scholarly journals Effect of Lentivirus-mediated GDF5 Transfection on Differentiation of Rabbit Nucleus Pulposus Mesenchymal Stem Cells

2020 ◽  
Author(s):  
kun zhu ◽  
Rui Zhao ◽  
Yuchen Ye ◽  
Gang Xu ◽  
Changchun Zhang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Nucleus Pulposus ◽  
Lentiviral Vector ◽  
Intervertebral Discs ◽  
Degenerative Disease ◽  
The Other ◽  
Control Group ◽  
Nucleus Pulposus Cells ◽  
Qrt Pcr

Abstract Background: Disc degenerative disease is a common senile degenerative disease, which seriously affects the quality of life of patients.The purpose of this study is to observe the biological and cytological characteristics of rabbit nucleus pulposus mesenchymal stem cells (NPMSCs), and to determine the effect of growth differentiation factor 5(GDF5) on the differentiation of rabbit NPMSCs by lentivirus transfection.Methods: In vitro culture model of rabbit NPMSCs was established and NPMSCs cells were identified by flow cytometry (FCM)and quantitative real-time PCR(qRT-PCR). Then NPMSCs were divided into three groups: lentiviral vector carrying GDF5 was used to transfect NPMSCs, to determine the transfection rate, which was recorded as transfection group, and the NPMSCs transfected with ordinary lentiviral vector was recorded as control group, NPMSCs without processing was recorded as normal group. FCM, qRT-PCR and Western Blot(WB) were used to detected the change of NPMSCs.Results: The transfected NPMSCs by GDF5 became longer and narrower, and the cell density decreased,and the positive rate of GDF5 in the transfected group was significantly higher than that in the other two groups (P<0.05). The mRNA expression of KRT8, KRT18, KRT19 in the transfected group was significantly higher than the other two groups(P<0.05),the result of WB were the same to qRT-PCR. Conclusions: GDF5 can induce the differentiation of NPMSCs and repair degenerative intervertebral discs. Lentiviral vector carrying GDF5 can be integrated into the chromosome genome of NPMSCs and promote differentiation of NPMSCs into nucleus pulposus cells(NPCs).

scholarly journals Effect of lentivirus-mediated growth and differentiation factor-5 transfection on differentiation of rabbit nucleus pulposus mesenchymal stem cells

2020 ◽  
Author(s):  
Kun Zhu ◽  
Rui Zhao ◽  
Yuchen Ye ◽  
Gang Xu ◽  
Changchun Zhang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Nucleus Pulposus ◽  
Lentiviral Vector ◽  
The Other ◽  
Nucleus Pulposus Cells ◽  
Disc Disease ◽  
Qrt Pcr ◽  
Differentiation Factor ◽  
Age Related

Abstract Background：Intervertebral disc degeneration (IDD) is a natural progression of age-related process. Degenerative disc disease (DDD) is a pathologic condition associated with IDD that has been one of the most common causes of chronic low back pain, which can have a severe impact on patients’ quality of life. The purpose of this study is to observe the biological and cytological characteristics of rabbit nucleus pulposus mesenchymal stem cells (NPMSCs), and to determine the effect of growth and differentiation factor-5 (GDF-5) on the differentiation of rabbit NPMSCs transducted with lentivirus vector.Methods: In vitro culture model of rabbit NPMSCs was established and NPMSCs were identified by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR). Subsequently, NPMSCs were randomly divided into three groups: the lentiviral vector carrying GDF-5 gene used to transfect NPMSCs was recorded as transfection group; the NPMSCs transfected with an ordinary lentiviral vector was recorded as control virus group; the NPMSCs alone was normal group. FCM, qRT-PCR and Western Blot (WB) were used to detect the change of NPMSCs.Results: The transfected NPMSCs by GDF-5 gene displayed elongated shape, the cell density decreased, and the positive rate of GDF-5 in the transfected group was significantly higher than that in the other two groups (P < 0.05). The mRNA expression of KRT8, KRT18, and KRT19 in the transfected group was significantly higher in comparison with the other two groups (P < 0.05), and the result of WB was consistent with that of qRT-PCR.Conclusions: GDF-5 can induce the differentiation of NPMSCs and repair degenerative intervertebral disc. Lentiviral vector carrying GDF-5 gene can be integrated into the chromosome genome of NPMSCs and promote differentiation of NPMSCs into nucleus pulposus cells (NPCs).

scholarly journals Effect of lentivirus-mediated growth and differentiation factor-5 transfection on differentiation of rabbit nucleus pulposus mesenchymal stem cells

