携带4-1BBL基因的胃癌细胞总RNA转染树突状细胞疫苗的研究

目的 观察携带4-1BBL基因的胃癌细胞总RNA转染树突状细胞疫苗,在小鼠体内诱导的抗肿瘤效应以及对小鼠免疫力的影响.方法 用脂质体法把pMKITneo/4-1 BBL质粒导入小鼠胃癌细胞MFC内,再提取此细胞的总RNA并转染至树突状细胞内制备MFC/4-1BBL/DC疫苗;把MFC细胞注射于20只615小鼠皮下建立荷瘤小鼠胃癌模型,并随机分为对照组、DC组、MFC/DC组和MFC/4-1BBL/DC组,每组5只;在MFC细胞接种后第7、14天给予相应DC疫苗治疗,于肿瘤细胞接种后第21天,处死实验动物,测量瘤重、测定肿瘤细胞的凋亡率及外周血的CD4+、CD8+T细胞和NK细胞的含量.结果 MFC/4-1BBL/DC组小鼠平均瘤重(2.06±0.39)g显著低于对照组(3.82±0.57)g、DC组(3.63±0.51)g、MFC/DC组(2.67±0.32)g(P＜0.05);且MFC/4-1BBL/DC组肿瘤细胞凋亡率(28.58±2.84)%明显高于MFC/DC组(25.03±1.88)%、DC组(20.66±2.71)%及对照组(19.09±2.73)%(P＜0.05);MFC/4-1BBI/DC组小鼠外周血CID4+T、NK细胞数明显高于对照组和其他治疗组(P＜0.05).结论 携带4-1BBL基因的胃癌细胞总RNA转染树突状细胞疫苗能够提高荷瘤机体的免疫能力、抑制肿瘤细胞的生长、促进肿瘤细胞的凋亡.

Abstract：

Objective To observe the induced anti-tumor effects and immune state in vivo by the transfection of dendritic cell vaccine into the total RNA of gastric cancer cells carrying 4-1BBL gene.Methods The liposome-mediated pMKITneo/4-1BBL gene was inserted into the MFC gastric cancer cells,and the cell total RNA was extracted and transfected into dendritic cells to make the MFC/4-1BBL/DC vaccine.After MFC cells were injected into the 615 mouse to establish tumor-bearing mouse model,and those 20 mice were randomly divided into control group,DC group,MFC/DC group,MFC/4-1BBL/DC group.The dendritic cell vaccine was subcutaneously injected on the day 7 and 14 after inoculation of tumor cells,and on the day 21 animals were killed and tumor weight was measured,tumor cell apoptosis rate was assayed and the contents of CD4 +,CD8 + T cells and NK cells in peripheral blood were analyzed.Results The mean tumor weight in MFC/4-1BBL/DC group (2.06 ±0.39) g was significantly less than that in control group (3.82 ±0.57) g,DC group (3.63 ±0.51 ) g,MFC/DC group (2.67 ±0.32) g (P ＜0.05).Moreover the apoptosis rate in MFC/4-1BBL/DC group (28.58 ± 2.84 ) % was significantly higher than that in MFC/DC group (25.03 ± 1.88)%,DC group (20.66 ±2.71 )% and control group ( 19.09 ±2.73 )% (P ＜0.05).The percentage of CD4 + T,NK cells in MFC/4-1BBL/DC group was significantly higher than in other groups ( P＜0.05).Conclusion The transfection of dendritic cell vaccine into the total RNA of gastric cancer cells carrying 4-1BBL gene can increase immunity of the bearing tumor mice,inhabit tumor growth and promote tumor cell apoptosis.     

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.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

GDDR位点突变载体的构建和稳转细胞系的建立

目的构建GDDR位点突变载体,使GDDR翻译蛋白无法通过二硫键与TFF1结合,建立稳定转染GDDR位点突变载体的人胃癌SGC-7901稳转细胞系。方法设计GDDR Cys38位点突变引物,构建GDDR位点突变的真核表达载体;脂质体法转染重组质粒至人胃癌细胞系SGC-7901;G418筛选阳性细胞并扩大培养;实时定量PCR检测突变GDDR mRNA在人胃癌细胞系SGC-7901的表达。结果经限制性内切酶酶切和DNA测序证实质粒中插入的为所需序列;实时定量PCR证实突变GDDR在人胃癌SGC-7901稳转细胞系高表达。结论 GDDR定点突变载体构建成功,建立了稳定转染GDDR位点突变载体的SGC-7901细胞系,为进一步研究GDDR的结构和功能提供了条件。     

