裸鼠肿瘤动物模型VEGF受体表达及其意义

目的　通过免疫组织化学染色了解flt 1与flk 1 KDR(VEGF的两个高亲和受体 )在人肿瘤细胞皮下接种肿瘤动物模型的血管内皮细胞与肿瘤细胞中的表达。方法　取荷瘤裸鼠皮下接种瘤块 ,漂洗、固定、石蜡连续切片 ,进行两种受体相应免疫组化检测。结果　在 13种荷瘤裸鼠血管内皮细胞及肿瘤细胞中flt 1的阳性率大部分为强阳性或中阳性 ,而只有在荷人胃腺癌MKN 4 5裸鼠的肿瘤细胞中flt 1的阳性率为弱阳性 ,在荷人卵巢癌SKOv3裸鼠的肿瘤细胞中flt 1的表达为阴性。相比较而言 ,在 13种荷瘤裸鼠血管内皮细胞及肿瘤细胞中KDR的阳性率大部分为中阳性或弱阳性 ,并且在荷人肝癌SMMC 772 1裸鼠 ,荷人胃腺癌SPC A1裸鼠 ,荷人高转移肝癌移植瘤裸鼠 ,荷人卵巢癌SKOv3裸鼠的肿瘤细胞中 ,荷人宫颈癌移植瘤裸鼠和荷人胃腺癌MKN 4 5裸鼠的肿瘤细胞中 ,KDR表达为阴性。结论　VEGF受体共同表达于肿瘤血管内皮细胞与肿瘤细胞 ,提示了VEGF与VEGF受体结合作用在肿瘤演化中的重要性 ,为靶向于VEGF受体的基因治疗策略选择裸鼠动物模型提供了参考依据     

Ang2-siRNA慢病毒干预裸鼠移植性恶性黑色素瘤的研究

目的研究血管生成素2小干扰RNA（Ang2-siRNA）对裸鼠移植性恶性黑色素瘤血管形成及其生长的影响。方法建立人恶性黑色素肿瘤裸鼠异种移植瘤模型。在体内使用pNL-EGFP—Ang2-siRNA慢病毒干扰裸鼠移植性恶性黑色素瘤Ang2基因的表达,观察统计各组肿瘤（空白组、空载组、实验组）生长情况。应用实时荧光定量RT—PCR检测肿瘤组织中Ang2基因的mRNA表达水平;CD34单克隆抗体作为血管内皮标志物,免疫组织化学法检测其表达,以评价肿瘤微血管密度。结果成功建立了恶性黑色素瘤裸鼠异种移植瘤模型,实验组肿瘤Ang2基因mRNA水平、微血管密度、肿瘤体积显著小于空白组和空载组（P〈0．01）,空白组和空载组之间无统计学意义（P〉0．05）。结论pNL—EGFP—Ang2-siRNA慢病毒能沉默Ang2的表达,显著抑制恶性黑色素瘤血管的形成和肿瘤的生长,可能为临床恶性黑色素肿瘤的基因治疗开辟新的途径。     

Adenovirus-mediated transfer of RA538 gene and its antitumor effect

The RA538 cDNA was transferred into human ovarian cancer cell line SK-OV-3 and human melanoma cell line WM-983A by its recombinant adenoviral vector constructed through homologous recombination. It was demonstrated that the recombinant adenovirus could transfer RA538 gene with high efficiency, and could obviously inhibit tumor growth, with the inhibiting rates of 85% and 73% respectively, at the same time greatly repress the colony forming ability of the cells. The therapeutic experiments on transplanted subcutaneous tumor model in nude mice demonstrated that RA538 could significantly inhibit tumor growth. Flow cytometry and DNA fragmentation analysis indicated that RA538 could induce the cell cycle G1 arrest/apoptosis of the tumor cells. The expression of c-myc gene was found pronouncedly reduced by Western blot analysis. These results suggest that the RA538 recombinant adenovirus could be a promising drug in cancer gene therapy.     

Detection of PIVKA II produced by human hepatoma cells in nude mice.

A novel experimental nude mouse model, which is useful for investigation of the mechanisms of PIVKA II synthesis, was established by inoculation with PIVKA II-producing human hepatoma cells (huH-1). We have found markedly elevated levels of PIVKA II in the plasma of nude mice transplanted with huH-1 cells and increased PIVKA II content in huH-1 tumor tissues. Whereas we have not found detectable level of PIVKA II neither in the plasma nor in tumor tissues of nude mice transplanted different human hepatoma cells (HLF) which is not producing PIVKA II. Histology of the tumor tissues produced by huH-1 cells revealed a thick trabecular pattern with blood spaces.     

