Transfection of adult degenerated intervertebral disc cells cultured in vitro with PCI-hTGF-beta 1 and determination of their expression product

Lei ZHANG; Shaowei SUN; Cong CHEN; Kang CHEN; Yougu HU; Hongjun ZHENG

ABSTRACT

BACKGROUND:

Human transforming growth factor-β1 gene can be used for gene therapy of the degeneration of intervertebral discs, but the key to the experiment is to construct its effective vector.

OBJECTIVE:

To determine whether or not adult degenerated intervertebrai disc cells cultured in vitro after transfected by eukaryotic expression vector can express the product of human transforming factor-βl, and to provide the experimental basis of gene therapy for intervertebral disc degeneration.

DESIGN:

Single sample experiment.

SETTING:

Traumatic Orthopedic Institute of Shandong Province and the Orthopedic Department of Weihai Municipal Hospital.MATERIALS The experiment was conducted at the laboratory of Traumatic Orthopedic Institute of Shandong Province between October 1999and January 2001. Intervertebral disc samples were from the operated patients with protrusion of irtervertebral disc after the patients were informed.Sample 1 was intervertebral disc at L4/5 from a 30-year-old woman; sample 2 was intervertebral disc at L5/S1from a 30-year-old woman.

METHODS:

① Culture of adult degenerated intervertebral disc cellsSamples ex vivo were taken back to the laboratory within 30 minutes; fibrous ring cells and myelin nucleus cells cultured primarily were collected.② Transfection Cells were put in the 24-well culture plate with 5.5×105cells in each well. Constructed PCI-hTGF-β1 eukaryotic expression vector was used to perform transfection, then transfected PCI group and nontransfected group were set. ③ The expression product of cells transfected for 48 hours was determined with immunohistochemical staining method.MAIN OUTCOME

MEASURES:

Comparison of absorbance of the positive cell product of eukaryotic expression vector PCI-hTGF-β1 in the primary fibrous ring cells and myelin nucleus cells in each group.

RESULTS:

① Sample 1 The absorbance of positive cell product of eukary otic expression vector PCI-hTGF-β1 in the primary fibrous ring cells and myelin nucleus cells was 3.49 and 3.69 times that in PCI group, and 3.55times that in non-transfected group. ② Sample 2 The absorbance of positive cell product of eukaryotic expression vector PCI-hTGF-31 in the primary fibrous ring cells and myelin nucleus cells was 3.56 and 3.46 times that in PCI group, and 3.43 times and 3.33 times that in non-transfected group.

CONCLUSION:

PCI-hTGF-31, as the effective eukaryotic expression vector in the transfection of transforming growth factor-31 gene to culture degenerated intervertebral disc cells in vitro, can transfect adult degenerated intervertebral disc cells cultured in vitro and obtain the high expression of human transforming growth factorβ1 gene.