La核糖核蛋白6对人血管内皮细胞增殖与凋亡的作用

Objective To investigate regulatory effect of Acheron (Achn) on proliferation and apoptosis of human vascular endothelial cell. Methods ( 1 ) Eahy926 cells were cultured in serum-free DMEM medium (96-well plates) and were divided into Achn inhibition group (transfected with plasmid psi-Achn), psi4.1 group (transfected with psi4. 1 empty vector), Achn induction group (transfected with pcDNA-Achn), pcDNA3.1 group (transfected with pcDNA3.1 empty vector), cotransfection group [cotransfected with pcDNA-Achn + psi-calcium/calmodulin-dependent serine protein kinase (CASK)] , blank control group (treated with PBS) according to the random number table (the same method below). The cell proliferation was determined by MTT assay at post transfection hour (PTH) 1, 24, 48, 72, with expression of absorbance value. (2) Total protein of Eahy926 cells were extracted and quantitated by BCA assay, and then they were divided into Achn antibody precipitation group (100 μg protein) , CASK antibody precipitation group ( 100 μg protein), IgG antibody group ( 100 μg protein), Western blot group (20 μg protein).Achn and CASK protein levels were determined by immunoprecipitation and Western blot. (3) Synchronously cultured Eahy926 cells were divided into LPS induction group (treated with 5 mol/L LPS), Achn transfection group (transfected with pcDNA-Achn), cotransfection group (cotransfected with psi-CASK and pcDNA-Achn) , KCl group (treated with 5 mol/L KCl), and blank control group (treated with 5 mol/LPBS). Cells in transfection groups were stimulated by LPS for 12 hours after PTH 24. Caspase-3 protein level was detected by immunohistochemistry. (4) Synchronously cultured Eahy926 cells were divided into Achn inhibition group (transfected with psi-Achn vector), Achn induction group ( transfected with pcDNA-Achn vector), and blank control group ( treated with PBS). Apoptosis rate was determined by FITC/PI with flow cytometry. Data were processed with one-way analysis of variance and t test. Results ( 1 ) The cell proliferation in Achn inhibition group was lower than that in psi4.1 group from PTH 24, and the differences were statistically significant at PTH 48, 72 (with t value respectively 10. 777, 6.112, P values all below 0. 05 ).The cell proliferation in Achn induction group during PTH 24-72 were higher that in pcDNA3. 1 group (with t value respectively 5. 367, 6. 053, 9. 831, P values all below 0.05 ). The cell proliferation in cotransfection group at PTH 48, 72 were significantly lower than that in Achn induction group ( with t value respectively 5.481, 9. 517, P values all below 0. 05). (2) Achn protein was detected in CASK antibody precipitation group while CASK protein was also detected in Achn antibody precipitation group. (3) Caspase-3 level in Achn transfection group was lower [( 15.6 ± 0. 5 ) %] as compared with that in LPS induction group [(32. 8 ±2.6)%, t = 10. 083, P ＜ 0. 05], and that in cotransfection group showed further inhibition [(7.0 ±2.0)%,t =9.827, P ＜0.01]. (4) Apoptosis rate in Achn inhibition group[(45.6 ± 10.9)%] was higher than that in blank control group [(13.2±4.3) %, t =7.043, P ＜0.05]; while that in Achn inductiongroup [(5.3 ±2.9)%] was lower than that in blank control group ( t =6.499, P ＜0.05).Conclusions Achn can promote human vascular endothelial cell proliferation, and inhibit its apoptosis induced by LPS or burn serum, and the effect is related to CASK.     

