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Effect of hydroxyurea and etoposide on transduction of human bone marrow mesenchymal stem and progenitor cell by adeno-associated virus vectors

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Acta Pharmacologica Sinica 2004年第2期25卷 70-76页

作者： Xiao-dong JU, Si-quan LOU2, Wei-guo WANG, Jian-qiang PENG3, Hua TIAN Department of Orthopaedics, Peking University Third Hospital, Beijing 100083 3Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100262, China Department of Orthopaedics Peking University Third Hospital 北京 100083 Institute of Virology Chinese Academy of Preventive Medicine 北京 100262

AIM: To study the effect of hydroxyurea and etoposide on transduction of human marrow mesenchymal and progenitor stem cells by adeno-associated virus (AAV). METHODS: Isolated human bone marrow mesenchymal stem and progenitor cells (hMSCs) were cultured in DMEM containing 10 % FBS or 5 % FBS and dexamethasone 1 μmol/L respectively. After being treated with hydroxyurea and etoposide, hMSCs were transduced by AAV-LUC. After two days luciferase activity (relative light unites per second or RLU/s) were tested, which indirectly reflected the relative transduction efficiency of different groups, and virus DNA was isolated by Hirt extraction for Southern hybridization. RESULTS: Transduction luciferase activity and transduction efficiency in cultures treated with hydroxyurea and etoposide were significantly higher than that in control cultures. Dividing cells had about 20-fold higher transduction efficiencycompared with control cells. Transduction efficiency in stationary cells was about 50 times higher than that in control cells. Southern analysis showed that hydroxyurea and etoposide enhanced second-strand DNA synthesis by rAAV. CONCLUSION: Hydroxyurea and etoposide could increase transduction efficiency of hMSCs by AAV vectors, and stationary cells were more sensitive to these drugs than dividing cells.

关键词： simian adenoviruses hydroxyurea etoposide mesoderm stem cells gene transfer techniques

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Efficient delivery of human clotting factor IX after injection of lentiviral vectors in utero

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Acta Pharmacologica Sinica 2004年第6期25卷 87-91页

作者： Xiao-guang CHEN1, Huan-zhang ZHU1, Ju-li GONG, Feng LI, Jing-lun XUE2 State Key Laboratory of genetic Engineering, Institute of genetics, Schoolof Life Sciences, Fudan University, Shanghai 200433,China Institute of genetics School of Life Sciences Fudan University State Key Laboratory of Genetic Engineering 上海 200433

AIM: To explore gene transfer feasibility for human clotting factor IX (hFIX) mediated by recombinant lentivirus in utero. METHODS: ICR mice fetus at 17-19 d gestation were received lentiviral vectors carrying hFIX cDNA under the control of liver specific promoter by intrahepatic injection. The expression and distribution of hFIX cDNA and possible immune responses against the hFIX were assessed by ELISA, PCR, RT-PCR, and immunohistochemistry, respectively. RESULTS: The serum hFIX protein were detected at different time points in all newborn mice, the highest level of hFIX was 50 μg/L and lasted for more than 30 d. Anti-hFIX antibody was not detected. hFIX cDNA was detected in liver, spleen, and heart. The expression of hFIX cDNA was only detected in liver. Besides, no germ line transmission was found at DNA and RNA levels, and no side effect associated with gene transfer was detected. CONCLUSION: The efficient delivery of hFIX can be achieved by prenatal gene transfer. It thus shows the feasibility of gene therapy for hemophilia in utero.

关键词： lentiviral vector factor IX gene transfer techniques in utero

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gene functional research using polyethylenimine-mediated in vivo gene transfection into mouse spermatogenic cells

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Asian Journal of Andrology 2006年第1期8卷 53-59页

作者： Li Lu Min Lin Min Xu Zuo-Min Zhou Jia-Hao Sha Laboratory of Reproductive Medicine Department of Histology and Embryology Nanjing Medical University Nanjing210029 China

Aim： To study polyethylenimine （PEI）-mediated in vivo gene transfection into testis cells and preliminary functional research of spermatogenic cell-specific gene NYD-SP12 using this method. Methods： PEI/DNA complexes were introduced into the seminiferous tubules of mouse testes using intratesticular injection. Transfection efficiency and speciality were analyzed on the third day of transfection with fluorescent microscopy and hematoxylin staining. The long-lasting expression of the GFP-NYD-SP12 fusion protein and its subcelluar localization in spermatogenic cells at different stages were analyzed with fluorescent microscopy and propidium iodide staining. Results： With the mediation of PEI, the GFP-NYD-SP12 fusion gene was efficiently transferred and expressed in the germ cells （especially in primary spermatocytes）. Transfection into Sertoli cells was not observed. The subcellular localization of the GFP-NYD-SP2 fusion protein showed dynamic shifts in spermatogenic cells at different stages during spermatogenesis. Conclusion： PEI can efficiently mediate gene transfer into spermatocytes. Thus, it might be useful for the functional research of spermatogenic-cell specific genes such as the NYD-SP12 gene. In our gtudy, the NYD-SP12 protein was visualized and was involved in the formation of acrosome during spermatogenesis. Our research will continue into the detailed function of NYD-SP12 in spermatocytes. （Asian J Androl 2006 Jan; 8： 53-59）

