| Background: Lumbar disc herniation is a common disease of bone to low back pain asthe main clinical manifestations, seriously affecting the quality of life of patients. Thelumbar disc herniation mainly due to disc degeneration caused by the degeneration ofthe intervertebral disc. There is a large number of apoptotic cells in the intervertebraldisc. Studies have shown that in vitro transfection of adenovirus vector with hIGF-1gene can inhibit the apoptosis of rabbit chondrocytes, suggesting that it could inhibit theapoptosis of the nucleus pulposus cells which is analogous with cartilage cells.Objective: In this study model of rabbit intervertebral disc nucleus pulposus cellsculture in vitro was established. The objective of the research was to investigate theeffects of insulin-like growth factor-1(IGF-1) gene on the apoptosis of rabbits nucleuspulposus cells induced by Tumor necrosis factor-α(TNF-α), and to reveal itsanti-apoptosis mechanism, through the transfection of adenovirus vector with hIGF-1gene. It would provide a theoretical basis for the treatment of lumbar disc degenerationby hIGF-1gene.Method:1. The intervertebral disc nucleus pulposus were taken out from the healthydomestic rabbit. First nucleus pulposus were cells isolated with collagenase Ⅱdigestion and cultured in monolayer. And they were purificated with differentialadhesion method and passaged with trypsin digestion.2. The adenovirus vector withhIGF-1gene were constructed by Biowit Technologies Ltd.3. Processing according tothe control group, Ad-hIGF-1group(Ad-hIGF-1,TNF-α co-processing) and TNF-αgroup (TNF-α processing).4. Results of transfection by adenovirus vector with hIGF-1gene were observed by fluorescence microscopy, hIGF-1protein was detected by Western-Blot, hIGF-1mRNA was detected by rt-PCR, TdT-mediated dUTP-biotin nickend labeling (TUNEL), and apoptosis rate of early apoptotic cells and late apoptosis rateof each group were detected by flow cytometry after Annexin-v/PI double stainning asindicators of apoptosis.Results:1. Most of the cells with green fluorescence under fluorescence microscopeshowed that the transfection of adenovirus vector with hIGF-1gene was succesful.cellstransfected, most of the cells with green fluorescence. The expression of hIGF-1proteinwas detected in Ad-hIGF-1group by Western-Blot, while the control group and TNF-αhad no expression of hIGF-1protein. The hIGF-1mRNA was detected in Ad-hIGF-1group by rt-PCR, while the control group and TNF-α had non-hIGF-1mRNA.2. Indetection of flow cytometry displayed: Early apoptosis rate of control group is (0.62±0.12)%. Apoptosis rate of non-viable apoptotic cells of control group is (0.29±0.06)%.Early apoptosis rate of TNF-α group is (34.40±4.42)%. Apoptosis rate of non-viableapoptotic cells of TNF-α group is (13.95±4.86)%. Early apoptosis rate of Ad-hIGF-1group is (6.47±1.13)%. Apoptosis rate of non-viable apoptotic cells of Ad-hIGF-1group is (10.68±3.42)%. Compared with TNF-α group, apoptosis rate of earlyapoptotic cells and non-viable apoptotic cells of Ad-hIGF-1group was significantlyreduced. There was a significant difference (P<0.05). Compared with control group,apoptosis rate of early apoptotic cells and non-viable apoptotic cells of TNF-α groupwas significantly reduced. There was a significant difference (P<0.01).3. In detection ofTUNEL displayed: Apoptosis rate of control group is (0.40±0.15)%, TNF-α groupapoptosis rate is (34.24±4.59)%, Ad-hIGF-1group of apoptosis rate is (6.59±1.02)%.Compared with TNF-α group, apoptosis rate of Ad-hIGF-1group cells apoptosis wassignificantly reduced. There was a significant difference (P<0.01). Compared with thecontrol group, apoptosis rate of TNF-α group was significantly increased, the differencewas significant(P<0.01).Conclusion:1. The transfection of adenovirus vector on rabbit nucleus pulposus cells invitro was successful in this study. Green fluorescent was strongest at24h, decreased at48h, and disappeared at72h after transfection.2. Apoptosis of nucleus pulposus cellscultured in vitro can be induced by TNF-α.3. It is indicated that Ad-hIGF-1could inhibit the apoptosis of nucleus pulposus cells induced by TNF-α. |
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