arresten对人胃癌细胞SGC-7901裸鼠移植瘤生长的抑制作用的研究

目的探讨arresten抑制人胃癌细胞SGC-7901裸鼠移植瘤生长的作用。方法构建包含arrestenc DNA的分泌型真核表达质粒pcDNA3．1（＋）-ss-arresten,并以脂质体法将重组质粒转染至SGC-7901细胞。蛋白印迹法（Western blot）检测SGC-7901是否分泌表达arresten,同时,通过生长曲线绘制及生长周期测定的方法了解上述转基因措施本身对SGC-7901细胞的体外生物学特性的影响。将转基因的SGC-7901细胞株接种至裸鼠皮下,使其在接种局部表达arresten,观察肿瘤生长情况。结果成功构建了分泌型真核表达载体pcDNA3．1（＋）-ss-arresten,并获得了可分泌表达arresten的人胃癌SGC-7901细胞株。质粒转染对SGC-7901细胞增殖特性无影响。转基因SGC-7901细胞的裸鼠移植瘤生长缓慢。结论arresten抑制人胃癌SGC-7901移植瘤的生长,其途径可能是通过抑制其滋养血管的生长而实现。     

腺病毒载体介导的早幼粒细胞白血病基因生长抑制因子诱导胃癌细胞凋亡的机制研究

目的 探讨腺病毒载体介导的早幼粒细胞白血病基因（PML）生长抑制因子诱导胃癌细胞凋亡作用的机制。方法 将人PML全长cDNA插入穿梭质粒pSGCMV，再与腺病毒骨架质粒pPE3共转染293细胞后获得重组病毒。用重组病毒感染胃癌细胞系SGC-7901，采用噻唑蓝比色法、流式细胞术以及免疫细胞化学法对p53和bcl-2在肿瘤细胞内的表达以及细胞凋亡的定性与定量指标进行检测。结果 经腺病毒介导的PML处理的SGC-7901细胞生长受到明显抑制，凋亡细胞发生率升高，且与MOI值呈正相关，MOI值由5增加到20，细胞的生长抑制率从22．4％增加到38．5％，而细胞凋亡率从37．2％增加到49．8％。经腺病毒介导的PML处理的SGC-7901细胞内p53蛋白表达较对照组明显增加，bel-2蛋白的表达则无明显变化。结论 腺病毒介导的PML对胃癌细胞的杀伤主要是通过诱导细胞凋亡，p53蛋白高表达为其诱发胃癌细胞凋亡的机制之一。     

抑癌基因WTX对胃癌SGC-7901细胞增殖、凋亡及细胞周期的影响

目的:探讨WTX基因对人胃癌SGC-7901细胞生物学行为的影响。方法:将WTX重组质粒或空载体质粒用Attractene法转染SGC-7901细胞,以无处理SGC-7901细胞为空白对照,检测不同时间e GFP标记的转染效率;RT-PCR法检测WTX m RNA水平;CCK-8法测定细胞增殖情况;流式细胞技术检测转染效率、凋亡及细胞周期的变化。结果:转染WTX基因48 h后,e GFP表达最强,转染效率达(33.10±4.16)%;与空白对照组和空载体组比较,WTX转染组WTX m RNA表达明显升高;细胞增殖能力明显降低,S期细胞明显增多,而G1期和G2/M期细胞减少(均P<0.05)。各组细胞均未见明显的细胞凋亡。结论:WTX可通过诱导S期阻滞抑制胃癌细胞SGC-7901生长,但不影响细胞凋亡。     

