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The Influence of Cationic LiposomeMediated Endostatin Gene on Colorectal Carcinoma Liver Metastasis

Objective To investigate the influence of cationic liposomemediated endostatin gene on colorectal cancer liver metastasis. Methods Animal model for colorectal carcinoma liver metastasis were established. The plasmid expressing endostatin genelipofectAMINE were injected in vein. Results After cationic liposomemediated endostatin gene were injected in vein, the incidence of liver metastasis and mean numbers of liver tumors were decreased, survival time of animal was significantly longer. Conclusion Intravenous injection of cationic liposomemediated endostatin gene can control the development of colorectal cancer liver metastasis effectively.

Mouse endostatin gene transfected lung cancer cells inhibit proliferation of endothelial cells in vitro

Objective To observe the expression of adenovirus vector coding for mouse endostatin gene（Ad-mES） in lung cancer cells and its antiangiogenic activity in human umbilical vein endothelial cells（ECV304） in vitro.Methods Lewis lung cancer（LLC） cells were transfected with Ad-mES at different multiplicity of infection（MOI）.The expression of mES in LLC cells and supernatant after 48 hours was detected by immunohistochemical staining and Western blot respectively.The inhibitory effect of supernatant at different MOI on ECV304 non-stamulated and stimulated by basic fibroblast growth factor（bFGF） was measured by methyl thiazolyl tetrazolium（MTT） assay.Results After transfected for 48 hours，endostatin was identified in the cell plasma of infected LLC and negative result was founded in non-infected LLC.Western blot revealed band of endostatin in 20 kDa in culture supernatant of infected LLC and negative results in non-infected LLC.The inhibitory effects on ECV304 cell proliferation were ber at higher MOI，and the difference was significant between stimulated and non-stamulated cells by bFGF（Plt;0.05）.Conclusion Ad-mES can transfect and express endostatin effectively in LLC with biological activity

Serum Levels of Endostatin and Vascular Endothelial Growth Factor in Different Therapy Stages of Mouse Lewis Lung Carcinoma

Objective To investigate the serum levels of endostatin and vascular endothelial growth factor ( VEGF) at different therapy stages of mouse Lewis lung carcinoma, and elucidate the relation to the progress and prognosis of tumor. Methods Forty-four Lewis lung carcinoma-bearing C57BL/6 mice were randomly divided into 4 groups, ie. a non-therapy group, a chemotherapy group, a gene therapy group, and a combination therapy group ( chemotherapy plus gene therapy) . Eleven healthy mice were included as normal control group. Serum was collected on the 0th, 5th, 19th day after therapy for measurement of endostatin and VEGF by ELISA. The correlations were analysed between endostatin and VEGF levels in each group. Results ( 1) The serum endostatin levels had no significant difference in all groups on the 0th day,but increased significantly on the 5th day in the gene and combination therapy groups than those in other three groups ( all P lt;0. 01) . Then the endostatin level decreased on the 19th day in the gene and combination therapy groups, but still higher than those in the chemotherapy group and the normal group. ( 2 ) On the contrary, serum VEGF levels of the gene and combination therapy groups decreased significantly on the 5th day and increased little on the 19th day, which were both significant lower than those in chemotherapy group on the 5th and 19th day( all P lt; 0. 05) . There were significant diferences between the three therapy groups and the non-therapy group( all P lt;0. 05) . ( 3) Negative correlations between VEGF and endostatin levels were revealed in the gene and combination therapy groups ( r = - 0. 77 and - 0. 761 respectively) .Correlation was not found in the non-therapy and chemotherapy groups. Conclusion The serum levels of endostatin and VEGF might be used as monitoring indices of antiangiogenesis therapy.

