【Abstract】 Objective To investigate the effect of IGF-1 on the growth of primary human embryonicmyoblasts. Methods The method of incorporation of 3H-TdR was used to evaluate the abil ity of prol iferation of myoblasts.The count per minute (CPM) values of myoblasts at different concentrations(1, 2, 4, 8, 16 and 32 ng/mL) of IGF-1 were measured,and dose-effect curves were drawn to choose the optional concentration of IGF-1 to promote the prol iferation. Then theexperimental group of myoblasts received the addition of the optional concentration of IGF-1 in the growth medium, the controlgroup just received the growth medium. The flow cytometry was used to detect the cell cycle . The method of incorporation of3H-TdR was used to measure the peak-CPM. The myotube fusion rate was measured in myoblasts with different concentrations(0, 5,10, 15, 20, 25 and 30 ng/ mL) of IGF-1 in fusion medium, the dose-effect curves were also drawn, so as to decided the optional concentrationof IGF-1 in stimulating differentiation. Fusion medium with optional concentration of IGF-1 was used in experimentalgroup, and the control group just with fusion medium. The fusion rate of myotube and the synthesis of creatine kinase(CK) weredetected in both groups. Results The optional concentration of 5 ng/mL IGF-1 was chosen for stimulating prol iferation . It was shown that the time of cell cycle of control was 96 hours, but that of the experimental group was reduced to 60 hours. The results of flow cytometry showed that the time of G1 phase, S phase and G2M phase was 70.03, 25.01 and 0.96 hoursrespectively in control group, and were 22.66, 16.47 and 20.87 hours respectively in experimental group. The time-CPM value curves showed that the peak-CPM emerged at 96 hours in control group and 48 hours in experimental group, which was in agreementwith the results of the flow cytometry. The optional concentration stimulating prol iferation was 20 ng/mL IGF-1. Compared with control, the quantity of CK was increased by 2 000 mU/mL and the fusion rate was elevated by 30% in experimental group. Conclusion The concentrations of 20 ng/mL IGF-1 can elevat obviously the fusion rate and the quantity of CK. IGF-1 can enhance the prol iferation and differentiation of myoblasts via inducing the number of myoblasts at G1 phase and increasing the number of myoblasts at S and G2M phases.
【Abstract】 Objective To investigate the secretion of target gene and differentiation of BMSCs transfected by TGF-β1 and IGF-1 gene alone and together into chondrocytes and to provide a new method for culturing seed cells in cartilage tissue engineering. Methods The plasmids pcDNA3.1-IGF-1 and pcDNA3.1-TGF-β1 were ampl ified and extracted, then cut by enzymes, electrophoresed and analyzed its sequence. BMSCs of Wistar rats were separated and purificated by the density gradient centrifugation and adherent separation. The morphologic changes of primary and passaged cells were observed by inverted phase contrast microscope and cell surface markers were detected by immunofluorescence method. According to the transfect situation, the BMSCs were divided into 5 groups, the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by TGF-β1 (Group C), the group transfected by IGF-1 (Group D) and the group transfected both by TGF-β1 and IGF-1 (Group E). After being transfected, the cells were selected, then the prol iferation activity was tested by MTT and expression levels were tested by RT-PCR and Western blot. Results The result of electrophoresis showedthat sequence of two bands of the target genes, IGF-1 and TGF-β1, was identical with the sequence of GeneBank cDNA. A few adherent cells appeared after 24 hours culture, typical cluster formed on the forth or fifth days, and 80%-90% of the cells fused with each other on the ninth or tenth days. The morphology of the cells became similar after passaging. The immunofluorescence method showed that BMSCs were positive for CD29 and CD44, but negative for CD34 and CD45. A few cells died after 24 hoursof transfection, cell clone formed at 3 weeks after selection, and the cells could be passaged at the forth week, most cells became polygonal. The boundary of some cells was obscure. The cells were round and their nucleus were asymmetry with the particles which were around the nucleus obviously. The absorbency values of the cells tested by MTT at the wavelength of 490 nm were0.432 ± 0.038 in group A, 0.428 ± 0.041 in group B, 0.664 ± 0.086 in group C, 0.655 ± 0.045 in group D and 0.833 ± 0.103 in group E. The differences between groups A, B and groups C, D, E were significant (P lt; 0.01). The differences between groups A and B or between C, D and E were not significant (P gt; 0.05)。RT-PCR and Western blot was served to detect the expression of the target gene and protein. TGF-β1 was the highest in group C, 0.925 0 ± 0.022 0, 124.341 7 ± 2.982 0, followed by group E, 0.771 7 ± 0.012 0, 101.766 7 ± 1.241 0（P lt; 0.01）; The expression of IGF-1 was the highest in group E, 1.020 0 ± 0.026 0, 128.171 7 ± 9.152 0, followed by group D, 0.465 0 ± 0.042 0, 111.045 0 ± 6.248 0 （P lt; 0.01）. And the expression of collagen II was the hignest in group E, 0.980 0 ± 0.034 0, 120.355 0 ± 12.550 0, followed by group C, 0.720 0 ± 0.026 0, 72.246 7 ± 7.364 0（P lt; 0.01）. Conclusion The repairment of cartilage defects by BMSCs transfected with TGF-β1 and IGF-1 gene together hasa good prospect and important significance of cl inic appl ication in cartilage tissue engineering.
