Objective To verify the potential of the recombinant adeno-associated virus 2 (rAAV2) vector as a strategy for human transforming growth factor β1 (hTGF-β1) gene transfer in degenerative intervertebral discs of rabbit, to investigate the gene transduction efficacy and to quantify the biologic effects on the proteoglycan level after gene transferring. Methods Rabbit models of disc degeneration were established by injecting the 25 μL fibronectin fragment (Fn-f, 1 mmol/ L), 4 weeks later,saline with or without virus was injected directly into 96 lumbar discs of 24 mature New Zealand white rabbits (male or female and weighing 1.7-2.2 kg) which were divided into 3 groups (n=8). Group A received the 25 μL rAAV2-hTGF-β1 (1 × 1012 vg/mL); group B received rAAV2-enhanced green fluorescent protein (rAAV2-EGFP); and group C received PBS. Two rabbits of groups A, C were killed 1 week after injection, the immunohistochemical staining for hTGF-β1 was performed on the sl ices of nucleus pulposus (NP) tissues. At 4, 8, and 12 weeks after gene transferring, NP tissues were harvested and cultured to quantify the changes of the proteoglycan level using 35S-sulfate incorporation assay. The expression of EGFP in group B was observed 12 weeks after injection. Results Immunohistochemical staining showed that extensive and intense positive immunohisochemical staining for hTGF-β1 were seen in group A when compared with group C 1 week after gene transferring. The nucleus pulposus tissues from the group A exhibited an increased synthesis of proteoglycan, which was significantly more than that from groups B and C (P lt; 0.05), and no significant difference was observed between group B and group C. The expression of EGFP in group B was high at 12 weeks. Conclusion The discs injected with rAAV2-hTGF-β1 can highly expressed the therapeutic proteins for more than 12 weeks, it is suggested that rAAV2 should be an valid vector for transferring exogenous genes in the degenerative disc. The therapeutic factors hTGF-β1 can efficiently increase the proteoglycan synthesis of the degenerative NP cells.
Objective Bone marrow mesenchymal stem cells (BMSCs) transplantation can potentially regenerate the degenerated intervertebral disc, with the underlying regenerating mechanism remaining largely unknown. To investigate the potential of human BMSCs protecting nucleus pulposus cells (NPCs) from oxidative stress-induced apoptosis in a coculturesystem, and to illustrate the possible mechanisms of BMSCs transplantation for intervertebral disc regeneration. Methods BMSCs collected by density gradient centrifugation in Percoll solution were cultured and sub-cultured till passage 3, and the surface molecules of CD34, CD45, and CD13 were identified. NPCs were isolated by collagenase digestion and the chondrocyte l ike phenotype was confirmed by morphologic observation after HE staining, inverted phase contrast microscope, proteoglycan, and collagen type II expression after toluidine blue and immunocytochemistry staining. The 3rd passage BMSCs and the 1st passage NPCs were divided into four groups: group A, NPCs (1 × 106 cells) were cultured alone without apoptosis inducing (negative control); group B, NPCs (1 × 106 cells) were co-cultured with BMSCs (1 × 106 cells) with apoptosis inducing; group C, NPCs (1 × 106 cells) were co-cultured with BMSCs (3 × 105 cells) with apoptosis inducing; group D, NPCs (1 × 106 cells) were cultured alone with apoptosis inducing (positive control). After 3 or 7 days of culture or co-culture, the NPCs in groups B, C, and D were exposed to 0.1 mmol hydrogen peroxide for 20 minutes to induce apoptosis. With DAPI staining cellular nucleus, Annexin-V/propidium iodide staining cellular membrane for flow cytometry analysis, the apoptosis of NPCs in each group was studied both qual itatively and quantitatively. Besides, the changes in Bax/Bcl-2 gene transcription and Caspase-3 protein content, were analyzed with semi-quantitative RT-PCR and Western blot. Results BMSCs were successfully isolated and CD34-, CD45-, and CD13+ were demonstrated; after isolated from degenerated intervertebral discs and sub-cultured, the spindle-shaped 1st passage NPCs maintained chondrocyte phenotype with the constructive expressions of proteoglycan and collagen type II in cytoplasm. DAPI staining showed the nucleus shrinkage of apoptosis NPCs. Co-cultured with BMSCs for 3 days and 7 days, the apoptosis rates of NPCs in groups B (29.26% ± 8.90% and 18.03% ± 