La核糖核蛋白6对人血管内皮细胞增殖与凋亡的作用

Objective To investigate regulatory effect of Acheron (Achn) on proliferation and apoptosis of human vascular endothelial cell. Methods ( 1 ) Eahy926 cells were cultured in serum-free DMEM medium (96-well plates) and were divided into Achn inhibition group (transfected with plasmid psi-Achn), psi4.1 group (transfected with psi4. 1 empty vector), Achn induction group (transfected with pcDNA-Achn), pcDNA3.1 group (transfected with pcDNA3.1 empty vector), cotransfection group [cotransfected with pcDNA-Achn + psi-calcium/calmodulin-dependent serine protein kinase (CASK)] , blank control group (treated with PBS) according to the random number table (the same method below). The cell proliferation was determined by MTT assay at post transfection hour (PTH) 1, 24, 48, 72, with expression of absorbance value. (2) Total protein of Eahy926 cells were extracted and quantitated by BCA assay, and then they were divided into Achn antibody precipitation group (100 μg protein) , CASK antibody precipitation group ( 100 μg protein), IgG antibody group ( 100 μg protein), Western blot group (20 μg protein).Achn and CASK protein levels were determined by immunoprecipitation and Western blot. (3) Synchronously cultured Eahy926 cells were divided into LPS induction group (treated with 5 mol/L LPS), Achn transfection group (transfected with pcDNA-Achn), cotransfection group (cotransfected with psi-CASK and pcDNA-Achn) , KCl group (treated with 5 mol/L KCl), and blank control group (treated with 5 mol/LPBS). Cells in transfection groups were stimulated by LPS for 12 hours after PTH 24. Caspase-3 protein level was detected by immunohistochemistry. (4) Synchronously cultured Eahy926 cells were divided into Achn inhibition group (transfected with psi-Achn vector), Achn induction group ( transfected with pcDNA-Achn vector), and blank control group ( treated with PBS). Apoptosis rate was determined by FITC/PI with flow cytometry. Data were processed with one-way analysis of variance and t test. Results ( 1 ) The cell proliferation in Achn inhibition group was lower than that in psi4.1 group from PTH 24, and the differences were statistically significant at PTH 48, 72 (with t value respectively 10. 777, 6.112, P values all below 0. 05 ).The cell proliferation in Achn induction group during PTH 24-72 were higher that in pcDNA3. 1 group (with t value respectively 5. 367, 6. 053, 9. 831, P values all below 0.05 ). The cell proliferation in cotransfection group at PTH 48, 72 were significantly lower than that in Achn induction group ( with t value respectively 5.481, 9. 517, P values all below 0. 05). (2) Achn protein was detected in CASK antibody precipitation group while CASK protein was also detected in Achn antibody precipitation group. (3) Caspase-3 level in Achn transfection group was lower [( 15.6 ± 0. 5 ) %] as compared with that in LPS induction group [(32. 8 ±2.6)%, t = 10. 083, P ＜ 0. 05], and that in cotransfection group showed further inhibition [(7.0 ±2.0)%,t =9.827, P ＜0.01]. (4) Apoptosis rate in Achn inhibition group[(45.6 ± 10.9)%] was higher than that in blank control group [(13.2±4.3) %, t =7.043, P ＜0.05]; while that in Achn inductiongroup [(5.3 ±2.9)%] was lower than that in blank control group ( t =6.499, P ＜0.05).Conclusions Achn can promote human vascular endothelial cell proliferation, and inhibit its apoptosis induced by LPS or burn serum, and the effect is related to CASK.     

顺铂联合加热对体外血液中肝肿瘤细胞的杀灭作用及对红细胞的影响

Objective To investigate the effectiveness of cis-diamminedichloroplatinum (DDP) combined with hyperthennia in killing liver tumor cells and its influence on erythrocytes in vitro. Methods Cultured liver tumor cells (2 ml) were mixed with erythrocyte suspension (10 ml) and then the mixture was separated into 6 centrifuge tubes with 2 ml in each one. The centrifuge tubes were randomly divided into A-F groups and the experiment was repeated for 30 times. Normal saline 2 ml was added in A and D groups. DDP 2 ml (200 μg/ml) was added in B and E groups. DDP 2 ml (400 μg/ml) was added in C and F groups. The cells were then incubated in warm bath of 37 ℃ for 30 min in A, B and C groups and in warm bath of 42 ℃ for 30 min in the other three groups.After hyperthermic treatment, tumor cells were isolated from erythrocytes using density gradient centrifugation, the inhibition rate of tumor cells was determined by MTT method and the clone formation of tumor cells was checked.The erythrocyte osmotic fragility and content of 2,3-diphosphoglyceric acid in erythrocytes were measured. Results The inhibition rate of tumor cells was gradually increased, while the rate of tumor cell clone formation decreased with the increase in the temperature and DDP concentrations ( P < 0.01) . The rate of tumor cell clone formation was more than 98% and no clone formation was tested in group F. There was no significant difference in the content of 2,3-diphosphoglyceric acid in erythrocytes between before and after hyperthermic treatment in group F ( P >0.05 ) . The rate of hemolysis of erythrocytes was less than 1 % in the 0.68 % sodium chloride solution in group F.Conclusion DDP 200 μg/ml combined with hyperthermic treatment with temperature of 42 ℃ for 30 min can make the liver tumor cells lose the capability of proliferation, however, it exerts slight effect on erythrocyte membrane and no influence on the oxygen-carrying capacity of erythrocytes.     