2022 ◽  
Vol 27 (1) ◽  
Author(s):  
Kun Zhu ◽  
Rui Zhao ◽  
Yuchen Ye ◽  
Gang Xu ◽  
Changchun Zhang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Nucleus Pulposus ◽  
Lentiviral Vector ◽  
The Other ◽  
Cell Phenotype ◽  
Mrna Levels ◽  
Qrt Pcr ◽  
Differentiation Factor ◽  
Gdf5 Gene

Abstract Background Intervertebral disc degeneration (IDD) is a natural progression of age-related processes. Associated with IDD, degenerative disc disease (DDD) is a pathologic condition implicated as a major cause of chronic lower back pain, which can have a severe impact on the quality of life of patients. As degeneration progression is associated with elevated levels of inflammatory cytokines, enhanced aggrecan and collagen degradation, and changes in the disc cell phenotype. The purpose of this study was to investigate the biological and cytological characteristics of rabbit nucleus pulposus mesenchymal stem cells (NPMSCs)—a key factor in IDD—and to determine the effect of the growth and differentiation factor-5 (GDF5) on the differentiation of rabbit NPMSCs transduced with a lentivirus vector. Methods An in vitro culture model of rabbit NPMSCs was established and NPMSCs were identified by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR). Subsequently, NPMSCs were randomly divided into three groups: a transfection group (the lentiviral vector carrying GDF5 gene used to transfect NPMSCs); a control virus group (the NPMSCs transfected with an ordinary lentiviral vector); and a normal group (the NPMSCs alone). FCM, qRT-PCR, and western blot (WB) were used to detect the changes in NPMSCs. Results The GDF5-transfected NPMSCs displayed an elongated shape, with decreased cell density, and significantly increased GDF5 positivity rate in the transfected group compared to the other two groups (P < 0.01). The mRNA levels of Krt8, Krt18, and Krt19 in the transfected group were significantly higher in comparison with the other two groups (P < 0.01), and the WB results were consistent with that of qRT-PCR. Conclusions GDF5 could induce the differentiation of NPMSCs. The lentiviral vector carrying the GDF5 gene could be integrated into the chromosome genome of NPMSCs and promoted differentiation of NPMSCs into nucleus pulposus cells. Our findings advance the development of feasible and effective therapies for IDD.

scholarly journals Marrow-Derived Mesenchymal Stem Cells Transfected with Survinvin Gene Protect Kidney from ischemia-reperfusion Injury in Rats

2021 ◽  
Author(s):  
Yang Xiaofeng ◽  
Qianqian Wang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Renal Tubule ◽  
Ischemia Reperfusion Injury ◽  
Lentiviral Vector ◽  
Ischemia Reperfusion ◽  
Renal Ischemia ◽  
The Self ◽  
The Other ◽  
Control Group

Abstract Objective: To investigate the survival ability of bone marrow Mesenchymal stem cells (MSCs) transfected with survinvin gene in the microenvironment of renal ischemia,and to study the ability and mechanism of repairing renal ischemia-reperfusion injury in rats.Method: Mesenchymal stem cells (MSCs) from bone marrow of male Sprague–Dawley rat were infected with the self‐inactive lentiviral vector and transfected with the Survinvin gene recombinant vector and then EGFP-tagged. After amplification and culture, they were detected by green fluorescence and then retained.48 specific pathogen-free C57BL/6J mice were randomly divided into 4 groups of 12 each. Rats in the control group were only surgically exposed. The other 3 groups were surgically exposed and the bilateral renal arteries were clamped for 45 minutes to restore blood supply, and models of renal ischemia-reperfusion were established. There were control group,ischemia reperfusion group(Marked as IR group), empty virus transfection transplantation group(Marked as MSCs group) or survinvin gene transfection transplantation group(Marked as SVV/MSCs group), and sequentially injected with normal saline,normal saline,1×106 MSCs infected with the self‐inactive lentiviral vector or 1×106 survivin gene-expressing MSCs. At different time points of 1d, 3d, 7d, 14d, collect serum to test blood urea nitrogen detection, to cut the rat kidney section for quantitative analysis, HE staining to observe renal issues changes and the degree of renal tubular damage and IL-10 by using ELISA detection. Result: The MSCs with resuscitation and expansion culture had strong proliferation and good fluorescence. The creatinine urea nitrogen level in the MSCs group and SVV/MSCs group was significantly lower than that in the IR group and control group (p<0.01 or p<0.05). The pathological damage score of HE staining in the kidney was lighter in the stem cell transplantation group, and the SVV/MSCs group was significantly lower than the other two groups (p<0.01 or p<0.05). On the 3rd and 14th day, the number of transplanted cells in the kidney tissue was much higher in the SVV/MSCs group than in the MSCs group. The MSCs expressing EGFP were mainly distributed around the glomerulus, the small vessel inner wall, and the interstitial between the renal tubule and the renal tubule. However, MSCs expressing EGFP were hardly seen on the inner wall of the renal tubule. The levels of protective factors IL-10 increased after renal ischemic injury. SVV/MSCs group was also significantly more than IR group or MSCs group (P<0.01 or p<0.05). And there was no statistical difference from the normal control group on the 14th day.Conclusion: Transfection of Survinifin gene can increase the survival ability of MSCs in ischemic kidney. After transplantation, MSCs are not directly differentiated into injured tubular endothelial cells, which further promote the repair of kidney damage through its strong paracrine effect.