2.25%) and C (37.10% ± 3.28% and 13.93% ± 1.25%) were lower than that in group D (54.90% ± 5.97% and 26.97% ± 3.10%), but higher than that of groupA (15.67% ± 1.74% and 8.87% ± 0.15%); all showing significant differences (P lt; 0.05). Besides, semi-quantitative RT-PCR showed Bcl-2 gene transcription up-regulated (P lt; 0.05) and no significant change of Bax (P gt; 0.05); Western blot result showed that the Caspase-3 protein expression of groups B and C was lower than that of group D, and was higher than that of group A; all showing significant differences (P lt; 0.05). Conclusion In a co-culture system without direct cellular interactions, the oxidative stress-induced apoptosis of human NPCs was amel iorated by BMSCs. The enhanced anti-apoptosis abil ity of NPCs preconditioned by co-culturing with BMSCs might come from the decreased Bax/Bcl-2 gene transcription ratio.
Objective Toreview theresearch progress of nucleus pulposus cells phenot ypic markers. Methods The domestic and international l iterature about nucleus pulposus cells phenotypic markers was reviewed extensively and summarized. Results Due to different biomechanical properties，nucleus pulposus cells and articular chondrocytes have differences in morphology and extracellular components such as the ratio of aggrecan to collagen type II α1. Nucleus pulposus cells can be identified by surface marker (CD24), gene markers (hypoxia inducible factor 1α, glucosetransporter protein 1, matrix metalloproteinase 2, vascular endothel ial growth factor A, etc), and various markers (keratin 19 and glypican 3，paired box 1, forkhead box F1 and integrin-binding sialoprotein, etc). Conclusion Nucleus pulposus cells and articular chondrocytes have different phenotypic markers, but nucleus pulposus cells are still lack of specific markers.
Objective The biological treatment of intervertebral disc degeneration becomes a research hotspot in recentyears. It is necessary to find an effective approach to induce bone marrow mesenchymal stem cells (BMSCs) differentiate to disc cells which could make appl ication of cell transplantation as a treatment of intervertebral disc degeneration. To investigate the effects of the recombinant plasmid pcDNA3.1IE-SOX9Flag on differentiation of rabbit BMSCs into nucleus pulposus-l ike cells. Methods The eukaryotic expression vector of pcDNA3.1IE-SOX9Flag was constructed. Rabbit BMSCs were isolated and cultured from one-month-old New Zealand white rabbits and were induced into osteogenetic cells in the osteogenesis supplement medium; and the cell surface markers were detected by flow cytometry. The cells at the 3rd passage were randomly divided into 3 groups: in transfected group, the cells were transfected with recombinant plasmid pcDNA3.1IE-SOX9Flag; in negative control group, the cells were transfected with plasmid pcDNA3.1; and in blank control group, the cells were treated with the media without recombinant plasmid. After selected by G418 for 7 days, the cells were harvested and RT-PCR was employed to assay SOX9 mRNA and collagen type II gene (Col2al) mRNA expressions in BMSCs. The expression of SOX9 protein was assayed by Western blot and collagen type II expression was also observed by immunohistochemical staining. Results The SOX9 eukaryotic expression vector was constructed successfully. The BMSCs after 5 days of osteogenetic induction were positive for the alkal ine phosphatase staining. What was more, CD44 expression was positive but CD34 and CD45 expressions were negative. The transfection efficiency was 34.32% ± 1.75% at 72 hours after transfection. After 2 weeks of transfection, BMSCs turned to polygonal and ell iptical. And the cell prol iferation was gradually slow which was similar to the growth characteristic of nucleus pulposus cells. RT-PCR identification showed that SOX9 mRNA and Col2al mRNA expressions were positive in transfected group, and were negative in 2 control groups. Western blot detection showed that SOX9 protein expressed in transfected group but did not express in the control groups. At 2 weeks after transfection, the result of the immunohistochemicalstaining for collagen type II was positive in transfected group. Conclusion The recombinant plasmid pcDNA3.1IE-SOX9Flag can be successfully transfected into rabbit BMSCs, the transfected BMSCs can differentiate into nucleus pulposus-l ike cells, which lays a theoretical foundation for treatment of intervertebral disc degeneration with BMSCs transplantation.
