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Updated: Aug 9, 2026
Retroviral Transduction of T-cell Receptors in Mouse T-cells
Published on: October 23, 2010
Anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection
Ke-qiang Li1, Wen-lin Li, Shu-you Peng
1Department of General Surgery, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China. lkq222jxmc@163.com
Background:
This study was designed to obtain a recombinant retroviral vector containing the human hepatocellular carcinoma-related gene ANGPTL4 (angiopoietin-like 4) cDNA and to evaluate the anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection.
Methods:
ANGPTL4 cDNA was cloned in vitro from normal human liver cells HL-7702 by using RT-PCR, and then subcloned into the plasmid vector pMSCV and sequenced. The retroviral plasmid vectors pMSCV-ANGPTL4, pVSV, and pGAG-POL were co-transfected into the packaging cell line 293 EBNA under mediation of lipofectamine. A high-titer retrovirus was obtained as a result, and HepG2 cells were infected with this retrovirus in vitro. Flow cytometry and fluorescence microscopy were used to detect expression of green fluorescence protein (GFP). The expression of ANGPTL4 mRNA in HepG2-ANGPTL4 cells was investigated using RT-PCR. The formation of tumors in nude mice and MTT assays were used to detect the growth of HepG2-ANGPTL4 cells in vivo and in vitro, respectively.
Results:
The cDNA sequence of the cloned ANGPTL4 gene was consistent with the recently reported sequence. Thus, the recombinant retroviral vector pMSCV-ANGPTL4 was constructed successfully. The titer of the packaged recombinant retrovirus was 1.4 x 10(6) infective viral grains/ml, and the rate of HepG2-ANGPTL4 cells expressing GFP was 68.45%, with an average intensity of fluorescence 31.67 times greater in HepG2-ANGPTL4 cells than in HepG2 cells. The expression of ANGPTL4 mRNA in HepG2-ANGPTL4 cells was higher than in HepG2-pMSCV cells (154% higher) or HepG2 cells (161% higher). The proliferation rate of HepG2-ANGPTL4 cells in vitro was obviously lower than those of HepG2-pMSCV cells and HepG2 cells (P <0.01). The mean volume and weight of tumors seeded from HepG2-ANGPTL4 cells were obviously lower than the mean volume or weight of tumors seeded from HepG2 cells and HepG2-pMSCV cells (P <0.01).
Conclusion:
A stable ANGPTL4-transfected human liver cancer cell line HepG2-ANGPTL4 has been created. The transfer of the human ANGPTL4 gene mediated by a retroviral vector is a possibly effective approach for liver cancer therapy.
Insights
This study successfully created a human liver cancer cell line (HepG2-ANGPTL4) using gene therapy. The angiopoietin-like 4 (ANGPTL4) gene transfer effectively inhibited liver cancer cell growth and tumor formation in mice.
Area of Science:
- Molecular Biology
- Gene Therapy
- Oncology
Background:
- Hepatocellular carcinoma (HCC) is a significant global health concern.
- Developing novel therapeutic strategies for HCC is crucial.
- The angiopoietin-like 4 (ANGPTL4) gene shows potential anti-tumor properties.
Purpose of the Study:
- To construct a recombinant retroviral vector carrying the human ANGPTL4 gene.
- To evaluate the anti-tumor efficacy of ANGPTL4 gene transfection in liver cancer cells.
- To establish a stable ANGPTL4-transfected human liver cancer cell line for further research.
Main Methods:
- Cloning of ANGPTL4 cDNA from human liver cells using RT-PCR.
- Subcloning into a retroviral vector (pMSCV) and sequencing.
- Co-transfection into packaging cells to produce high-titer retrovirus.
- Infection of HepG2 liver cancer cells with the retrovirus.
- Assessment of gene expression via RT-PCR and fluorescence microscopy.
- Evaluation of cell proliferation using MTT assays and tumor growth in nude mice.
Main Results:
- Successful construction of the pMSCV-ANGPTL4 recombinant retroviral vector.
- High titer retrovirus production (1.4 x 10^6 infective viral grains/ml).
- Significant expression of ANGPTL4 mRNA in transfected HepG2-ANGPTL4 cells (154-161% higher than controls).
- Marked inhibition of HepG2-ANGPTL4 cell proliferation in vitro (P <0.01).
- Significant reduction in tumor volume and weight in vivo (P <0.01).
Conclusions:
- A stable ANGPTL4-transfected human liver cancer cell line (HepG2-ANGPTL4) was successfully established.
- Retroviral vector-mediated transfer of the human ANGPTL4 gene demonstrates significant anti-tumor effects.
- This gene therapy approach holds promise as a potential treatment strategy for liver cancer.
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