顺铂联合加热对体外血液中肝肿瘤细胞的杀灭作用及对红细胞的影响

Objective To investigate the effectiveness of cis-diamminedichloroplatinum (DDP) combined with hyperthennia in killing liver tumor cells and its influence on erythrocytes in vitro. Methods Cultured liver tumor cells (2 ml) were mixed with erythrocyte suspension (10 ml) and then the mixture was separated into 6 centrifuge tubes with 2 ml in each one. The centrifuge tubes were randomly divided into A-F groups and the experiment was repeated for 30 times. Normal saline 2 ml was added in A and D groups. DDP 2 ml (200 μg/ml) was added in B and E groups. DDP 2 ml (400 μg/ml) was added in C and F groups. The cells were then incubated in warm bath of 37 ℃ for 30 min in A, B and C groups and in warm bath of 42 ℃ for 30 min in the other three groups.After hyperthermic treatment, tumor cells were isolated from erythrocytes using density gradient centrifugation, the inhibition rate of tumor cells was determined by MTT method and the clone formation of tumor cells was checked.The erythrocyte osmotic fragility and content of 2,3-diphosphoglyceric acid in erythrocytes were measured. Results The inhibition rate of tumor cells was gradually increased, while the rate of tumor cell clone formation decreased with the increase in the temperature and DDP concentrations ( P < 0.01) . The rate of tumor cell clone formation was more than 98% and no clone formation was tested in group F. There was no significant difference in the content of 2,3-diphosphoglyceric acid in erythrocytes between before and after hyperthermic treatment in group F ( P >0.05 ) . The rate of hemolysis of erythrocytes was less than 1 % in the 0.68 % sodium chloride solution in group F.Conclusion DDP 200 μg/ml combined with hyperthermic treatment with temperature of 42 ℃ for 30 min can make the liver tumor cells lose the capability of proliferation, however, it exerts slight effect on erythrocyte membrane and no influence on the oxygen-carrying capacity of erythrocytes.     

乳腺癌小鼠动物模型中白细胞介素-17的表达及其意义

Objective To explore the impact of interleukin (IL) -17 expression on tumor growth in experimental models of murine mammary carcinoma and potential mechanisms. Methods Two murine cell lines, MA782/5S28102 and 4T1 were used to establish experimental models of murine mammary carcinoma. The IL-17 expression in tumor tissues derived from MA782-bearing mice or 4T1-bearing mice was detected in early and late stages of the tumor by Western blotting. The tumor cells and tumor-infiltrated-lymphocytes were separated from tumor tissues and cultured for 5 days with stimulation of PMA, anti-CD3 antibody and anti-CD28 antibody. The supernatants of culture media of stimulated tumor cells or tumor-infiltrated-lymphocytes were harvested and tested for IL-17 concentration by enzyme linked immunosorbent assay (ELISA). To evaluate the effect of IL-17 on the proliferation of tumor cells, 4T1 cells were culture in media with or without IL-17 and the cell number was counted on the day 5. For ire vivo assay, 4T1-bearing mice were injected with IL-17 or culture media via tail vein, and the tumor volume was measured. To assay the angiogenesis, the tumor tissues from 4T1-bearing mice with or without injection of IL-17 were stained with anti-CD31 antibody by immunohistochemistry. Results The IL-17 expression was significantly higher in late stage than in early stage of tumor in two experimental models. The tumor expression of IL-17 was secreted by tumor infiltrated lymphocytes (P ＜0.01). IL-17 could not increase the generation of tumor cells in vitro (1. 11 ±0. 11, 1. 28 ±0. 21 ,P ＞0. 05). But IL-17, injected into 4T1 -bearing mice, markedly enhanced in vivo tumor growth and significantly increased tumor vascularity (35. 79 ±9. 49, 13. 52 ±3. 55,P ＜0.01). Conclusion IL-17 in tumor tissue probably promotes tumor growth through enhancing angiogenesis.     