Objective To introduce the research of nucleus pulposus cells for treating intervertebral disc degeneration. Methods The original articles in recent years about nucleus pulposus cells for treating intervertebral disc degeneration were extensively reviewed, and retrospective and comprehensive analysis was performed. Results Nucleus pulposus cells are not only simply a remnant of embryonic notochordal cells, but have also an important influence on the well-being of the whole disc. The biological treatment strategies aim to regenerate the disc by either trying to improve the micro-enviroment within the disc or to increase the popoulation of the nucleus pulposus, which includes transplanting mesenchymal stem cellsto differentiate into nucleus-l ike cells in the degenerated intervertebral disc. Conclusion Nucleus pulposus cells or ucleus pulposus l ike cells based cell transplantation methods prove to be a promising and real istic approach for the intervertebral disc regeneration.
Objective The senescence and death of nucleus pulposus (NP) cells are the pathologic basis of intervertebral disc degeneration (IVD). To investigate the molecular phenotypes and senescent mechanism of NP cells, and to identify the method of alleviating senescence of NP cells. Methods The primary NP cells were harvested from male SpragueDawley rats (8-10 weeks old); the hypoxia inducible factor 1α (HIF-1α), HIF-1β, matrix metalloproteinase 2 (MMP-2), andcollagen type II as phenotypic markers were identified through immunocytochemical staining. RT-PCR and Western blot were used to test the silencing effect of NP cells after the NP cells were transfected with p53 and p21 small interference RNA (siRNA). Senescence associated-β-galactosidase (SA-β-gal) staining was used to test the senescence of NP cells, flow cytometry to test the change of cell cycle, the growth curve analysis to test the NP cells prol iferation. Results Immunocytochemical staining showed that NP cells expressed HIF-1α, HIF-1β, MMP-2, and collagen type II. RT-PCR and Western blot showed that the relative expressions of mRNA and protein of p53 and p21 were significantly inhibited in NP cells at passage 35 after transfected with p53 and p21 siRNA. The percentage of SA-β-gal-positive NP cells at passage 35 was significantly higher than that at passage 1 (P lt; 0.001). And the percentage of SA-β-gal-positive NP cells in the p53 siRNA transfection group and p21 siRNA transfection group were significantly lower than that in control group (Plt; 0.001). The flow cytometry showed that the G1 phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group was significantly shorter than that in control group (P lt; 0.05), but the S phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group were significantly longer than that in control group (P lt; 0.05). In addition, the growth curve showed that the growth rate of NP cells could be promoted after transfection of p53 and p21 siRNA. Conclusion The senescence of NP cells can be alleviated by silencing of p53 and p21. The effect of alleviating senescence can even ameliorate the progress of IVD and may be a useful and potential therapy for IVD.