Adenovirus-mediated transfer of RA538 gene and its antitumor effect

The RA538 cDNA was transferred into human ovarian cancer cell line SK-OV-3 and human melanoma cell line WM-983A by its recombinant adenoviral vector constructed through homologous recombination. It was demonstrated that the recombinant adenovirus could transfer RA538 gene with high efficiency, and could obviously inhibit tumor growth, with the inhibiting rates of 85% and 73% respectively, at the same time greatly repress the colony forming ability of the cells. The therapeutic experiments on transplanted subcutaneous tumor model in nude mice demonstrated that RA538 could significantly inhibit tumor growth. Flow cytometry and DNA fragmentation analysis indicated that RA538 could induce the cell cycle G1 arrest/apoptosis of the tumor cells. The expression of cmyc gene was found pronouncedly reduced by Western blot analysis. These results suggest that the RA538 recombinant adenovirus could be a promising drug in cancer gene therapy.     

Up-Regulation of Hepatoma-Derived Growth Factor Facilities Tumor Progression in Malignant Melanoma

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200) showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn) expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16–F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.     

Effect of chemotherapy on human tumor heterografts and lymphocytes in athymic mice

Sensitivity of human serially transplanted tumours (melanoma, hepatoma, lung, stomach and bladder cancer) to monochemotherapy was studied in nude mice. The following groups of nude mice were examined: intact and those with human xenografts (control and mice treated with alkylating agents). Changes in peripheral blood morphology have been revealed and functional activity of the energetic enzymes (SDH and alpha-GPDH) has been cytochemically discovered.     

Angiogenesis and vascular growth factor receptor expression in malignant melanoma.

The growth and metastases of many solid tumors are dependent on the recruitment of new blood vessels. Tumor angiogenesis is most likely initiated by paracrine release of growth factors that bind to their corresponding endothelial cell surface receptors. To determine whether angiogenesis and growth factor receptor expression are consistent findings in malignant melanoma, primary human melanomas were examined for mRNA expression of receptors for fibroblast growth factors (FGFR-1, FGFR-2), vascular endothelial growth factor (VEGFR-1, VEGFR-2), and the receptors Tiel and Tie2. Charts were reviewed and archival formalin-fixed, paraffin-embedded primary tumors were obtained from patients with thin (<1 mm; n = 10), intermediate (1 to 4 mm; n = 10), or thick malignant melanoma (>4 mm; n = 8). Also examined was whether melanoma cell lines could induce endothelial growth factor receptor synthesis by metabolic labeling. It was found that tumor vascularity did not correlate with clinical stage, melanoma thickness, or clinical outcome. It was also found that melanoma cell lines were not capable of directly regulating endothelial cell synthesis of growth factor receptors. However, expression of Tiel and VEGFR-2 mRNA by the tumor vasculature in select stage IA-IIB patients, and FGFR-1 mRNA expression by the tumor cells in the same clinical stages was found. The expression of these growth factor receptors did not correlate with clinical outcome. These data suggest that angiogenesis is not a prominent characteristic of primary malignant melanoma lesions and that the endothelial cell expression of Tiel and VEGFR-2 in vivo is probably not directly induced by the tumor.     

Suicide gene therapy of human hepatoma and its peritonitis carcinomatosis by a vector of replicative-deficient herpes simplex virus

Herpes simplex virus type 1 (HSV-1) deleted for the immediate-early gene was applied for treatment of hepatoma cells of SKHep 1 and Huh-7. Hepatoma cells were cultured in medium containing HSV1 expressing GFP gene (QOZ/HG) to determine its transfection rate, and both cell lines infected by MOI 1 of QOZ/HG were found to have high expression of GFP without cytotoxicity. Subcutaneous growth of SKHep 1 cell tumor in nude mice was significantly reduced by injection of replicative-deficient herpes virus (TOZ.1) containing Tk-gene with administration of GCV, in comparison with that of noninjected tumor. SCID mice of peritonitis carcinomatosis due to Huh-7 hepatoma cells infected with TOZ.1 could survive longer under administration of GCV than those without TOZ.1. Therefore replicative-deficient HSV1 is a useful vector for treatment of human hepatoma cells, and TOZ.1 with GCV may be applied to suicide gene therapy for hepatoma and peritonitis carcinomatosis of hepatoma cells.     

