Experimental Study On Construction Of Eukaryotic Expression Vector Of Insulin-like Growth Factor 1 Gene And Its Action On Rat Tendon Cells

It has been shown that recombinant human insulin-like growth factor 1(rhIGF-1) could stimulate tendon cells to proliferation and increase the production of collagen and proteoglycan in a dose-dependent manner. And promote flexor tendon healing in vivo. However, the short half-life of rhIGF-1 in vivo and lack of suitable stable vehicle for IGF-1 administration limited its potential application in tendon repair. Gene mediated directional transfection of tendon tissue will be a good way to fullfill the function of IGF-1. Primary rat flexor tendon cells were cultured by improving pieces culture technique and passaged successfully. The plasmids pAdlox-IGF-1 and pcDNA3.1(+) were transformed and then extracted from the competent DH5α strain. After digested by Hind III and BamHⅠ,the short fragment of pAdlox-IGF-1 and the long fragment of pcDNA3.1(+) were recovered and linked to construct pcDNA3.1(+)-IGF-1 with T4 DNA ligase in vitro. The vector was transferd to the secondary passage rat tendon cells mediated by lipopolyamine DOGS. 48 hours after the transfection, the total RNA and culture medium of tendon cells were collected for measurement of mRNA by RT-PCR and IGF-1 active protein by enzyme-linked immunoabsorbent assay(ELISA) respectively. To identify the influence of IGF-1 active protein on tendon cells, 3H-TdR incorporation and collagen type Ⅰ production of tendon cells were detected. The tendon cells which density was 2×105/ml were passaged to 96 well plate as 150μl per well. After 24 hours, 48 hours and 72 hours of the transfection, 1 μCi 3H-TdR per well was added to the cells and cultured continuously for six hours, then the cells were collected to assay the incorporation of the 3H-TdR respectively. Null vector transfection as the control. Moreover, both of the transfected tendon cells and the untransfected cells were passaged to grow on small slide for immunofluoresence of collagen typeⅠ. The results are below:1. Eukaryotic expression vector of pcDNA3.1(+)-IGF-1 was constructed successfully By digestion, recovery and ligation in vitro, the recombined eukaryotic expression vector pcDNA3.1(+)-IGF-1 was constructed and identified by digestion with the same two restriction endonuclease and sequencing with T7 promotor primer. Both of the results show that the vector was successfully constructed.2. Primary rat flexor tendon cells were cultured and passaged successfullyPrimary cultured rat flexor tendon cells were fusiform shape, deltoid and polygonal. In early days more of them were long fusiform shape. And finally most of them became polygonal and the antennas were very long and connect to each other. After passaging two generations, the cells began to aging and grow more and more weaker. Immunocytochemistry of collagen type Ⅰand type Ⅲ show that collagen type Ⅰ staining was very stronger than that of collagen type Ⅲ. It proved that the cultured cells were tendon cells. 3. The expression of IGF-1mRNAThe electrophoresis results of 1.5% sepharose of RT-PCR products show that there were two fragments that had been amplifyied in the transfected group. One is IGF-1mRNA(365 bp) and the other isβ-actin(441 bp). However, there was only oneβ-actin(441 bp) fragment found in the control group. The results manifested that IGF-1mRNA has expressed successfully in the transfected tendon cells. 4. The expression of IGF-1 biological active proteinEnzyme-linked immunoabsorbent assay(ELISA) of insulin-like grouth factor 1(IGF-1) of the cultured medium show that the content of IGF-1 of the experimental group was 111.71±18.60 ng/ml(±s), which was significant higher than that of the control 74.23±25.99 ng/ml(±s) (P﹤0.05). 5. 3H-TdR incorporation of the tendon cellsSix hours after adding the 3H-TdR to the tendon cells, 3H-TdR incorporation of the cells was assaied by 3H-TdR method. The results show that both of the experimental group and control group's incorporation peak of 3H-TdR occur at 48 hours after the transfection. The CPM of the transfected group was 2355.75±541.98(?...