RESUMEN
This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models of intervertebral disc degeneration (IDD). Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix (ECM)-related genes (aggrecan and type II collagen) were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding (P<0.05). The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance (A) value at passages 4-7 was obviously decreased as compared with that at passage 1 (P<0.05). Cell cycle analysis showed that the proportion of normal NP cells at Gl phase was 65.4%±3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase with the value being 77.6%±4.8%. The degenerated NP cells were predominantly arrested at G1 phase and failed to enter S phase. The expression of type II collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.
RESUMEN
This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models of intervertebral disc degeneration (IDD). Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix (ECM)-related genes (aggrecan and type II collagen) were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding (P<0.05). The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance (A) value at passages 4-7 was obviously decreased as compared with that at passage 1 (P<0.05). Cell cycle analysis showed that the proportion of normal NP cells at Gl phase was 65.4%±3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase with the value being 77.6%±4.8%. The degenerated NP cells were predominantly arrested at G1 phase and failed to enter S phase. The expression of type II collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.
Asunto(s)
Animales , Conejos , Apoptosis , Fisiología , Ciclo Celular , Fisiología , Proliferación Celular , Tamaño de la Célula , Supervivencia Celular , Fisiología , Células Cultivadas , Disco Intervertebral , Biología Celular , Degeneración del Disco Intervertebral , Patología , Valores de ReferenciaRESUMEN
<p><b>OBJECTIVE</b>To construct the HPV16 L1 prokaryotic expression plasmid and to optimize its expression.</p><p><b>METHODS</b>A pair of primers was designed according to plasmid sequences of pGEX-KG and the HPV16L1 genes published by GeneBank. The DNA fragment of 1500 bp was amplified by PCR from the HPV recombinant plasmid with HPV16L1 gene, then cloned into pGEX-KG and transformed into the host E.coli strain JM109. The pGEX-KG-HPV16L1 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degree, the expression product of HPV16L1 gene was identified by SDS-PAGE and Western blot.</p><p><b>RESULTS</b>HPV16L1 fusion protein was expressed successfully in the form of inclusion bodies. The molecular weight was 83 kD. Meanwhile, the optimum condition of HPV16L1 fusion protein expression was induced with 1.0 mmol*L(-1) IPTG for 4 h. The fusion protein reacted specifically with antibodies against HPV16L1.</p><p><b>CONCLUSION</b>The prokaryotic expression vector of HPV16L1 gene has been constructed and expressed in E.coli successfully.</p>