CO2气腹环境对胃癌细胞黏附与侵袭转移的影响

目的 探讨不同压强持续性CO2气腹环境对胃癌细胞黏附与侵袭转移的影响.方法 建立体外CO2气腹模型,选用3种不同分化程度的胃癌细胞株MKN-45(低分化腺癌细胞)、SGC-7901(中分化腺癌细胞)和MKN-28(高分化腺痛细胞),分别在9 mm Hg、15 mm Hg以及常规条件下(0 mm Hg,对照组)作用2 h和4 h后,用RT-PCR法、CytoMatrixTM细胞黏附试剂盒和ECMatrixTM细胞侵袭试剂盒检测黏附分子E钙黏蛋白(E-cadherin)和细胞间黏附分子1(intercellular adhesionmolecule-1,ICAM-1)以及侵袭分子基质金属蛋白酶2(matrix metalloproteinases,MMP-2)和血管内皮生长因子A(vascular endothelial growth factor A,VEGF-A)的表达.将胃癌细胞注入裸鼠腹腔(2×106个细胞/只),每组10只.4周后每组取5只处死,记录腹腔成瘤情况,观察其余裸鼠的生存时间.结果 RT-PCR结果显示3种胃癌细胞株经CO2处理后,随着时程的延长及压强的升高,E-cadherin表达有下降的趋势(MKN-45:2.26→2.19、SGC-7901:2.16→2.09、MKN-28:2.06→1.99),而ICAM-1(MKN-45:2.20→2.28、SGC-7901:2.10→2.18、MKN-28:2.00→2.08)、MMP-2(MKN-45:2.05→2.13、SGC-7901:1.95→2.03、MKN-28:1.85→1.93)和VEGF-A(MKN-45:2.10→2.16、SGC-7901:2.00→2.06、MKN-28:1.90→1.96)则有升高的趋势,但是各实验组之间比较或实验组与对照组之间比较差异均无统计学意义(P>0.05).黏附侵袭实验也得出类似的结果.裸鼠模型显示3种胃癌细胞株在不同CO2气腹环境及对照条件下,腹腔成瘤个数随着时程的延长及压强的升高而增加(MKN-45:22→23、SGC-7901:20→22、MKN-28:21→22),存活天数则减少(MKN-45:23→21、SGC-7901:22→21、MKN-28:22→21),但各组的腹腔成瘤个数和存活时间之间相比差异均尤统计学意义(P>0.05).结论 在不高于15 mm Hg 压强以及不超过4 h的情况下,不同压强与时程的CO2对3种胃癌细胞株的黏附和侵袭能力并无显著影响,且不增加肿瘤的转移风险.     

shRNA靶向抑制COX-2基因表达对胃癌细胞SGC-7901增殖的影响

目的 探讨环氧化酶-2(COX-2)短发夹RNA(shRNA)对胃癌细胞SGC-7901的生长和细胞周期的影响.方法 构建COX-2基因的特异性小RNA干扰质粒,构建靶向抑制COX-2基因的重组表达载体pshRNA-COX-2.实验分3组:未转染胃癌细胞组,阴性对照HK组,pshRNA-COX-2转染组.用质脂体lipofectamine~(TM) 2000转染胃癌细胞SGC-7901,RT-PCR和Western blot分别检测COX-2基因mRNA和蛋白表达情况,流式细胞术检测细胞周期,细胞计数检测细胞的生长变化.结果 与阴性对照组相比,pshRNA-COX-2重组表达载体抑制COX-2 mRNA及蛋白表达的抑制率分别为70.1%和43.2%;G_0～G_1期细胞由61.5%上升至70.2%,S期细胞由27.3%下降至21.7%;细胞生长明显减慢.结论 pshRNA-COX-2重组表达载体能显著抑制COX-2的表达,从而抑制胃癌细胞生长.     

吡咯烷二硫代氨基甲酸盐增强肿瘤坏死因子α诱导人胃癌细胞株SGC-7901凋亡作用的研究

目的研究核转录因子-κb（NF—κb）抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）对肿瘤坏死因子α（TNF—α）诱导的人胃癌细胞株SGC-7901生长抑制及凋亡的影响,并探讨其作用机制。方法应用噻唑蓝（MTr）法检测不同浓度的PDTC和TNF-α以及两者联合应用对SGC-7901细胞增殖的抑制率：采用Hoechst检测SGC-7901细胞凋亡情况：Westernblot检测SGC-7901细胞survivin和easpase-3蛋白的表达。结果PDTC在15、30、60和100μmol／L浓度时．对SGC-7901的细胞生长抑制率分别为（12．14±0．91）％、（20．00±1．11）％、（37．63±1．01）％和（41.46±1．07）％．均可抑制细胞增殖（P〈0．01）。TNF-α为25mg／L时,对SGC．7901细胞的生长抑制率为（2．38±0．67）％,与对照组（1．50±0．81）％相比,差异无统计学意义（F=28．28,P〉0．05）：而在50、100和150mg／L浓度时,对SGC-7901细胞的生长抑制率分别为（4．53±0．85）％、（4．43±0．70）％和（4．74±1．07）％,与对照组相比,差异有统计学意义（P〈0．05）。PDTC15μmol／L分别与25、50、100和150mg／L的TNF．仅联合应用时,对SGC-7901的细胞生长抑制率则分别为（18．94±1．10）％、（30．23±0．89）％、（41．55±0．94）％和（53．34±0．98）％,与单用TNF—α或单用15μmol／LPDTC比较,细胞生长抑制率增加（P〈0．01）。Hoechst检测结果显示,TNF-α100mg／L组、PDTC15μmol／L组及两者联合应用组细胞凋亡率均显著增加（P〈0．01）,且联合用药组细胞凋亡率增高最为显著（P〈0．01）。PDTC（15μmol／L）与TNF-α（100mg／L）联合用药与单用TNF—α（100mg／L）比较,细胞survivin蛋白表达明显降低（P〈0．01）,与单用PDTC（15μmol／L）比较,差异无统计学意义（P〉0．05）;但caspase-3蛋白的表达联合用药组较两者分别单用时显著增加（P〈0．01）。结论PDTC可增强TNF-α对人SGC-7901细胞的促凋亡作用,其机制可能与PDTC阻断TNF-α诱导的NF—κb活性、下调survivin表达并最终上调凋亡蛋白easpase-3的表达有关。     