Objective To investigate the effects of human insulin-like growth factor 1 (hIGF-1) gene transfected by recombinant adenovirus vector (Ad-hIGF-1) on the apoptosis of rabbit nucleus pulposus cells induced by tumor necrosis factor α (TNF-α). Methods The intervertebral disc nucleus pulposus were harvested from 8 healthy adult domestic rabbits (male or female, weighing 2.0-2.5 kg). The nucleus pulposus cells were isolated with collagenase II digestion and the passage 2 cells were cultured to logarithm growing period, and then they were divided into 3 groups according to culture condition: DMEM/F12 medium containing 10% PBS, DMEM/F12 medium containing 10% PBS and 100 ng/mL TNF-α, and DMEM/ F12 medium containing 10% PBS, 100 ng/ mL TNF-α, and Ad-hIGF-1 (multiplicity of infection of 50) were used in control group, TNF-α group, and Ad-hIGF-1 group, respectively. The results of transfection by adenovirus vector carrying hIGF-1 gene were observed by fluorescent microscopy; the expression of hIGF-1 protein was detected by Western blot, hIGF-1 mRNA expression by RT-PCR, and the cell apoptosis rate by TUNEL and flow cytometry. Results Green fluorescence was observed by fluorescent microscopy in Ad-hIGF-1 group, indicating that successful cell transfection. The expressions of hIGF-1 protein and mRNA were detected in Ad-hIGF-1 group by Western blot and RT-PCR, while the control group and TNF-α group had no expression. The cell apoptosis rates of TNF-α group, Ad-hIGF-1 group, and control group were 34.24% ± 4.60%, 6.59% ± 1.03%, and 0.40% ± 0.15%, respectively. The early apoptosis rates of TNF-α group, Ad-hIGF-1 group, and control group were 22.16% ± 2.69%, 5.03% ± 0.96%, and 0.49% ± 0.05%, respectively; the late cell apoptosis rates were 13.96% ± 4.86%, 10.68% ± 3.42%, and 0.29% ± 0.06%, respectively. Compared with TNF-α group, the cell apoptosis rates of Ad-hIGF-1 group and control group were significantly reduced (P lt; 0.05); the cell apoptosis rate of Ad-hIGF-1 group was significantly higher than that of control group (P lt; 0.05). Conclusion Ad-hIGF-1 could inhibit the apoptosis of nucleus pulposus cells induced by TNF-α.