乳腺癌小鼠动物模型中白细胞介素-17的表达及其意义

Objective To explore the impact of interleukin (IL) -17 expression on tumor growth in experimental models of murine mammary carcinoma and potential mechanisms. Methods Two murine cell lines, MA782/5S28102 and 4T1 were used to establish experimental models of murine mammary carcinoma. The IL-17 expression in tumor tissues derived from MA782-bearing mice or 4T1-bearing mice was detected in early and late stages of the tumor by Western blotting. The tumor cells and tumor-infiltrated-lymphocytes were separated from tumor tissues and cultured for 5 days with stimulation of PMA, anti-CD3 antibody and anti-CD28 antibody. The supernatants of culture media of stimulated tumor cells or tumor-infiltrated-lymphocytes were harvested and tested for IL-17 concentration by enzyme linked immunosorbent assay (ELISA). To evaluate the effect of IL-17 on the proliferation of tumor cells, 4T1 cells were culture in media with or without IL-17 and the cell number was counted on the day 5. For ire vivo assay, 4T1-bearing mice were injected with IL-17 or culture media via tail vein, and the tumor volume was measured. To assay the angiogenesis, the tumor tissues from 4T1-bearing mice with or without injection of IL-17 were stained with anti-CD31 antibody by immunohistochemistry. Results The IL-17 expression was significantly higher in late stage than in early stage of tumor in two experimental models. The tumor expression of IL-17 was secreted by tumor infiltrated lymphocytes (P ＜0.01). IL-17 could not increase the generation of tumor cells in vitro (1. 11 ±0. 11, 1. 28 ±0. 21 ,P ＞0. 05). But IL-17, injected into 4T1 -bearing mice, markedly enhanced in vivo tumor growth and significantly increased tumor vascularity (35. 79 ±9. 49, 13. 52 ±3. 55,P ＜0.01). Conclusion IL-17 in tumor tissue probably promotes tumor growth through enhancing angiogenesis.     

AdKDR-CDglyTK融合基因系统治疗胰腺癌的实验研究

Objective To investigate the curative effect of the adenovirus-mediated fusion gene system driven by KDR promoter (AdKDR-CDglyTK) on a model of pancreatic cancer. Methods By using transplantation of the cultivated cells, human pancreatic cell line Capan-2 was injected subcutaneously on the back of nude mice to establish the animal model of the pancreatic cancer. Twenty nude mice were divided randomly and equally into four groups. The mice in group Ⅰ were injected with AdKDR-CDglyTK and 5-FC/GCV, those in group Ⅱ were injected with 5-FC/GCV, those in group Ⅲwere injected with AdKDR-CDglyTK and those in group Ⅳ received no any injection. AdKDR-CDglyTK was injected directly into the tumor and 5-FC/GCV was given by intraperitoneal injection. The observing parameters included common status, tumor bulk, tumor weight, inhibition rate of tumor growth, pathology, immunohistochemistry and treatment effect in each group. Electron microscopy was performed to observe the pathological changes of cells. The apoptotic cells in tumor were detected using the TUNEL assay. The expression of CDglyTK in tumors from each group was examined by RT-PCR. Results Tumor growth was dramatically inhibited in group Ⅰ. Tumor growth has no significant difference among groupⅡ , group Ⅲ and group Ⅳ. The apoptotic rate (34.20±4.60)% was significantly increased in group Ⅰ (F= 243. 22, P= 0. 00) and it had no significant difference among groupⅡ , group Ⅲ and group Ⅳ (P＞0.05). Conclusion AdKDR-CDglyTK with 5-FC/GCV can obviously inhibit the growth of human KDR-expressing pancreatic cell line Capan-2 and induce the cell apoptosis in vivo. The probable molecular mechanism lies in the facts that the system can cause a decline in the level of Bcl-2.     

AdKDR-CDglyTK融合基因系统治疗胰腺癌的实验研究

Objective To investigate the curative effect of the adenovirus-mediated fusion gene system driven by KDR promoter (AdKDR-CDglyTK) on a model of pancreatic cancer. Methods By using transplantation of the cultivated cells, human pancreatic cell line Capan-2 was injected subcutaneously on the back of nude mice to establish the animal model of the pancreatic cancer. Twenty nude mice were divided randomly and equally into four groups. The mice in group Ⅰ were injected with AdKDR-CDglyTK and 5-FC/GCV, those in group Ⅱ were injected with 5-FC/GCV, those in group Ⅲwere injected with AdKDR-CDglyTK and those in group Ⅳ received no any injection. AdKDR-CDglyTK was injected directly into the tumor and 5-FC/GCV was given by intraperitoneal injection. The observing parameters included common status, tumor bulk, tumor weight, inhibition rate of tumor growth, pathology, immunohistochemistry and treatment effect in each group. Electron microscopy was performed to observe the pathological changes of cells. The apoptotic cells in tumor were detected using the TUNEL assay. The expression of CDglyTK in tumors from each group was examined by RT-PCR. Results Tumor growth was dramatically inhibited in group Ⅰ. Tumor growth has no significant difference among groupⅡ , group Ⅲ and group Ⅳ. The apoptotic rate (34.20±4.60)% was significantly increased in group Ⅰ (F= 243. 22, P= 0. 00) and it had no significant difference among groupⅡ , group Ⅲ and group Ⅳ (P＞0.05). Conclusion AdKDR-CDglyTK with 5-FC/GCV can obviously inhibit the growth of human KDR-expressing pancreatic cell line Capan-2 and induce the cell apoptosis in vivo. The probable molecular mechanism lies in the facts that the system can cause a decline in the level of Bcl-2.     

