In Vitro Pellet Bilaminar Coculture Of Human Adipose-derived Stem Cells And Nucleous Pulposus Cells From Degenerative Disc Tissue And The Effect Of LMP-1Gene On Their Interactions

Objective: To explore the methods of ilsolating,culturing and identification of nucleous pulposus cellsfrom degenerative disc tissue and the effect of pellet culturing on its biologic activity. Methods: discsamples from25patients with degenerative disc diseases were collected to extract NPCs by use of0.025%collagenase â…¡for4hours. During primary culturing and passaging, the morphological changesof nucleous pulposus cells were observed with inverted microscope. The chondrogeniccharacter(collagenâ…¡ and aggrecan) of nucleous pulposus cells were identified by immnofluorescencestaining. The gene expression of aggrecan,â…¡ a nd SOX-9were detected by RT-PCR assay. Result:human nucleous pulposus cells from degenerative disc tissue can be achieved by digestion of0.025%collagenase â…¡. It took an average of8days for the spindle or polygonal primary NPC cells to achieveadherence and about4weeks to reach a confluence of80%. All NPC cells had an expression ofcollagen â…¡a nd aggrecan as detected by immnofluorescence staining. NPC cells cultured under pelletcondition had more mRNA level expression of aggrecan,collageâ…¡ and SOX-9gene than monolayerculturing. Conclusion: Human NPC cells can be isolated from degenerative disc tissue by use of0.025%collagenase â…¡. They had expression ofthe chdrogenic gene markers such as aggrecan,collageâ…¡and SOX-9. Pellet culturing can promote the maintance of chondrogenic phenotype by NPCcells. Objective: To explore the methods of ilsolating,culturing and identification of mesenchymal stem cellsfrom fat tissue and the effect of pellet culturing condition on its chondrogenic differentiation potentials.Methods: human ADSCs (Adipose derived stem cells)from subcutaneous fat tissue of10patients withcesarean section surgery were extracted using0.075%collagenase I for1hour. Morphologic charactersof ADMSC cells were observed with inverted microscope. Flow cytometry assaying were used to detectthe expression of cell surface markers such as CD90,CD29,CD105,CD34,CD45and HLA-DR. Afteridentification, human ADSCs were induced by specific induding medium under monolayer cultureconditions to detect its differentiating potentials towards adipose, osteocyte.For chondrogenicdifferentiation, two kinds of culture methods were used: monolayer or pellet culturing. After induction,Oil red,Alizarin red S,Safranin'O,Toluidine blue staining and immunofluorescence staining ofaggrecan, collagen â…¡ were used to evaluate its differentiation. Besides, RT-PCR assay were taken toinvestigate the expression of chondrogenic marker genes by ADSCs. Result: human adipose-derivedmesenchymal stem cells from subcutaneous fat tissue can be extracted by digestion of0.075%collagenase â… . It took an average of3days for the polygonal primary ADSCs to achieve adherence andabout9days to reach a confluence of80%. After3passagings, the polygonal cells gradually changedinto spindle-like cells, which demonstrated a swirling clustering character. They had positive expressionof CD90,CD29,CD105and low or negative expression of CD34,CD45,HLA-DR. The cells undermonolayer culturing conditions had positive results of Oil red,Alizarin red S,Safranin'O staining afteran induction period of21days. The cells which underwent chondrogenic differentiation under pelletculturing conditions had positive results of HE, Masson trichrome, Toluidine blue staining andimmunofluorescence staining of aggrecan, collage â…¡. RT-PCR demonstrated that ADSCs had moreexpression of chondrogenic gene when induced under pellet culture. Conclusion: Humanadipose-derived mesenchymal stem cells can be isolated from subcutaneous fat tissue by use of0.075%collagenase â… . They had the multilineage differentiation poetentials such as adipose,osteocyte andchondrocyte. Pellet culturing can promote the chondrogenic differentiation of ADSC cells. Objective: To design and produce lentivirus carrier containing human LMP-1gene for its use intransduction of human adipose stem cell. Methods: Based on mRNA sequence of human LMP-1gene inGeneBank(NM₀05451), human LMP-1gene coding region fragment was obtained by PCR methodand then cloned into