表达miR-10a的重组腺病毒的构建与鉴定

Objective To develop a miR-10a-expressing recombinant adenoviral vector. Methods The EGFP gene in shuttle plasmid pDC316-EGFP-U6 was replaced by red fluorescent protein mCherry gene. Long DNA sequence coding miR-l0a was synthesized. After annealing, the double-stranded miR-10a-coding sequence was inserted into pDC316-mCherry-U6. The resultant pDC316-mCherryU6-miR-10a was co-transfected with adenoviral skeleton plasmid pBHGlox△E1, 3Cre into HEK293 cells. The replication-defective adenovirus Ad-miR-10a was purified, amplified, and tittered by plaque assay. The mCherry expression was detected by fluorescence microscope. The expression of miR-10a by Ad- miR- 10 a in HeLa cells was determined by RT-qPCR. Results The sequence of pDC316-mCherryU6-miR-10 a was confirmed by restriction enzyme digestion and sequencing. mCherry expression could be detected under fluorescence microscope. The titers of Ad-miR-10a was 1.8 × 107 pfu/mL, and the level of miR-10a expression in HeLa cells infected with Ad-miR-10a was 40 times higher than that with Admock. Conclusion A miR-10a-expressing recombinant adenovirs Ad-miR-10a has been successfully constructed. The strategy for artificial expression of siRNA is applicable for expression of miRNA.     

Recombinant adenovirus vector-mediated human MDA-7 gene transfection suppresses hepatocellular carcinoma growth in a mouse xenograft model

Hepatocellular carcinoma is one of the most common tumors in the world.The purpose of the present study was to investigate the inhibitory effects of adenoviral transduction of human melanoma differentiation-associated gene-7(MDA-7)gene on hepatocellular carcinoma,so as to provide a theoretical basis for gene therapy of the disease.The human MDA-7 gene was cloned into replication-defective adenovirus specific to HepG2 cells using recombinant virus technology.RT-PCR and Western blotting assays were used to determine the expression of human MDA-7 mRNA and MDA-7 protein in HepG2 cells in vitro.Induction of apoptosis by overexpression of the human MDA-7 gene was determined by flow cytometry.In-vivo efficacy of adenoviral delivery of the human MDA-7 gene was assessed in nude mice bearing HepG2 cell lines in vivo by determining inhibition of tumor growth,VEGF and CD34 expression,and microvascular density(MVD).The results showed that AdGFP/MDA-7 induced apoptosis of HepG2 cells in vitro and significantly inhibited tumor growth in vivo(P < 0.05).The intra-tumoral MVD decreased significantly in the treated tumors(P < 0.05).We conclude the recombination adenovirus AdGFP/MDA-7 can effectively express biologically active human MDA-7,which leads to inhibition of hepatocellular carcinoma growth.     

Construction and identification of a human liver specific microRNA eukaryotic expression vector

MiR-122 is one of the non-coding RNAs which showed its effects on the lipo-metablism, virus infection and HCC forming through regulation of liver gene expression. Its eukaryotic expression vector was constructed by using pSuper which was widely applied in the siRNA expression. The precursor of human miR-122 gene was amplified by polymerase chain reaction （PCR） from the human genomic DNA. The positive clones were screened by PCR and restriction enzyme digestion. The new expression vector of miR-122 was named pHsa-m122. PHsa-m122 and its controls were transfected to HepG2 cells. The miR-122 expression activity was evaluated by GFP122i sensor reporter plasmid through fluorescence detection and Western blot. It was shown that the fluorescence intensity of GFP122si and pHsa-m122 co-transfection group was weaker than that of the controls, so the functional activity of expressed miR-122 was detected. When HepG2 cells were co-transfected with HBV1.3 and pHsa-m122 plasmids, the results showed miR-122 may down-regulate the gene expression of HBV. The human liver specific microRNA eukaryotic expression vector of miR-122 was constructed successfully, which may facilitate further study of its function in the development of liver virus infection diseases and HCC. Cellular ＆ Molecular Immunology.     