scholarly journals Fibroblast Transplantation Results to the Degenerated Rabbit Lumbar Intervertebral Discs

2017 ◽  
Vol 11 (1) ◽  
pp. 404-416 ◽  
Author(s):  
Ibrahim Halil Ural ◽  
Kerem Alptekin ◽  
Aysegul Ketenci ◽  
Seyhun Solakoglu ◽  
Hasan Alpak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Intervertebral Discs ◽  
Degenerative Disease ◽  
Apoptotic Cells ◽  
Histological Findings ◽  
Cultured Fibroblasts ◽  
Radiological Changes

Background: Our study is an analysis of the histological and radiological changes in degenerated lumbar intervertebral discs, after transplantation of fibroblasts in rabbits. With that study we aimed to show the viability of the fibroblasts injected to the degenerated discs, and observe their potential for further studies. Method: The apoptosis of the cell is one of the factors at the disc degeneration process. Fibroblasts may act as mesenchymal stem cells at the tissue to which they are injected and they may replace the apoptotic cells. The nucleus pulposus of the discs from eight rabbits were aspirated under scopic guidance to induce disc degeneration. Results: One month later, cultured fibroblasts, which had been taken from the skin, were injected into the disc. The viability and the potential of the injected cells for reproduction were studied histologically and radiologically. Cellular formations and organizations indicating to the histological recovery were observed at the discs to which fibroblasts were transplanted. The histological findings of the discs to which no fibroblasts were transplanted, did not show any histological recovery. Radiologically, no finding of the improvement was found in both groups. The fibroblasts injected to the degenerated discs are viable. Conclusion: The findings of improvement, observed in this study, suggest that fibroblast transplantation could be an effective method of therapy for the prevention or for the retardation of the degenerative disease of the discs.

scholarly journals Bi-Directional Exchange of Membrane Components Occurs during Co-Culture of Mesenchymal Stem Cells and Nucleus Pulposus Cells

PLoS ONE ◽  
2012 ◽  
Vol 7 (3) ◽  
pp. e33739 ◽  
Author(s):  
Sandra Strassburg ◽  
Nigel W. Hodson ◽  
Patrick I. Hill ◽  
Stephen M. Richardson ◽  
Judith A. Hoyland
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Nucleus Pulposus ◽  
Nucleus Pulposus Cells ◽  
Membrane Components

Transplantation of mesenchymal stem cells and nucleus pulposus cells in a degenerative disc model in rabbits: a comparison of 2 cell types as potential candidates for disc regeneration

2011 ◽  
Vol 14 (3) ◽  
pp. 322-329 ◽  
Author(s):  
Ganjun Feng ◽  
Xianfeng Zhao ◽  
Hao Liu ◽  
Huina Zhang ◽  
Xiangjun Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Signal Intensity ◽  
Disc Height ◽  
Nucleus Pulposus Cells ◽  
Disc Regeneration ◽  
Disc Model

Object The aim of this study was to compare transplanted mesenchymal stem cells (MSCs) with nucleus pulposus cells (NPCs) in a degenerative disc model in rabbits to determine the better candidate for disc cell therapy. Methods Mesenchymal stem cells and NPCs were transplanted in a rabbit model of disc degeneration. Changes in disc height, according to plain radiography, T2-weighted signal intensity on MR imaging, histology, sulfated glycosaminoglycan (sGAG)/DNA, and associated gene expression levels, were evaluated among healthy controls without surgery, sham-operated animals in which only disc degeneration was induced, MSC-transplanted animals, and NPC-transplanted animals for a 16-week period. Results Sixteen weeks after cell transplantation, in the MSC- and NPC-transplanted groups, the decline in the disc height index was reduced and T2-weighted signal intensity increased compared with the sham-operated group. Safranin O staining showed a high GAG content, which was also supported by sGAG/DNA assessment. Disc regeneration was also confirmed at the gene expression level using real-time polymerase chain reaction. However, no significant differences in expression were found between the NPC- and MSC-transplanted groups. Conclusions Study data showed that MSC transplantation is effective for the treatment of disc degeneration and seems to be an ideal substitute for NPCs.