血管形成因子在非小细胞肺癌中的表达及其临床意义

目的 探讨血管形成因子在非小细胞肺癌(NSCLC)中的表达及其在NSCLC发生、生长和转移中的作用。 方法 在光学显微镜下计数微血管密度(MVD)，用免疫组织化学链霉素抗生物素蛋白-过氧化酶(S-P)法对血管内皮细胞生长因子（VEGF）和内皮抑素进行染色观察,分析它们之间的相关关系。 结果 有淋巴结转移者MVD、VEGF和内皮抑素表达均较无淋巴结转移者高（Plt;0.05），肺癌组织MVD、VEGF和内皮抑素表达均较正常肺组织高（Plt;0.05）;但与肺癌患者的性别和年龄无明显的关系（Pgt;0.05）;T3+T4的原发肿瘤VEGF表达水平较T1+T2者高（Plt;0.05）。肺癌组织MVD、VEGF积分光密度和内皮抑素积分光密度在Ⅰ期、Ⅱ期和Ⅲ期间两两比较差异均有统计学意义(Plt;0.01)。不分期时MVD与VEGF呈正相关（r=0.680，P=0.023）,MVD与内皮抑素呈负相关（r=-0.700，P=0.015）。正常肺组织中VEGF与内皮抑素呈正相关（r=0.620，P=0.027）。 结论 新生血管的形成在NSCLC的发生、生长和转移过程中发挥着重要作用，其形成可能是由于VEGF和内皮抑素的平衡被破坏所致。

Microencapsulation of a genetically engineered cell line secreting human endostatin

　　Objective　To microencapsulate a genetically engineered cell line which stably secrete human endostatin (hES).Methods　Endostatin gene fragment was amplified from plasmid pcDNA3-Endo by polymerase chain reaction, and inserted into mammalian eukaryotic expression vector pEGFPN1, resulting into recombinant plasmid pEGFP-N1-ES.Hek-293 cells were transfected with pEGFP-N1-ES via cationic liposome and selected by G418, and were measured by Western blot for endostatin protein expression.The hES/293 cells were further entrapped by alginate-chitosan-alginate (ACA) microcapsules, and the expression of endostatin in the supernatant of cultured hES/293 cell microcapsules was examined by western blot at different time points.Results　Recombinant plasmid pEGFP-N1 endostatin was digested by HindIII and BamHI, and resulted into 2 DNA fragments of 7 kb and 600 bp. The sequence of the 600 bp fragment was identical to human endostatin. Western blot of the supernatant of cultured hES/293 cells or hES/293 cell microcapsules detected a positive band with the relative molecular mass of 20times;103.Conclusion　The hES protein was expressed in HeK-293 transfected with pEGFP-N1-endostatin, and secreted to the culture medium,and can freely diffused outside the micro-capsule.

Gene expression of ECV304 cells after treated with endostatin detected by microarray analysis

　　Objective　To observe the altered gene expression of ECV304 cell after treated with endostatin (ES) using cDNA microarray, and to investigate its molecular mechanism.Methods　BiostarH40S gene expression DNA microarrays were used to profile changes in gene expression of ECV304 cells after treated with ES for 36 hours. To prepare the probes, mRNA from both control and treated cells were isolated and purified, and then reversely transcribed to cDNA with the incorporation of fluorescentlabeled dUTP: Cy3 and Cy5 respectively. The probes were hybridized with expressed cDNA microarray, the fluorescent signals of Cy3 and Cy5 were scanned and analyzed by a computer system.Results　ES inhibited the proliferation of ECV304 cells. The result of electrophoresis indicated that the extracted total RNA had high quality.Approximately 5.96% of all human genes examined on 4096 gene microarrays showed altered expression, with 225 downregulated genes and 19 upregulated genes.Conclusion　The expression of some genes of ECV304 cells decrease after treated with ES, which may be related to the mechanism of inhibition of neovascularization.

应用转基因技术体外培养表达内皮抑素的Brown Norway大鼠视网膜色素上皮细胞

The Synergistic Anti-tumor Effect of Endostatin on a Tumor-progression Murine Lung Cancer Model