结肠癌与直肠癌根治术后复发的比较研究

Objective To explore the difference in tumor biological behaviors and prognosis between recurrent colon cancer and recurrent rectal cancer after radical operation. Methods Complete clinical and follow-up data of 132 patients with colorectal cancer developed recurrence,including 36 colon cancers and 96 rectal caners, after curative resection were retrospectively analyzed and compared with respect of clinical pathological features and prognosis between colon and rectal cancer.Results Significant differences were found in primary tumor gross type, histological type, tumor differentiation and lymph node metastasis between colon and rectal cancer (P＜0.05). Colon cancer recurred earlier than rectal cancer after radical surgery with the median time to recurrence being 14.0 months and 21.5 months, respectively (P=0.028). The difference in multiple sites recurrence was also found between colon (n=16,44.4%) and rectal cancer (n=65,67.7%)(P=0.014). The 3-year survival rate of recurrent rectal cancer was better than that of colon cancer (24.8% vs 15.6% ,P=0.026).Conclusion There are some differences in tumor biological behaviors between colon and rectal cancer,and the prognosis of rectal cancer with recurrence is better than that of colon cancer.     

抗PSA/抗CD3两种双特异性单链抗体的活性研究

目的:研究并比较已成功构建的抗PSA/抗CD3两种双特异性单链抗体的生物学活性。方法:利用流式细胞仪和51Cr释放试验评价抗PSA/抗CD3双特异性单链抗体的抗原亲和活性和体外介导杀伤靶细胞的效果。建立前列腺癌裸鼠模型,分为非治疗组、对照组、双特异性单链抗体(BsAb)组和多价双特异性单链抗体(mBsAb)组,分析两种双特异性单链抗体在体内介导细胞毒T细胞对肿瘤细胞杀伤的能力。结果:流式细胞仪结果显示:BsAb与LNCaP细胞和Jurkat细胞的阳性结合率分别为56.3%和55.4%;mBsAb与LNCaP细胞和Jurkat细胞的阳性结合率分别为74.0%和83.0%。在体外,有细胞毒T细胞存在时两种抗体均可引起前列腺癌细胞的裂解,裂解效率分别与抗体浓度和T细胞与靶细胞比例呈正相关。与对照组比较,接种前列腺癌细胞的裸鼠在体内注射激活的细胞毒T细胞的同时分别接受两种抗体的治疗后,肿瘤生长均明显受到抑制(P<0.05)。在亲和力、体外杀伤和体内抑制肿瘤生长等方面,mBsAb的活性明显优于BsAb。结论:抗PSA/抗人CD3 BsAb及其四聚体均具有良好的生物学活性,单链抗体四聚体的形成可以明显改善抗体的生物学活性。     

人端粒酶反转录酶启动子调控NK4基因靶向治疗结肠癌的实验研究

目的 构建人端粒酶反转录酶（HTERT）启动子调控的NK4基因重组腺病毒表达系统,探讨NK4基因对结肠癌生长、侵袭转移的抑制作用.方法 以Ad HTERTp-NK4重组腺病毒载体感染结肠癌细胞HTC116,分别以RT-PCR方法和Western blot检测细胞内NK4基因mRNA及蛋白表达量：MTT和体外侵袭实验观察NK4对结肠癌细胞增殖及侵袭转移能力的抑制作用.建立结肠癌裸鼠皮下移植瘤模型,观察重组腺病毒Ad HTERTp-NK4对裸鼠结肠癌移植瘤生长的影响.结果 Ad HTERTp-NK4重组腺病毒载体感染HCT116细胞后,HCT116细胞内NK4 mRNA及蛋白表达水平明显增高：Ad HTERTp-NK4重组腺病毒感染后,HCT116细胞生长抑制率和侵袭抑制率分别为47.14%和40.63%,与Ad HTERTp-GFP（空载体）组（2.75%和2.31%）比较,差异均有统计学意义（均P＜0.05）.局部注射Ad HTERTp-NK4重组腺病毒载体后,结肠癌裸鼠皮下移植瘤模型肿瘤生长受抑,抑瘤率为47.3%,与空载体组（4.6%）比较,差异有统计学意义（P＜0.05）.结论 HTERT启动子调控NK4基因靶向治疗系统可有效抑制结肠癌的增殖及侵袭转移,在结肠癌的基因治疗具有较好的应用前景.     

