腹腔不同渗透压液体温热化疗对小鼠腹水瘤的防治作用

探讨温热、化疗药物、低渗液及其联合应用对小鼠腹水瘤细胞生长的防治作用,并探索结合热化疗药物达到最佳疗效的灌注液介质浓度。方法:以昆明小鼠腹水瘤为模型,观察43℃重蒸馏水(DDW)、37℃丝裂霉素(MMC)生理盐水溶液、43℃MMC不同浓度氯化钠溶液和43℃DDW MMC等不同处理方法对干扰腹水瘤细胞生长、杀伤作用及其毒性作用。结果:以上方法均能破坏小鼠腹水瘤细胞,减少瘤细胞数量,抑制腹水产生,延长小鼠生存时间,使S期肿瘤细胞百分率减少,瘤细胞DNA指数(DI)降低(P<0.05),其中以43℃DDW及MMC腹腔内灌注的方法疗效最佳。结论:低渗液结合热化疗药物行腹腔灌注是防治进展期消化道肿瘤患者发生腹膜种植转移的一种有效方法。     

高热重蒸馏水加顺铂腹腔灌注治疗癌性腹水及其在胃癌根治术中的应用

选用70只LACA小白鼠于其腹腔内植入上H_22瘤细胞(每鼠瘤细胞数2×10~7个)后第5天,分别进行43℃重蒸馏水(低渗液)、等渗液,顺铂及43℃重蒸馏水加顺铂腹腔内灌注治疗癌性腹水。结果发现:以上方法均能破坏小鼠腹水瘤细胞,抑制癌性腹水生长,阻止腹膜癌转移的发生,延长动物生存时间,并以高热重蒸馏水加顺铂的作用最强(P<0.01)。在实验研究基础上,对32例进展期胃癌根治术后的患者采用预防性高热重蒸馏水加顺铂腹腔内灌注治疗,疗效满意。     

p53重组腺病毒联合顺铂或三氧化二砷对人膀胱癌EJ细胞的作用

目的：探讨p53重组腺病毒（Ad-p53）与顺铂（CDDP）或三氧化二砷（As2O3）联合应用提高膀胱癌疗效的可能性及其机制。方法：将Ad-p53[100感染强度（MOI）]与CDDP（0．5mg/L)或As2O3(2.0μmol/L）联合应用，通过细胞生长抑制实验、克隆形成实验、细胞周期分析、免疫组织化学分析以及裸鼠皮下移植瘤模型，观察其对膀胱癌EJ细胞的作用及机制。结果：与单独应用相比，Ad-p53与低剂量的CDDP或As2O3联合可明显抑制EJ细胞体外生长，诱导EJ细胞凋亡，EJ细胞G2／M期阻滞更明显；裸鼠体内肿瘤发生时间延迟，4周后肿瘤体积与单独应用时相比，差异有显著性（P＜0．05）。结论：基因治疗与化疗联合应用，可进一步提高膀胱癌的疗效。     

1,25二羟维生素D3与5-氟尿嘧啶对乳腺癌细胞生长及凋亡的影响

目的研究1，25二羟维生素D3[1，25(OH)2D3]与5-氟尿嘧啶(5-Fu)对人乳腺癌细胞株MCF-7生长及凋亡的影响。方法采用噻唑蓝(MTT)比色法分析细胞生长抑制作用，流式细胞术测定细胞周期和凋亡率，免疫组织化学法检测bcl-2蛋白表达。结果1，25(OH)2D3与5-Fu均可抑制MCF-7细胞生长、1，25(OH)2D3阻滞细胞周期于G0／G1期，5-Fu阻滞细胞周期于S期，并可诱导细胞凋亡；当两药联合应用时，上述作用得到显著加强，凋亡率明显上升。两药均可下调bcl-2蛋白表达，当两药联合应用时，bcl-2蛋白几乎不表达。结论1，25(OH)2D3与5-Fu联合应用对乳腺癌细胞具有协同抑制生长和诱导凋亡作用。     