FasL、B7-1联合基因修饰的胃癌细胞诱导抗胃癌主动免疫的研究

目的 探讨FasL、B7-1联合基因转移是否具有协同的抗肿瘤效应。方法 采用重组腺病毒载体将FasL、B7-1基因导入人胃癌细胞SGC-7901,G418阳性克隆筛选,流式细胞分析,逆转录-聚合酶链反应(RT-PCR),显示FasL和B7-1的表达,并且通过Hoechst33342染色检测胃癌细胞凋亡;将携带 FasL和 B7-1基因的胃癌细胞(命名为 SGC-7901/FB-11)接种于 C57BL/6小鼠背部皮下,观测其致瘤性能;SGC-7901/FB-11致敏的小鼠对野生型瘤细胞是否具有免疫保护作用;用SGC-7901和 SGC-7901/FB-11细胞分别经腹腔免疫小鼠,得到腹腔浸润淋巴细胞及致敏脾细胞,MTT法检测其体外杀伤实验。结果 FasL和B7-1基因在胃癌细胞中获得高表达并可诱导胃癌细胞的凋亡,双基因转染的胃癌细胞的致瘤性明显下降;SGC-7901/FB-11诱导的细胞毒T淋巴细胞(CTL)对SGC-7901的杀伤活性显著高于野生型SGC-7901诱导的CTL对相同靶细胞的杀伤活性(P<0.05);SGC-7901/FB-11诱导的 CTL对 SGC-7901/FB-11的杀伤率显著高于对野生型 SGC-7901的杀伤率(P<0.05)。结论 FasL促进胃癌细胞凋亡,B7-1促进抗胃癌CTL的增殖、活化,在效应阶段两基因发挥着重要的协同作用。     

谷氨酰胺酶反义基因对裸鼠胃癌移植瘤生长的抑制作用

目的研究反义核酸技术封闭谷氨酰胺酶(GA)基因对裸鼠体内胃癌生长的抑制作用。方法将携带GA反义基因的质粒转染胃癌细胞SGC-7901,并将转染的胃癌细胞接种于裸鼠皮下,观察其在裸鼠体内的生长情况,通过病理学检查和免疫组化方法评价细胞增殖活性的改变;RT-PCR技术检测移植瘤内GA mRNA的含量。结果GA反义基因质粒载体成功转染胃癌细胞后,癌细胞成瘤能力下降,肿瘤生长速度减慢,肿瘤血管生成减少,肿瘤组织GA mRNA的含量减少。结论GA反义基因有效抑制GA基因的表达并抑制胃癌细胞在裸鼠体内的生长。     

p21WAF1与p27KIP1真核双表达载体的构建鉴定及在胃癌细胞中的表达

目的 构建p21WAF1与p27KIP1真核双表达载体,体外转染胃癌7901细胞,观察其在胃癌细胞中的表达及相关特性.方法 提取人全血总RNA,利用逆转录-聚合酶链反应(RT-PCR)扩增p21WAF1和p27WAF1基因并将其分别克隆到真核双表达载体pIRES中构建重组质粒pIPES-p27KIP1-p21WAF1,经酶切和测序鉴定重组质粒.脂质体介导重组质粒plRES-p27KIP1.p21WAF1"转染胃癌7901细胞,收集并提取转染后12、24、48、72 h和5 d各细胞的总RNA,逆转录成cDNA,荧光定量聚合酶链反应(FQ-PCR)检测各细胞中p21WAF1和p27KIP1在RNA水平的表达.带有绿色荧光蛋白的pIRES-EGFP作为对照.结果 RT-PCR扩增分别获得约500和600 bp大小的产物,重组质粒pIRES-p27KIP1.p21WAF1经酶切和测序鉴定证实为p21WAF1和p27KIP1基因.转染胃癌7901细胞48 h时转染率为68%.与空质粒对照组比较,重组质粒转染胃癌7901细胞12、24、48、72 h和5 d后FQ-PCR证实p27KIP1的Ct值(cycle threshold,Ct)分别减少8.2、10、10、7.4和6.7,p21WAF1的Ct值分别减少7.4、8.2、8.2、6.3和5.70其中24 h和48 h的Ct值减少最大.结论 真核双表达载体pIRES-p27KIP1.p21WAF1构建成功并能在胃癌7901细胞内高效表达.