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.