特异性阻断Shh信号通路对胰腺癌细胞系SUIT-2增殖和凋亡的影响

目的： 研究组抑胚胎发育相关信号通路Sonic hedgehog (Shh)对人胰腺癌细胞系SUIT-2增殖和凋亡的影响。方法：用四唑蓝（MTT）比色试验检测Shh信号通路特异性抑制剂cyclopamine对胰腺癌细胞系SUIT-2增殖的抑制作用；流式细胞术检测细胞增殖指数(PI)和凋亡指数(AI)；用裸鼠移植瘤模型检测cyclopamine对移植瘤生长的抑制作用。 结果：cyclopamine对SUIT-2细胞株增殖的抑制作用呈剂量和时间依赖性。cyclopamine作用后使胰腺癌细胞周期阻滞在G0／G1期，细胞凋亡增加；SUIT-2的AI为14.3±0.35， PI为36.1±0.44，对照组分别为1.3±0.24和52.3±0.28（均P<0.05）。在裸鼠移植瘤模型中，cyclopamine同时给药组肿瘤生长明显受到抑制，而延期给药组对肿瘤生长的抑制作用较弱。结论：shh信号分子对胰腺癌细胞的增殖起维持作用；通过特异性阻断该信号通路，可以抑制胰腺癌细胞增殖，并促进细胞凋亡。     

细胞周期素D1反义cDNA治疗肝癌的研究

目的 通过基因反义封闭技术抑制细胞周期素D1（CydinD1）的表达，研究其对肝癌细胞增殖以及成瘤性的影响。方法 以肝癌HepG2细胞株为研究对象，通过转染可表达CyclinD1反义互补脱氧核苷酸（AScDNA）的质粒后，观察CyclinD1反义cDNA对肝癌细胞CyclinD1基因表达、体外增殖活性及裸鼠体内成瘤性的影响。结果 噻唑蓝（MTT）法检测细胞增殖活性显示转染表达反义CydinD1的质粒后，HepG2细胞的增殖受到抑制．抑制作用在48h左右最强；逆转录-聚合酶链反应（RT-PCR）检测显示CyclinD1 mRNA基因的表达明显被抑制；间接免疫荧光检测结果显示CyclinD1蛋白表达显著降低；流式细胞仪检测结果显示G0／G1期的细胞比例增高，G2＋M和S期的细胞比例下降，HepG2细胞周期在G1期被阻滞；裸鼠成瘤试验显示肝癌HepG2细胞的成瘤性受到明显抑制。结论 CyclinD1反义cDNA可以特异性的抑制肝癌HepG2细胞株CyclinD1蛋白的表达，从而调控细胞周期，抑制肝癌细胞增殖及体内成瘤性。CyclinD1反义cDNA对于肝细胞癌的生物治疗具有一定的应用前景。     

Flavopiridol对尤文肉瘤SK-N-MC细胞增殖的抑制作用

目的探讨细胞周期蛋白激酶抑制剂（flavopiridol）在体内外抑制尤文肉瘤细胞增殖的作用及其机制。方法以不同浓度的flavopiridol作用于培养的SK-N-MC细胞,用噻唑蓝法（MTT）检测细胞增殖,流式细胞分析仪测定细胞周期。采用DNA凝胶电泳检测法,观察flavopiridol诱导细胞凋亡的情况;蛋白质电泳观察Bcl-2及Mcl-1表达变化;建立SK-N-MC细胞裸小鼠皮下移植瘤模型,腹腔内给药,观察肿瘤生长情况;肿瘤标本石蜡包埋切片,免疫组织化学原位末端标记法（TUNEL）检测flavopiridol在体内对肿瘤细胞的凋亡诱导情况。结果（1）Flavopiridol对人尤文肉瘤细胞增殖有抑制作用,时间越长、浓度越高,抑制作用越强。（2）Flavopiridol可改变尤文肉瘤细胞周期,随药物浓度增加Sub-G1期百分比逐渐上升。尤文肉瘤细胞经flavopiridol中、高浓度给药后,DNA凝胶电泳可见典型的梯状带。（3）Flavopiridol给药前后,Bcl-2蛋白表达无变化,而Mcl-1蛋白表达明显下降。（4）TUNEL染色结果显示实验组阳性细胞比例上升,与对照组比较差异有统计学意义。结论Flavopiridol能在体内外抑制尤文肉瘤细胞的增殖,其机制主要是阻滞细胞周期的G1/S期,诱导细胞凋亡。     