scholarly journals Bioinformatics analysis and identification of circular RNAs promoting the osteogenic differentiation of human bone marrow mesenchymal stem cells on titanium treated by surface mechanical attrition

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9292
Author(s):  
Shanshan Zhu ◽  
Yuhe Zhu ◽  
Zhenbo Wang ◽  
Chen Liang ◽  
Nanjue Cao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Human Bone ◽  
Cell Counting ◽  
Circular Rnas ◽  
Control Group ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Mechanical Attrition

Background To analyze and identify the circular RNAs (circRNAs) involved in promoting the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs) on titanium by surface mechanical attrition treatment (SMAT). Methods The experimental material was SMAT titanium and the control material was annealed titanium. Cell Counting Kits-8 (CCK-8) was used to detect the proliferation of hBMSCs, and alkaline phosphatase (ALP) activity and alizarin red staining were used to detect the osteogenic differentiation of hBMSCs on the sample surfaces. The bioinformatics prediction software miwalk3.0 was used to construct competing endogenous RNA (ceRNA) networks by predicting circRNAs with osteogenesis-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The circRNAs located at the key positions in the networks were selected and analyzed by quantitative real-time PCR (QRT-PCR). Results Compared with annealed titanium, SMAT titanium could promote the proliferation and osteogenic differentiation of hBMSCs. The total number of predicted circRNAs was 51. Among these, 30 circRNAs and 8 miRNAs constituted 6 ceRNA networks. Circ-LTBP2 was selected for verification. QRT-PCR results showed that the expression levels of hsa_circ_0032599, hsa_circ_0032600 and hsa_circ_0032601 were upregulated in the experimental group compared with those in the control group; the differential expression of hsa_circ_0032600 was the most obvious and statistically significant, with a fold change (FC) = 4.25 ± 1.60, p-values (p) < 0.05.

scholarly journals A20 of nucleus pulposus cells plays a self-protection role via the nuclear factor-kappa B pathway in the inflammatory microenvironment

2020 ◽  
Vol 9 (5) ◽  
pp. 225-235
Author(s):  
Xin Peng ◽  
Cong Zhang ◽  
Jun-Ping Bao ◽  
Lei Zhu ◽  
Rui Shi ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Messenger Rna ◽  
Nuclear Factor Kappa B ◽  
Intervertebral Discs ◽  
Control Group ◽  
Nuclear Factor ◽  
Nucleus Pulposus Cells ◽  
Necrosis Factor Alpha ◽  
Nuclear Factor Kappa ◽  
Tnf Α

Aims Inflammatory response plays a pivotal role in the pathophysiological process of intervertebral disc degeneration (IDD). A20 (also known as tumour necrosis factor alpha-induced protein 3 (TNFAIP3)) is a ubiquitin-editing enzyme that restricts nuclear factor-kappa B (NF-κB) signalling. A20 prevents the occurrence of multiple inflammatory diseases. However, the role of A20 in the initiation of IDD has not been elucidated. The aim of the study was to investigate the effect of A20 in senescence of TNF alpha (TNF-α)-induced nucleus pulposus cells (NPCs). Methods Immunohistochemical staining was performed to observe the expression of A20 in normal and degenerated human intervertebral discs. The NPCs were dissected from the tail vertebrae of healthy male Sprague-Dawley rats and were cultured in the incubator. In the experiment, TNF-α was used to mimic the inflammatory environment of IDD. The cell viability and senescence were examined to investigate the effect of A20 on TNF-α-treated NPCs. The expression of messenger RNA (mRNA)-encoding proteins related to matrix macromolecules (collagen II, aggrecan) and senescence markers (p53, p16). Additionally, NF-κB/p65 activity of NPCs was detected within different test compounds. Results The expression of A20 was upregulated in degenerate human intervertebral discs. The A20 levels of NPCs in TNF-α inflammatory microenvironments were dramatically higher than those of the control group. TNF-α significantly decreased cell proliferation potency but increased senescence-associated beta-galactosidase (SA-β-Gal) activity, the expression of senescence-associated proteins, the synthesis of extracellular matrix, and G1 cycle arrest. The senescence indicators and NF-κB/p65 expression of A20 downregulated group treated with TNF-α were significantly upregulated compared to TNF-α-treated normal NPCs. Conclusion A20 has a self-protective effect on the senescence of NPCs induced by TNF-α. The downregulation of A20 in NPCs exacerbated the senescence of NPCs induced by TNF-α. Cite this article: Bone Joint Res. 2020;9(5):225–235.

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