plasmid of Pwpxl-MOD to produce Pwpxl-MOD/LMP-1. After identification ofsequencing and restriction analysis, the plasmid of Pwpxl-MOD/LMP-1was co-transfected withlentivirus-packaing plasmid into293T cells to obtain recombinant virus containing LMP-1gene. Afterthat, viruses containing LMP-1-GFP gene or GFP gene were used in the infection of human adiposestem cell, which give rise to human adipose stem cells expressing LMP-1-GFP gene or GFP gene. Thenthe two kinds of cells were induced by chondrogenic medium containing TGF-β3or basal chondrogenicmedium without TGF-β3. The total induction period was21days. Result: By use of the methodsmentioned above, a high titer of2x109TU/ml lentivirus containing human LMP-1gene can be obtained.The human adipose stem cells expressing LMP-1-GFP gene induced by chondrogenic mediumcontaining TGF-β3had the most expression of chondrogenic genes, the second was the adipose stemcells expressing LMP-1-GFP gene induced by basal chondrogenic medium without TGF-β3, and theweakest was the the adipose stem cells expressing GFP gene induced by chondrogenic mediumcontaining TGF-β3. Conclusion: Lentivirus can be used as an ideal carrier of human LMP-1gene. TheLMP-1gene had more potentials in the chondrogenic induction of human adipose stem cells whencompared to TGF-β3. Besides, their potentials can be combined to get the most effect. Objectives: To investigate the effect of LMP-1gene on the interactions between human adipose mesenchymal stem cells and nucleous pulposus cells in bilaminar pellet coculture system in vitro.Mehods: Human ADSCs cells of3th passage were modified with LMP-1-GFP or GFP gene by use oflentivirus. ADSCs cells expressing LMP-1-GFP or GFP gene and human NP cells were centrifuged in15ml centrifuge tubes in succession(ADSCs inside and NPCs outside) with a ratio of75%:25%. Sevenexperimental groups were designed as follows:100%NPCs pellets, pellets containing100%ADSCsexpressing LMP-1-GFP gene, pellets containg25%ADSCs expressing LMP-1-GFP gene in/75%NPCsout,100%ADMSCs pellets expressing GFP gene, pellets containg25%ADSCs expressing GFP genein/75%NPCs out, pellets containg25%ADSCs expressing LMP-1-GFP gene mixed with75%NPCs andpellets containing25%ADSCs expressing GFP gene mixed with75%NPCs. The sGAG content of eachgroup were measured by Alican blue assay. ADSCs expressing GFP or LMP-1-GFP gene in coculturesystem were isolated by FACS method. After that, expression of chondrogenic marker genes such asaggrecan,collagenâ…¡ a nd SOX-9were detected by RT-PCR and Western-blot assay. Result: By thesecond week, The sGAG content of the bilaminar pellet coculture group was significantly greater thanthat of mixed coculturing groups(P<0.05). The coculture groups containing ADSCs expressingLMP-1-GFP gene had more sGAG content than ADSCs expressing GFP gene (P<0.05). By the firstweek of culture, the NPCs in pellet coculture had more expression of genes such as typeâ…¡collagen,SOX-9,aggrecan than NPCs in single pellet culture groups(P<0.05), but no evidence of difference waspresent between mixed and bilaminar pellet coculture (P>0.05). However, the NPCs in pellet coculturecontaing ADSCs expressing LMP-1-GFP gene had more expression of typeâ…¡collagen,SOX-9,aggrecan genes than NPCs in pellet coculture containg ADSCs expressing GFP gene(P<0.05). Besides,NPCs in all pellet culture groups had a gradual decrease in expression of typeâ… collagengene(P<0.05).On the other hand, by the second week, the Human ADSCs in pellet coculture groups hadmore expression of typeâ…¡collagen, SOX-9,aggrecan genes than ADSCs in single pellet culturegroups. Among these coculture groups, human ADSCs in bilaminar pellet coculture groups had moreexpression of above genes than ADSCs in mixed pellet coculture groups(P<0.05). Besides, ADSCsexpressing LMP-1-GFP gene in coculture groups had more expression of typeâ…¡,collagen SOX-9,aggrecan genes than ADSCs expressing GFP gene(P<0.05). A marked increase was noted inexpression of type â… gene by100%ADSCs pellets whereas a significant increase was noted in ADSCs from coculturing system.(P<0.05). Conclusion: Human ADSCs can be differentiated towards nucleouspulposus-like cell in pellet coculture with NPCs and NPCs can be feeded by ADMSCs. Both Bilaminarstructure and LMP-1gene can promote this interaction.