ObjectiveTo investigate the expression and correlation of hypoxia inducible factor 1α (HIF-1α) and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells under hypoxia in vitro.MethodsThe nucleus pulposus cells were extracted from the nucleus pulposus of healthy adult Sprague Dawley rats and passaged. The 3rd generation cells were identified by HE staining and collagenase type Ⅱ immunofluorescence staining and randomly divided into 4 groups. The cells in group A were cultured for 8 hours under normal oxygen condition (37℃, 5%CO2, 20%O2); the cells in group B were cultured for 8 hours under hypoxia condition (37℃, 5%CO2, 1%O2); the cells in group C were transfected with HIF-1α-small interfering RNA and cultured for 8 hours under hypoxia condition; and the cells in group D were cultured with autophagy inhibitor 3-MA for 8 hours under hypoxia condition. Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to detect the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in all groups.ResultsHE staining of the 3rd generation nucleus pulposus cells showed that the cytoplasm was light pink and the nucleus was blue black, and the collagenase type Ⅱ immunofluorescence staining was positive. Western blot and qRT-PCR results showed that the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group B were significantly higher than those in group A (P<0.05); the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group C were significantly lower than those in group B (P<0.05). There was no significant difference in the relative expression of HIF-1α protein and gene between groups B and D (P>0.05); while the relative expressions of Beclin1 and LC3B proteins and genes in group D were significant lower than those in group B (P<0.05).ConclusionHypoxia can induce the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells, and HIF-1α in rat nucleus pulposus cells under hypoxia is related to the expression of autophagy related molecules, that is, down-regulation of HIF-1α can significantly reduce the expression of autophagy related molecules, while the down-regulation of autophagy levels under hypoxia has no or little effect on the expression of HIF-1α.
Objective To investigate the effects of in-vitro monolayer culture and three-dimensional (3-D) alginate microsphere culture on the differentiation of normal human nucleus pulposus cells (NPCs), and to discuss the regulatory mechanism of restoring the phenotype of dedifferentiated NPCs by culturing resveratrol (RES) in 3-D alginate microsphere. Methods Normal human nucleus pulposus tissues were harvested for culture and identification of NPCs from 6 patients with burst lumbar vertebra fracture. NPCs at passages 1, 3, 5, and 7 in the in-vitro monolayer culture were harvested to observe the morphology, cell aging, and proteoglycan expression. The cell proliferation rates of NPCs at passage 1 in-vitro in monolayer culture and in 3-D alginate microsphere culture were detected. NPCs at passage 7 were randomly divided into 3-D alginate microsphere control group (group A), RES group (group B), silent mating type information regulation 2 homolog 1 (SIRT1)- small interfering RNA (siRNA) + RES group (group C), and negative control-siRNA + RES group (group D); and NPCs in the in-vitro monolayer culture was monolayer control group (group E). After corresponding treatment, Western blot was used for determining the protein expressions of SIRT1, Aggrecan, and collagen type II; real-time fluorescence quantitative PCR was used for detecting SIRT1 mRNA expression. Results The cultured cells were identified to be NPCs. Morphological observation, senescence-associated β-galactosidase (SA-β-gal) staining, and toluidine blue staining showed that dedifferentiation of normal NPCs tended to occur under continuous in-vitro monolayer culture, which was more obvious with increase of passage number. NPCs in 3-D alginate microsphere culture showed significantly lower proliferation rate than NPCs in the in-vitro monolayer culture (P lt; 0.05), but it could significantly improve the protein expressions of collagen type II and Aggrecan in dedifferentiated NPCs, showing significantly difference between groups E and A (P lt; 0.05). The protein expressions of SIRT1, collagen type II, and Aggrecan in group B were significantly improved when compared with that in group A (P lt; 0.05). Real-time fluorescence quantitative PCR and Western blot showed that the expressions of SIRT1 mRNA and proteins in group C were significantly inhibited after transfected with SIRT1-siRNA when compared with those in groups B and D (P lt; 0.05), and the protein expressions of collagen type II and Aggrecan in group C were significantly lower than those in groups B and D (P lt; 0.05). Conclusion Continuous in-vitro monolayer culture could efficiently cultivate numerous seeding NPCs, but it is liable to dedifferentiate. In 3-D alginate microsphere culture, RES could restore the phenotype of dedifferentiated NPCs and synthesize more extracellular matrix, which is related to the regulation of SIRT1.