金雀异黄素与5-氟尿嘧啶对SGC-7901细胞的抑制作用

目的研究金雀异黄素（genistein，Gen）与5-氟尿嘧啶（5-fluorouracil，5-FU）对人胃癌细胞SGC-7901的抑制作用。方法采用MTT法检测Gen和5-FU对SGC-7901细胞的抑制作用，流式细胞术检测细胞周期分布，并在透射电镜下观察细胞超微结构改变。结果Gen和5-Fu单用或联合应用对胃癌SGC-7901细胞的生长均有显著的增殖抑制作用，且呈剂量和时间依赖性。两药联合应用时，当药物效应在0．2～0．8时，具有协同或相加作用（CI≤1）；18．8btmol／L的Gen作用后，62．97％的SGC-7901细胞阻滞在G2／M期；8．84μmol／L的5-FU作用后，63．76％的SGC-7901细胞阻滞在s期；3．06μmol／L的Gen与7．96μmol／L的5-Fu联合应用后，67．46％的SGC-7901细胞阻滞在G0／G1期。Gen和5-FU均可诱导SGC-7901细胞凋亡。结论Gen与5-FU通过阻滞细胞周期和诱导细胞凋亡而抑制胃癌细胞的生长，对胃癌细胞的增殖抑制具有协同作用。     

Gp96多肽复合物激活树突状细胞诱导抗肝癌免疫的研究

目的 研究热休克蛋白Gp96肽复合物修饰树突状细胞(DC)后的体内外特异性抗瘤作用。方法 从小鼠肝癌细胞株H22细胞中提取Gp96肽复合物,采用细胞培养方法,从小鼠骨髓中获得DC,体外大量培养。用Gp96肽复合物修饰DC,刺激小鼠脾淋巴细胞,以51Cr释放法测定细胞毒性T淋巴细胞(CTL)杀伤活性。制作小鼠肝癌模型,分别予以修饰后的DC、Gp96蛋白、灭活的H22肝癌细胞治疗,检测血清中自细胞介素(IL)-10、干扰素(IFN)-γ水平;CD_4、CD_8和IFN-γ、IL-10双阳性细胞比例。观察其抑制肿瘤的效果。结果 Gp96肽复合物修饰的DC可激活小鼠脾淋巴细胞;后者能对H22瘤细胞进行特异性杀伤,而对艾氏腹水瘤细胞无效;经修饰的DC可明显改善机体的免疫状态,使Th1细胞比例上升,抑制体内肿瘤的生长。结论 Gp96肽复合物能够很好的修饰体外诱导获取的DC,使后者成为一种有效的瘤苗。     

玉米蛋白粉中玉米黄素对人口腔鳞癌细胞株KB增殖的抑制

研究了玉米蛋白粉中玉米黄素的抗肿瘤活性。采用体外培养的人癌细胞株——人口腔鳞癌KB细胞作为对象，观察玉米黄素对抑瘤活性、细胞周期的影响，发现玉米黄素对KB细胞24h的半数抑制浓度IC50为35μmol／L，在20μmol／L时即明显抑制KB细胞的对数生长；细胞周期分析显示时相左移，G1期细胞百分率增多，S期、G2＋M期百分率降低．DNA凝胶电泳显示30μmool／L玉米黄素作用72h能使KB细胞的DNA发生明显的降解．因此从玉米蛋白粉中提取的玉米黄素能明显抑制KB细胞生长，其抗肿瘤活性强，对细胞周期的影响提示G0期阻滞．     

Management of malignant ascites by intraperitoneal injection of OK-432. Possible mechanism of the reduction of original tumor mass volume

