule formation nodes, and relative expression level of miR-552 in the HUVEC-miR-552 inhibitor exosome group decreased (P<0.05). Conclusion The exosomes of gastric cancer cells carrying miR-552 can significantly promote the proliferation, migration, and angiogenesis of HUVEC cells.
Objective To investigate whether the growth of the human experimental hepatocellular carcinoma (HCC) can be suppressed by the antibody against vascular endothelial growth factor (VEGF). MethodsThe monoclonal antiVEGF antibody was injected to nude mice nearby the xenograft tumour foculs of the human SMMC7721 HCC. The changes of the tumour size were measured at different times. The intratumoural microvessels were showed by immunohistochemical staining of CD31 antigen; the apoptotic cells in the tumour tissues were detected in situ by terminal deoxynucleotidyl transferasemediated dUTPbiotin nick end labeling (TUNEL) assay. ResultsIn the first and second week after finishing the injection procedure, the tumour sizes were compared as the length (mm) multiplying width (mm) between the two groups, the tumour sizes as the test group vs the control group were (26.46±19.81) mm2 vs (105.77±17.40) mm2 (P＜0.001) and (45.20±23.02) mm2 vs (150.77±77.41) mm2 (P＜0.05), respectively. After 2 weeks the intratumoural microvessel density (iMVD) and the apoptotic index (AI) were compared between two groups with iMVD being (2 311±120)/mm2 vs (3 900±328)/mm2(P＜0.001 ) and AI being (15.31% vs 6.83%), P＜0.005. Conclusion The antiVEGF antibody can suppress the xenograft tumour growth of the human SMMC7721 HCC by antiangiogenesis.In fact, its antitumour effect is produced by elevating the incidence of apoptosis in tumour tissues.
Objective To study the expression of thymidine phosporylase (TP) and the counts of lymph vessels in pancreatic cancer and chronic pancreatitis tissues, and to explore their clinicopathologic significances and correlation in the course of pancreatic cancer. Methods SP immunohistochemical method was used to detetct the expression of TP and the locations of lymph vessels on the routinely paraffin-embedded sections of the specimens from 51 cases pancreatic cancer and 10 cases of chronic pancreatitis. Results The positive rate of TP and the counts of lymph vessels were significantly higher (P＜0.05 and P＜0.01 respectively) in pancreatic cancer 〔54.9%, (12.5±4.3)/HP〕 than those in chronic pancreatitis 〔20.0%，(5.2±2.4)/HP〕. The positive rate of TP and the counts of lymph vessels were significantly lower （P＜0.05, P＜0.01） in well-differentiated adenocarcinoma cases and cases without metastasis compared with poor-differentiated adenocarcinoma cases and cases with metastasis. The counts of lymph vessels were significantly higher in the positive cases of TP than those in the negative ones in pancreatic cancer 〔(13.8±3.4)/HP vs (10.9±3.2)/HP〕, P＜0.01.Conclusion The expression of TP and counts of lymph vessels might be important markers reflecting the progression, biological behaviors, metastatic status and prognosis of pancreatic cancer. TP might promote lympoangiogenesis in pancreatic cancer tissues.
Objective To develop an in vitro three-dimensional angiogenesis system and analyze the expression and function of CD105 in angiogenesis. Methods After primary human umbilical vein endothelial cells (HUVEC) were purified and cultured, the microcarriers were coated with HUVEC and then embedded and cultured into fibrin gel. The angiogenesis process of HUVEC on the microcarriers was formed. The expression of CD105 during this process was detected by reverse transcription polymerase chain reaction (RT-PCR). Antisense oligodeoxynucleotide (ASODN) was used to inhibit the expression of CD105 and the changes of the angiogenesis process were analyzed quantitatively. Results HUVEC on the microcarriers which were embedded into the fibrin gel, occurred the angiogenesis process of sprouts, branches and capillary networks with lumina. During this process, CD105 was over expressed in the periods of forming sprouts and branches, and depressed in the relatively steady periods including the periods before forming sprouts and after forming capillary networks. While the expression of CD105 was inhibited by ASODN, the angiogenesis process was significantly inhibited. Conclusions The expression of CD105 is varied within the angiogenesis process, over expressing during the sprouts and branches forming periods. Inhibiting the expression of CD105 could efficiently inhibit angiogenesis.
This study aims to investigate the effect of substances secreted or metabolized by vascular endothelial cells on epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma cells under indirect co-culture condition. Human hepatocellular carcinoma cell line QGY-7703 was cultured in vitro, and then was co-cultured with conditioned medium of human umbilical vein endothelial cells (HUVEC). The morphological changes of QGY-7703 cells were observed by inverted phase contrast microscopy. The migration ability of QGY-7703 cells was analyzed by scratch-wound assays. The effect of conditioned medium on the expression and distribution of EMT related proteins was detected by Western blot and immunofluorescence assays, respectively. The results showed that the QGY-7703 cells gradually changed from polygonal to spindle shape, the migration ability promoted significantly, and both the expression and distribution of EMT related marker changed in a time-dependent manner after co-culturing. The results confirm that vascular endothelial cells can induce EMT in hepatocellular carcinoma cells under indirect co-culture condition.