目的：构建hTERT启动子和保留HBV同源序列的缺失点变hTERT启动子（pGL3del445）的真核表达载体，为进一步研究HBV与hTERT启动子的关系奠定实验基础。方法：通过双酶切及PCR方法从pCRTRTP载体上将全长及含有HBV同源序列的hTERT启动子的基因序列克隆到pPLbasic多克隆位点上，构建真核表达载体pGL3TRTP与pGL3Bdel445，将该两质粒与pRLtk共转染不同细胞系，评估其端粒酶启动子活性。结果：经测序证明成功构建了全长及含有HBV同源序列的hTERT启动子的真核表达载体pGL3TRTP和pGL3Bdel445，共转染实验中，证实在永生细胞系中重组子高效表达，在非永生正常细胞中重组子不表达。结论：构建的两种重组表达载体具有端粒酶启动子活性，在不同端粒酶表型的细胞系中其表达具有选择性。

AIM: To evaluate the inhibitory effect of antisense phosphorothioate oligonucleotide (asON) complementary to the initiator of human telomerase catalytic subunit (hTERT) on the growth of hepatoma cells. METHODS: The as-hTERT was synthesized by using a DNA synthesizer. HepG2.2.15 cells were treated with ashTERT at the concentration of 10 mmol/L. After 72 h, these cells were obtained for detecting growth inhibition, telomerase activity using the methods of MTT, TRAP-PCRELISA, respectively. BALB/c(nu/nu) mice were injected HepG2.2.15 cells and a human-nude mice model was obtained. There were three groups for anti-tumor activity study. Once tumors were established, these animals in the first group were administered as-hTERT and saline. Apoptosis of tumor cells was detected by FCM. In the 2nd group, the animals were injected HepG2.2.15 cells together with as-hTERT. In the third group, the animals were given as-hTERT 24 hours postinjection of HepG2.2.15 cells. The anti-HBV effects were assayed with ELISA in vitro and in vivo. RESULTS: Growth inhibition was observed in cells treated with as-hTERT in vitro. A significant different in the value of A570-A630 was found between cells treated with as-hTERT and control (P<0.01) by MTT method. The telomerase activity of tumor cells treated with as-hTERT was reduced, the value of A450 nm was 0.42 compared to control (1.49) with TRAP-PCR-ELISA. The peak of apoptosis in tumor cells given as-hTERT was 21.12%, but not seen in saline-treated control. A prolonged period of carcinogenesis was observed in the second and third group animals. There was inhibitory effect on the expression of HBsAg and HBeAg in vivo and in vitro. CONCLUSION: As-hTERT has an anti-tumor activity, which may be useful for gene therapy of tumors.

AIM: To establish a mice model of hepatitis B by using HBV-transgenic mice, and to transfer HBV-specific cytotoxic T lymphocytes (CTL) induced from syngeneic BALB/c mice immunized by a eukaryotic expression vector containing HBV complete genome DNA. METHODS: HBV DNA was obtained from digested pBR322-2HBV and ligated with the vector pcDNA3. Recombinant pcDNA3-HBV was identified by restriction endonuclease assay and transfected into human hepatoma cell line HepG2 with lipofectin. ELISA was used to detect the expression of HBsAg in culture supernatant, and RT-PCR to determine the existence of HBV PreS1 mRNA. BALB/c mice were immunized with pcDNA3-HBV or pcDNA3 by intramuscular injection. ELISA was used to detect the expression of HBsAb in serum. MTT assay was used to measure non-specific or specific proliferation ability and specific killing activity of spleen lymphocytes. Lymphocytes from immunized mice were transferred into HBV-transgenic mice (2.5

目的：探索利用内皮抑素（Endostatin）进行肿瘤抗血管基因治疗的效果与安全性。方法：以pVAX1为载体，构建含IgGγ链信号肽序列和Endostatin基因的重组载体pVAX2sEN。重组子经KpnI、EcoRI双酶切及测序鉴定。建立小鼠肝癌细胞H22动物模型，分别瘤内注射重组载体pVAX-sEN、空载体、生理盐水（NS），每周2次，测量肿瘤体积，ELISA检测肿瘤局部Endosatin的表达量，流式细胞术检测肿瘤细胞的凋亡。同时，100μg高剂量pVAX-sEN 裸DNA注射实验动物，观察动物的一般状况，20d后处死动物取其内脏病理切片H2E染色。结果：测序结果表明正确构建了含有信号肽及Endostatin的真核表达载体。瘤内注射pVAX-sEN后，ELISA检测到实验组肿瘤局部Endostatin表达量为（201±311）ng/ml，而空载体及NS对照组瘤内未见Endostatin特异性表达；瘤体测量证明pVAX-sEN可以抑制肿瘤的生长，重组载体组肿瘤的平均体积为（0.4±0.3）cm3，显著低于对照组[空载体和NS对照组分别为（1.6±0.4）cm3、（1.9±0.6）cm3，P<0.05。pVAX-sEN可以增加肿瘤细胞的凋亡，实验组、空载体和NS对照组肿瘤细胞的凋亡率分别为（51.9±3.16）%、（24.6±4.43）%、（18.8±1.2）%，实验组与两组差异均有统计学意义（P<0.01）。高剂量pVAX-sEN注射小鼠后，一般状况良好，病理切片H-E染色未见炎症及其他损伤表现。结论：pVAX-sEN载体体内应用可显著抑制小鼠种植性H22肝癌的生长，促进肿瘤细胞凋亡，高剂量应用无可见毒副作用，是一种具有开发潜力的安全有效基因治疗载体。