Objective To investigate the effect of hydrostatic pressure and insulin-like growth factor 1 (IGF-1) on the expression of filamentous actin (F-actin) of temporomandibular joint disc cells in goats, and to analyze the F-actin changes of temporomandibular joint disc cells in vitro under hydrostatic pressure and IGF-1 stimulation. Methods The bilateral temporomandibular joint discs were harvested from 4 1-month-old goats, and temporomandibular joint disc cells were isolated with collagenase. Immunohistochemical staining for collagen type I and collagen type II was performed for identification. The cells at passages 2-3 were used; the experiment was divided into 4 groups according to different interventions: the cells were cultivated with complete medium in group A as control; the cells were intervened by hydrostatic pressure (0.2 MPa and 1 Hz for 3 hours) in group B, by complete medium containing IGF-1 (10 ng/mL) in group C, and by a combination of hydrostatic pressure (0.2 MPa and 1 Hz for 3 hours) and complete medium containing IGF-1 (10 ng/mL) in group D. The changes of F-actin at 24 and 72 hours after cultivation were observed by immunofluorescence staining. The cell fluorescence intensity was measured. Results The cultivated cells were identified to be temporomandibular joint disc cells by morphological observation and immunohistochemical staining. At 24 hours, fluorescence intensity of groups A and C was b and clear, with normal morphology of temporomandibular joint disc cells; F-actin arranged in disorder in group B, and F-actin was thinner with arrangement disorder in group D. At 72 hours, the F-actin arranged regularly in groups A and C; however, some F-actin became blurry with irregular arrangement, breakage, and pseudopodia in group B; and F-actin was thinner and ruptured formed in group D. With time passing, the fluorescence intensity of F-actin in groups A, B, and D had an increasing trend, showing significant differences between 24 hours and 72 hours (P lt; 0.05); but there was no significant difference between 24 and 72 hours in group C (t=0.284, P=0.781). At 24 hours, fluorescence intensity of F-actin was highest in group C and was lowest in group B, showing significant difference when compared with groups A and D (P lt; 0.05). At 72 hours, fluorescence intensity in groups B and D was significantly lower than that in groups A and C (P lt; 0.05), but there was no significant difference between groups B and D, and between groups A and C (P gt; 0.05). Conclusion Hydrostatic pressure may cause the F-actin breakage and rearrangement of temporomandibular joint disc cells, and IGF-1 can up-regulate the F-actin expression. Such effects may be correlated with the biological behavior of the temporomandibular joint disc cells.
Objective To study the effect of chitosan (CS) mediated insul in-l ike growth factor 1 gene (igf-1) transfection on the repair of articular cartilage defect. Methods Twelve 3-month-old healthy male rabbits weighting 2.0-2.5 kg were randomly divided into 2 primary groups, control and intervention groups (n=6 per group). Control group was further divided into normal control (left knee) and normal saline (NS) control (right knee) groups. While, intervention group was divided into CS (left knee) and CS/igf-1 intervention (right knee) groups. Cartilage defects were created in the knee joints except normalcontrol. Intra-articular injections of CS/igf-1 complex was administrated 2 times a week for 4 weeks in CS/igf-1 interventiongroup, 0.5 mL CS in CS intervention group, and 0.5 mL sal ine solution in normal control and sal ine control groups. At 28days after treatments, the cartilage samples were collected for histological observation and collagen type II and aggrecan mRNA evaluation. Results HE staining and toluidine blue staining revealed that CS/igf-1 and CS intervention could significantly stimulated cartilage regeneration accompanied with fibrosis and inflammatory cell infiltration, however, CS/igf-1 treatment resulted in the best repair of cartilage defect. In contrast, sal ine control group only showed fibrous tissue prol iferation and inflammatory cell infiltration without significant cartilage repairing. In terms of collagen type II and aggrecan gene expression, significant differences were observed in each pairwised comparison among 4 groups in the order of CS/igf-1 gt; CS gt; NS gt; normal control (P lt; 0.05). Conclusion In situ CS/ifg-1 complex transfection can enhance the formation of mesochondrium by upregulating collagen type II or aggrecan expression, which might enhance the repair of articular cartilage defect.
