Quantitation of alpha-fetoprotein messenger RNA in peripheral blood of nude mice and its relationship with tumor recurrence and metastasis after curative resection of hepatocellular carcinoma]

OBJECTIVE: To assess the level of alpha-fetoprotein (AFP) messenger RNA (mRNA) in peripheral blood of nude mice, and to study its relationship with tumor recurrence and metastasis after curative resection of hepatocellular carcinoma (HCC). METHODS: The metastatic model of human HCC in nude mice LCI-D20 was used in this study. Curative resection was performed at 10th day after tumor implantation in 28 nude mice. Interferon alpha-1b (IFN alpha-1b) was administered subcutaneously from the next day after resection, at doses of 3 10(7)U/kg/d (8 nude mice), 1.5 10(7) U/kg/d (8 nude mice) respectively in the treatment groups, and normal saline alone in the controlled group (12 nude mice). Thirty-five days after treatment, one milliliter of peripheral blood was taken and AFP mRNA was quantitatively analyzed using TaqMan real-time quantitative RT-PCR. The mice were sacrificed. The size of recurrent tumor was measured and the presence of lung metastases was observed. RESULTS: The liver recurrent rate, lung metastatic rate and positivity of AFP mRNA in the controlled group were all 100% (12/12), whereas it was 62.5% (5/8), 0% (0/8), 87.5% (7/8) respectively in the IFN alpha-1b 1.5 10(7)U/kg/d treated group. The recurrent tumor in liver of the IFN alpha-1b 1.5 10(7)U/kg/d treated group was much smaller than that of the controlled group (25 mm(3) 2 mm(3) vs 1143 mm(3) 3 mm(3), t =9.27, P<0.01), and the level of AFP mRNA was also lower than that of the controlled group [(85 6)copies/mug vs (955 2) copies/mug, t =4.33, P<0.01]. In the IFN alpha-1b 3 10(7)U/kg/d treated group, there was only one recurrent tumor (0.5 mm(3)), no lung metastasis, and the positivity of AFP mRNA was 0% (?(2)=11.67, P<0.01). CONCLUSIONS: These results suggest that the level of AFP mRNA in peripheral blood may indicate recurrence and/or metastasis after curative resection of HCC. TaqMan real time quantitative RT-PCR is a very sensitive and convenient method for detecting circulating cancer cells.     

重组人内皮抑素腺病毒抑制肝癌裸鼠移植瘤生长

目的 观察重组人内皮抑素腺病毒(Ad/hEndo)对人肝癌裸鼠移植瘤生长的影响。方法 人脐静脉内皮细胞ECV-304经Ad/hEndo感染后,western印迹检测人内皮抑素的表达。人肝癌BEL-7402细胞移植到裸鼠背脊部后,检测Ad/hEndo对肝癌移植瘤生长的抑制作用。逆转录聚合酶链反应(RT-PCR)检测肿瘤组织中内皮抑素mRNA的表达。分析人内皮抑素在裸鼠体内的表达分布。结果 Western印迹检测到人内皮抑素基因在ECV-304细胞内高效表达。Ad/hEndo明显抑制人肝癌BEL-7402裸鼠移植瘤生长(F=4.061,P<0.05)。Ad/hEndo组血管密度计数为6.88±1.08,DMEM组为13.60±1.71(t=9.216,P<0.01)。瘤内注射Ad/hEndo后3d,RT-PCR在肿瘤组织检测到内皮抑素mRNA的表达,7d后表达不明显。人内皮抑素蛋白主要分布在肿瘤组织。结论 腺病毒介导的人内皮抑素基因在体内、体外获得高效表达,并明显抑制肝癌裸鼠移植瘤的生长与血管生成。     