We studied the effect of intraperitoneal injection of OK-432 on the growth of original tumor mass in patients with malignant ascites and a possible mechanism of reduction of tumor volume. Sixteen patients with valuable original tumor mass and a large amount of ascites caused by gastro-intestinal cancer were studied. Tumor cells were separated from ascitic fluids and cultured in vitro before the study. Lymphocytes were collected from the fluids at varying intervals after intraperitoneal injection of OK-432 and cultured 24 hours in vitro. Effect of the culture supernatant on ascites-derived autologous tumor cell growth was examined in vitro using microplate assay. The results were as follows. 1) Reduction of tumor mass more than four weeks was found in 4 of 16 cases. 2) Before OK-432 injection, the culture supernatant from ascites-derived lymphocytes did not inhibit autotumor cell growth in vitro. But, the supernatant from lymphocytes which were collected from the ascites after OK-432 injection markedly inhibited tumor growth in all of 4 tumor mass reduction cases. In 12 non-reduction cases the supernatant slightly inhibited tumor growth only in 2 cases. 3) A similar growth inhibitory factor was detected in the mixed culture-supernatant of peripheral blood lymphocytes and OK-432 in vitro. 4) Preliminary studies indicated that the tumor growth inhibitory factor might be different from tumor necrosis factor and interferons. These results indicate that ascites-derived lymphocytes-producing factor may play an important role in reduction of tumor mass volume in patients with cancerous ascites.     

Dependence of efficient adenoviral gene delivery in malignant glioma cells on the expression levels of the Coxsackievirus and adenovirus receptor

OBJECT: Recombinant adenovirus is used as a competent vector in a wide spectrum of cancer gene therapies because of its high efficiency in gene delivery. To study the feasibility of gene therapy in malignant gliomas, the authors examined the antiproliferative effect of the adenovirally transduced wild-type p53 tumor suppressor gene by using 15 different high-grade glioma cell lines. METHODS: Although growth suppression in association with a high adenoviral p53 transduction efficiency was seen in five of 15 cell lines, it was not observed in the remaining 10 cell lines. To clarify the underlying mechanism, we examined the expression levels of the Coxsackievirus and adenovirus receptor (CAR), which is the primary receptor for adenovirus, and of the integrins alpha vbeta3 and alpha vbeta5, which promote adenoviral internalization. The expression level of the CAR gene showed a close correlation to adenoviral gene transduction efficiency in the tested cell lines, whereas the expression levels of the integrins did not. The CAR expression was decreased by wild-type p53 transduction in U251MG cells harboring mutant p53 and increased by antisense inhibition of p53 in LN443 cells with endogenous wild-type p53. CONCLUSIONS: The results of this study indicate that CAR expression is a critical determinant of transduction efficiencies in adenovirus-based gene therapy for human malignant gliomas.     

Interferon-beta gene therapy improves survival in an immunocompetent mouse model of carcinomatosis

BACKGROUND: Interferon-beta (IFNbeta) has multiple antitumor effects; however, its use has been limited by its short half-life in vivo. This limitation may be overcome by IFNbeta gene therapy. We evaluated adenovirus-IFNbeta therapy in an immunocompetent mouse model of carcinomatosis. METHODS: Mice that were treated intraperitoneally 5 days after tumor (mouse ovarian teratoma) inoculation with an adenoviral vector that contains the mouse IFNbeta gene (Ad-IFNbeta), control adenoviral vector or saline solution. Mice were monitored for multiple outcome measures and toxicity. To determine the mechanism of antitumor effect, flow cytometry of ascites fluid was performed to differentiate immune cell populations. Nitric oxide in ascites fluid was measured with an electrochemical microsensor. RESULTS: Tumor burden was decreased and survival was prolonged (P<.001) in the Ad-IFNbeta group after a single treatment of 3.3 x 10(8) plaque-forming units, with acceptable toxicity. By flow cytometry, an increase in the proportion of natural killer cells (from less than 2% of the gated population to more than 8%; P=.024) and an increase in macrophages were seen in the treated animals. Although there was a trend toward increased levels of nitric oxide in Ad-IFNbeta treatment groups, it was not statistically significant. CONCLUSION: IFNbeta gene therapy results in decreased tumor burden and improved survival in an aggressive, immunocompetent mouse model of carcinomatosis. This therapy warrants further evaluation as a treatment for disseminated peritoneal cancer.     