ObjectiveTo investigate the effect of phosphorylatable short peptide (pSP) conjugated chitosan (CS) (pSP-CS) mediated insul in-l ike growth factor 1 (IGF-1) gene and human interleukin 1 receptor antagonist (IL-1Ra) gene local transfection on the repair of articular cartilage defect. MethodsCo-expression plasmid pBudCE4.1-IL-1Ra+IGF-1, single gene expression plasmid pBudCE4.1-IL-1Ra and pBudCE4.1-IGF-1 were constructed and combined with pSP-CS to form pSP-CS/ pDNA complexes. Thirty 3-month-old healthy male New Zealand white rabbits, weighing 2.0-2.5 kg, double legs were randomly divided into 5 groups (n=12). Lateral femoral condyle articular surface was only exposed in sham-operated group (group A); full-thickness cartilage defects were created in the articular surface of the lateral femoral condyle of the knee in 4 intervention groups: pSP-CS/pBudCE4.1 (group B), pSP-CS/pBudCE4.1-IL-1Ra (group C), pSP-CS/pBudCE4.1-IGF-1(group D), and pSPCS/ pBudCE4.1-IL-1Ra+IGF-1 (group E). At 1 week after operation, intra-articular injection of pSP-CS/pDNA complexes was administrated 2 times a week for 7 weeks in each intervention group, the same volume normal sal ine in group A. The general condition of animal was observed after operation, and rabbits were sacrificed at 8 weeks. Knee joint synovial fluid was collected to measure the concentrations of the IL-1Ra and IGF-1 by ELISA; mRNA expressions of Aggrecan, matrix metalloproteinase 3 (MMP-3), and MMP inhibitor 1 (TIMP-1) were detected by real-time fluorescent quantitative PCR; the chondrogenic phenotype of nascent cells in the damage zone was identified by alcian blue-periodic acid/schiff (AB-PAS) histochemistry and Aggrecan immunohistochemistry staining. ResultsThirty experimental rabbits all survived to the end of experiment, without infection and death. Large amounts of exogenous proteins of IGF-1 and IL-1Ra were detected in the synovial fluid of 4 intervention groups. There were significant differences between groups D, E and group A in IGF-1 protein expression, and between goups C, E and group A in IL-1Ra protein expression (P < 0.05). Aggrecan and TIMP-1 mRNA expressions were significantly up-regulated in group E, simultaneously MMP-3 mRNA expression was significantly down-regulated when compared with groups C and D (P < 0.05). Varying degrees of cartilage repair appeared in groups C, D, and E, showing positive staining of AB-PAS and Aggrecan, and group E had better results than groups C and D (P < 0.05); inflammatory cell infiltration and fibrous tissue prol iferation were seen in the defect region of group B, without significant cartilage repairing. ConclusionpSP-CS is an ideal gene del ivery system for cartilage defect gene therapy; IL-1Ra and IGF-1 double gene transfection has better biologic effect on cartilage defect repair.
ObjectiveTo investigate the effects of micro-fracture and insul in-l ike growth factor 1 (IGF-1) in treatment of articular cartilage defect in rabbits. MethodsTwenty-four New Zealand white rabbits (aged, 4-6 months; weighing, 2.5-3.5 kg) were randomly divided into 4 groups (n=6):micro-fractures and recombinant human IGF-1 (rhIGF-1) treatment group (group A), micro-fracture control group (group B), rhIGF-1 treatment control group (group C), and blank control group (group D). Full thickness articular cartilage defects of 8 mm×6 mm in size were created in the bilateral femoral condyles of all rabbits. The micro-fracture surgery was performed in groups A and B. The 0.1 mL rhIGF-1 (0.01 μg/μL) was injected into the knee cavity in groups A and C at 3 times a week for 4 weeks after operation, while 0.1 mL sal ine was injected in groups B and D at the same time points. At 4, 12, and 24 weeks, the gross, histological, and immunohistochemical observations were performed, and histological score also was processed according to Wakitani's score criteria. The collagen contents in the repair tissues and normal patellofemoral cartilage were detected by the improved hydroxyproline (HPR) method at 24 weeks. Electron microscope was used to observe repair tissues of groups A and B at 24 weeks. Results All animals were survival at the end of experiment. At 24 weeks after operation, defect was repaired with time, and the repair tissue was similar to normal cartilage in group A; the repair tissue was even without boundary with normal cartilage in group B; and the repair tissue was uneven with clear boundary with normal cartilage in groups C and D. Histological staining showed that the repair tissues had no difference with normal cartilage in group A; many oval chondrocytes-l ike cells and l ight-colored matrix were seen in the repair tissues of group B; only a few small spindle-shaped fibroblasts were seen in groups C and D. Moreover, histological scores of group A were significantly better than those of groups B, C, and D (P<0.05) at 4, 12, and 24 weeks. Electron microscope observation showed that a large number of lacuna were seen on the surface of repair tissue in group A, and chondrocytes contained glycogen granules were located in lacunae, and were surrounded with the collagen fibers, which was better than that in group B. Collagen content of the repair tissue in group A was significantly higher than that in groups B, C, and D (P<0.05), but it was significantly lower than that of normal cartilage (P<0.05). Conclusion Combination of micro-fracture and rhIGF-1 for the treatment of full thickness articular cartilage defects could promote the repair of defects by hyaline cartilage.