目的　建立重组人内皮抑素（恩度）联合顺铂一线治疗肿瘤进展的小鼠模型，继续应用内皮抑素联合紫杉醇二线治疗，研究内皮抑素协同紫杉醇抗肿瘤的作用及其机制。 方法　建立小鼠Lewis 肺癌移植瘤动物模型，内皮抑素联合顺铂治疗后观察肿瘤生长情况，遴选出肿瘤进展小鼠16只，随机留取1只，余15只小鼠随机分成紫杉醇组和联合用药组治疗，观察疗效。另取上述肿瘤进展小鼠1只，剥离肿瘤组织，重新接种，将成瘤小鼠随机分成生理盐水组，紫杉醇组及联合用药组治疗，观察疗效。治疗结束后24 h处死所有小鼠，采用免疫组织化学CD31单克隆抗体标记检测微血管密度（MVD），采用原位末端转移酶（TUNEL）检测细胞凋亡。 结果　只肿瘤进展小鼠中，联合用药组相比紫杉醇组生存时间无明显延长，但肿瘤体积增长较慢；而在重新接种成瘤的小鼠中，联合用药组较其余各组微血管密度明显减低（P＜0.05），凋亡指数明显增加（P＜0.05），肿瘤体积抑制明显。 结论　在内皮抑素联合顺铂治疗肿瘤进展的小鼠模型中，继续应用内皮抑素治疗与紫杉醇有较明显的协同抗肿瘤作用。

Study on Expressions and Significances of Endostatin, bFGF and CD34 in Gallbladder Cancer

ObjectiveTo study the effects of the expressions of endostatin, basic fibroblast growth factor (bFGF) and CD34 on oncogenesis and progression of gallbladder cancer, and to explore some valuable criterias for its biotherapy. Methods The expressions of endostatin, bFGF and CD34 were studied by means of immunohistochemistry (SP) in 61 cases of gallbladder cancer and 10 cases of normal cholecystic tissue, and microvessel density (MVD) was calculated by the expression of CD34. Their relationships with clinical pathological features were also investigated. Results The expression rates of endostatin in normal cholecystic tissue and in gallbladder cancer tissue were 40.00% (4/10) and 77.05% (47/61) respectively, which had statistical difference (P＜0.05). The expression of endostatin in 61 cases of caner was relational to clinical stage and metastasis of lymph nodes (P＜0.05), while no significant correlation was detected with sex and age of patient, location of tumor, size of tumor and histologic grade (P＞0.05). The expression rates of bFGF in normal cholecystic tissue and in gallbladder cancer tissue were 20.00%（2/10） and 67.21% （41/61） respectively, which had statistical difference (P＜0.05). The expression of bFGF in 61 cases of caner was relational to clinical stage and metastasis of lymph nodes (P＜0.05), while no significant correlation was detected with sex and age of patient, location of tumor, size of tumor and histologic grade (P＞0.05). MVD in gallbladder cancer tissue and in normal cholecystic tissue was （76.66±20.15） piece/HP and (29.53±5.03) piece/HP respectively, showing significant difference (P＜0.01). In 61 cases of cancer, MVD in clinical stage Ⅲ～Ⅴ 〔(80.53±17.98) piece/HP〕 was much higher than that in stage Ⅰ+Ⅱ 〔(46.79±5.38) piece/HP〕, P＜0.01; MVD was higher in those with lymph nodes metastasis 〔(94.60±7.28) piece/HP〕 than those without metastasis 〔(58.12±9.24) piece/HP〕, P＜0.01; and MVD was (60.59±14.71) piece/HP in histologic grade G1， (83.08±15.30) piece/HP in G2， and (96.53±6.92) piece/HP in G3, the difference was significant among them （P＜0.01）. There was no significant correlation between MVD and sex and age of patient, location of tumor and size of tumor (P＞0.05). There were statistically significant correlations between expressions of endostatin and MVD （P＜0.01）, expressions of bFGF and MVD （P＜0.01）. Conclusions The result suggests that endostatin, bFGF and CD34 play roles in oncogenesis and progression of gallbladder cancer. Detection of these proteins has positive effects on diagnosis, malignant degree determination and treatment of gallbladder cancer.

Construction of Human Endostatin in Yeast Eukaryotic Expression Vector

Objective　To construct yeast eukaryotic expression vector carrying human endostatin (ES) cDNA.　 Methods　The functional fragment of endostatin gene in human hepatic tissue was amplified by using RT-PCR technology, and cloned into yeast pPIC9 expression vector. The positive clone was sequenced by using automatized sequencer. Results　The endostatin cDNA was successfully cloned. The positive ES clone gene in pPIC9 expression vector was sieved, and its coding sequence was identified to be as same as the previously reported sequence. Conclusion　The successful construction of ES gene in pPIC9 expression vector using molecular biological method maybe helpful for the high expression of ES protein, which may lay the foundation for the treatment of malignant tumor through anti-angiogenesis appoach.