Adenovirus-mediated wild-typep53 tumor suppressor gene therapy induces apoptosis and suppresses growth of human pancreatic cancer

Background: Thep53 tumor suppressor gene is mutated in up to 70% of pancreatic adenocarcinomas. We determined the effect of reintroduction of the wild-typep53 gene on proliferation and apoptosis in human pancreatic cancer cells using an adenoviral vector containing the wild-typep53 tumor suppressor gene. Methods: Transduction efficiencies of six p53-mutant pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, and PANC-1) were determined using the reporter gene construct Ad5/CMV/-gal. Cell proliferation was monitored using a³H-thymidine incorporation assay, Western blot analysis forp53 expression was performed, and DNA laddering and fluorescence-activated cell sorter analysis were used to assess apoptosis.p53 gene therapy was tested in vivo in a subcutaneous tumor model. Results: The cell lines varied in transduction efficiency. The MIA PaCa-2 cells had the highest transduction efficiency, with 65% of pancreatic tumor cells staining positive for beta-galactosidase (-gal) at a multiplicity of infection (MOI) of 50. At the same MOI, only 15% of the CFPAC-1 cells expressed the -gal gene. Adenovirus-mediatedp53 gene transfer suppressed growth of all human pancreatic cancer cell lines in a dose-dependent manner. Western blot analysis confirmed the presence of the p53 protein product at 48 hours after infection. DNA ladders demonstrated increased chromatin degradation, and fluorescence-activated cell sorter analysis demonstrated a four-fold increase in apoptotic cells at 48 and 72 hours following infection with Ad5/CMV/p53 in the MIA PaCa-2 and PANC-1 cells. Suppression of tumor growth mediated by induction of apoptosis was observed in vivo in an established nude mouse subcutaneous tumor model following intratumoral injections of Ad5/CMV/p53. Conclusions: Introduction of the wild-typep53 gene using an adenoviral vector in pancreatic cancer withp53 mutations induces apoptosis and inhibits cell growth. These data provide preliminary support for adenoviral mediatedp53 tumor suppressor gene therapy of human pancreatic cancer.Presented at the 51st Annual Cancer Symposium of The Society of Surgical Oncology, San Diego, California, March 26–29, 1998.     

Restoration of p53 function in anaplastic Wilms' tumor

BACKGROUND/PURPOSE: Recent studies have reported a high incidence of p53 mutations in anaplastic Wilms' tumors (WT). Restoration of the normal p53 state by current gene therapy techniques is thus an attractive potential mode of therapy for this tumor, which is poorly responsive to standard therapy. The purpose of this study is to determine whether gene delivery of normal p53 is possible and to characterize the subsequent effect of restoring the wild-type p53 state. METHODS: Anaplastic WT RM1 cells (mutant p53) were transduced with replication-deficient adenoviral vectors containing either the wild-type p53 gene (rAd-p53) or the gene encoding a green fluorescent protein (rAd-GFP). The transduction efficiency of adenovirus for RM1 cells was determined by flow cytometric analysis of rAd-GFP-transduced cells. The effect of p53 transduction on cell viability was evaluated using a colorimetric proliferation assay. Apoptosis was evaluated by labeling DNA breaks using a TUNEL assay (Apo-Direct kit). RESULTS: Cells treated with increasing concentrations of viral particles relative to tumor cells (multiplicity of infection-MOI) showed a dose-dependent increase in the number of cells transduced. Twenty-four hours after viral treatment, the percentage of cells transduced for MOIs of 10, 50, 100, and 500 was 29.5, 60.9, 74.6, and 92.4, respectively; at 48 hours the percentage of cells transduced increased to 70.8, 90.7, 93. 7, and 96.3, respectively. Viral treatment at an MOI of 50 reduced cell proliferation by 10% at 17 hours and 97% at 5 days; at an MOI of 100, the relative reduction in proliferation was 15% and 99.8%, respectively. When assayed, 30% of cells became apoptotic at an MOI of 50, and 48% at an MOI of 100. CONCLUSIONS: Highly efficient delivery of the p53 tumor suppressor gene by adenoviral vector to anaplastic WT is possible. Subsequent restoration of the normal p53 state results in reduced viability and increased apoptosis. Gene replacement of p53 may represent a novel therapeutic agent for anaplastic Wilms' tumors.     