Human lymphomas transplanted in nude mice

This study was carried out to determine the take rate of malignant lymphomas transplanted subcutaneously in nude mice. Lymphoid tissue from patients with a variety of lymphoproliferative and lymphomatous disorders was employed, but only four (8.3% of total) non-Hodgkin's lymphomas of various types grew in the nude mice. These transplants were from patients with lymphoplasmacytic, lymphoblastic, immunoblastic and centroblastic lymphomas; two of them were serially transferred. High grade malignant lymphomas seem easier to transplant than low grade ones. In each case, the tumor growing in nude mice was similar to the patient's tumor. In one case, metastases were found in mesenteric lymph nodes and lung. Electron microscopic study showed that neoplastic cells were associated with peculiar dark cells the nature of which is discussed. Furthermore, in two transplanted lymphomas, neoplastic cells contained C type virus particles, indicating contamination by the murine xenotropic type C virus.     

G6PD缺陷抑制人黑色素瘤细胞裸鼠移植瘤的生长

敲减葡糖6-磷酸脱氢酶(G6PD)表达的人黑色素瘤A375细胞(A375-G6PDΔ) 呈现生长增殖抑制和凋亡率升高. 为明确G6PD缺陷对裸鼠体内成瘤的影响及其可能机制,用A375-WT与A375-G6PDΔ细胞制作裸鼠荷瘤模型,观察体内瘤体生长,real-time PCR、免疫组织化学染色与紫外分光光度法分别检测瘤体组织G6PD mRNA、G6PD蛋白及酶活性,Western 印迹分析凋亡相关蛋白,分光光度法测定NADPH和GSH/GSSG水平. 结果显示,A375-G6PDΔ细胞注射组的裸鼠成瘤时间延长,瘤体生长明显减慢,瘤体的体积与质量显著低于A375 WT细胞注射组（P <0.01）;与A375-WT细胞注射组相比,A375 G6PDΔ细胞注射组的裸鼠瘤体组织中G6PD mRNA表达、G6PD阳性细胞数与G6PD活性分别降低了87.10%、77.20%与75.77%（P<0.01）,G6PD、p53和Bcl-2的表达分别降低了67.92%、65.54%和62.32%（P<0.01）,Fas升高了86.38%（P<0.01）,NADPH和GSH/GSSG分别降低了74.37%和86.02%（P<0.01）. 结果提示,G6PD缺陷可能通过减少核酸等合成的原料、改变细胞内氧化还原状态及凋亡相关蛋白表达抑制裸鼠瘤体生长与增殖,这为黑色素瘤发生和治疗研究提供了新的线索.     

Interfering growth of malignant melanoma with Ang2-siRNA

To investigate the intervention therapy effect on the growth of malignant melanoma, we have made an observation of expression levels of Ang2 in malignant melanoma cells, which was transduced by small interfering RNA (Ang2-siRNA) of Ang2 in vitro and in vivo. We successfully constructed Ang2-siRNA lent virus, and constructed nude mice model by transplanting malignant melanoma. Ang2-siRNA lent virus inhibited Ang2 mRNA of malignant melanoma in vitro and in vivo, and inhibited malignant melanoma tumor growth at the same time. Ang2-siRNA lent virus can interfere expression levels of Ang2 in malignant melanoma cells, inhibit tumor growth, and silent Ang2 gene expression, which may pave a new way for clinical gene therapy of malignant melanoma.     

New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line.

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.     