ObjectiveTo investigate the expression of p16INK4a in nucleus pulposus (NP) and to clarify its relationship with intervertebral disc degeneration so as to provide evidence for biological repair of intervertebral disc. MethodsThe NP specimens were obtained from 17 patients with intervertebral disc degeneration undergoing discectomy, who aged 40-50 years (mean, 45.4 years). Based on the preoperative MRI, there were 10 cases of grade Ⅲ degeneration, and 7 cases of grade IV degeneration. Cell senescence was evaluated by detecting senescence-associated β-galactosidase (SA-β-gal) activity. Senescence marker (p16INK4a) and disc degeneration markers [A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS 5), Aggrecan, and Sryrelated HMG box transcri ption factor 9 (Sox-9)] were determined in the NP specimens with immunohistochemistry and Western blot. The correlation between ADAMTS 5 and p16INK4a was analyzed. ResultsClustered distribution of green SA-β-gal-positive cells was seen in the NP with grade Ⅲ and IV degeneration. A few single round SA-β-gal-positive NP cells (NPCs) wrapped by the layered extracellular matrix were also seen in the NP with grade Ⅲ degeneration. It was difficult to see single distribution of NPCs in the NP with grade IV degeneration. The percentage of SA-β-gal-positive cells was 22.7%±5.4% and 37.1%±7.6% in the NP with grade Ⅲ and IV degeneration respectively, showing significant difference (t=-9.666, P=0.000). The percentages of p16INK4a-positive and ADAMTS 5-positive NPCs in the NP with grade IV degeneration were significantly higher than those with grade Ⅲ degeneration (P<0.05). The percentages of Aggrecan-positive and Sox-9-positive NPCs in the NP with grade IV degeneration were significantly lower than those in the NP with grade Ⅲ degeneration (P<0.05). The protein expressions of Aggrecan and Sox-9 in the NP with grade IV degeneration were significantly lower than those in the NP with grade Ⅲ degeneration (P<0.05). The NP with grade IV degeneration showed significantly higher protein expressions of p16INK4a and ADAMTS 5 (P<0.05). Importantly, there was a good correlation between p16INK4a and ADAMTS 5 protein expressions (r=0.908, P=0.000). ConclusionPremature senescent NPCs increase in the NP with the advancing disc degeneration. The expression of p16INK4a and its association with degeneration grades suggest that the p16INK4a may play a significant role in the pathogenesis of intervertebral disc degeneration.
ObjectiveTo isolate nucleus pulposus cells (NPCs) from the caudal and lumbar intervertebral disc of rat, and to identify the morphology and to compare the characteristics. MethodsThe whole spine was separated from 8-week-old Sprague Dawley rats under the sterile conditions. NPCs of different segments (lumbar group: L1,2-L6, S1; caudal group: C1,2-C17,18) were cultured by adherent cultivation approach. Cellular morphologic change was noted by HE staining and continuous observation under inverted phase contrast microscope. Besides, the aggrecan and collagen type Ⅱexpression were examined by toluidine blue and immunocytochemistry staining respectively. The total protein contents, senescence level, and the cell viability of passage 1-5 (P1-5) were detected. The growth curves of the P1 cells in lumbar and caudal groups were determined by cell counting kit 8. ResultsThe NPCs were isolated and identified successfully. The adherence time of the primary cells (the cell fusion reached 90%) in lumbar group was significantly longer than that in caudal group in primary generation (P<0.05). HE staining showed that cytoplasm was pink with the blue nucleus. Lumbar disc NPCs were spindle. The larger caudal disc NPCs were polygonal or irregular. Toluidine blue staining showed that the proteoglycan was stained as blue. In the cytoplasm of cells, collagen type Ⅱwas stained as brown surround the blue-black nucleus. The cell viability had no significant difference between lumbar and caudal groups and between different passages in the same group (P>0.05). The caudal disc NPCs reached their logarithmic growth phase after 3 days of culture, while the cells in lumbar segments did after 4-5 days of culture. The cell proliferation in caudal segments was more than that in lumbar segments at 3-9 days (P<0.05). The difference in the total protein contents was not significant between cells at P1-5 in 2 groups (P>0.05), and the caudal disc NPCs had higher protein contents than lumbar disc NPCs (P<0.05). There was no significant difference in cell senescence rate between cells at P1, P2, and P3 in 2 groups (P>0.05), but significant difference was shown in senescence rate between 2 groups in cells at P4 and P5 (P<0.05). ConclusionCaudal disc NPCs have a better status, which is more suitable for experiment as a seed cell than the lumbar disc NPCs in the same generation.