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Retrovirus-Mediated Transfection of the Tissue-type Plasminogen Activator Gene Results in Increased Thrombolysis of Blood Clots

  • Xianhua Wu
  • Yongsheng Gong
  • Xuebing Ding
  • Gang Cheng
  • Weiya Yan
  • Xiaowei She
  • Changxing Wang
  • Xia Li
Original Article
  • 1 Downloads

Abstract

Tissue-type plasminogen activator (tPA) is involved in the lysis of blood clots. In this study, we attempted to target thrombolysis and enhance blood clot lysis by generating a construct (pLEGFP-N1-tPA) to integrate tPA gene into the genome of different cell lines. pLEGFP-N1-tPA construct was generated and used to target the tPA gene in different cell lines. The thrombolytic effects mediated by the supernatant from transfected HeLa cells and Linx cells were assessed using plasma thrombus plates. Furthermore, enhanced green fluorescent protein (EGFP), which was fused to the tPA gene in the pLEGFP-N1-tPA construct, was analyzed under the fluorescent microscope to assess tPA localization. We also monitored tPA activity and expression in the transfected cell lines. As part of the study, we successfully generated the pLEGFP-N1-tPA construct. The sequence of this construct was verified and the construct was subsequently used to generate the PT67/pLEGFP-N1-tPA cell line. The pLEGFP-N1-tPA construct was also used to transfect HeLa cells and Linx cells. We observed that supernatants from transfected cells were capable of lysing thrombi. In addition, tPA activity and tPA concentration were elevated in the latter supernatants and tPA was rapidly and stably expressed in the transfected cell lines. These results reveal a potentially important thrombolytic role for tPA-targeted gene therapy following cardiac valve replacement.

Keywords

Retroviridae tPA Heredity vector Thrombus Thrombolysis 

Notes

Acknowledgements

This study was funded by Natural Science Foundation of Jiangsu Province (grant number BK20141176). We thank Professor Xiangdong Fu (Department of Cellular and Molecular Medicine and Department of Pathology University of California, San Diego, La Jolla, CA, USA).

Funding

Funding was provided by Natural Science Foundation of Jiangsu Province (Grant No. BK20141176).

Compliance with ethical standards

Conflict of Interest

The authors declare that they have no conflict of interest.

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Thoracic-Cardiovascular SurgerySuzhou Municipal Hospital, Nanjing Medical UniversitySuzhouPeople’s Republic of China
  2. 2.Department of NephrologySuzhou Municipal Hospital, Nanjing Medical UniversitySuzhouPeople’s Republic of China

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