胡桃醌不同给药途径对人肝癌细胞BEL-7402裸鼠皮下移植瘤生长的影响

目的采用裸鼠皮下移植瘤模型,通过不同给药途径对胡桃醌抗肿瘤活性和毒性进行评价。方法建立人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,通过腹腔注射和局部注射两个给药途径观察胡桃醌抑制肿瘤生长的效果。结果①以600、300和150μg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,发现该剂量胡桃醌对肿瘤生长没有明显的影响;NK细胞活性检测发现,600、300μg/kg胡桃醌对裸鼠免疫功能有影响(P均<0.01),150μg/kg胡桃醌则没有影响(P>0.05);与阳性对照组(5-Fu)相比,600μg/kg胡桃醌组NK细胞活性差异无显著性(P>0.05),300和150μg/kg胡桃醌组NK细胞活性差异有显著性(P<0.05,P<0.01),结果提示胡桃醌对小鼠免疫系统有一定的损伤作用。②以4.5、3和1.5 mg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,抑瘤率分别为为78.24%、66.57%、48.94%;4.5、3 mg/kg胡桃醌的抑瘤作用可与阳性对照组比拟(P均>0.05)。但4.5 mg/kg胡桃醌组裸鼠出现明显的皮下脂肪减少、消瘦,并有死亡现象。③以pH 7.4和pH 4.0的600、300和150μg/kg胡桃醌人肝癌BEL-7402细胞裸鼠皮下移植瘤模型局部给药,结果发现不同pH(pH 7.4或4.0)600、300μg/kg的胡桃醌局部注射抑瘤作用与阳性对照组(5-Fu)组差异无显著性(P>0.05),而不同pH的150μg/kg胡桃醌抑瘤作用不明显。同一浓度不同pH药物的抑瘤作用差异无显著性(P均>0.05),但pH 4.0的胡桃醌组肿瘤细胞肝转移较少。结论胡桃醌不同给药途径均可抑制人肝癌BEL-7402细胞裸鼠皮下移植瘤的生长,但有一定的毒副作用,药物安全范围较小。     

Inhibition of the growth of human hepatoma cell line both in vitro and in vivo by transducing CKI gene p21~(WAF-1) with GE7 targeting gene delivery system

The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous gene pCEP-p21WAF-1 into human hepatocellular carcinoma cell both in vitro and in vivo. After in vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. The in vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untrans-fected control. The average tumor weight of the experiment group was (0.083 ?0.043) g, while that of the control group was (0.28110.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous gene pCEP-p21WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growt     

Inhibition of metastasis of syngeneic murine melanoma in vivo and vasculogenesis in vitro by monoclonal antibody C11C1 targeted to domain 5 of high molecular weight kininogen

Metastasis of malignant tumors is a major cause of morbidity and mortality. Inhibition of tumor growth in distant organs is of clinical importance. We have demonstrated that C11C1, a murine monoclonal antibody to the light chain region of high molecular weight kininogen (HK), reduces growth of murine multiple myeloma in normal mice and human colon cancer in nude mice. C11C1 inhibits angiogenesis by reducing tumor microvascular density by blocking binding of HK to endothelial cells. We now evaluate the anti-metastatic effect of C11C1 on C57BL/6 mouse lung metastatic model using B16F10 melanoma cells. The tail veins of mice were injected with 0.5 × 10⁶ cells of melanoma B16F10. One group received C11C1 and the other received saline (control) intraperitoneally. When mice were killed at 28 days, 6 of 10 control mice had detectable metastatic pulmonary nodules which stained positive with an antibody against S-100 protein, a tumor antigen present in malignant melanoma cells. In the C11C1 groups, none of the mice showed metastatic foci in their lungs. We showed that C11C1 inhibits endothelial cell tube formation in a 3-D collagen fibrinogen gel model by inhibiting the rate of cleavage of HK by plasma kallikrein without changing the binding affinity for HK. These studies demonstrate that a monoclonal antibody to HK has the potential to prevent metastasis with minimal side effects.     

苦杏仁苷介导人肝癌细胞HuH-7凋亡的实验研究

摘要 目的：通过体内体外实验探讨苦杏仁苷在肝癌中的抗肿瘤作用。方法：MTT 法检测不同浓度的苦杏仁苷对肝癌 HuH-7细胞存活率的影响;DAPI染色法观察苦杏仁苷介导HuH-7细胞的凋亡形态学变化;流式细胞术检测苦杏仁苷干预后HuH-7细胞凋亡率变化;Western Blot法检测细胞凋亡相关蛋白Bax、Bcl-2的表达,计算Bax/Bcl-2的比值。建立裸鼠HuH-7细胞移植瘤模型,观察苦杏仁苷对荷瘤裸鼠移植瘤体积的影响。结果：体外实验结果证实苦杏仁苷能够诱导人肝癌HuH-7细胞凋亡的发生（P<0.05）。随着苦杏仁苷浓度的增加,HuH-7细胞的存活率降低,凋亡率升高,干预后Bax/Bcl-2比值明显升高（P<0.05）。体内实验结果表明,裸鼠HuH-7细胞移植瘤的体积增长速度减慢（P<0.05）。结论：苦杏仁苷能够诱导人肝癌HuH-7细胞和裸鼠HuH-7细胞移植瘤细胞发生凋亡,减缓肿瘤生长,从而发挥抗肿瘤作用。