基质金属蛋白酶-9及其抑制物-1在原发性肝癌中的表达及临床意义

探讨肝细胞癌(HCC)中MMP-9及TIMP-1mRNA的表达,及其与HCC侵袭、转移等生物学行为的关系。使用RT-PCR法检测20例HCC及癌周组织手术切除标本的MMP-9、TIMP-1mRNA表达。结果显示HCC组的MMP-9、TIMP-1mRNA的相对含量均高于癌周对照组。有包膜浸润的HCC的MMP-9 mRNA表达明显高于无包膜浸润HCC。提示MMP-9、TIMP-1在HCC的浸润和转移中起重要作用。MMP-9 mRNA表达与肝癌细胞包膜浸润密切相关。     

Metastatic human hepatocellular carcinoma models in nude mice and cell line with metastatic potential

Metastatic human HCC model is needed for the studies onmechanism and intervention of metastatic recurrence,Byusing orthotopic implantation of histologically intacttissues of 30 surgical specimens,a patient-likemetastatic model of human HCC in nude mice (LCI-D20)and a low metastatic model of human HCC in nude mice(LCI-D35) have been established.All mice withtransplanted LCI-D20 tumors exhibited extremely highmetastatic ability including spontaneous metastasis toliver,lungs,lymph nodes and peritoneal seeding.Remarkable difference was also found in expression ofsome of the invasiveness related genes and growthfactors between the LCI-D20 and LCI-D35 tumors.PAI-1increased gradually following tumor progression in LCI-D20 model,and correlated with tumor size and AFP level.Phasic expression of tissue intercellular adhesionmolecule-1 in this model was also observed.Using cornealmicropocket model,it was demonstrated that the vascularresponse induced by LCI-D20 tumor was stronger than thatinduced by LCI-D35 tumor.Similar report on metastatichuman HCC model in nude mice and human HCC cell linewith metastatic potential was rarely found in theliterature.This LCI-D20 model has been widely used forthe studies on intervention of metastasis,including anti-angiogenesis,antisense approach,metalloproteinaseinhibitor,differentiation inducer,etc.It is concluded thatthe establishment of metastatic human HCC model in nudemice and human HCC cell line with metastatic potentialwill provide important models for the in vivo and in vitrostudy of HCC invasiveness,angiogenesis as well asintervention of HCC recurrence.     

Phase tissue intercellular adhesion molecule-1 expression in nude mice human liver cancer metastasis model

AIM To study the phase cancer tissue intercellular adhesion molecule-1 (ICAM-1) expression of human cancer metastasis model in nude mice, and to analyze the relationship between ICAM-1 expression and the metastasis and recurrence of hepatocellular cancinoma (HCC).METHODS HCC tissues from liver cancer metastasis model in nude mice (LCI-D20) was orthotopically implanted, and ICAM-1 expression in HCC tissues at different growing time were detected by immunodot blot. Tumor size, intrahepatic and extrahepatic metastasis foci were observed by naked eyes and under light microscope.RESULTS ICAM-1 was positively correlated to the tumor growing time (r=0.88, P＜0.01) and tumor size r=0.5, P＜0.05). It was higher in metastatic HCC than in nonmetastatic HCC (8.24±0.95 vs 3.03±0.51, P＜0.01). ICAM-1 content in cancer tissues increased suddenly after metastasis occurred and then maintained in a high level. ICAM-1 was also higher in multimetastasis group than in monometastasis group (10.05±1.17 vs 5.48±0.49, P＜0.05).CONCLUSION Tissue ICAM-1 could predict not only the metastasis of human liver cancer metastasis model in nude mice early and sensitively, but also the metastasis degree. So tissue ICAM-1 may be a potential index indicating the status of metastasis of HCC patients.     