AIM: To explore a safe and efficient strategy of tumor therapy using anti-angiogenetic agents. METHODS: Endostatin gene with a signal sequence of human IgG γ chain was amplified by PCR and cloned into pVAX1 plasmid which was the only vector authorized by FDA in clinical trial to construct a recombinant plasmid named as pVAX-sEN. The recombinant plasmid was detected with EcoRI/KpnI and DNA sequencing. BALB/c mice bearing hepatocarcinoma cell line H22 were treated with naked pVAX-sEN or liposome-DNA complex in which the dose of DNA and the ratio of DNA and liposome were different from each other. To compare the efficiency of gene transfection, expression of endostatin at the treated tumor site was assayed with ELISA. To investigate the effect of pVAX1-sEN on hepatocellular carcinoma, pVAX-sEN either naked or in liposome-DNA complex was injected into BALB/c mice bearing H22, then the diameter of tumors was measured, microvessel density was detected by immunohistochemistry, endostatin expression in vivo was assayed at different time points. RESULTS: DNA sequencing showed the endostatin gene with the signal peptide was correctly cloned. In situ gene expression assay indicated that both the ratio of DNA and liposome and the dose of DNA could affect the gene transfection efficiency. Interestingly, naked pVAX-sEN had a similar in situ endostatin expression to pVAX-sEN with liposome. Animal experiments showed that pVAX-sEN together with pVAX-sEN-liposome complex could efficiently suppress the growth of mouse hepatoma cells. CONCLUSION: Naked endostatin plasmid intratumoral injection can get a similar gene transfection efficiency to liposome-DNA complex when used in situ.

AIM: To construct a novel HBV antisense RNA delivery system targeting hapatocellular carcinoma and study its inhibitory effect in vitro and in vivo. METHODS: GE7, a 16-peptide specific to EGFR, and HA20, a homologue of N-terminus of haemagglutinin of influenza viral envelope protein, were synthesized and conjugated with polylysin. The above conjugates were organized into the pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system, named AFP-enhancing 4-element complex. Hepatocelluar carcinoma HepG2.2.15 cells was used to assay the in vitro inhibition of the complex on HBV. Expression of HBV antigen was assayed by ELISA. BALB/c nude mice bearing HepG2.2.15 cells were injected with AFP-enhancing 4-element complex. The expression of HBV antisense RNA was examined by RT-PCR and the size of tumor in nude mice were measured. RESULTS: The AFP-enhancing 4-element complex was constructed and DNA was completely trapped at the slot with no DNA migration when the ratio of polypeptide to plasmid was 1:1.The expression of HBsAg and HBeAg of HepG2.2.15 cells was greatly decreased after being transfected by AFP-enhancing 4-element complex. The inhibitory rates were 33.4% and 58.5% respectively. RTPCR showed HBV antisense RNA expressed specifically in liver tumor cells of tumor-bearing nude mice. After 4 injections of AFP-enhancing 4-element complex containing 0.2mg DNA, the diameter of the tumor was 0.995cm

Objectives To construct a hepatoma di rected gene delivery system which could transfer presantisense RNA to liver cancer cells specifically, and to explore a new therapeutic strategy for hepatocellular carcinoma by blocking hepatitis B vi rus(HBV)with antisense RNA targeting hepatocellular carcinoma. Methods GE7 and HA20 were synthesized and mixed with pEBAF-as-preS2, a heDatocarcinOma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system named AFP-enhancing 4-element complex Nude mice bearing hepatocelluar carcinoma cells HepG2 2 1 5 were injected with AFP-enhancing 4-element complex via a tail vein Total RNA from tissues was extracted, and reversal transcription-ploymerase chain reaction(RT-PCR)was used to detect the expression of preS2 Different doses of AFP-enhancing 4-element complex was injected into nude mice at different time points, and tumor diameter was measured. Results AFP-enhancing 4-element complex was constructed successfully RT-PCR showed preS2 antisense RNA delivered by AFP-enhancing 4-element complex only expressed in liver tumor HepG2 2 1 5 cells of the mice After the treatment of AFP-enhancing 4-element complex with dose of 0 2 ug per mouse(once a week for 4 weeks), the mean tumor diameter of nude mice was significantly shorter than that of the control groups(0.995