过表达人抗菌肽LL37腺病毒构建及转染阴道上皮细胞

BACKGROUND: LL37, the only antimicrobial peptide of the cathelicidin family identified from the human, not only promotes the proliferation of endothelial cells, but also plays an important role in angiogenesis and re-epithelialization. OBJECTIVE: To construct a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene, and to detect the expression and secretion of LL37 after transfected into the canine vaginal epithelial cells. METHODS: The cDNA encoding LL37 was amplified by PCR. Recombinant adenovirus expression plasmid encoding LL37 and green fluorescent protein (EGFP) was constructed, and identified using restriction endonuclease technology and DNA sequencing method. Adenovirus particles were generated by cotransfecting the 293-packaging cell line. The adenovirus were collected, amplified and concentrated, and viral titers were determined by end-point dilution assay by applying serial dilutions of the purified viruses to 293 cells. Primary cultured canine vaginal epithelial cells were transfected by the recombinant adenovirus. The transfection efficacy was observed by fluorescence microscope, and the cultured supernatant was collected to determine the expression of LL37 by ELISA method at 1, 2, 3, 5 and 7 days after transfection. RESULTS AND CONCLUSION:The adenovirus vector GV314-LL37 with the titer of 3×109 pfu/mL was successfully constructed and identified by DNA sequencing methods. Canine vaginal epithelial cells were successfully isolated and cultured and grew stably. After transfection, vaginal epithelial cells could express the EGFP and LL37 efficiently in a time-dependent manner detected by fluorescence microscope and ELISA method. The transfection efficacy of EGFP reached to 89% at 72 hours. The level of LL37 in the cell culture supernatant in the transfection group was significantly higher than that in the control group, the highest expression of LL37 was found at 3 days that lasting for 7 days. In conclusion, the recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene is successfully constructed, which can express and secrete LL37 after transfected into canine vaginal epithelial cells, providing a foundation for constructing the tissue-engineered vagina possessing anti-infection and neovascularization. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

鼠IL-28重组腺病毒体外抗肺癌活性研究

目的 将已成功构建的mIL-28A重组腺病毒载体转染至肺腺癌细胞LA795,并对其抗肺癌细胞生物学活性进行研究.方法 将Ad-pshuttle-cmv-mIL-28A转染至LA795细胞,用PCR、免疫细胞荧光、Tunel、Annexin V及MTT法等进行检测.结果 LA795细胞转染Af-mIL-28A后,MiL-28A的mRNA基因表达明显增加,且细胞内明显表达IL-28蛋白,LA795细胞凋亡增多,细胞生长明显抑制.结论 成功构建的mIL-28A重组腺病毒载体转染至肺腺癌细胞LA795后表达IL-28,且可能通过促进细胞凋亡而抑制其一定程度的生长.

Abstract：

Objective To transfect the recombinant mIL-28A adenovirus vector into lung adenocarcinoma cell line LA795 and research its anticancer activity. Methods Transfected the constructed mouse IL-28(mIL-28) recombinant adenovirus vector into LA795 cell line, detected with PCR, immunocytal fluorescence, Tunel, Annexin V and MTT. Results Transfected with rAd-mlL-28A into the LA795 cells, mIL-28A gene expression products mRNA increased obviously, IL-28 expression was detected in cells obviously,apoptosis cell number increased, and the growth of LA795 cells transfected with rAd-mIL-28A were inhibited obviously. Conclusion The recombinant miL-28A adenovirus vector we have constructed, which expresses IL-28 when transfected to lung adenocarcinoma cell line LA795, inhibits growth of carcinoma cell to some extent, and may work by promoting the apoptosis of cancer cells.     

大鼠C-sis基因克隆及其真核表达载体的构建

BACKGROUND: C-sis proto-oncogene can promote tissue repair by inducing cell proliferation and inhibiting cell apoptosis. Therefore, C-sis may play a positive role in the repair of damaged liver tissue and the treatment of fulminant hepatic failure. OBJECTIVE: To construct pcDNA3.1/C-sis eukaryotic expression vector and detect its expression in BRL cells (the normal liver cells of rats) and rat liver cells in vivo. METHODS: The full-length coding sequence of C-sis gene was cloned through real time-PCR. pcDNA3.1/C-sis eukaryotic expression vector was constructed and sequenced, followed by transfected into BRL cells using liposome and injected into the rat liver via tail vein. Finally, its expression in BRL cells and rat liver cells in vivo was identified by fluorescence quantitative PCR and western blotting. RESULTS AND CONCLUSION: (1) The full length of encoding region of C-sis gene was successfully cloned. Sequencing proved that pcDNA3. 1/C-sis recombinant eukaryotic expression vector was constructed successfully. (2) The expression of C-sis was increased after transfected into BRL cells and rat liver. (3) These results provide basis for the subsequent study of the effect of C-sis gene on fulminant hepatic failure in rats. 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程     