ObjectiveTo observe the effect of lentivirus-mediated cyclooxygenase 2 (COX-2) and Aggrecanase-1 silencing and insulin-like growth factor 1 (IGF-1) in BMSCs after injecting into the knee joint cavity in cynomolgus monkeys with knee osteoarthritis (OA). MethodsBMSCs were isolated from the bone marrow of 10 donors. The lentivirus vector expressing genes of COX-2, Aggrecanase-1, and IGF-1 were constructed, and transfected into the third generation human BMSCs at 40 multiplicity of infection (virus group); BMSCs transfected with lentivirus-empty vector served as blank-virus group. The growth status and number of BMSCs were observed under inverted phase contrast microscope, and normal BMSCs were used as normal control group. At 1 week after transfected, the mRNA expressions of COX-2, Aggrecanase-1, and IGF-1 were detected with RT-PCR. Nine 3-year-old cynomolgus monkeys were selected to establish the OA model according to Hulth modeling method, and were randomly divided into 3 groups (n=3). At 6 weeks after remodeling, the right knee joint cavity was injected accordingly with 1 mL BMSCs (about 1×107 cells) in virus group and blank-virus group, with 1 mL of normal saline in the blank control group; the left knee served as normal controls. The general condition was observed after injection; at 1, 4, and 6 weeks, the concentrations of prostaglandin E2 (PGE2), IL-1, Aggrecanase-1, and IGF-1 of double knee liquid were detected with ELISA; at 6 weeks, MRI, general observation, histology method, and immunohistochemistry method were used to detect the knee cartilage changes and the expressions of COX-2, Aggrecanase-1, and IGF-1 were measured with RT-PCR. ResultsNo significant difference was found in cell morphology and growth curve between 2 groups after transfection. By RT-PCR, COX-2, and Aggrecanase-1 expressions were significantly reduced, IGF-1 expression was significantly increased in virus group when compared with normal control group and the blank-virus group (P < 0.05). All monkeys survived to the end of the experiment after injection. When compared with blank-virus group and blank control group, the concentrations of PGE2, Aggrecanase-1, and IL-1 significantly decreased and the concentration of IGF-1 significantly increased in the virus group (P < 0.05), but the indicators in 3 groups were significantly higher than those in the normal control group (P < 0.05). MRI showed that abnormal articular surface with high density could be found in virus group, blank-virus group, and blank control group, while the virus group had the minimum area. Gross observation and histological observation showed that the cartilage morphology of virus group, blank-virus group, and blank control group was accordance with early OA articular cartilage changes, but virus group was better than blank-virus group and blank control group in repair degree, whose improved Pineda score was significantly lower (P < 0.05). Immunohistochemical staining showed that the virus group had deeper dyeing with occasional brown particles and more chondrocytes than blank-virus group and blank control group. By RT-PCR, COX-2 and Aggrecanase-1 mRNA expressions of cartilage in virus group were significantly decreased, and IGF-1 expression was significantly increased when compared with blank control group and the blank-virus group (P < 0.05). ConclusionLentivirus-mediated multi-genes co-transfection in BMSCs can inhibit the expressions of COX-2 mRNA and Aggrecanase-1 mRNA, and enhance the IGF-1 mRNA expression, which decreases the concentration of inflammatory factors, and protects the joint cartilage effectively.