抗表皮生长因子受体单克隆抗体对人胰腺癌裸鼠模型的化疗增敏作用

目的 观察鼠抗人EGFR单克隆抗体联合5-氟尿嘧啶(5-Fu)、健择对裸鼠胰腺癌化疗效果的影响.方法 将人胰腺癌肿瘤细胞悬液注射于裸鼠背部皮下,建立肿瘤模型.干预组腹腔分别注射鼠抗人EGFR单克隆抗体(MMAb-2)、5-Fu、健择及其配伍联合用药;对照组腹腔注射生理盐水.干预4周后处死裸鼠,切取肿瘤,测量瘤体积,取肿瘤组织送病理学检查;免疫组化法检测肿瘤组织中bax、bcl-2的表达;免疫荧光法检测肿瘤组织中细胞凋亡指数,评价干预效果.结果 5-Fu、健择联合应用MMAb-2对裸鼠胰腺癌细胞增殖的抑制作用明显增强(P＜0.05),且能提高裸鼠胰腺癌细胞凋亡率(P＜0.05).结论 抗EGFR单克隆抗体能有效提高化疗药物5-Fu、健择对裸鼠胰腺癌细胞抑制作用的敏感性,可明显提高胰腺癌的化疗效果.     

抗原致敏树突细胞增强细胞因子诱导杀伤细胞抗胃癌的效应

目的 观察抗原致敏树突细胞(DC)与细胞因子诱导的杀伤细胞(CIK)共培养体内外抗胃癌(SCC-7901)的效应.方法 取健康供者外周血体外诱导培养DC和CIK,应用噻唑蓝(MTT)法测定比较(每组n=3)单纯CIK(CIK组)、DC诱导CIK(DC-CIK组)和经胃癌抗原致敏的DC诱导CIK(抗原-DC-CIK组)体外对SGC-7901的杀伤活性,并利用SGC-7901建立胃癌裸鼠模型,设立PBS对照组,比较各组(每组n=5)瘤体体积、抑瘤率和肿瘤坏死面积评分.结果 成熟DC与CIK共培养第7天时,DC-CIK扩增倍数为17.8±2.0,约为单纯CIK扩增倍数(10.9±1.8)的1.63倍(P＜0.05);抗原-DC-CIK组、DC-CIK组、CIK组,体外实验中效靶比为20:1时对SGC-7901的杀伤活性分别为:(72.3±0.5)%、(53.9±0.7)%、(46.4±0.4)%(P均＜0.01);体内动物实验中抑瘤率分别为30.02%、18.11%、15.94%(P均＜0.01).结论 SGC-7901抗原致敏DC能增加CIK的扩增数量,增强CIK对SGC-7901的杀伤作用.     

穿膜融合多肽TAT-N24对结肠癌荷瘤小鼠肿瘤生长的抑制作用

目的 观察穿膜融合多肽TAT-24对结肠癌荷瘤小鼠肿瘤生长的抑制作用.方法 以结肠癌荷瘤小鼠为研究对象,观察静脉系统给予TAT-N24或TAT-N24m(无关肽对照)对肿瘤生长的影响,比较肿瘤重量变化,应用BrdU掺入的方法检测TAT-N24对细胞DNA合成的影响,体内实验证实TAT-N24的抗肿瘤作用.结果 免疫组织化学检测,在TAT-N24及对照肽TAT-N24m组均看到HAtag的阳性表达,提示融合多肽体内能够有效进入肿瘤细胞;TAT-N24组肿瘤生长明显减慢,各组肿瘤重量分别为:称量各组肿瘤平均重量,Blank组:(0.34±0.09)g;TAT-N24m组:(0.27±0.06)g;TAT-N24组:(0.12±0.02)g,提示TAT-N24能够抑制HT29细胞裸鼠移植瘤生长(P＜0.05).BrdU掺入显示TAT-N24组BrdU阳性细胞显著低于对照组.结论 融合多肽TAT-24能有效抑制结肠癌荷瘤小鼠肿瘤生长,抑制肿瘤内细胞DNA合成.     