p53N15融合肽转染肺腺癌细胞系H1299的抗肿瘤作用

目的 探讨p53N15融合肽转染对肺腺癌细胞H1299体外生长的抑制作用.方法 通过腺体相关病毒(adeno-associated virus AAV)载体把p53N15融合肽高效导入p53缺失肺腺癌H1299细胞系,应用相差倒置显微镜观察、MTT细胞活性试验及流式细胞仪技术分析p53N15对肺癌细胞的抑制作用.结果 ...     

p53N15融合肽转染肺腺癌细胞系H1299的抗肿瘤作用

目的 探讨p53N15融合肽转染对肺腺癌细胞H1299体外生长的抑制作用.方法 通过腺体相关病毒(adeno-associated virus AAV)载体把p53N15融合肽高效导入p53缺失肺腺癌H1299细胞系,应用相差倒置显微镜观察、MTT细胞活性试验及流式细胞仪技术分析p53N15对肺癌细胞的抑制作用.结果 p53N15融合肽在H1299细胞中得到了高效表达并发挥明显效应,表现为显微镜下,重组病毒转染72 h可见大片细胞死亡.MTT试验分析,重组病毒转染组细胞活性明显降低.流式细胞仪技术显示,重组病毒转染组可见大量死亡细胞.结论 p53N15融合肽转染可明显抑制肺腺癌细胞H1299的体外生长.     

p53N15融合肽转染肺腺癌细胞系H1299的抗肿瘤作用

目的 探讨p53N15融合肽转染对肺腺癌细胞H1299体外生长的抑制作用.方法 通过腺体相关病毒(adeno-associated virus AAV)载体把p53N15融合肽高效导入p53缺失肺腺癌H1299细胞系,应用相差倒置显微镜观察、MTT细胞活性试验及流式细胞仪技术分析p53N15对肺癌细胞的抑制作用.结果 p53N15融合肽在H1299细胞中得到了高效表达并发挥明显效应,表现为显微镜下,重组病毒转染72 h可见大片细胞死亡.MTT试验分析,重组病毒转染组细胞活性明显降低.流式细胞仪技术显示,重组病毒转染组可见大量死亡细胞.结论 p53N15融合肽转染可明显抑制肺腺癌细胞H1299的体外生长.     

Adenovirus encoding soluble tumor necrosis factor alpha receptor immunoglobulin prolongs gene expression of a cotransfected reporter gene in rat lung

OBJECTIVE: Because almost all pulmonary diseases are not caused by one gene, multiple gene transfection is required for current gene therapy. Adenovirus is an important gene therapy vector, but a short duration and the inability of repeated administration remain limitations. The aims of this study were to evaluate whether adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolongs gene expression and facilitates repeated vector administration to investigate the feasibility of a cotransfection strategy. METHODS: F344 rats received intratracheal administration of 1 x 10(9) plaque-forming units of adenoviral vector encoding beta-galactosidase or both adenoviral vector encoding beta-galactosidase and adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin. In the expression study beta-galactosidase gene expression in the lung was examined by means of enzyme-linked immunosorbent assay on days 2, 7, 14, 28, and 56 (n = 4/day). In the repeated transfection study, soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase were readministered once (7 days after the first adenovirus administration) or twice (on days 7 and 14; n = 4/day). A 2-way factorial analysis of variance was used for statistical analysis. RESULTS: Soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolonged the duration of beta-galactosidase expression. However, antiadenovirus antibody production was significantly increased in the cotransfection group. In addition, there was no increase in beta-galactosidase expression after readministration of soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase. CONCLUSION: Adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolongs beta-galactosidase expression but does not increase beta-galactosidase expression after repeated administration. These results suggest that tumor necrosis factor alpha is one of the most important factors in regulating the duration of gene expression. The cotransfection approach is feasible, but the increase of antiadenovirus antibodies might make repeated cotransfection unfeasible.