Expression of TIMP-3 gene by construction of a eukaryotic cell expression vector and its role in reduction of metastasis in a human breast cancer cell line

The present study is aimed at studying the gene for TIMP-3,a mammalian tissue inhibitor,by constructing arecombinant eukaryotic cell vector for gene therapy in human breast cancer.We obtained the TIMP-3 genefrom the human placent by RT-PCR.TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vectorby means of gene cloning to construct pcDNA3.1 recombinant vector.Human breast cancer cell lineMDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent.Thenthe expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined.The correctconstruction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis,PCR amplication andnucleotide sequencing.Western blotting showed that the transfected cells were able to express TIMP-3,indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructedsuccessfully.Our experiments further indicated that the potential of metastasis was significantly reduced forthe transfected cell line MDA-MB-453.Cellular & Molecular Immunology.2004;1(4):308-310.     

人骨形成蛋白7重组腺病毒载体的构建及其在牙周膜细胞中的表达

Objective To investigate the expression of adenovirus-transfected human bone morphogenetic protein-7 (BMP-7) in human periodontal ligament cells(PDLCs) and its effect on PDLCs proliferation after transfection. Methods The BMP-7 fragment was amplified by PCR according to the pCMV-SPORT6-BMP-7, and the fragment was cloned into pShuttle-CMV-BMP-7, then it was homogenously recombined with pBHGE3 in 293 cells to obtain adenovirus expression vector containing BMP-7 (Ad-BMP-7). Ad-BMP-7 was identified and the titer of virus was measured after amplification and purification. Ad-RMP-7 transfected human PDLCs in vitro. The BMP-7 protein expression and proliferation of transfected PDLCs were detected by Western blot and MTT assay respectively. Results PCR analysis confirmed that the human BMP-7 gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 1.785×1012 pfu/ml. Expression of BMP-7 protein was detected in PDLCs transfected with Ad-BMP-7. The MTT test showed no significant difference between PDLCs transfected with or without Ad-BMP-7. Conclusions Adenovirus expression vector containing BMP-7 can transfect human PDLCs successfully with high expresion of BMP-7 in PDLCs in vitro.     

IL-24基因修饰的树突状细胞促进CIK细胞对肺癌细胞的杀伤作用

Objective To study the antitumor effect and mechanism of co-cultured cytokine-induced killer(CIK) cells and autologous DC modified with IL-24 gene on A549 cells in vitro. Methods DC and CIK cells were prepared routinely from human peripheral blood mononuclear cells(PBMC). Recombinant adenovirus vector pAdEasy-1-pTrack-CMV-IL-24 was extracted from DH5α, it was lineared with Pac I and transfected into A293 cells, and then the IL-24 recombined adenovirus(Ad-IL-24) was obtained. Ad-IL-24 was used to infect DC. The cells obtained were named DC-IL-24. RT-PCR and ELISA were used to evaluate the expression of IL-24 gene in transfected DC. The phenotypes change of DC were identified by flow cytometry analysis, the concen-tration of IL-12 and TNF-α in supernatant of DC were determined by EIJSA. The ability of CIK producing per-forin was measured by homolysis method. FCM was used to determine the cytotoxicity of cocultured CIK cells and autologous DC modified with IL-24 gene to A549 cells. Results We obtained the high titre of Ad-IL-24.IL-24 gene was transfered into DC successfully via Ad-IL-24. The green fluorescence was observed on DC by fluorescence microscope. The expression rate of CD80, CD83, HI.A-DR, CD40, CXCR4 on DC-IL-24 was sig-nificantly increased compared with that of the control group. DC-IL-24 produced markedly higher levels of IL-12 and TNF-α as compared with DC. DC-IL-24 can enhance the ability of CIK cells producing perforin. On com-parison with non-transfected DC co-cultured with CIK cells, transfected DC co-cultured with CIK cells had a sig-nificantly higher lytic activity against A549 cells. Conclusion IL-24 gene modification can enhance the anti-tu-moral immunity of DC. The mechanism of which might be related to the increased secretion of IL-12 and TNF-α, up-regulation expression of co-stimulatory molecules and MHC Ⅱ class molecules on DC, promoting the acti-vation and maturation of DC, and then enhancing CIK cells to generate specific anti-tumoral immunity.