Endostatin gene therapy for liver cancer by a recombinant adenovirus delivery

AIM: To investigate the expression of adenovirus-mediated human endostatin (Ad/hEndo) gene transfer and its effect on the growth of hepatocellular carcinoma (HCC) BEL-7402 xenografted tumors. METHODS: Immunohistochemistry analysis with an anti-endostatin antibody was preformed to detect endostatin protein expression in HCC BEL-7402 cells infected with Ad/hEndo. MTT assay was used to investigate the effects of Ad/hEndo on proliferation of human umbilical vein endothelial cells (HUVEC). Intra-tumoral injections of 1X10(9) pfu Ad/hEndo was given to treat BEL-7402 xenografted tumors in nude mice once weekly for 6 wk. Mice received injections of Ad/LacZ and DMEM were regarded as control groups. After intra-tumoral administration with Ad/hEndo, the endostatin mRNA expression in tumor tissue was analyzed by Northern blotting, and plasma endostatin levels were determined using enzyme-linked immunosorbent assay (ELISA). RESULTS: High level expression of endostatin gene was detected in the infected HCC BEL-7402 cells. Ad/hEndo significantly inhibited HUVEC cell proliferation by 57.2% at a multiplicity of infection (MOI) of 20. After 6-week treatment with Ad/hEndo, the growth of treated tumors was inhibited by 46.50% compared to the Ad/LacZ control group (t=2.729, P<0.05) and by 48.56% compared to the DMEM control group (t=2.485, P<0.05). The ratio of mean tumor volume in treated animals to mean tumor volume in the control animals (T:C ratio) was less than 50% after 24 d of treatment. Endostatin mRNA in tumor tissue was clearly demonstrated as a band of approximately 1.2 kb, which was the expected size of intact and functional endostatin. Plasma endostatin levels peaked at 87.52+/-8.34 ng/mL at d 3 after Ad/hEndo injection, which was significantly higher than the basal level (12.23+/-2.54 ng/mL). By d 7, plasma levels dropped to nearly half the peak level (40.34+/-4.80 ng/mL). CONCLUSION: Adenovirus-mediated human endostatin gene can successfully express endogenous endostatin in vitro and in vivo, and significantly inhibit the growth of BEL-7402 xenografted liver tumors in nude mice.     

大肠癌血管内皮生长因子的表达及其意义

目的：探讨血管内皮生长因子（VEGF）mRNA在大肠癌和癌周组织中的表达及其与临床特征之间的关系,为在分子水平干预肿瘤血管形成,预防大肠癌复发和转移打下基础。方法：采用逆转录－聚合酶链反应（RT－PCR）检测方法,对68例大肠癌手术标本癌和癌周组织中VEGF mRNA的表达水平进行了相对定量研究。结果：肿瘤组织中VEGF mRNA的表达率为67．6％（46／68）,癌周组织表达率为32．4％（22／68）;肿瘤组织中VEGF mRNA表达水平在浆膜浸润组,淋巴结转移,远处转移和Dukes D期组分别显著高于未侵及浆膜组,无淋巴结转移组,无远处转移和Dukes A,B,C期组;肿瘤组织中VEGF mRNA表达水平在肿瘤直径大的大肠癌组（＞5cm)与大小肠癌组（≤5cm)以及不同组织学分组组之间相比差异无显著性。结论：VEGF在大肠癌浸润和转移过程中发挥重要作用,肿瘤血管形成与大肠癌浸润和转移关系密切。     

Clinical value of apoptosis and angiogenesis factors in estimating the prognosis of hepatocellular carcinoma