BMMSC输注对脐血CIK/NK细胞在荷瘤小鼠体内抗肿瘤效应的影响

目的 利用荷瘤小鼠来探讨不同的输注途径以及不同时间输注骨髓间充质干细胞( BMMSC)对脐血来源细胞因子诱导的杀伤(CIK)/自然杀伤(NK)细胞在体内抗肿瘤效应的影响。方法 NOD/SCID小鼠共56只,分为7组,每组小鼠经尾静脉输注K562细胞后12 h分别进行以下实验：同时经尾静脉输注人BMMSC+ CIK/NK细胞（A组）;经尾静脉相隔48 h分别输注人BMMSC和CIK/NK细胞（B组）;经小鼠胫骨骨髓腔输注入BMMSC,同时经尾静脉输注CIK/NK细胞(C组);经小鼠胫骨骨髓腔输注人BMMSC,48 h后经尾静脉输注CIK/NK细胞;经小鼠胫骨骨髓腔输注人BMMSC,48 h后经尾静脉输注CIK/NK细胞（E组）：较其他组延迟48 h经尾静脉输注CIK/NK细胞（F组）;除输注K562细胞外,不输注其他细胞（G组）。计算各组小鼠的生存曲线,检测小鼠外周血、骨髓、肝、脾和肺等脏器的肿瘤细胞负荷情况。结果 A、B、G组小鼠的存活时间短于C、D、E、F组(P＜0.05);A、B、G组间以及C、D、E、F组间小鼠的存活时间的差异均无统计学意义(P＞0.05);A、B、G组小鼠外周血、骨髓涂片中肿瘤细胞的比例高于C、D、E、F组(P＜0.05)。A、B、G组小鼠外周血、骨髓及肝、脾、肺组织匀浆中人肿瘤细胞标记CD33的表达率高于C、D、E、F组(P＜0.05);A、G组小鼠肝、脾、肺组织匀浆中CD33表达率高于B组(P＜0.05);C组小鼠肺组织匀浆中CD33表达率高于D组(P＜0.05)。结论 同部位注射BMMSC可明显抑制CIK/NK细胞在荷瘤小鼠体内的抗肿瘤作用,而分部位注射,则BMMS的抑制作用明显减弱。提前输入的BMMSC在体内仍会削弱CIK/NK细胞的抗肿瘤作用。     

Vitamin E succinate inhibits colon cancer liver metastases

BACKGROUND: Vitamin E succinate (VES) is a promising anti-cancer micronutrient. In this study, we tested the hypothesis that VES will promote colon cancer tumor dormancy and inhibit liver metastases in colon cancer. METHODS: CT-26 colon cancer cells were treated with VES in vitro and in an in vivo model of liver metastases. The impact of VES on cellular proliferation and apoptosis was measured in vitro by MTS assay and sandwich ELISA and in vivo by PCNA staining and TUNEL assay, respectively. Correlation coefficients and independent t tests were used for statistical analysis. RESULTS: VES significantly and specifically inhibited cell proliferation (P = 0.011) and promoted apoptosis (P < 0.0074) of cancer cells in vitro. VES produced a 40% reduction of liver metastases (P = 0.037). Five of the eight mice had an excellent response to VES. Subsequent analysis of these five mice revealed a 75% reduction in the number of liver metastases (P < 0.05). VES significantly promoted tumor cell apoptosis (P < 0.0003) and inhibited cell proliferation (P = 0.0069) in vivo. CONCLUSIONS: VES inhibits the growth of colon cancer cells in vitro and in vivo. This is the first report of VES inhibition of colon cancer tumor metastases. The mechanism of VES anti-tumor and anti-metastatic activity in vivo appears to involve promotion of tumor apoptosis and inhibition of cell proliferation. These findings support further investigation of VES as a micronutrient to promote colon cancer tumor dormancy and prevent metastases.     

消炎痛抑制肝细胞肝癌生长转移的研究

目的 观察消炎痛(Indomethacin)对有转移潜能的人肝癌MHCC97L细胞增殖侵袭的影响和对裸鼠肝癌生长和转移的抑制作用.方法 (1)体外实验:采用0.2 mmol/L的消炎痛分别作用于MHCC97L细胞,观察细胞增殖、侵袭实验、运动实验和血管内皮生长因子(VEGF)和基质金属蛋白酶2(MMP-2)蛋白表达[酶联免疫吸附试验(ELISA)].(2)体内实验:建立转移性人肝癌裸鼠原位模型后,将裸鼠随机分为对照组和消炎痛组.6周后处死动物,测量肿瘤体积,计算抑瘤率、肺转移灶数目及肺转移率.免疫组织化学方法检测VEGF、MMP-2、环氧合酶-2(COX-2)蛋白的表达.结果 (1)体外实验:0.2mmol/L消炎痛明显抑制MHCC97L细胞增殖(P值均＜0.01),消炎痛组穿过人工基底膜(侵袭实验)和上室底膜(运动实验)的细胞数分别为2.2±1.3和4.4±1.1,明显低于对照组(11.4±1.9和12.8±1.8,P值均＜0.01);ELISA法检测发现,消炎痛组VEGF蛋白和MMP-2蛋白含量和对照组比较明显降低(P值均＜0.01).(2)体内实验:对照组、消炎痛组肿瘤体积分别为(1700 ±422)mm3 和(1170±585)mm3(P＜0.05),肺转移率分别为75%和50%(P＞0.05),平均肺转移灶数目分别为2.92±2.07和1.33±1.56(P＜0.05);与对照组比较,消炎痛组的抑瘤率为31.2%.免疫组织化学染色显示,消炎痛组VEGF、MMP-2、COX-2蛋白的表达和对照组比较均有降低(P值均＜0.01).结论 在一定条件下,消炎痛可抑制肝细胞肝癌的生长转移,其作用和抑制VEGF蛋白和MMP-2蛋白的表达有关.