目的 研究乙型肝炎病毒（HBV）转染后,细胞对肿瘤坏死因子（TNF）相关的凋亡诱导配体（TRAIL）诱导的凋亡的敏感度变化及作用机制。通过人肝癌细胞HepG2及转染了HBV全基因组的HepG212115细胞对TRAIL诱导凋亡的敏感度研究，探讨HBV感染对细胞凋亡的影响及其机制。方法 流式细胞仪检测TRAIL诱导的凋亡反应和细胞表面TRAIL蛋白的表达，酶联免疫吸附试验检测细胞培养上清中分泌型TRAIL蛋白质的表达，半定量逆转录聚合酶链反应检测TRAIL受体和其转导通路中凋亡拮抗分子FLIP的表达。结果 HepG2和HepG212115对TRAIL诱导的凋亡率分别为51.60%和9.12%。与HepG2相比，HepG212115细胞表面TRAIL蛋白质及其受体的表达都有不同程度的下调，而抵抗细胞凋亡的分子—FLIP的表达却显著上调（P<0.01），两细胞系上清中分泌型TRAIL的表达量差异无显著意义（P>0.05）。结论 HBV转染可以抵抗TRAIL诱导的细胞凋亡，可以下调细胞表面TRAIL蛋白质及其受体的表达，但使FLIP的表达显著上调。这能从一定程度上解释HBV感染逃逸机体免疫监视，持续存活，导致相应病理变化的机制。

AIM: To detect the expression of soluble TRAIL (TNF-related apoptosis inducing ligand, TRAIL) in the peripheral blood of HBV infected patients and try to elucidate whether the expression level of sTRAIL have any correlativity with the clinical staging, the expression level of HBV markers and the degree of liver damage. METHODS: 52 cases of HBV infected patientswere investigated, including 8 HBV carriers, 30 chronic hepatitis B, 11 cirrhotics and 3 HBV infection related hepatocellular carcinoma. Expression of soluble TRAIL and markers of the hepatitis B were mearsured by enzyme-linked immunosorbent assay. RESULTS: The expression level of sTRAIL in the peripheral blood of the HBV infected patients was significantly higher than that of healthy controls (1378.35 540.23 pg/ml vs 613.75 175.80 pg/ml, P<0.001). In the group of chronic hepatitis, the expression level of sTRAIL was coincident with the status of the disease and was significantly correlated with the level of ALT. In the group of cirrhosis and liver cancer, its expression level was significantly higher than that of the healthy persons and HBV carriers, but lower than that of the hepatitis B patients; meanwhile, the expression of sTRAIL did not have any correlativity with the functional indexes of the liver. CONCLUSION: The soluble TRAIL in the HBV infected people may participate in the liver damage. Our results indicated that the expression level of soluble TRAIL may reflect the ravage of liver caused by host immune reaction to a certain degree.

目的：观察针对乙型肝炎病毒（HBV）PreS2基因特异性反义寡核苷酸（asON）对转基因细胞HepG2.2.15表面的人类白细胞Ⅰ类抗原（HLA2I）基因表达的影响。方法：设计合成互补于HBV PreS2翻译起始区的反义寡核苷酸即序列Ⅰ，并设非互补序列作对照即序列Ⅱ，免疫细胞化学染色观察asON对HepG2.2.15表面表达的HLA-I类抗原的影响，用ELISA法动态检测细胞上清液中HBsAg和HBeAg的变化，放射免疫测定法检测asON对宿主细胞自身分泌蛋白2血清铁蛋白的影响。结果：HepG2.2.15细胞表面HLA-I类抗原表达降低，用药组和对照组相比有统计学意义（P<0.05）。asON能够抑制HBV表达HBsAg和HBeAg，对HBsAg和HBeAg的抑制率分别为66%和91%，而序列Ⅱ对HBsAg和HBeAg的抑制率均为11%。asON对宿主细胞自身分泌蛋白无影响，即对宿主细胞无毒性作用。结论：asON以序列特异性方式抑制HBV基因表达而对宿主细胞无毒性作用，HLA-I类抗原表达降低，这对减轻过度炎症反应，阻止慢性肝炎至重症肝炎的发生具有重要意义。