Purpose Whereas some studies have indicated that the prognosis of hepatocellular carcinoma (HCC) was correlated to some apoptosis and angiogenesis factors: p53, survivin, matrix metalloproteinases (MMPs, including MMP-2 and MMP-9) and vascular endothelial growth factor (VEGF), other studies have failed to confirm this. The aim of the present study is to investigate the expression of p53, survivin, MMPs and VEGF in HCC and the relationship between these factors and the prognosis of HCC patients.Methods The expression of p53, survivin, MMP-2, MMP-9 and VEGF was measured by immunohistochemical assays in the liver resection specimens of 90 patients with HCC.Results The positive rate of p53, survivin, MMP-2, MMP-9 and VEGF was 33.3, 51.1, 60.0, 37.8 and 76.7%, respectively. The expression of MMP-2, MMP-9 and VEGF was correlated to the recurrence of HCC patients, respectively (P < 0.01). No correlation was found between the expression of apoptosis factors (p53 and survivin) and the recurrence of HCC patients, respectively (P > 0.05). The positive correlations were found between MMP-2 and VEGF (r = 0.32, P < 0.01), MMP-9 and VEGF (r = 0.24, P < 0.05). Significant differences of disease-free survival rates occurred among subgroups according to the expression of MMP-2, MMP-9 and VEGF (P < 0.01). Multivariate analysis revealed that macroscopically disseminated nodules, tumor micrometastasis, high serum alpha-fetoprotein level, positive expression of MMP-9 and VEGF were independent recurrence risk factors.Conclusions Our investigation revealed that p53 and survivin could not estimate the prognosis of HCC patients. Angiogenesis factors (MMPs and VEGF) positively correlated to the prognosis of HCC patients. The expression of MMPs and VEGF in HCC tissues could be regarded as a valuable indicator in estimating the prognosis of HCC patients.     

Relationship between expression of α-fetoprotein messenger RNA and some clinical parameters of human hepatocelular carcinoma

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Expression of platelet-derived endothelial cell growth factor and vascular endothelial growth factor in hepatocellular carcinoma and portal vein tumor thrombus

Purpose: Both platelet-derived endothelial cell growth factor (PD-ECGF) and vascular endothelial growth factor (VEGF) are known to promote the development of new blood vessels, which are fundamental to tumor growth and metastasis. We aimed at evaluating the gene expression of PD-ECGF and VEGF in hepatocellular carcinoma (HCC) and portal vein tumor thrombus (PVTT). Patients and methods: Surgical specimens (28 HCC, 28 nontumorous liver tissues and 18 PVTT) were studied by Northern blot analysis. The levels of PD-ECGF mRNA and VEGF mRNA expression were measured by densitometric scanning of the autoradiographs, and they were normalized to the level of expression of an internal control (glyceraldehyde-phosphate dehydrogenase) mRNA. Results: The expression rates of PD-ECGF mRNA in PVTT, HCC and nontumorous liver tissues were 77.8% (14/18), 67.9% (19/28) and 35.7% (10/28), being 88.9% (16/18), 75.0% (21/28) and 17.9% (5/28) respectively for VEGF mRNA. The expressions of PD-ECGF mRNA and VEGF mRNA were higher in HCC with PVTT than when PVTT was absent (P < 0.05). The PVTT was more often seen in patients with positive expression of both PD-ECGF mRNA and VEGF mRNA in HCC than in patients who were positive for only one of these factors or negative for both (P < 0.05). Conclusion: Both PD-ECGF and VEGF correlated well with the formation of PVTT of HCC. Received: 20 June 1999 / Accepted: 20 July 1999     

达肝素钠对肝癌生长转移抑制的实验研究

目的研究低分子肝素达肝素钠对肝癌生长转移的抑制作用。方法采用人肝癌裸鼠转移模型(LCI-D20)。40只模型鼠随机分成4组即对照组、化疗组(顺铂 氟尿嘧啶),达肝素钠组、联合组(顺铂、氟尿嘧啶与达肝素钠)。观察肿瘤大小和转移、抑瘤率、测血清甲胎蛋白(AFP)、肿瘤微血管密度(MVD)、CD31。结果对照组、化疗组、达肝素钠组、联合组的肿瘤体积分别为(25245±13367)mm3、(1610 ±1217)mm3、(5883±3131)mm3和(5556±2570)mm3;抑瘤率分别为0%、93.6%、76.7%和78.0%;MVD 分别为20.7±6.8、18.2±2.6、4.8±1.8和6.5±2.4;CD31分别为31.8±5.7、25.5±5.1、21.6±4.8和19.6±2.4;AFP分别为(121.8±31.4)ng/ml、(21.5±13.3)ng/ml、(75.6±29.7)ng/ml 和(55.8±38.0)mg/ml;肝转移率分别为80%、70%、20%和10%;肺转移率分别为70%、60%、20%和10%; 腹壁转移率分别为90%、60%、30%和30%;腹水形成率分别为20%、10%、0%和0%。化疗组、达肝素钠组、联合组分别与对照组比,对肝癌生长的抑制作用差异有统计学意义,F=9.191,P<0.01。达肝素钠对肝癌血管形成和转移有良好的抑制作用,与对照组及单纯化疗组比差异有统计学意义,F=4.937,P<0.01。结论低分子肝素达肝素钠通过抗肿瘤血管形成,对肝癌的生长与转移有抑制作用。     