关键词： gene transfer techniques polyethylenimine NYD-SP12 gene spermatogenic cells spermatogenesis

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BPI_(700)-Fc∴1_(700) chimeric gene expression and its protective effect in a mice model of the lethal E. coli infection

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Chinese Medical Journal 2006年第6期119卷 474-481页

作者： KONG Qing-li GUAN Yuan-zhi JING Xue-fang LI Chen GUO Xiang-hua LU Zhe AN Yun-qing Department of Immunology Capital University of MedicalSciences Beijing 100069 China Department of Microbiology ＆ Parasitology Peking UnionMedical College Beijing 100005 China

Background Infections caused by gram-negative bacteria （GNB） often lead to high mortality in common clinical settings. The effect of traditional antibiotic therapy is hindered by drug-resistant bacteria and unneutralizable endotoxin. Few effective methods can protect high risk patients from bacterial infection. This study explored the protection of adeno-associated virus 2 （AAV2）-bacteriacidal permeability increasing protein 700 （BPI700） -fragment crystallizable gamma one 700 （Fcγ1700） chimeric gene transferred mice against the minimal lethal dose （MLD） of *** and application of gene therapy for bacterial infection. Methods After AAV2-BPI700-Fcγ1700 virus transfection, dot blotting and Western blotting were used to detect the target gene products in Chinese hamster ovary-K1 cells （CHO-Klcells）. Reverse transcription-polymerase chain reaction and immunohistochemical assay were carried out to show the target gene expression in mice. Modified BPI-enzyme linked immunosorbent assay was used to identify the target gene products in murine serum. The protection of BPI700-Fcγ1700 gene transferred mice was examined by survival rate after MLD E. coli challenge. Colony forming unit （CFU） count, limulus amebocyte lysate kit and cytokine kit were used to quantify the bacteria, the level of endotoxin, and proinflammatory cytokine. Results BPI1-99-Fcγ1 protein was identified in the CHO-K1 cell culture supernatant, injected muscles and serum of the gene transferred mice. After MLD E. coli challenge, the survival rate of AAV2-BPI700-Fcγ1700 gene transferred mice （36.7%） was significantly higher than that of AAV2-enhanced green fluorescent protein （AAV2- EGFP） gene transferred mice （3.3%） and PBS control mice （5.6%）. The survival rate of AAV2-BPI700-Fcγ1700 gene transferred mice treated with cefuroxime sodium was 65.0%. The bacterium number in main viscera, the levels of endotoxin and proinflammatory cytokine （tumor necrcsis factor-α and interleukin-1β） in serum of the AAV2-BPI

关键词： bacteriacidal permeability increasing protein fragment crystallizable gene transfer techniques minimal lethal dose Escherichia coli

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Erythropoietin gene transfer into rat testes by in vivo electropo-ration may reduce the risk of germ cell loss caused by cryptorchidism

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Asian Journal of Andrology 2005年第4期7卷 369-373,页

作者： Masaki Dobashi Kazumasa Goda Hiroki Maruyama Masato Fujisawa Division of Urology Department of Organs Therapeutics Faculty of Medicine Kobe University Graduate School of Medicine Kobe 650-0017 Japan Division of Nephrology and Rheumatology Niigata University Graduate School of Medical and Dental Sciences Niigata 951-8120 Japan

Aim： To investigate the effects of rat Erythropoietin （Epo） on spermatogenesis by transferring rat Epo gene into cryptorchid testes by means of in vivo electroporation. Methods： Sprague-Dawley rats with surgically-induced unilateral cryptorchidism were divided into three groups： the first group was given intratesticular injections of pCAGGS-Epo （pCAGGS-Epo group）, the second group was given intratesticular injections of pCAGGS （pCAGGS group）, and the third group were given intratesticular injections of phosphate-buffered saline （PBS group）. At the same time, square electric pulses of 30 V were applied six times with a time constant of 100 ms. One or two weeks after injection, each testis was weighed and the ratio of the total number of germ cells to that of Sertoli cells （G/S ratio） was calculated to evaluate the impairment of spermatogenesis. Ten testes taken from each of the three groups were examined at each time point. Results： The testicular weight after the injection of pCAGGS-Epo or pCAGGS control plasmid was （0.85 ± 0.08） g and （0.83 ± 0.03） g, respectively, at week 1 （P = 0.788） and （0.62 ± 0.06） g and （0.52 ± 0.02） g, respectively, at week 2 （P = 0.047）. At week 1, spermatids and sperm were more abundant in testes with pCAGGS-Epo than those in the control testes. At week 2, spermatids and sperm were hardly detected in either group. The G/S ratio was 23.27 ± 6.80 vs. 18.63 ± 5.30 at week 1 （P = 0.0078） and 7.16 ± 3.06 vs. 6.05 ± 1.58 at week 2 （P = 0.1471）, respectively. Conclusion： The transfer of Epo to rat testes by in vivo electroporation may reduce the risk of the germ cell loss caused by cryptorchidism.

关键词： electroporation gene transfer techniques erythropoietin spermatogenesis Epo

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