Abstract：

Objective To study the effects of indomethacin on proliferation and invasion of hepatocellular carcinoma (HCC) cell line MHCC97L with metastatic potential and the effect of indomethacin on the growth and metastasis of HCC. Methods (1) In vitro; Proliferation, Transwell invasion assay, cell motility assay, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) protein activity were evaluated after cells were treated with 0. 2 mmol/L indomethacin. (2)In vivo: Mice bearing xenografts in the liver were randomly divided into control and indomethacin groups. At the end of sixth week, the mice were killed and tumor volume, inhibitory rate, immunohistochemistry assay (IHA) and metastasis were evaluated. Results (1)In vitro; 0. 2 mmol/L indomethacin could inhibit the proliferation of MHCC97L cells markedly (P ＜0. 01). The average amount of invading cells per field in cell invasion assay and motility assay was 2. 2 ± 1. 3 and 4.4 ± 1. 1 respectively in indomethacin group, significantly less than in control group ( 11. 4 ± 1. 9 and 12. 8 ± 1. 8 respectively, P ＜0. 01). The expression of VEGF and MMP-2 in cells treated with indomethacin was significantly lower than in control group (P ＜0. 01). (2)In vivo; Tumor volume, incidence and number of lung metastases in control and indomethacin groups were (1700 ±422) mm3 and (1170 ± 585) mm3 (P ＜ 0. 05), 75% and 50% ( P ＞ 0.05), 2. 92 ± 2. 07 and 1.33 ±1.56 (P＜0. 05) , respectively. Inhibition rate in indomethacin group was 31.2%. IHA showed that the expression of VEGF, MMP-2, and cyclooxygenase-2 ( COX-2) was down-regulated in indomethacin group (P ＜0.01). Conclusion Indomethacin could inhibit the growth and metastasis of HCC, which was in part mediated by down-regulation of VEGF and MMP-2.     

人MUC1重复序列与GM-CSF融合表达的重组卡介苗疫苗对乳腺癌生长的抑制作用

目的 构建一种新的基于人MUC1重复序列与GM-CSF融合表达的重组卡介苗疫苗,并观察其对乳腺癌生长的预防性抑制作用.方法 分步克隆人MUC1重复序列1、4、8串联体基因(MVNTR1/4/8),构建人MUC1重复序列串联体与GM-CSF基因融合的大肠-分枝杆菌表达载体pDE22-MVNTR1/4/8-CSF,酶切鉴定及序列分析后,将构建的穿梭质粒电穿孔转染卡介苗,构建重组卡介苗疫苗rBCG-MVNTR1/4/8-CSF,SDS-PAGE和Western Blot检测MVNTR与GM-CSF融合蛋白的表达.在hu-PBL-SCID鼠模型评价其对乳腺癌生长的抑制作用,并通过免疫组化染色检测其免疫诱导的T细胞反应.结果 SDS-PAGE和Western Blot结果说明:人MUC1重复序列与GM-CSF基因融合的重组卡介苗疫苗分别有MVNTR1/4/8与GM-CSF融合蛋白的表达.rBCG-MVNTR4/8-CSF免疫组在MCF-7乳腺癌细胞接种42 d后,肿瘤成瘤率分别为37.5%和25%,而PBS和BCG-pDE22对照组均为100%(P＜0.05).与对照组相比,rBCG-MVNTR4/8-CSF预防接种显著抑制MCF-7乳腺癌细胞生长(P＜0.01),且生长抑制作用随着VNTR的增加而增强.肿瘤细胞接种70 d后,rBCG-MVNTR4/8-CSF组小鼠生存率分别为75%和87.5%,而BCG-pDE22对照组的生存率为12.5%(P＜0.05).rBCG-MVNTR4/8-CSF免疫组瘤体组织中可见CD4+和CD8+T淋巴细胞浸润.结论 重组卡介苗疫苗rBCG-MVNTR4/8-CSF预防接种可显著抑制乳腺癌生长.