循环血单核细胞GPC-3mRNA和甲胎蛋白对肝癌诊断与转移监测的潜在价值

目的分析肝癌患者外周血单核细胞中磷脂酰肌醇蛋白多糖(GPC)-3 mRNA表达及甲胎蛋白(AFP)对肝癌诊断与转移监测的价值。方法收集住院肝病患者外周血,分离单核细胞,制备总RNA,经逆转录合成GPC-3cDNA,以荧光定量PCR扩增并分析其对肝癌诊断与转移监测的价值。结果外周血单核细胞中GPC-3 mRNA阳性仅见肝癌患者,55例肝癌患者中39例阳性(70.9%);肝硬化、肝炎、转移性肝癌、非肝肿瘤患者及正常对照组中未检出阳性(χ2=26.773,P<0.001)。外周血单核细胞GPC-3 mRNA阳性表达与HBsAg阳性(χ2=14.601,P<0.001)、肝癌的TNM分期(χ2=15.252,P<0.001)、门静脉癌栓(χ2=45.658,P<0.001)及伴肝外转移显著相关(χ2=22.273,P<0.001),与瘤体直径、数目、AFP浓度及分化程度间无明显相关;与血AFP可互补诊断,提高肝癌诊断阳性率(90.9%)。结论循环血单核细胞GPC-3 mRNA分析,有助于肝癌诊断和肝外转移监测,对AFP阴性肝癌具互补诊断价值。     

Phase tissue intercellular adhesion molecule-1 expression in nude mice human liver cancer metastasis model

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p27kip1腺病毒重组体对人食管癌裸鼠移植瘤生长的抑制作用

D27kip1是新近发现的一种抑癌基因，研究表明p27kip1基因转移能显著抑制食管癌和胃癌细胞生长，该基因疗法在体内实验中是否同样有效值得进一步研究。目的：研究p27kip1腺病毒重组体对人食管癌裸鼠移植瘤生长的抑制作用。方法：将携带人p27kip1基因的重组腺病毒载体导人人食管癌裸鼠移植瘤中．测定肿瘤生长抑制率．免疫组化方法检测移植瘤中p27kip1和生存素(survivin)的表达。结果：经p27kip1基因治疗的裸鼠，肿瘤生长抑制率达64．1％，移植瘤中p27kip1呈高表达．生存素呈低表达。结论：p27kip1腺病毒重组体能显著抑制人食管癌裸鼠移植瘤的生长．上调p27kin1和下调生存素的表达可能是其雷要作用机制。     

Histologic features of venous invasion, expression of vascular endothelial growth factor and matrix metalloproteinase-2 and matrix metalloproteinase-9, and the relation with liver metastasis in pancreatic cancer