ObjectiveTo investigate the effect of daphnetin (DAP) combined with insulin-like growth factor 1 (IGF-1) gene transfection on chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) in rats.MethodsRat ADSCs were isolated and amplified by enzymatic digestion. The third generation ADSCs were treated with IGF-1 gene transfection as experimental group and normal ADSCs as control group. The cells of the two groups were treated with different concentrations of DAP (0, 30, 60, 90 μg/mL), respectively. Cell proliferation was detected by cell counting kit 8 (CCK-8) after cultured for 72 hours. After 14 days, real-time fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expressions of chondrocyte markers (collagen type Ⅱ and Aggrecan) in each group; and toluidine blue staining and collagen type Ⅱ immunohistochemical staining were performed.ResultsCCK-8 assay showed that with the increase of DAP concentration, the cell absorbance (A) value of the control group and the experimental group increased gradually (P<0.05). At the same DAP concentration, the cell A value of the experimental group was significantly higher than that of the control group (P<0.05). Real-time fluorescence quantitative PCR and Western blot showed that with the increase of DAP concentration, the relative mRNA and protein expressions of collagen type Ⅱ and Aggrecan in the control group did not change significantly, and there was no significant difference among the different concentration groups (P>0.05). But the mRNA and protein expressions of collagen type Ⅱ and Aggrecan in the experimental group increased gradually, and the 60 and 90 μg/mL DAP concentration groups were significantly higher than 0 μg/mL DAP concentration group (P<0.05). At the same DAP concentration, the relative mRNA and protein expressions of collagen type Ⅱ and Aggrecan were significantly higher in the experimental group than in the control group (P<0.05). Toluidine blue staining showed that with the increase of DAP concentration, there was no significant difference in cell staining between the control group and the experimental group. At the same DAP concentration, the cells in the experimental group were slightly darker than those in the control group. Immunohistochemical staining of collagen type Ⅱ showed that with the increase of DAP concentration, there was no significant difference in the cytoplasmic brown-yellow coloring of the cells in the control group. The cytoplasmic brown-yellow coloring of the cells in the experimental group gradually deepened, with 60 and 90 μg/mL DAP concentration groups significantly deeper than 0 μg/mL DAP concentration group. At the same DAP concentration, the color of the cells in the experimental group was significantly deeper than that in the control group.ConclusionDAP can promote the proliferation of ADSCs in rats. The differentiation of ADSCs into chondrocytes induced by DAP alone was slightly, but DAP combined with IGF-1 gene transfection has obvious synergistic effect to promote chondrogenic differentiation of ADSCs.
ObjectiveTo observe and compare the cytological and biological differences between human normal and degenerated nucleus pulposus (NP), and to investigate the repair effect of insulin-like growth factor 1 (IFG-1) and platelet derived growth factor (PDGF) on human degenerated NP.MethodsHuman degenerative and normal NP tissues were obtained from operative patients, a portion of which were processed into tissue sections and HE staining was performed to observe the morphological changes of nucleus pulposus cells (NPCs) before and after degeneration of NP. Immunohistochemistry staining was used to determine the expression levels of collagen type Ⅰ, collagen type Ⅱ, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax) proteins. Another portion of tissues were isolated and cultured and NPCs morphology was observed under inverted microscope. Western blot analysis was used to detect collagen type Ⅱ protein expression. Then, the gene transfection experiments were launched, including 4 groups, with group A designed as degenerated NPCs only, and groups B, C, and D of degenerated NPCs transfected with IGF-1 gene lentiviral particles, PDGF gene lentiviral particles, and lentiviral particles carrying IGF-1 and PDGF double genes, respectively. At 21 days after transfection, the cell morphology of each group was observed under inverted microscope, the positive rates of IGF-1 and PDGF of each group were measured by flow cytometry, and the expression of collagen type Ⅱ protein was detected by using immunohistochemistry staining and Western blot.ResultsHE staining showed that there were a large number of notochordal cells and a small number of chondrocytes in the central NP tissue of normal group, while the NPCs in degeneration group were significantly reduced, and a large proportion of fibrocartilage tissues were found in NP tissue. Immunohistochemistry staining showed that the percentages of collagen type Ⅰ and Bax protein-positive cells in degeneration group were significantly higher than those of normal group, while the percentages of collagen type Ⅱ and Bcl-2 protein-positive cells were significantly lower than those of normal group (P<0.05). Western blot showed that the relative expression level of collagen type Ⅱ protein in degeneration group was significantly lower than that in normal group (t=65.493, P=0.000). At 21 days after gene transfection, compared with group A, the cell viability of groups B, C, and D increased and the morphology became more regular. Flow cytometry showed that the percentages of IGF-1-positive cells in groups B and D were significantly higher than that in group A, and the percentages of PDGF-positive cells in groups C and D were significantly higher than that in group A (P<0.05). Immunohistochemistry staining showed that the positive stainings of collagen type Ⅱ in groups A, B, C, and D was (±), (+), (+), and (++), respectively. Western blot showed that the relative expression of collagen type Ⅱ protein in groups A, B, C, and D increased by degrees, and the differences between groups were significant (P<0.05).ConclusionBoth IGF-1 and PDGF can reverse the degeneration of intervertebral discs NPCs and they have synergistic effects, providing experimental basis for its application in clinical treatment approaches for degenerative disc disease.