INTRODUCTION: Pancreatic cancer frequently is associated with venous invasion and hematogenous metastasis. AIMS: To determine morphologic features of invaded veins, intratumoral vascular composition, the correlation with liver metastasis, and expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9, and the mechanism of development of hematogenous metastasis. METHODOLOGY: We examined 32 patients with resected pancreatic cancer: 18 had postoperative liver metastasis, and 14 had no liver metastasis. Specimens were examined to determine the composition of veins and microvessels by staining of victoria-blue and CD34. We also investigated expression of VEGF, MMP-2, and MMP-9 by immunohistochemical staining. RESULTS: Venous invasion was detected in 31 of 32 patients. Invaded venous densities of middle- and large-sized veins were significantly higher in patients with liver metastasis than in those with nonliver metastasis, and they were related to MMP-2 and MMP-9 overexpression. Invaded veins with fragmentation of the lumen through cancer cells were considered to be an intravasation of cancer (destroyed type vein), and their numbers were significantly related to liver metastasis, and MMP-2 and MMP-9 overexpression. CONCLUSION: In conclusion, almost all the patients with pancreatic cancer showed venous invasion, indicating that invasion into large veins and destroyed type veins could be a risk factor for liver metastasis and that increased expression MMP-2 and MMP-9 were related to such invasion.     

Relationship between expression of alpha-fetoprotein messenger RNA and some clinical parameters of human hepatocellular carcinoma

AIM:To certify the relationship between AFP mRNA and some pathological parameters of hepatocellular carcinoma (HCC).METHODS:We detected the expression of AFP in mRNA level in tissue samples from 52 patients suffered from HCC by RT-PCR method.RESULTS:The positive rate of AFP mRNA was 76.9% in the HCC tumor tissues,and 69.4% in the paratumor tissues from the HCC patients with severe cirrhosis.However, in HCC patients without cirrhosis, the positive rate reached 50% in tumor tissues, but no AFP mRNA expression was found in the related paratumor tissues.CONCLUSION:The AFP protein was specially expressed by HCC cells and mutated hepatocytes.The AFP mRNA was positively related with cirrhosis, but no significant relationship was found between AFP mRNA and tumor size, capsule status and tumor metastasis.     

HBX蛋白、骨桥蛋白、基质金属蛋白酶-9在肝细胞癌组织中的表达及意义

目的探讨乙肝病毒X（HBX）蛋白、骨桥蛋白（OPN）、基质金属蛋白酶-9（MMP-9）表达在肝癌发生、发展中的作用。方法采用免疫组化法检测49份原发性肝细胞癌（HCC）组织及癌旁组织与10份正常肝组织中HBX蛋白、OPN、MMP-9的表达。结果三者在癌组织、癌旁组织中阳性率均明显高于正常肝组织（P均〈0.05）,均呈线性相关,癌组织转移者高于无转移者（P〈0.05）。结论HBX蛋白、OPN、MMP-9高表达可促进HCC的发生、发展与转移。     

~(125)Ⅰ粒子植入联合内皮抑素对A549肺腺癌裸鼠移植瘤生长抑制作用

目的研究放射性125I粒子植入联合重组人血管内皮抑素(Endostatin,ES)对A549肺腺癌移植瘤生长的抑制作用。方法建立移植瘤模型,24只裸鼠随机分成治疗组:单纯粒子内照射组,单纯内皮抑素组,内照射+内皮抑素联合组和空白对照组。治疗结束,免疫组化检测微血管内皮CD31及血管内皮生长因子(VEGF)的表达,蛋白免疫印迹方法测定VEGF的表达,TUNEL法检测肿瘤细胞凋亡。结果治疗15天后,内皮抑素组、125Ⅰ粒子组及联合治疗组瘤体积抑制率依次为69.65%、92.64%、116.4%;联合治疗组MVD较粒子组下降明显(P0.05);联合治疗组的细胞凋亡明显增多;内皮抑素对VEGF表达的影响无显著差异(P0.05)。结论 125Ⅰ粒子植入近距离放疗联合内皮抑素能较早抑制A549移植瘤的生长,通过减少放射治疗后肿瘤血管的再生及增加细胞的凋亡起到抑瘤作用。